key: cord-0006771-sx4f2jl8 authors: nan title: 6th Congress of the GTH (Gesellschaft für Thrombose- und Hämostaseforschung): Kiel, February 21–24, 1990 date: 1990-02-01 journal: Blut DOI: 10.1007/bf01720517 sha: 510927a83b5fefbef1dddc065aec27693c609a8d doc_id: 6771 cord_uid: sx4f2jl8 nan Actin determination in cells requires lysis of cells which is carried out more easily in cell suspensions compared to lysis of cells attached to artificial surfaces. Here we report our data of actin analysis in platelets adhered to plastic and of cultured endothelial cells (ec) . Analysis of the actin pools in platelets and ec was performed using the ONase I inhibition assay. Ouri~g adhesion of platelets to plastic wells (Nunclon , Denmark) a shift in the G-/F-actin equilibrium occured. An increase of 22.0 + 6.7~ (n = 5) F-actin was found compared to control washed platelets. This increase during adhesion is moderate in comparison to that found during thrombin-induced platelet aggregation. Ec from human umbilical veins were cultured on gelatin-coated p~astic dishes. Ihey contain 12.9 + 2.3 /ug actin/lO ec (n : iO), 57.4 + 9.1% were found to be G-actin and 42.6 + 9.1% F-actin. For one single determination ~ne petri dish (90 mm diameter) with about 4.5 x lO ec was necessary. Whereas lysed ec were scraped off for actin determination from the dish and transferred into Eppendorf tubes, actin analysis in platelets was quantitatively only after depolymerization of the filaments on the surface, indicating that filaments stick tightly to the plastic. Aotin exists in platelets in two main forms, the monomer or G-aotin and the filamentous polymeric form, F-actin. The equilibrium of G-to F-astin in resting platelets is maintained at a constant ratio, but upon stimulation G-actin can readily polymerize to form filaments. After irreversible aggregation induced by AOP, collagen, adrenaline, arachidonic acid, PAF or thrombin, a significant increase in F-actin was found (30-50~ more cellular F-actin compared to controls). Actin polymerization is a very fast process reaching a significant level already with shape change and well before aggregation commenced or release of serotonio was signilicant. During reversible aggregation the degree of actin polymerization is smaller and at the end of deaggregation the F-actin level nearly returned to values found in unstimulated platelets. A 23187 or ionomycin also induce actin polymerization, but the degree is smaller (12-25~ more cellular F-a~$in compared to controls). The increase in cytosolic Ca ~ induced by the ionophores precedes the actin polymerization. EDTA inhibits partially ADP-or collagen-induced actin polymerization. From both results and the finding that phorbol ester (O.B/uM PMA) leads to an increase of about l~ F-actin in pl@telets we conclude tentatively Z+ that Ca and proteinkinase C seem to be involved at least in part into the initial actin assembly process in platelets. Platelet functions in a 63 years old woman with autoantibodies against the GP IIb-IIIa complex (see A. Schirmer et al., part l) were severely impaired. Aggregation could not be induced by AOP and collagen, thrombin caused only a weak aggregation. Agglutination by ristocetin as well as adhesion were also defective. Patient's serum (i-5~ final concentration) inhibits thrombin-or collageninduced aggregation of normal platelets, q Patient's platelets contain only 473/ug actin/lO ~ platelets and an extremely low F-actin value (3Q of total platelet actin). Stimulation of these platelets with 0.i U/ml thrombin for 3 min and measurement of the G-/Factin equilibrium resulted in an increase of only 5% F-actin, whereas ADP and collagen did not induce any actin polymerization. Addition of patient's serum to normal platelets leads to an inhibition of thrombin-induced actin polymerization. Both effects of patient's serum on normal platelets (inhibition of aggregation and actin polymerization) points to receptor blockade by platelet autoantibodies found in th%patient's serum. Ca ~ movement in the patient's platelets is severely impaired after AOP or collagen stimulation whereas a normal Ca movement was induced by O.l U/ml thrombin. How-Z+ ever, normal Ca movement by thrombin but inhibited actin polymerization by this agonist does not necessarily+point to an independence of actin polymerization from Ca ~ ions, but emphasizes the receptor-mediation for actin polymerization. Classical hereditary thrombocytopathies (Glanzmann's thrombasthenia, Bernard-Sculler syndrome) are caused by deficiencies of certain platelet membrane glycoproteins (GPs). He report on patients from three families with excessive bleeding. Bleeding time was prolonged or at the upper normal limit. Patients exhibited variable degrees of defective platelet aggregation: either a complete lack of response with ADP and collagen or a decreased aggregation with desaggregation. Platelet GPs were analysed by one-and two-dimensional electrophoresis, lectin-and immunoblotting, and immunoelectrophoretic techniques. In one family the patients' platelets showing decreased aggregability in response to AOP contain only 25 -50 of GP IIb-IIIa complex. In addition, the complex migrated with an altered mobility in crossed immunoelectrophoresis. In a second family a structural abnormality of the GP IIb-IIIa complex is suggested dy the finning that platelets although containing 40 -60 ~ of the normai amount of GP IIb-IIIa complex show a complete lack of aggregation. In the third family the patient's platelets contain an abnormal GP idemtifieo as a variant of GP Ib. This variant is different from the polymorphic GP Ib forms reported so far. In conclusion, hereditary thrombopathies with similar functional defects may be caused by a variety of structural abnormalities of platelet GPs. (UFH) . In anlmal models f.he anti--tic effect is oor~ar~hle to UFH %~uLle the risk of bleeding is ~ller. indu~ by UF~ =ay be stop~,:t by ~=o~. In clotting tests a noznmlizaticn of thrombi~ time and activated partial thrcmboplastin t~ (al~) can be observed. In this study it Was investigated ix) what degree protamlne chloride antagonizes the effect of LU 47311 on plasma clotting (aPTT, bleeding t/ms a~Z ~ time) and on factor Xa. ~he investigation8 performed in 12 healthy subjects showed that the anticoagulant effects of It/ 47311 on the al~, thrombin time (%~f) and bleeding t~ are immediately and cxmpZstely antegonised by intrav~mou~ injection of prots~ chloride. The anti-factor Xa activity is neutralized h~ about 40 %, A rebound phencmsnmn of the ~Lnticc~CJu_lant effect of heparin was not observed. The results are o3m~le to other trials with Lv~H and Bhow that protsmine chloride can be applied as an antidote for the anticoagulant effects of the I~ molecular weight hel:arL~ LU 47311. The fluorescent dye thiazole orange (TO) is characterized by a large fluorescence enhancement and high quantum yield upon binding to nucleic acids, particularly RNA. In addition, the dye readily permeates live cell membranes. Whole blood samples from hematologically normal subjects and from patients with quantitative platelet disorders were stained with thiazole orange and the platelet populations were analyzed by flow eytometry. The mean percentage (+ SD) of TO-positive platelets in 50 control subjects was 8.6 + 2.8 %. In 21 thrombocytopenic patients whose bone marrow contained normal or increased numbers of megekaryooytes, the percentage of TO-positive platelets was significantly elevated to 26.9 + 10.9 % (p < 0.0001). In contrast, the percentage of positively stained platelets in 23 patients with thrombocytopenza due to underproduction of platelets did not significantly differ from controls (7.1 ~ 3,2 %). Both the sensitivity and the specificity of the assay in differentiating between these two categories of thrombocytopenia was > 95 %. In patients with thrombocythemza/thrombocytosis (n = 13), the percentage of TO-positive plateleta averaged 7.2 + 2.9 %. We conclude, that flow cytometrlc analysis of platelets after staining with TO is a sensitive and spec±fic test that rapidly prov].des information on the adequacy of platelet production and, thus, on the mechanism of thrombocytopenza. of North Carolina at Chapel Hi I I ; and M uni ci pal Hospi t al Fel dki r ch, Aust r i a Factor X(FX) "Vorarlberg" is acongenital CRM+ FX deficiency characterized clinically by a mild bleeding ten den cy. Horn ozy gou s i n di v i du als hav e a FX-act i v i t y of-less than 10% in the extrinsic system and 32% in the intrinsic system. FXantigen is 20%. To elucidate the genetic defect in FX"Vorarlberg" genomic DNAwas prepared from the leucocytes of an affected family member. Southern blot analysis of the genomio DNA cut with EcoRI and probed with a FX cDNA gave the same restriction pattern as normal DNA. The enzy m at i c am p l i f i cat i on t echni que was used t o i sol at e theexonscodingfor the FX protein. Thesewere subcloned in M13 and sequenced. Two point mutations were detected: a G4Aat bp 160 in exon II resulting In a change from Gla +14 (GAA) to Lys(AAA) ; a G-~A at bp 424 in exon V resulting in a change from Glu + f 02(GAG) to Lys( AAG). The mutations abol i shed a Taql r est r i ction sitein exon II anda Mnll sitein exon V. Todetermine wether these mutation werepresent on one or on both allels, restriction analysis of amplified exonll and exon Vfragmentswasperformed. In three members of the kindred with bleeding episodes and a FX activity of less than 10%, ahomozygoyusstatefor the defect in exon II was detected. These individuals were heterozygous for the defect in exon V. Six individuals with no bleeding tendency and only a slightly reduced FXactivity exhibited a heterozygous slate for both mutations. We therefore conclude thai the mutation in exon II, which alters aGlaresidue, is responsible for the functional defect in FX"Vorarlberg'. strate reagent containing CaCt and a thrombin specific chromogenic substrate. After a 3 min. incubation with the activator at 370C the reaction is started by adding the substrate reagent. For CTS-Neothromtin a preliminary referance range of 28-40 sec. was caLculated, intra and inter assay CVIs were found to be b~ween 1,5 and 3 % in normal plasma and 3-4 % in control plasma with prolonged aPTT. The chromogenic aPTT assay shows a good heparin sensitivity of 0.08 IU/ml heparin. The factor VIII sensitivity of CtS-Neothromtin is comparable with the estabtisted coagu[ometric reagents Pathrom-R R tin and Neothromtin . The chromogenic aPTT method, especiatLg developed for the Chromotimer s c~n also be perfomed on the clinical chemistry analyzer Cobas Fara . In conclusion, CTS-Neothromtin offers following features: 1) fibrinogen-inpendent results, 2) smaLL sample volumes 3) easy performance, 4) good comparability to coagutometric routine methods. The efficacy and safety of Low Molecular Weight Heparin (LMWH, Sandoz, FRG, -i500 aPTT units once daily + 2 placebo injections daily) and a convential Unfractionated Heparin (UFH-3xS000 I.U. daily) were compared. In a prospective double blind investigation of the prophylaxis of deep vein thrombosis undergoing major gynecologic surgery, 300 patients were randomized into two groups. Treatment was initiated 2h preoperatively in both groups and continued for 7 days. Screening for the Deep Vein Thrombosis (DVT) was performed by impedance plethysmography. The following parameters were investigated: Anti Xa-activity, aPTT, fibrinogen, D-Dimer, F VIII, AT III, Protein C, Erythrocyte aggregation and plasma visosity. Results: Two patients (1.2%) treated with LMWH and 6 patients (4%) receiving UFH developed a DVT. Haemorrhagic complications occured more olten in the UFH-group: Intraoperative high blood loss g% vs. 7.3%, blood transfusion 24~1% vs. 2!.3% drainage '31eedins. ?0.t5% vs. 27.t~% and wound haematoma lgg" vs. 12.6%. ""ne acceetance of 9-e '.MV7H-regimen was decisivly better. Concerning the results of the laboratory tests, the most striking effect of the LMWH was a significantly 2 -3 times higher Anti Xa-activity in the postoperative period in comparison to the UFH. The aPTT was only minimally affected by both groups. There results show that once daily prophylaxis is equally as effective and as safe as the three daily regimen using UFH in patients undergoing elective, major gynecologic surgery. Abteilung for Gyn~kologie und Geburtshilfe, Akademisches Lehrkrankenhaus der Universit~t Mainz, August-Bebel-Str. 5% D-6090 R ~sselsheim 16 Antithrombin III-Heoantigene als s e n s i t i v e r Marker e i n e r A k t i v i e r u n g des Gerinnungssystems E. Zimmer und E. Spanuth Boehringer Mannheim GmbH, 6800 Mannheim 31 Antithrombin III-Neoantigene sind Komplexverbindungen zwischen Antithrombin I l l (AT I l l ) und aktivierten Gerinnungsfaktoren (Proteasen wie z.B. F IIa, F Xa, F IXa etc.) Sie treten als Folge der Regulation eines Aktivierungsprozesses des H~mostasesystems durch AT I l l in der Zirkulation auf. Die Bestimmung dieser AT III-Protease-Komplexe erfolgt mit Hilfe yon monoklonalen Antik~rpern, die spezifisch das im Komplex vorliegende, modifizierte AT I l l erfassen. Eine mit F (ab')2-Fragmenten von monoklonalen Antik6rpern beschichtete Mikroelisaplatte wird mit Citratplasmaprobe inkubiert. Die AT III-Neoantigene (AT-N) werden durch diese Antik6rper gebunden. Die Menge der Antigen-AntikSrper-Komplexe wird mit Hilfe peroxidase markierter polyclonaler AT III-Antik6rper, die an noch freien antigenen Determinanten binden (Sandwhich-Komplexe) bestimmt. Mit dem ELISA AT-N werden im Gegensatz zu TAT-Bestimmungen sEmtliche Komplexe erfaBt, die im Zuge einer Aktivierung der plasmatischen Gerinnung von AT I l l mit den proteolytischen Faktoren gebildet werden. D.h. intravasale Aktivie-rungsvorg~nge sollten frQher mit hEherer Spezifit~t und Sensitivit~t erfaSt werden. Ein entsprechender Methodenvergleich zwischen ELISA AT-N und der TAT-Bestimmung an Patienten mit unterschiedlichen Grunderkrankungen wird vorgestellt. strate reagent containing CaCt and a thrombin specific chromogenic substrate. After a 3 min. incubation with the activator at 370C the reaction is started by adding the substrate reagent. For CTS-Neothromtin a preliminary referance range of 28-40 sec. was caLculated, intra and inter assay CVIs were found to be b~ween 1,5 and 3 % in normal plasma and 3-4 % in control plasma with prolonged aPTT. The chromogenic aPTT assay shows a good heparin sensitivity of 0.08 IU/ml heparin. The factor VIII sensitivity of CtS-Neothromtin is comparable with the estabtisted coagu[ometric reagents Pathrom-R R tin and Neothromtin . The chromogenic aPTT method, especiatLg developed for the Chromotimer s c~n also be perfomed on the clinical chemistry analyzer Cobas Fara . In conclusion, CTS-Neothromtin offers following features: 1) fibrinogen-inpendent results, 2) smaLL sample volumes 3) easy performance, 4) good comparability to coagutometric routine methods. The efficacy and safety of Low Molecular Weight Heparin (LMWH, Sandoz, FRG, -i500 aPTT units once daily + 2 placebo injections daily) and a convential Unfractionated Heparin (UFH-3xS000 I.U. daily) were compared. In a prospective double blind investigation of the prophylaxis of deep vein thrombosis undergoing major gynecologic surgery, 300 patients were randomized into two groups. Treatment was initiated 2h preoperatively in both groups and continued for 7 days. Screening for the Deep Vein Thrombosis (DVT) was performed by impedance plethysmography. The following parameters were investigated: Anti Xa-activity, aPTT, fibrinogen, D-Dimer, F VIII, AT III, Protein C, Erythrocyte aggregation and plasma visosity. Results: Two patients (1.2%) treated with LMWH and 6 patients (4%) receiving UFH developed a DVT. Haemorrhagic complications occured more olten in the UFH-group: Intraoperative high blood loss g% vs. 7.3%, blood transfusion 24~1% vs. 2!.3% drainage '31eedins. ?0.t5% vs. 27.t~% and wound haematoma lgg" vs. 12.6%. ""ne acceetance of 9-e '.MV7H-regimen was decisivly better. Concerning the results of the laboratory tests, the most striking effect of the LMWH was a significantly 2 -3 times higher Anti Xa-activity in the postoperative period in comparison to the UFH. The aPTT was only minimally affected by both groups. There results show that once daily prophylaxis is equally as effective and as safe as the three daily regimen using UFH in patients undergoing elective, major gynecologic surgery. Antithrombin III-Neoantigene sind Komplexverbindungen zwischen Antithrombin Ill (AT Ill) und aktivierten Gerinnungsfaktoren (Proteasen wie z.B. F IIa, F Xa, F IXa etc.) Sie treten als Folge der Regulation eines Aktivierungsprozesses des H~mostasesystems durch AT Ill in der Zirkulation auf. Die Bestimmung dieser AT III-Protease-Komplexe erfolgt mit Hilfe yon monoklonalen Antik~rpern, die spezifisch das im Komplex vorliegende, modifizierte AT Ill erfassen. Eine mit F (ab')2-Fragmenten von monoklonalen Antik6rpern beschichtete Mikroelisaplatte wird mit Citratplasmaprobe inkubiert. Die AT III-Neoantigene (AT-N) werden durch diese Antik6rper gebunden. Die Menge der Antigen-AntikSrper-Komplexe wird mit Hilfe peroxidase markierter polyclonaler AT III-Antik6rper, die an noch freien antigenen Determinanten binden (Sandwhich-Komplexe) bestimmt. Mit dem ELISA AT-N werden im Gegensatz zu TAT-Bestimmungen sEmtliche Komplexe erfaBt, die im Zuge einer Aktivierung der plasmatischen Gerinnung von AT Ill mit den proteolytischen Faktoren gebildet werden. D.h. intravasale Aktivie-rungsvorg~nge sollten frQher mit hEherer Spezifit~t und Sensitivit~t erfaSt werden. Ein entsprechender Methodenvergleich zwischen ELISA AT-N und der TAT-Bestimmung an Patienten mit unterschiedlichen Grunderkrankungen wird vorgestellt. In the course of this study, separation of plasma using the systems 'Ptasmapur Monitor' (Fa. Organon), 'Autopheresis-C' (Fa. Baxter) and 'PCS' (Fa. Haemonetios) was compared with the conventional blood bag cantrifugatlon. In 16 apherasls per method, parameters mainly reflecting the blood coagulation were examined In addition to other criteria. Blood/plasma samples were drawn from the donor before, during and after the separation run as well as from the used device during the separation and from the collected plasma. Thrombin-antithrombin-oomplexes and the fibdn(ogen)-derivatNes, soluble fibrin and flbrinopeptid A, proved to be sensitive indicators of a slight activation of the coagulation system. This activation was less pronounced by separation through cantrifugation than through filtration; however, even the filtration devices activated the coagulation system tess than common bag centrifugatlon. Furthermore, a decrease in blood coagulation factors and inhibitors were most pronounced using bag centrifugation. The number of cells remaining in the collected plasma was highest after separation by centrifugation alone, whereas less than 1/10 as many were seen using filtration methods. Considering the fact that presently most of the dally transfused fresh frozen plasma Is collected by centrlfugatlon of blood bags, the comparatively slight activation of blood coagulation by plasma separation machines seems to be negligible. However, the number of remaining cells in the collected plasma may be of greater interest: a total elimination of cells from transfused plasma should be the goal to avoid activation of blood coagulation in recipients. Abtlg.f. Transfusionsmedizin der Universit&t, 3400 G6ttingen / FRG 18 U ROKINASE-TYPE PLASMINOGEN ACTIVATOR GENE: TRANSCRIPTIONAL CONTROL OF PLASMINOGEN ACTIVATOR SYNTHESIS AND REGULATION OF EXPRESSED ACTIVITY. D. vonder Ahe, Y. Nagamine--and G. MOIler-Berghaus The genes coding for the two plasminogen activators urokinasetype (uPA) and tissue-type (tPA) have been identified. They are located on different chromosomes. Both genes are organized with separate exons. The homology between the two PAs at the amino acid level is only 40%, but the enzymes are highly similar in their basic structures, which demonstrates a close evolutionary relationship, uPA is implicated in various aspects of cellular functions such as fibrinolysis, cell migration, tissue reorganization during morphogenesis and invasive growth in both normal and pathological conditions linked to increased plasmin activity, uPA synthesis is modulated by a variety of effector molecules such as phorbol esters, growth factors, peptide and steroid hormones retinoids and others. The regulation of uPA synthesis occurs mainly at the level of gene transcription. To elucidate the mechanisms of regulation, we studied the human and pig uPA gene including their putative regulatory 5'flanking regions. For this purpose, we constructed a series of hybrid genes with uPA-5'-flanking deletion mutants and the E. coil chloramphenicol acetyl-transferase as a reporter gene. Using gene transfer techniques, this hybrid gene was stably integrated in the host cell genome and the activity of the reporter gene measured. This analysis revealed that sequences proximal to the transcription start site as well as sequences far upstream play an important rol e in hormonal regulation through the protein kinaee A pathway and by phorbol esters using the protein kinase C pathway. Furthermore, we identified the protein-binding sequences within the regulatory regions by DNAase I footprint and gel band shift analysis. A comparison of regulatory sequences of human and pig uPA genes will be discussed. Germany) and Fnednch Thrombosis, D-6300 Giessen (West * " " Miescher Institute, CH-4002 Basel, Switzerland Seven glycosaminoglycans (GAGs) were studied for t h e i r anticoagulant effects in v i t r o , platelet adhesion to bovine e xt r a c e l l u l a r matrix (ECM) and antithrombotic effects in a r a t thrombosis model. Unfractionated heparin (UFH), low molecular weight heparin (CY 216), a synthetic heparin pentasaccharide (PS) and dermatan sulfate IDS) were obtained from I n s t i t u t e Choay, Paris, France and a supersulfated low molecular weight heparin (GAG 869) and heparin in which the binding site for antithrombin I I I had been inactivated (GAG 262) were obtained from Prof. Casu ( I s t . Scientifico di Chimica "G. Ronzoni", Milano, I t a l y ) . A series of in v i t r o assays including aPTT, TT, heptest, anti Xa and anti l l a assays using chromogenic substrates were performed in both human and rat platelet poor plasma. All agents inhibited thrombus formation in venules at minimal effective doses from 0.01 to 5 mg/kg 30 min after a single i . v . injection. The dose-dependent antithrombotic effect was not p o s i t i v e l y correlated with the inhibition of F. lla or F. Xa in human or rat plasma. These data suggest that i n h i b i t i o n of thrombosis at the endothelial surface does not d i r e c t l y correlate with current ex vivo coagulation assays. Higher doses were required to observe antithrombotic effects after subcutaneous injections and their duration lasted far longer after s.c. administration than after i . v . injection. The antithrombotic effects persisted longer than detectable ex vivo anticoagulant a c t i v i t i e s . The low molecular weight heparins, pentasaccharide and dermatan sulfate showed in v i t r o i n h i b i t o r y effects on platelet adhesion to bovine ECM. This i n h i b i t o r y effect was not observed with two unfractionated heparins (GAG 262 and Liquemin) . Further studies are needed to c l a r i f y a possible correlation between inhibition of platelet adhesion and antithrombotic effects of GAGs. The exact regulation of synthesis of yon Willebrand factor by endothelial cells is s t i l l unclear. The effect of the vWF content of the culture medium on the production of vWF by cultured human ECs was studied. The amount of vWF produced and released by ECs in the presence of culture medium containing normal human serum was significantly higher than that produced in the presence of medium containing serum from patients with severe Willebrand syndrome. The latter correlated well with the amount of vWF produced in the presence of a serum-free culture medium. The multimeric structure of vWF released by ECs was the same in all cases. I f human ECs were cultivated in a medium containing normal serum the adhesion of platelets to the matrix produced by these cells was higher than the adhesion to matrix produced by ECs which had been cultivated in a mediumcontaining serum from a patient with severe vWS. This finding may be correlated with the higher vWF content of ECM. A stimulating effect of yon Willebrand factor contained in the culture medium on the vWF synthesis of cultured ECs is supposed. Center of Internal Medicine, Division of Angiology, J.W. Goethe-University, Theodor-Stern-Kai 7, 6000 Frankfurt/M., FRG We investigated dose-, time-and temperaturedependent effects of urokinase (UK) on normal citrated plasma in vitro. When 5 ~g/ml UK were added to pooled normal plasma and incubated for 30 minutes at an ambient temperature (25 ° C), ~2-antiplasmin decreased to 8% of the control value. Incubation on ice yielded a decrease to 45% of control, whereas ~2-antiplasmin was fully consumed at 37 ° C. Fibrinogen and plasminogen fell to 46% and 39%, respectively, after a 30 minutes incubation at 25 ° C. Thrombin time prolonged to 182% of control. Various inhibitors were studied with respect to their suitability and efficacy to prevent these in vitro effects. Aprotinin exhibited a good protective effect on fibrinogen at concentrations exceeding 500 KIU/ml plasma. Its use however was limited due to interferences with some haemostatic assays. We could demonstrate that L-Glutamyl-L-Glycyl-L-Arginyl chloromethylketone (GGACK) and a specific polyclonal anti-UK-antibody effectively inhibited urokinaseinduced plasmin generation without interfering with haemostatic assays. The anti-UK-antibody afforded full protection of ~2-antiplasmin at therapeutic levels of UK. It is concluded that UK in plasma samples from patients during thrombolytic therapy may induce in vitro effects which should be prevented by the use of a suitable inhibitor such as GGACK or specific anti-UK-antibody. Medizinische Klinik und Poliklinik, University of Ulm, Robert-Koch-Str. 8, D -7900 Ulm, F.R.G. Changes of the fibrinolytic system were investigated in twenty adult polytrauma patients with a mean injury severity score (ISS) of 37 (20 -50) and a NACA-score between IV and VI. Citrated blood samples were taken without delay at the site of the accident with a median time interval between trauma and sampling of 18 min (range: i0 -29 min). No patient had received more than 500 ml of cristalloid or colloidal fluid before sampling. Blood samples were centrifuged and snap frozen immediately using a portable centrifuge and cool aggregate. A second blood sample was taken in the emergency room at hospital. The following parameters were determined in both blood samples (mean results are given, sample taken in hospital in brackets): Plasminogen 93% (72); Antiplasmin 68 %(48); t-PA-antigen 14,5 ng/ml (3,3); t-PA-activity 21,6 ng/ml (4,7); PAI 8,0 AU/ml (19,1); D-dimer 13,7 ~g/l (15,4); FgDP 8,9 ~g/ml (6,7); FbDP 15,4 ~g/ml (19,2); TDP 27,4 ~g/ml (28,1). The meaning of these changes of the fibrinolytic system are unclear. Longitudinal studies of the fibrinolytic system after polytrauma have to be performed to answer the question whether therapeutic use of inhibitors of fibrinolysis i.e. aprotinin have a positive effect on the outcome of these patients, especially with regard to the frequency of acute respiratory distress syndrom (ARDS). Medizinische Klinik und Poliklinik, University of Ulm, Robert-Koch-Str. 8, D -7900 Ulm, F.R.G. Changes of the blood coagulation system were investigated in twenty adult polytrauma patients with a mean injury severity score (ISS) of 37 (20 -50) and a NACA-score between IV and VI. Citrated blood samples were taken without delay at the site of the accident with a median time interval between trauma and sampling of 18 min (range: i0 -29 min). No patient had received more than 500 ml of cristalloid or colloidal fluid before sampling. Blood samples were centrifuged and snap frozen immediately using a portable centrifuge and cool aggregate. A second blood sample was taken upon admission to hospital. The following parameters were determined in both blood samples (median results are given, sample taken in hospital in brackets): Quick 91% (64); aPTT 31 s (38); TT ii s (12); fibrinogen 1,6 g/l (0,95); AT III 81% (52); TATcomplex 138,7 ~g/l (132,5); protein C activity 60% (21); protein S antigen 70% (50); F.V 137% (86); F.VIII:C 225% (121); hematocrit 39% (28); platelets 269 000/~i (166 000). We conclude that distinct alterations in the coagulation system occur including a decrease of inhibitors of coagulation. Prospective studies need to be done in order to evaluate the clinical importance of these effects and the benefit of early therapeutic substitution. Medizinische Klinik und Poliklinik, University of Ulm, Robert-Koch-Str. 8, D -7900 Ulm, F.R.G. The patient developed a coumarin necrosis at the right breast. Second case: A 21 year old obese woman (weight: i00 kg, height: 160 cm) presented with an acute DVT which was complicated by a subsequent pulmonary embolism of grade III. The patient had a longstanding history of severe allergic bronchial asthma. She was a heavy smoker and took the o./c. pill. I.v. heparin was started, followed by Marcumar R. After a few days, the patient developed a large necrosis at the anterior abdominal wall which finally had to be treated with skin grafting. Third case: A 25 year old obese patient was admitted with PE. He had a i0 years history of hay fever and allergic bronchial asthma and smoked 40-60 cigarettes/day. The patient received heparin followed by Marcumar R. On the 5th day a large coumarin necrosis developed on the right hip with involvement of the muscles; the patient had to undergo five operations. No abnormalities in the haemostatic system were detected in any of the patients, in particular no protein C-deficiency. It is concluded that an allergic diathesis may play an important role in the pathogenesis of coumarin necrosis. Medizinische Klinik und Poliklinik, University of Ulm, Robert-Koch-Str. 8, D -7900 Ulm, F.R.G. and total DP (TDP) in plasma were assessed by specific ELISA. FDP in serum were also measured. In MI-patients D-dimer, FgDP, FbDP, TDP and FDP in serum increased from 0.i, 0.4, 0.4, 0.8 and 6.0 to 2.3, 20, 8, 30 and 120 ~g/ml at the end of the infusion, respectively. In normal subjects FbDP and D-dimer increased from 0.5 to 0.9 and from 0.i to 0.5 ~g/ml. Mean fbg levels were 51%, 61% (Clauss and photometric assay) and 81% (ELISA) of the preinfusion values. After 3 days all fibrin(ogen) degradation product values were normal whereas fbg levels were 120 -140% (Clauss, photometric) and 105% (ELISA). The discrepancies between the functional and ELISA levels of fbg suggest that about 25% of fbg is converted to (slowly clotting, i.e. less functional) LMW'fbg and that rt-PA induces less fbg degradation than expected from functional assays The levels of FbDP and D-dimer are too high to be derived exclusively from a coronary thrombus and probably originate from another fibrin pool. Medizinische Klinik und Poliklinik, University of Ulm, Robert-Koch-Str. 8, D -7900 Ulm, F.R.G. ROLE OF FIBRINOGEN-MEDIATED CONTACTS FOR THE ACTIVATION OF POLYMORPHONUCLEAR LEUKOCYTES (PMNLs) BY STIMULATED PLATELETS. A. Ruf, R. Schlenk, A. Maras, E. Mor,qenstern* and H. Patscheke. Cell suspensions containing human washed platelets (2 x 108/ml) and human PMNLs (2 x 106/ml) were stimulated with the platelet agonists thrombin 0.06 IU/ml, U 46619 1 pM, or ADP 10 pM. Platelet aggregation and the luminolenhanced chemiluminescence (CL) of the PMNLs were simultaneously monitored in a Lumiaggregometer. Thrombin and U 46619 were able to trigger the platelet aggregation and the CL in the absence of exogenous fibrinogen, whereas ADP required the presence of added fibrinogen to aggregate aspirin-treated platelets and to induce the CL. EDTA and no stirring suppressed the platelet aggregation and the CL in the mixed suspensions. Interference and electron microscopy revealed mixed platelet-PMNL aggregates. Cell-bound fibrinogen was localized by immunofluorescence and immunoelectron microscopy. It was found in the contacts between platelets, between platelets and PMNLs, at the free surfaces of both cell species and in the platelet C-granules. Neither thrombin, U 46619 and ADP nor the supernatant or a filtrate of thrombin-stimulated platelets induced any CL in pure PMNL suspensions. In asplrin-treated whole blood, U 46619 4 IJM or ADP 10 pM also led to an aggregation as measured by the impedance method and to a strong CL. The CL and the aggregation were again suppressed by EDTA, no stirring and RGD peptides. In contrast, ristocetin-induced platelet agglutination was not associated with CL. It is concluded that the platelet-dependent activation of PMNLs requires a close intercellular contact that is mediated by fibrinogen-binding to the surfaces of both cell species. ( During the acute disease as well as after she had completely recovered 10 weeks later the patient showed significant protein C deficiency. In the mother who had already gone through a thrombosis protein C was also found deficient. Thus the girl is most likely to have an inborn protein C deficiency. It is suggested that DIC and the extremely severe and fulminant course of the disease were initiated by protein C deficiency. To date there is no clear evidence why in an only small number of patients meningococcosis takes such a fulminant, mostly fetal course. The question arises wether, more frequently than known so far DIC in this cases must be attributed to an inborn ipclination to thrombosis due for example to protein C or AT Ill deficiency. To evaluate the possible interactions between phenprocoumon and either metoclopramide or cisapride the following open crossover trial was carried out: 24 healthy volunteers were given 0.22 mg/kg phenprocoumon as a single oral dose on day 4 of a 10 day lasting metoclopramide or cisapride administration and, after a washout period of 4 weeks, alone without those drugs. Several coagulation parameters (prothrombin tlme, factor X-and factor VII-activity, protein C activity and protein C antigen) and the plasma levels of phenprocoumon were determined at certain intervals up to 7 days after administration. Metoclopramide decreased the area under the curve (AUC) (about I6%) and the halflife of phenprocoumon (from 132 to 111 hours), but did not alter peak concentration or the time to peak concentration. Cisapride had no effect on the pharmacocinetics of phenprocoumon. These findings fit for an increase in the elimination of phenprocoumon through metoclopramide. Except for a significant decrease in factor X activity between 2 and 36 hours after phenprocoumon intake during metoclopramide coadministration there were no significant changes in the coagulation parameters through coadministration of eisapride or metoclopramide. As the pharmacokinetlc and the coagulation parameters varied to a large extent intra-and interindividually we conclude that both drugs should be used with caution in combination with phenprocoumon. Tissue-type plasminogen activator (t-PA) produced by recombinant DNA technology in eucaryotic ceils is well known as a thrombolytic agent. Recombinant t-PA produced in E. coli and renatured tacks carbohydrate moieties in contrast to t-PA produced in eucaryotic cells. The aim of our studies was to investigate if there were differences between recombinant tissue-type piasmlnogen activator (rt-PA) derived from eucaryotic cells (Aiteplase, Thomae GmbH, glberach, FRG) and rt-PA from E.coli (BM 06.021) regarding their pharmacoklnetic and pharmacodynamic characteristics. Rabbits were infused with 200 OOO lU/kg b.w. of DM 06.021 or Altepiase for 30 minutes. Plasma samples were assayed for t-PA-activity by a spectrophotometric method with ChromozymRpL. In comparison with AIteplase (n=6), BM 06.021 (n=5) showed an about three-fold longer initial half-life (2.1+_0.6vs.5.6±2.6 mln, mean+-So) a three-fold slower plasma clearance (22.2±7.6vs. 7.5+1.7 ml • min -1 • kg -1) and a three-fold increased area under the curve (133+_44 vs. 452+_105 IU • m1-1 " min). In a rabbit model of jugular vein thrombosis, BM 06.021 or Atteptase was infused for 4 hours at at least 3 dose levels. The specific activity of RM 06.021 was comparable with that of Alteplase. The analysis of the dose-response curves showed that BM 06.021 has a two-fold increase in thrombotytlc potency (ED~ 0.26 mg/kg b.w. vs. 0.55 mg/kg b.w.). At equieffective doses there were no statistically significant differences either in the plasma levels of t-PA-activity, fibrinogen, plasminogen or alpha~antiplasmln between BM 06.021 and Alteplase at the end of the experiments or in the hemodynamics during the infusion of the two agents. It is concluded that unglycosyLated rt-PA produced in E. coli is biologically active. Furthermore, BM 06.021 shows improved pharmacokinetic properties and an increase in thrombolytic potency. At equieffective thrombolytic doses BM 06.021 is comparable with ALteplase in its effect on the hemostatic and hemodynamic systems. Therefore, one might anticipate that non-gtycosyLated recombinant t-PA from E. coti exhibits superior pharmacological properties. and 250BI of a buffered (Tris/HCl,100 mmol/l, pH 8.1) thrombin solution (0.024 U/l) are incubated for 5 minutes. Then the chromogenic substrate solution is added (50BI) and the increase of absorbance is measured kinetically during a time intervall of 2 minutes after another minute. At 37°C reaction temperature half of the sample volume and half of the thrombin concentration are used. Results(25°C):Within run CVs (n=20) ranged from 1% to 3.3% between 14 and 5.6 IU/ml (112-45% AT III), day to day CVs (n=10) from 1.2% to 3.7% using the same specimens (control plasma and frozen human plasma). No deviation from linearity was observed between 3 and 25 IU/ml (25-200% AT III) using +/-5% as acceptance criteria. The recovery of assigned values in two controls (PreciChrom ® I and II) was 98% and 101%. Method comparison in 50 deep frozen plasma specimens with AT III activities from 2 to 25 IU/ml showed an excellent agreement with the manual method: y= -0.2 + 1.00 x. The median AT III activity in 15 fresh frozen plasma samples from healthy volunteers was 12.8 IU/ml (102%).Using the same reagent in the analyzer the calibration factor was found to be stable for at least 3 weeks. Results of the same quality were obtained at 30 and 37°C. We conclude, that the described procedure is well suited for routine analysis. Patients with coronary heart disease undergoing coronary-artery bypass surgery represent a well-studied group in which changes of a number of hematological, mostly platelet parameters have been described. New electronic cell counting and sizing techniques in hematology enable us to analyze most of these partially uncommon parameters such as mean platelet volume (MPV), platelet volume distribution width (PDW) and RBC-distribution width (RDW) on a fast scale. It is important to be aware of the extent of normally occurring changes so that misinterpretation of findings can be avoided. In this study, our first aim was to generate diagrams showing the average response (and 99% confidence intervals) of those hematological parameters that showed characteristic postoperative changes in patients undergoing coronary-artery bypass grafting without complications (n = 50). Secondly, we examined if by means of hematological parameters postoperative disease states such as thrombosis, reinfarction or infections could be differentiated from changes that normally occur during the postoperative period. Our investigation shows that the monitoring of plate!et count and MPV during the postoperative phase with the aid of the mentioned diagrams helped identify thrombotic disease and infections in all patients with these postoperative complications (7 out of a total of 83) up to 48 h before clinical signs were apparent. The monitoring of platetet parameters was clearly superior to leucocyte monitoring. Leucocyte parameters were misleading in 16 cases (5 false negative, 11 false positive). Furthermore, this study was able to confirm recent findings that an abnormal increase in PDW and low platelet counts found preoperatively in patients with coronary heart disease may serve as good indicators for the prethrombotic state and the risk of myocardial infarction. Hypercoagulabilitv and thrombotic episodes are well-known Eeatures oE nephro~ic svndrome(NS),but the relative contribution o~ -alledgedlv impaired~ Gibrinolysis(FI> has not been elucidicated. We. therefore examined FI in NS in more detail under basal conditions and following two stimulatory manoevers(vemous occlusion(VO)~in~lating cuf~ ~or 15 min between systol, and diastol DP; ingusion o4 DDVAP o,4 ug/kg over 30 min;blood sampling 3o min thereafter). 13 pat with NS( proteinuria > 3,5g/ d/1,73 m =, normai GFR, prior to any medication) and 8 controlswere analysed. RESULTS: Nephrotics showed the e×p~cted elevation of 9ibrinogen, GVIII(Ag,C) and a=-macroglobulin; no FDP: no sign changes oF TPA, UPA( urokinase type PA),a,antitrypsim, a=-antiplasmin, plasminogen (protein and chromogen, substr.), TAT-(thromPln/antithrombin 3) complexes, prekallikrein and F ×II levels. PAI-I was decreased. VO sign shortened euglobulin ivsis time, hematocrit rose less, TPA, PAI 1 and FDP rose sign.,whereas UPA and TAT remained unchanged in NS in comparison to controls. AFter DDAVP f VIII rose less in NS than in controls. Other fibrinolvtic parameters were not diFFerent. CONCLUSION: In NS provocation manoeuvers demonstrate unexpected hyperresponsivenesm i.e.augmented release oF FIcontrolling proteins 9tom endothelial cells. Studies are underway to clarigy whether this is due to an increaspd pool oE stored proteins. Comparing the young (20-35 years, n=18) with the oldest participants (75 years and more, n=20), plasminogen, ~-2-antiplasmin and Cl-inhibitor did not change. Antithrombin III (AT III) sank from 107 + 11 to 94 + 12 % (p20 nin. Coa@datery and inhibitory factors of hemestasis (TPZ : AT Ill, factor VII : protein C ) correlated positively ( r _< 0.7) A new balance of hemestasis results. Also parameters of bemostasis and liver function correlated in different phases of portal and functional decolpensation. Bleeding events followed portal hypertension and were indepeedunt of parameters of hemestesis. Prolongation of ICG tl/2 is an indirect parameter of portal shunt flow. Probabillity of bleeding evunts is si30 lin colpared with ICG tl/2 <20 nin.Osing a score of TPZ and AT III <40% aim factor VlI <30% pro(]mesis is very bad (*liver failure). This situation is an urgent indication of liver trzmspluntation. Substituation of fresh frozen plesme was without benefit concerning pro~esis of these patients. Cosolusion: Score of Child-Pup-classification, ICG tl/2 and parameters of he~tasis (TPZ, factor VII, AT 111) should enshle one to estisots pro91esis in course of disease and find out the occasion of elective liver transpluntstiun. Ber~Bn,sheil-~niversititsklinik -463 Bochun, Gilsingstr. 14 (1) Abt.~strounterol. u. Hepatol. Initiation of coagulation on cell surfaces is triggered by Tissue Factor (TF) an integral membrane protein with a wide tissue distribution. TF serves as a receptor for factor VII/VIIa forming a catalytic TF:VlIa bimolecular complex. We attempted to define the functional regions in TF using moneelonal antibodies (FLAbs) as probes for the surface of the TF protein. MAbs were shown to be specific for factor VII interactive regions by functional characterization of their inhibition pattern and by competition studies with factor VII. The recognition sites on TF were characterized by Western-blot analysis of MAb reactivity with proteolytic fragments of TF and with carboxy terminal deletion mutants of TF produced in a prokaryaotie expression system. One group of MAbs did not react with proteolytic fragments of TF and reacted with a deletion mutant spanning amino acids 1-157. No reactivity with shorter mutants was observed suggesting that the interaetlve region of the MAbs is located in exon 5 or requires this part of the molecule for proper folding. A second group of MAbs was shown to react with proteolytic fragments which bound anti peptide antibodies to amino terminal sequences of TF. These MAhs reacted with a deletion mutant spanning amino acid 1-83 but not with shorter mutants. These data therefore implicate the amino terminal half of the TF extracellular domain in factor VII binding. Site directed mutagenesis and peptidyl mimicry will help to define key amino acids in the factor VII interactive region. Since pre-kinins and their inhibitors under the administration of heparin fragments have not been very much taken into account until now, comparative investigations with high molecular heparin (HMW) will contribute to learn more about them. The clinical relevance is going to be discussed in this context and investigated with the help of the activators prekallikrein, of the kallikrein-like-activity, of the functional kallikrein-inhibition and @-factor XIIa-inhibition before the chirurgical intervention and on the third and seventh postoperative days. The investigation was carried out on 122 patients who had to be submitted to different elective abdominal surgical operations. 60 patients were treated with standard-heparin (HMW); 62 with LMW-heparin. In patients after abdominal surgical interventions, a compensed activation of the contact phase was to be noted. This could be proved by a remarguable PKK d~crease which however was not combined with a more intensive kallikrein-like-activity. The mecanisms of compensation could be ascribed to higher levels of the capacity of inhibition by kallikrein and ~-factor XIIa. The release of PMN-elastase can be followed in plasma with the elastase alphal-proteinase inhibitor complex. Two series of-recirculations with heparinized whole blood in a HLM-system were done. The testing-length was each time 120 minutes; blood was taken at nine defined sample times. In another series 1500 plasma units of the Cl-esterase-inhibitor was each time before the recirculation added to the filling volume of the HLM. The concentration of elastase revealed a steady increase up to an end value which exceeded after two hours the initial value by twenty times. Compared with the control group, the application of CI-INH did not lead to a significant inhibition of elastase. Despite a significant increase of the inhibition of kallikrein and ~-factor XIIa no effects on the activation of the contact phase could be established. The reactions of the coagulation system and of complement system were ivestigated ex vivo in a closed HLM modell. Testing time was over 90 minutes. In order to stop the coagulation of blood efficiently, 6 IU/ml of CY-216 (Fraxiparin) had to be administrated. This is, despite a higher anti-FXa-activity, equivalent to twice the dosage of KABI 2156 (Fragmin). Possible explanations for this are: first, an inactivation ++ caused by the bond to Ca -ions; second, a slight affinity of heparin with ATIII. During the course of recirculation the concentration of heparin steadily decreased. This was mainly caused by the inactivation of heparin by platelet factor 4, combined with a significant decrease of the number of thrombocytes. A comparison of CY-216 and KABI 2156 shows that CY-216 is less bonded to platelet factor 4 and also less neutralized by platelet factor 4; on a larger scale, the aggregation of thrombocytes undergoes an inhibition. This reaction could have a positive effekt on the daily dosage, but a negative one on the arising of hemorrhage complications. At the beginning of the test, a confused activation of coagulation occurs intrinsically and extrinsically. This can be established mainly by the decrease of factor XII and Cl-inhibitor activities, as well as by the increase of the activity of factor X. Coagulation is caused by the release of specific proteins by the thrombocytes. As a result of this study, it is to be noted that CY-216 is absolutely capable to stop efficiently the coagulation, but also that crucial differences with the LMW-heparin KABI 2156 do exist. Chir. Univ. Klinik, 7400 T~bingen, Calwerstr.7 In the present study a HLM machine was used to investigate to what degree the observed in vivo activation of the contact phase factors (prekallikrein, kallikrein-like-activity, kallikrein and @-factor XIIa-inhibitor), as well as that of the plasma heparin level depend upon purely mechanical influences. The single parameters obtained by varying the aprotinin dosages during the administration of HMW-heparin (150 and 300 KIU/ml), as well as during the administration of varying molecular weight heparin preparations were statistically compared with those obtained by the administration of constant doses of aprotinin. The prekallikrein activity was not effectively inhibited by HMW and I24W-heparin, whereas with a low dosage of aprotinin (150 KIU/ml) it is extremely effectively inhibited and with a high dosage it.is completely inhibited. The kallikrein-like-activity curve confirms these findings independ4nt of the molecular weight. The kallikrein formation was increasingly reduced with increasing doses of proteinase inhibitor; however, a total prevention could not be attained. In the present study a consumption of kallikrein inhibitors, independent of the aprotinin dosage and the heparin administered cannot be demonstrated. One can, therefore, proceed on the assumption that the activity of the physiological kallikrein inhibitors -mostly Cl-esterase inhibitor -decreases in corresponding proportion to the decrease in the @-factor XIIa-inhibition activity. A higher aprotinin dosage as compared to a lower dosage revealed only a slight fall in the ~-factor XIIa-inhibition activity. Chir. Univ. Klinik, 7400 T~bingen, Calwerstr. 7 Because of the positive effects of the proteinase inhibitor aprotinin in the field of heart surgery, this study was concerned with Cl-esterase inhibitor which is probably the most efficient inhibitor in the contact activation phase of the coagulation. The investigation was carried out in an ex-vivo model. Anti-coagulant CY 216 (Fraxiparin) was used at a dosage 4 IU/ml. In a initial experiment, Cl-inhibitor was dosed at i000 U/500-700 ml. The extra corporal circulation was kept up at over 120 minutes. The determined parameters at each sample time were co~ pared to the respective results of the runs without Cl-esterase inhibitor, but with the same dosage of heparin. During the filling of stored blood into the reservoir of the HLM, a contact activation of the coagulation and of the prekallikrein-kallikrein-kinin systems was to be noted, produced by the intensified contact with artificial surfaces, and in spite of heparinisation. After the addition of Cl-eWterase inhibitor, further increasing coagulation processes were measured despite significantly increased activity values of ~-factor XIIa-inhibition and Cl-esterase inhibitor. The prekallikrein-kallikrein-kinin system showed as well a further act~ vation. The heparin activity decreased during the circulation. The proteolytical processes of the contact activation phase, as well as the activation of the prekallikrein-kallikrein-kinin system could not be suffiently inhibited. Chir. Univ. Klinik, 7400 Tiibingen, Calwerstr. 7 In the present study, low respectively high molecular heparin was administered to patients during and after abdominal chirurgical interventions. In this process, low molecular heparin appeared to be slightly superior with regard to the emergence of thromboembolism complications. In order to check the effectiveness of the different heparins, patients were not only clinically observed; before and after the operation, relevant parameters concerning the coagulation were analyzed. Blood samples were taken on the first preoperative day, and on the seventh postoperative day. Besides the determination of the activity the inhibitor ATIII, the factor XII-levels and the capacity of inhibition of Cl-esterase inhibitor were measured. The results did not point at any difference between low respectively high molecular heparin. In almost every case, a reduction of the activity of ATIII was confirmed in the postoperative period. In patients with big abdominal operdtions a more important decrease of this activity was to be noted. The factor XII-levels reacted similarly. Since -as our results reveale -factor XII has an important part to play in the fibrinolysis, both the consumption of inhibitor ATIII and the reduction of factor XII could be responsible for the greater incidence of thromboembolism complications in big abdominal chirurgical interventions. The only difference we could find out at the comparison of both groups was in the remaining activities of heparin before the morning subcutaneous application: patients with low molecular heparin indicated then clearly higher prime activities. chit. Univ. Klinik, 7400 T~bingen, Calwerstr. 7 The release of PMN-elastase can be followed in plasma with the elastase alphal-proteinase inhibitor complex. Two series of recirculations with heparinized whole blood in a HLM-system were done. The testing-length was each time 120 minutes; blood was taken at nine defined sample times. In another series 1500 plasma units of the Cl-esterase-inhibitor was each time before the recirculation added to the filling volume of the HLM. The concentration of elastase revealed a steady increase up to an end value which exceeded after two hours the initial value by twenty times. Compared with the control group, the application of CI-INH did not lead to a significant inhibition of elastase. Despite a significant increase of the inhibition of kallikrein and @-factor XIIa nQ effects on the activation of the contact phase could he established. Slightly reduced plasma levels of prekallikrein, as well as a clear increase of the kallikrein-like-activity, reveal an activation of the plasmakallikrein system at the very beginning of the experiment. Other results show a start of the endogene coagulation and a slight activation of the complement cascade. That is why the place value of the activated Hageman-factor, plasmakallikrein and anaphylatoxins C5, respectively C3a, is of secondary importance in the series of experiments. The similarly increasing runs of the concentration of platelet factor 4 and PMN-elastase show that Pf 4 has intensified the release of elastase from thrombocytes. At the end of the experiment we found reduced heparin levels. Chir. Univ. Klinik, 7400 T~binqen. Calwerstr. 7 It is a special clinical interest to have screening tests for the diagnosis "high risk for DIC" in individual patients. There should be screening tests which can be performed during day and night without special knowledge. The quantitative measurements of fibrino-peptide A, thrombin-AT lll-complex and other substances, which arise through coagulation, may give reasonable informalions of the beginning of DIC, but such assays are time consuming or they need special knowledge or special equipment. Semiquantitative latex-agglutination tests can be done by every lab assistant. Therefore we initiated the semiquantitative determination of fibrin(ogen) degration products (FDP) or of a neoantigen (D-D) in all patients on an intensive care unit, who were thought to have early DIC by the physician on duty. We present a retrospective analysis of the data from 300 patients. About 2/3 show elevated FDP or D-D. The results of these screening tests are compared a) with the results of other coagulation tests performed often (PTZ, fibrinogen, AT III, platelets), b) with the results of lab screening for organ failure (creatine, P%, 3/GT, leucocytes) and c) with some clinical paramet@rs (heart rate, blood pressure, temperature, main diagnosis). The comparisons show that in patients, who have no bleeding or other positive signs of DIC, no relations exist between FDP or D-D value and other coagulation tests. But even in these patients there is a relation between FDP or D-D and the results of the lab screening for organ failure. High temperature and/or the combination of low blood pressure and high heart rate lasting longer than 4 h are associated with high FDP or D-D respectively. Among 4,077 autopsies of the years 2/1979 to 1/1989, 966 cases with vein thromboses and lung thrembembolism were demonstrated (corresponding to 24 %). From these we found thrombembalism in 758 cases (78.5 %). The rate of fatal and fulminant embolism was 78.9 %. We found in 16.3 ~ thromboses of the peplitea] veins, in nearly 60 % thromboses of the femoral and pelvic veins, in 4 % thromboses of the caval vein and 2.7 % were located in cardiac auricle. We compared the results with 350 autopsies of the year 1985 without thromboses or embolism. In bath groups, the average age is 74 years. Nearly 60 % was spreaded between 70 and 85 years. In the control group 52.3 % were male, whereas only 44.2 % of cases with thromboses or embolism were male. The annual rate varied between 17.8 % in 1988 and 28.4 ~ in 1981. Monthly rate had its maximum with 29.1% in november, and its minimum with 21.9 % in july. The average weight differed nearly 5 kg in favour of thromboses. A decisive factor was immebi}ation, measured in days of confinement to bed. Average immobitation in thromboses was 19, in control group 10.4 days. A further important factor was the form and period of entieoagulation therapy. Refering to the different diseases, we found that cardiovascular diseases were more frequent in control group and malignant tumors were more freqent in the group with thromboses. Proportions of gastrointestinal tumors were increased in group with thromboses , whereas patients ~ith bronchial carcinoma had the same rate. We do not find a significant difference in right heart failure, reasoned by the high average age in both groups. Hematocritas a parameter for exsieeosis was in the group with thrombembolism two points higher. The difference in thromboeytocrit was irrelevant. Abteilung for Patholegie des Marienkrankenhauses Hamburg, AlfredstraBe 9, 2000 Hamburg 76, F.R.G. Platelet aggregation screening is now becoming increasing important. It is not only necessary in clinical research to testify the laboratory effects of new substances for primary and secondary preventation of thromboembolic disease, but also to control the quality of therapeutical plasma preparations and to check side effects of pharmaceutical products on platelet function. P1atelet function assays should be designed in such a way that they can be performed in normal clinical labs without special knowledge. They should enable direct data comparisons of the different labs. We present a standardized measurement of collagen and ADP induced ptatelet aggregation with the help of a personal computer. The measuring data are registered on line, laid out graphically and calculated in accordance with suitable points given. The dose effect relationships between aggregants (6 different concentrations) and calculated parameters (5 after ABP induction, 4 after collagen induction) are demonstrated (n = 40). The comparison of the results of this normal group with those of 8 volunteers 24 hours after they had taken 250 mg ASS and of 5 patients respectively with typical illnesses associated with type-and hyperaggregability, shows that the analysts of the aggregation with 2 reagent-concentrations respectively (1.2 and 4 #g/ml collagen; 1.2 and 4 pHol/ml ADP) and the resulting calculation parameters can differ between normal, type-and hyper-aggregable platelets, We believe that the presented standardized and simplified platelet aggregation meas[:rements are suitable for multi centre trials. In a large unselected autopsy series (N=4,461) 35 retroperitoneal haematomas (0,78 %) were found (table 1) To discriminate between these factors, we prospectively studied PF in 191 men, 78 PTS with angiographically documented coronary heart disease and 113 healthy subjects (HS). In HS, stepwise multiple linear regression identified age to be a major determinant of PF, Platelet aggregability after induction with both ADP and collagen increased with age (p ~ 0.01, respectively). Possibly due to counterregulation, the levels of c-AMP in platelet rich plasma increased with age, both basally (p < 0.05) and after stimulation with prostaglandin E 1 (PGEI, p< 0.01). In PTS, PF was not dependent on age. In multivariate analysis of variancet hypercholesterolemia (~240 mg/dl) was not a determinant of PF in neither group. In HS, smokers had lower levels of PGE 1 stimulated C-AMP than non-smokers (p ~ 0.01), but aggregation did not differ. Elevated blood pressure (~160/95) was associated with inhibited ADP induced aggregation (p~ 0.01) and increased c-AMP after PGE 1 (p<0.O01) in HS. ADP and collagen induced aggregation were markedly stimulated in PTS compared to HS (p ~ 0.001). Basal C-AMP was reduced (p< 0.01) and throrgOoxane formation after collagen 5 ~g/ml enhanced in PTS compared to HS (p < 0.01). Thus, by multivariate analysis, age was a major determinant of PF in HS, but not in PTS. In HS, some tendency to a stimulation of PF was observed in smokers, and hypertension tended to be associated with inhibited PF. When compared to HS, PTS had markedly stimulated PF. The interaction of heparins of varying moleca:lar weight has been investigated in vitro using high performance liquid chromatography (HPLC) method, antifactor IIa and Xa tests and a whole blood coagulation system. Unfractionated heparin (UFH) and 5 low molecular weight heparins (LMWH) were added to increasing doses of protamine in vitro. The heparin-protamine complex was sedimentated by centrifugation. The supernatant was analyzed by HPLC. Fractions from HPLC were collected and aXa and alia activities were measured using commercial assays. The data show that UFH and all LMWH's were quantitatively bound by protamine. 1 mg protamine totally bound i mg of all heparins. No aXa or alIa activity was detected in the eluates from the HPLC column. We also examined the neutralization of UFH and the LMWH Kabi 2165 by protamine after intravenous injection of both substances in man on an ex vivo whole blood clotting test. The results show that the effect of protamine on aPT/and thrombin time is completely inhibited by protamine although the coagulation of whole blood was still impaired. There was no difference in the prolongation of coagulation using UFH and LMWH. Clotting of whole blood was monitored in serial subsamples with measurement of FpA and PF4. No relation between FpA and PF4-values and antifactor Xa activity was found. The data demonstrate that protamine binds UFH and all LMWH preparations. After administration into man UFH and LMWH both are bound by protamine. However, some anticoagulant activitiy can still be detected after administration of protamine. This suggests that UFH and LMWH cause release of heparin-like compounds into the blood stream which do not react with pmtamine. Four groups of patients received prophylaxis of thromboembolism because of prior venous thromboembolism (n=51), artificial heart valve replacement (n=13), atrial fibrillation with arterial embolism (n=l 1) and cardiomyopathy (n=ll). They received the LMWH Kabi 2165 in a dose ranging from 2500 to 15.000 aXa units 1 x daily s.c. The dose was adjusted according to the bodyweight and the statistical risk of developing thromboembolism. The treatment period ranged from 1 to 48 months. Occurrence of thromboembolism and haemorrhage, laboratory parameters (Heptest, capillary blood, whole blood, and plasma, S 2222 factor Xa method, aPTT, thrornbin clotting time and AT III) and safety parameters were controlled every 4-6 weeks. The mean treatment period was 0,5 years summing up to a total observation period of 50 years. No fatal thromboembolism occured. 2 patients of group 1 experienced rethrombosis. One major gastrointestinal bleeding occured in a patient with a so far unknown colon carcinoma. Seven minor haemorrhages occured which did not re-occur after a 15 % dose reduction in these patients. All plasma samples were taken 2-4 hours after the s.c. injection. Heptest values from capillary blood and whole blood ranged from 30-60 sec., values in plasma ranged from 45-100 sec. S 2222 aXa method showed values ranging from 0.2 to 1.0 aXa units/ml, aPTT and TCT were only slightly prolonged. AT III and safety parameters remained unchanged during the observation period. Osteoporosis was not detected. 8 pregnant women were included in the study. No anti Xa activity was detected in blood drawn from the umbilical cord after delivery. The data suggest that LMWH can be effectively and safely used for prophylaxis of thromboembolism in patients with venous thromboembolism, artificial heart valve replacement, atrial fibrillation with arterial embolism, cardiomyopathy, and in pregnancy. Ist Medical Clinic, Faculty of Clinical Medicine Mannheim, University of Heidelberg and 3rd* Medical Clinic, University of Heidelberg, 6800 Mannheim, FRG. Two cases of heparin induced thrombocytopenia (HIT) typell with severe thrombo-embolic complications are reported. Thrombocytopenia occured 13(15) days after the onset of heparin therapy and platelet count dropped to the lowest value of 13 000(40 O00)/~l at the 16(24)th day. Thromboembolic complications under heparin treatment included pulmonary embolism and venous thrombosis of the legs in both patients and additionally thrombosis of arm veins in one patient. Cessation of heparin resulted in normalization of platelet count within 8(3) days. Diagnosis of HIT was confirmed by in vitro plat~let aggregation tests. At the time of the patients thrombocytopenia normal donor platelets were used and mixed with the patients platelet poor plasma(PPP). Two commercial standard heparin preparations (from a minimum concentration of 0,2 IU/ml upwards) led to a strong aggregation of platelets in presence of either patients plasma. No remarkable aggregation could be seen with one of five tested LMWH fractions. This preparation and a second LMWH failed to aggregate platelets with the plasma of the second patient. Controls of all preparations were carried out in normal platelet rich plasma(PRP) and the use of donor platelets with a notable spontaneous or heparin dependent aggregation was excluded. Aggregation studies with the patients PRP were performed after normalization of their platelet count and no change in aggregation behaviour compared to the results with the mixture of donor platelets and patients PPP was observed. These results indicate that in patients with HIT i t might be very helpful to perform in vitro platelet aggregation studies with various LMWH fractions in order to select one preparation without aggregatoric potency that could be applied therapeutically. Inst. f. Exp. Haematologie und Bluttransfusionswesen der Universit~tsklinik Bonn, 5300 Bonn 1 68 ACTIVATION OF COAGULATION BY LUNG CARCINOMA CELLS R. Seitz, H. Heidtmann, M. Maasberg, R. Egbring, K. Havemann It is well known that fibrin formation takes place within tumor tissues. In a randomized trial with eoumarin treatment, prompted by the assumption that the activation of coagulation favours tumor growth and metastasis, a significant prolongation of survival of small cell (SCLC) but not non-small cell lung cancer (NSCLC) patients was found (Zacharski et al.,Cancer 45:2046; 1984) . The coagulation process in tumor tissue may be triggered by adjacent macrophages, but also by tumor cells themselves. They may express tissue factor-like activity, prothrombinase-active surfaces or a vitamin K dependent cysteine proteinase, termed 'cancEr procoagulant', activating factor X directly. The aim of this study was to assess the activation of coagulation in lung cancer patients and by cell lines in vitro. We found in 87% of unselected lung cancer cell patients a striking elevation of thrombin-antithrombin III complex (TAT) levels up to 120 ~g/l (normal < 4 ~g/l). It was found in both SCLC and NSCLC patients and was sustained over months. In vitro, sonicated c e l l s u s p e n s i o n of 6 NSCLC and I of 2 SCLC lines, kept in serum free media, caused marked reduction of" the recalcification time in normal pool plasma. Using an assay system with purified factor X (Behringwerke) and the chromogenic substrate S-2222 (Kabi Vitrum), we found a Ca ++ dependent factor X activation, particularly strong in 2 NSCLC lines. The data suggest that the biological role of coagulation activators should be re-evaluated particularly in NSCLC. is the most potent compound of this series. It has a molecular weight of 2388 daltons and exhibits low anticoagulatory activity compared to heparin and low molecular weight heparins. The activity in the aPTT-test is highest for LW 10082 and decreases gradually when the number of methylene groups in the alkylene chain increases. In the Heptest, his-lactobionic acid amides demonstrate still lower, but definite activities. At concentrations up to 0.i mg/ml in platelet rich plasma, no effect on agent (ADP, collagen, arachidonic acid etc.) induced platelet aggregation was observed with these compounds. In different animal models in rabbits and rats (Wessler-test, Harbauer-model, laser induced thrombosis and a venous thrombosis model) the antithrombotic activity was dose and time dependently. In contrast to its low anticeagulatory activity LW 10082 proved equipotency to a low molecular weight heparin in a dose range of 0.5 -5.0 mg/kg b.w.. In the rat tail bleeding model and the rabbit ear bleeding model LW 10082 was compared with Heparin and low molecular weight heparins. In doses below 5.0 mg/kg, LW 10082 induced only a slight prolongation of the bleeding time. At 5.0 mg/kg the bleeding time was prolonged by 83 %. In contrast Heparin and low molecular weight heparins prolonged the bleeding time by about i00 % at doses of 0.5 and 1.0 mg/kg b.w., respectively. Therefore, bleeding risks seem to be a minor problem in the action of the lactobionic acid amides. On the basis of a midpoint evaluation of more than 10.000 patients, a large multicenter Gemea trial with enoxaparin (Clexane) will be presented. In this study, in which 211 hospitals are participating, the safety and tolerance, as well as the incidence of pulmonary embolism and thraN)osis following administration of enoxaparin 2(ling (Clexane 20) once daily in patients undergoing general surgery are investigated. The general surgical procedures included are: abdcminal, gynaecologic (with the exception of mastectonw) and urologic operations lasting at least 30 minutes under general anesthesia and/or spinal/peridural anesthesia. All potential complications with an incidence of greater than 2 per 1.000 will he documented. This number was chosen because it reflects the observed incidence of fatal pulmonary enbolism with unfractionated heparin prophylaxis. -Occurrence of thromboses and pulmonary embolism, which, in case of clinical signs, are to be confirmed by objective techniques. -Bleeding and blood loss as well as changes in platelet count, hematology and transaminases. -Adverse drug reactions. RESULTS: The safety and tolerance of thromboembolic prophylaxis with Clexane 20 in general surgical procedures are shown to be excellent (no injection hematoma, thrc~imocytopenia, and low incidence of bleeding). With respect to the incidence of fatal and non-fatal pulmonary embolism, the reported midpoint evaluation shows a superiority of Clexane 20 in the prevention of thrc~looe~bolic ca~olications when retrospectively compared with trials using uofractienated heparin (e.g. Kakkar: International Multicenter Trial). Reports of bleeding within the enoxaparin trial were statistically below those in the above-mentioned trial. Compared to Heparin, LW 10082 exhibits moderate aPTT activity but very low anti-Xa and anti-iIa activities when measured in Heptest and thrombin clotting time, respectively. There was a marked difference in the anticoagulant potency of LW 10082 when human or bovine thrombin were used in thrombin clotting time measurements. LW 10082 was about 20 times more potent as inhibitor of human thrombin than of bovine thrombin. LW 10082 proved to be a potent activator of heparin cofactor II in a system using purified factors. In contrast, purified AT III did not inhibit thrombin in the presence of LW 10082. In the presence of plasma and LW 10082 thromhin was inhibited, however, probably by the action of heparin cofactor II. The anticoagulant activity of LW 10082 in the aPTT-test was shown to be independent of AT IiI by the use of anti-AT III antibodies. In the presence of the antibodies the potency of LW 10082 was not reduced, while the activity of heparin was clearly attenuated. The anticoagulant activity of LW 10082 was readily neutralized by protamine and polybrene. Depending on the assay, different relative amounts of antidote were necessary to normalize the clotting times. In contrast to protamine Experimental studies in our laboratories have shown that the procoagulant action of non-ionic contrast media is related to their ability to release thromboplastic material from red cell membranes and to their weaker anticoagulant nature in comparison to ionic contrast media. Thus, these agents are unable to inhibit the activation of the coagulation process at the blood-cell interphase in the angiographic catheter. Incubation of non-ionic contrast agents such as Omnipaque s and Isovue s with whole blood resulted in marked generation of fibrinopeptide A and thrombinantithrombin complexes. In contrast, Hexabrix R and Angiovist R produced much weaker generation of the two markers. Supplementation of a low molecular weight heparin (CY 216D) at 50 #g/ml or of hirudin at 5.0 #g/ml totally inhibited the generation of these markers. The in rive systemic procoagulant effects of these contrast agents were also inhibited by prior administration of CY 216D or hirudin as studied in primates. Our studies suggest that the procoagulant effects of non-ionic contrast agents can be minimized with low molecular weight heparin or hirudin. Loyola University Medical Center, Maywood, IL USA. PHARMACOLOGIC VALIDATION OF THE ANTITHROMBOTIC AND VASCULAR EFFECTS OF DEFIBROTIDE J. Fareed, G. Kindel, J. Walenga, D. Hoppensteadt, *K. Krupinski and *K. Breddin Defibrotide (D) a polydeoxyribonucleotide based drug has been used clinically for the prevention of occlusive disorders of varying origin. Several pharmacologic models have been used to investigate the mechanism of action of this agent. D produced a dose-dependent antithrombotic action in rabbit (venous stasis) and rat (arterial) models of thrombosis with the intravenous EDs0 ranging from 2.5 -i0.0 mg/kg IV. Ex vivo analysis of blood did not reveal any alteration of the PT, PTT, TT and Heptest times. However, the thrombelastograph patterns of bloods obtained from animals 30 minutes after IV injection of D showed a hypocoagulable state. Although the ex vivo clotting of blood from rabbits treated with defibrotide were not significantly different from control animals, sera from the treated group did not produce any contractile action of the isolated rabbit aortic strip suggesting the absence of procontractile agents. In animals with induced clot occlusions, pretreatment of animal with D augmented the thrombolytic efficacy of urokinase and t-PA. Repeated IV administrations of D into rabbits resulted in increased levels of 6-keto-PFG1~ production. In a primate model of hypercoagulable state, intravenous D induced a mild fibrinolytic state as measured by an increase in total fibrinogen degradation products. These studies suggest that D exerts its pharmacologic effects at multiple sites which involve endogenous cellular and humoral components which are not measurable by routine ex vivo tests. The WHO has recently introduced a LMWH standard which is a nitrous acid depolymerized product provided by KabiVitrum (Stockholm, Sweden). In a well-defined, uniform, biochemical and pharmacological screening, 8 LMWHs were compared at equivalent WHO standard-adjusted potencies in terms of anti-Xa activity using valid doseresponse curves. As expected, the standard minimized the AT III affinity differences in the anti-Xa assay. However,a marked elevation of the anti-IIa component of all of the assayed LWMHs ocurred, and the relative HC II activation profile between these agents was widened. In animal models of thrombosis and bleeding, potency adjusted LMWHs exhibited individually different safety/efficacy ratios. When the 8 LMWHs were titrated with PF-4 and protamine, their neutralization profiles showed marked differences. These data suggest that utilizing the WHO standard to adjust the potency of LMWHs in terms of anti-Xa activity leads to distinctly different safety/efficacy profiles of each LMWH which may not be related to their observed in vitro (anti-Xa) potency. Thus cross-referencing of LMWHs, which are expected to have more than one effect, by one in vitro assay is of questionable value as this may result in serious dosimetric errors. These problems will exist for subcutaneous administration of these agents but will be magnified when they are used via an intravenous route for therapeutic anticoagulation. should not be considered the optimal antithrombotic agent because the determinant of their biological activities are not known. Dermatan sulfate preparations exhibit varying in vitro, ex vivo and in vivo activities should be taken into consideration in developing these agents as antithromboties. We have compared native dermatan (Mm = 33,000) with its subfractions (Mr range 2,000-22,000) to study the effect of molecular weight and chemical on the in vitro and in vivo action of dermatan. Our data showed that some of the anticoagulant and antiprotease properties were not molecular weight dependent. The bioavailability of each of these agents was dependent not only on the molecular weight but also on the charge density characteristics. In purified systems, these agents showed marked variation in interactions with HC II and AT III. Similarly, ability to inhibit the generation of thrombin showed differences between the farctions. The intravenous and subcutaneous antithrombotic actions of each of these agents showed marked variations which were not explainable on the basis of molecular weight alone. The individual properties of dermatans are determined by several factors such as inhibitor interactions, endothelial uptake and presence of ATIII affinity components. Charge density may be very important for interactions with HC II and endothelium. When beef mucosal dermatan was compared with porcine mucosal dermatan of similar molecular weight, differences were noted in their biological properties. These studies show that dermatans, are a heterogeneous group of glycosaminoglycans which exhibit marked variations in both the molecular and biological profiles. (3): [357] [358] 1989) . We have used several biochemical systems to characterize their mechanism of antithrombotic actions. In the amidolytic assays these agents were found to produce strong activation of human, bovine and a recombinant heparin cofactor II as measured by thrombin inhibition. In the AT Ill depleted plasma system these agents produced significant anticoauglant effects. No interaction with human AT III was noted in several biochemical systems using fluorescence quenching and thrombin inhibitor based assays. These agents also inhibited IXa-PL-Ca+Z/VIII mediated generation of thrombin suggesting that direct inhibition of intrinsic system may be involved. In undiluted plasma systems at micromolar amounts these agents produced a strong inhibition of prothrombin activation as measured by immunoquantitation of TAT complex. Although at the submicromolar levels these agents did not inhibit prothrombinase activation, they catalyzed HC II thrombin complex formation. No direct inhibition of thrombin in fibrinogen supplemented and purified antithrombin III based systems at >20 #M was noted. In prothrombin complex based Xa and thrombin generation assays, these agents produced marked inhibitory effects which were partially mediated by HC-II. These studies suggest that sulfated lactobionic acids produce their antithrombotie effects via heparin cofactor II and modulating serine protease generation in the intrinsic pathway. Additional studies on the interactions of these agents with the blood/vascular system are needed to understand their prolonged antithrombotic actions. Loyola University Medical Center, Maywood, IL USA and *Luitpold-Werke, West Germany. From its ability to specifically bind to crosslinked fibrin, but not to fibrinogen, rt-PA gains its fibrin specificity. In 28 patients undergoing thrombolysis for myocardial infarctions with rt-PA doses ranging from 0.625 to 1.875 mg/kg or 24.5 to 63 mg/m 2, we have investigated the dose dependency of fibrinogen (Fg)-, plasminogen (PLG)-, and~-2-antiplasmin (AP) consumptions and the simultaneous increases of fibrinogen (FgOP), fibrin (FbDP) tion products. and total (TDP) degrada-.,.:. Conclusionsl There is a strong dose dependency for the systemic effects of rt-PA (fibrinogenolysis and plasminaemia). In contrast, the dose dependency of the clot directed lytic activities of rt-PA as reflected from the release of fibrin specific degradation products (FbDP), however, is only week. Abteilun 9 Kardiologie (1) und H~matologie (2) Although neonatal asphyxia relating to intracranial hemorrhage may have several reasons, the etiology of the most common type, intraventricular (IVH) is not yet known. There is histological evidence of disturbance of blood circulation in cases of intrecranial bleeding in the aaphyxiated newborn and lowbirthweight, end in addition there are changes in both coagulation and fibrinolytic systems. Recent information demonstrating thet the initial lesion is due to hemorrhagic infarction raises the possibility that hypercoagulability may have something to do with an etiologic roles. Material and Methods 48 cases of umbilical cord blood were divided by Apgar score, asphyxiated group (Q7) and normal group (8,9, and 10) 1) ADP-and Collagen-Aggregation (Platelet) were tested by whole-Blood Aggregometer(Chrono-Log) with impedance channel. 2) Platelet volume and Platelet count were determined by Baker 810, and MPV was calculated as well as Mode and Mean. The value of MPV in asphyxiated group was 8.1 cu and it was greater than that of normal (6.8 cu). It has been demonstrated in recent dose-finding studies, that thrombolysis in acute myocardial infarction may success fully be performed with urokinase (UK) preactivated pro-urokinase (PUK), without depleting the fibrinogen levels in the blood. In 35 patients undergoing thrombolysis with 250 000 IU of UK and 42 to 118"103 U/kg or 2.1 to 4.2 mega U/m 2 of PUK, we have investigated the dose dependency of the fibrinogen (Fg), plasminogen (PLG), and~-2-antiplasmin (AP) consumptions and the simultaneous release of fibrinogen (FgDP), fibrin (FbDP), and total (TOP) degradation products. A 63 years old woman suffered from excessive bleeding diagnosed as idiopathic thrombocytopenio purpura because of moderately decreased platelet count and shortened platelet survival. After splenectomy platelet count increased to normal or subnormal values but bleeding symptoms persisted. Platelet function was severely impaired: lack of platelet aggregation in response to AOP and collagen, defective ristocetin-induced agglutination and adhesion. Membrane glycoproteins (GPs) of the patient's platelets were analysed by one-and two-dimensional gel electrophoresis and blotting in combination with concanavalin A-peroxidase staining. A normal GP pattern including normal amounts of GPs IIb and Ilia was detected. Quantification of GP IIb-IIIa complex by electroimmunoassay indicated apparently reduced amounts. In addition, in crossed immunoelectrophoresis the complex migrated with lower mobility. A similar shift in the position o£ the GP IIb-IIIa precipitate of normal could be induced by incubation with serum of the patient. Strongly increased amounts of IgG associated with the patient's platelets were detected. This IgG-type immunoglobulins coprecipitared with the GP IIb-IIIa complex. The results provide evidence for the assumption that clinical symptoms and defective platelet function in the patient are caused at least in part by autoantibodies against the GP IIb-IIla complex. Abteilung for Angiologie, Zentrum der Inneren Nedizin, Klinikum der J. W. Goethe Universit~t Frankfurt/M., Theodor-Stern-Kai 7, 0-6000 Frankfurt/H. Ear.ly restenosis following successful angioplasty is a big problem in interventional cardiology. Till now there exsist no treatment regime that alter the rate of restenosis. In the present study the incidence of restenosis was examined under the treatment with the low molecular weight heparin (LMWH) FRAGMIN versus aspirin (ASS, 100-300 mg/d). An adjusted LMWH dose was given that led to peak levels of .3-.6 anti-Xa-activity (mean 6000 U/d). The definition for restenosis based on the guidelines of the American National Heart, Lung and Blood Institute. The rate of restenosis was proven by coronary angiography 3 month after angioplasty. The amount of stenosis was estimated by using a semiautomatical com-puterized slide analyzing system. The incidence of restenosis in the ASS group was higher (6 stenosis out of ii in i0 patients) than in the LMWH group (3 out of ii stenosis in 9 patients). There was no significant difference in mean area stenosis pre-(LMWH: 85.1%, ASS: • 84.7%) and initially post-PTCA (LMWH: 39.5%, ASS: 47.3%). None of the patients suffered myocardial infarction in the follow up period. Bleeding complications or other drug related side effects did not occur. Conclusion: The treatment with LMWH seems to be an effective and safe treatment after PTCA with advantages in comparison to aspirin. Till now,after an initial bolus of about 10% of the total dosage rt-PA was given as a prolonged intravenous infusion because of its short half-life and in order to avoid early reocclusions. A single bolus would simplify the therapeutic regimen.+Therefore 20 patients (3 female, 17 male; mean age 55 -11 years) wish acute myocardial infarction (duration of symptoms 125 -58 min) were treated with a single rt-PA bolus of 50 mg intravenously (over 2 min).The basic therapy before lysis consisted in 5000 IU heparin, 500 mg ASA and nitroglycerin intravenously. Results: T~e fibrinogen lev~l {Clauss method) decreased from 2.7 -0.5 g/1 to 1.5 I 0.9 g/l after 2-4 hours and reached the initial value within 24 hours. Pharmacokinetic parameters were obtained in 7 patients by measuring rt-PA antigen levels in multiple plasma samples. Mean peak rt-PA concentration was 9.9 -3.8 ~g/ml, ~otal plasma clearance 490 ± 150 ml/min and half-life 4.9 -1.9 min. Coronary angiography 60 min after rt-PA bolus revealed an open infract-related artery (TIMI 2,3) in 15/20 patients (=75%); in the remaining patients reperfusion was achieved by angioplasty (PTCA),intracoronary thrombolysis and twice an aorto-coronary bypass was necessary. Control angiography at 24 hours showed a reocclusion in 4/18 patients; during hospital stay PTCA and bypass grafting was performed in 10 patients.One female patient died 17 days after rt-PA bolus because of fatal intracranial hemorrhage. Conclusions: A single intravenous rt-PA bolus of 50 mg seems to provide similar patency rates and kinetics as the usual infusion;the incidence of bleedings requires further studies. Recombinant Hirudin (rH, CGP 39393) is a potent thrombin inhibitor. In addition to its anticoagulant effect, we were interested in its profibrinolytic activity in vitro and ex vivo. For the in vitro experiments the fibrinolytic activity of the euglobulin fraction (EGL) of 15 normal plasmas with or without urokinase (Uk) was tested in a fibrin plate assay both before and after addition of rH. The same cycle was repeated in 8 plasmas with heparin (Hep) instead of rH. For the test system EGL was obtained from 0.5ml plasma containing 5U Uk and 6.6 ttg rH or 0.5 U Hep and applied on human fibrin plates. For the ex vivo evaluation 12 healthy volunteers received 0.1-0.3 mg/kg/h rH for 6h. Blood samples were drawn at Oh, 6h, 12h and 24h. Fibrinogen (Fbg), plasminogen (Pig) and antiplasmin activity (AP), t-PA antigen and PAI activity, thrombin-antithrombin III complexes (TAT), rH levels (Elisa) and EGL fibrinolytic activity were assayed. RESULTS: rH induced more intensive fibrinolysis in synergism with Uk measured as lysis surface (mm 2) (Uk: 194.2~_46.6 vs Uk+rH: 245_+11.7, p<0.0001), rH enhanced the fibrinolytic activity of plasma (P) in the absence of Uk also (P: 55.2_+10.9 vs P+rH: 62.6+5.2, p<0.02). Heparin had a similar but slightly weaker effect with Uk (Hep+Uk: 180.1+55.7 vs rH+Uk: 188.4+_52.4, p<0.06) and without Uk (Hep: 67.5+-38.9 vs rH: 72.2_+44.4, p<0.2). Ex vivo on the other hand, Fbg (p<0.02), Pig (p<0.2) and AP (p<0.1) were slightly reduced at the end of 6h. From Oh to 6h PAI decreased from 24.7+8.6 to 13,1_+1.9 AU/ml (p<0.005) and t-PA from 4.5_+1.9 to 3.2_+1.0 ng/ml (p<0.001), a variation that parallels the normal circadian rhythm. TAT did not show Significant changes. The EGL of the proband plasmas spiked with Uk showed a stronger fibrinolytic activity (p<0.03) at 6h compared to those of Oh and 24h (Oh: 156.6_+76.6, 6h: 216.8_+33.9, 24h: 173.3_+61.5), but this activity did not correlate well with the rH levels. CONCLUSIONS: 1. In the presence of rH a slightly stronger fibrinolytic effect is observed measured as fibrin plate lysis activity both if rH is added to plasma in vitro and if it is administered intravenously. But this may be well explained by the specific anticoagulant action of rH and Hep in the dynamic equilibrium of fibrinolysis and clotting in the test system. 2. Under rH treatment the observed changes of Fbg, Pig, AP, t-PA and PAI although suggestive of enhanced fibrinolysis cannot be differentiated from the normal circadian rhythm of these proteins, since "the highest ex vivo fibrinolytic activity coincides with the lowest PAI levels. We investigated the blood of 64 patients suffering from liver cirrhosis (Cl) or chronic active hepatitis (CAH) on the levels of protein C (ag and act.), antithrombin III (ag and act.), ~2-antiplasmin (~2-AP), plasminogen, fibrinogen, prothrombin time (PT), f.V, f.Vll, f.VIII:C and v.WF-Ag. The results of the coagulation tests were compared with the levels of cholinesterase (CHE) and albumin. In addition the levels of the liver enzymes: GGT, GOT, GPT and AP were determined. All the results were compared with the findings in 34 healthy persons. In the patients suffering form Cl we found the best significant correlation between PC and CHE (p<0.001), between PC and albumin (p<0.01), between AT III and CHE (p<0.001) and between AT III and albumin (p<0.01). By differentiation in compensated and decompensated groups of liver cirrhosis the correlations were still more pronounced. In the patients suffering from CAH a positive correlation could be demonstrated between PC and CHE (p<0.05) and between PC and albumin (p< 0.05). In this group we found neither a significant correlation between AT III and CHE nor between AT III and albumin. We could demonstrate that the decreased levels of the other parameters: plasminogen,~2-AP, fibrinogen, PT (in %), f.V, f.Vll and the increased levels of f.VIII:C and v.WF-Ag corresponded not significantly with the decrease of liver synthesis. PC and AT III proved to be the best prognostic markers for decreased liver synthesis in cirrhosis and CAH. (a) is an LDL-like particle to which the characteristic apolipoprotein (a) is attached by disulfide bonds to apo B-IO0. Human apo(a) cDNA contains domains homologous to kringles 4 and 5 and the protease domain of plasminogen. Recent reports suggest that Lp(a) in vitro inhibits streptokinase induced plasminogen activation and enhances ADP and epinephrine mediated platelet aggregation. Therefore we investigated the Lp(a) concentrations in 2 unrelated patients suffering from recurrent deep vein thromboses with proven abnormal plasminogen molecules and in their affected relatives. Furthermore we studied the levels of Lp(a) in 203 young patients below the age of 45 suffering from venous thromboses. Lp(a) was measured with a sandwich immunoradiometric assay using 2 different monoclonal antibodies. The assay proved specific for Lp(a) in that on immunoblotting analysis only the solid phase antiapo(a), but not the tracer Ab crossreacted with purified human plasminogen. The Lp(a) concentrations of the patients with abnormal plasminogen molecule corresponded to that of a healthy reference population. The levels of Lp(a) in the young patients suffering f;'om venous thromboses coincided with that in the normal controls (84 healthy blood donors). Therefore we concluded that there is no direct correlation between the Lp(a) concentration and the risk of venous thrombosis. A new abnormal plasminogen, Frankfurt II, has been identified in the plasma of a 27 year-old male patient who has a long history of recurring thromboembolic disease since age 6. He has a plasminogen functional deficiency, also the antigen concentration was slightly reduced. His plasmin generation rates were lower than normal as were those of other family members. His isolated Plg had a specific proteolytic activity of 15.0 IU/mg protein, about 50% of normal; family members values ranged from 17.5 to 23.5 IU/mg protein. Maximum active site generation rates in the equimolar Plg-Sk complex (0-60 min. at 37°C) ranged from 60% to 80% of normal for all family members, the propositus and his father were at the lower levels. The Plg defect appears to be in the formation of the equimolar PIg-SK complex and the generation of plasmin (PIg)-SK. The formation of the PIg-SK complex and generation of PIn-SK analyzed in SDS-PAGE showed a) intact Plg, b) slow formation of PIg-SK with two PIg-SK complexes, c) slow conversion of PIg-SK to PIn-SK with two PIn-SK complexes, d) slow fragmentation of SK in the complex. Reduction of the 0-60 min. complexes in SDS at I00°C, and at O°C, showed degradated A chains, two sets of A and B chains, and SK complexes. Other causes of thrombophilia could be excluded by repeated investigations. Therefore we presume a correlation between the plasminogen abnormality and the recurrent thrombosis in this young patient. patients were treated with streptokinase followed by urokinase in 56 cases. Recanalisation was documented by phlebography in 12 patients with thrombosis of the' upper extremity. SK-therapy was effective in 55,3~, UK in 30,2~ in thrombosis of the lower extremity. Effectivity of thrombolytic therapy depended on localisation and extent of thrombosis. Therapy was less effective when clinincal symptoms of phlebothrombosis were present for more than 2 weeks before initiation of treatment. Phlebographic follow-up was performed in 34 patients up to 4 years after UK/SK-therapy. Further improvement was observed in 62~ indicating that spontaneous recanalisation may occur in spite of unsuccessful thrombolysis. DUB to its steric configuration fib~inogen interacts with red blood cells -mainly in the small vessel sustem, wh~ce shear stress is lowcausing revecsible aggn~gation, well known as "rculeaux" or "sludge" phenomenon, Hence sludging can be the reason o£ severe ismhemia, and the tendency to aggregate cotTelstas well with concentrations cf £i~rinogen, there is a need to qcantifg the corce~trstion of £ibFirogem. Since blood serum is defined as plasmB without £ibFinc~n, it seems to make Sm~lse -when testirQ the visoosit~ 06 both -to calculate the coneentFatioq of f i b r i n c~n from the diFFerence betmeen these two values. Using ~ common kit ( M L~T I F I~A P bj Behmingwerk@, MerbL[~g, FRG) as r~e r~r~ method, the time needed to ooagulat8 plasma ( " t " i n equation; dimension: seconds) is eeeeuced. Alterations of the fibrinolytic system in coronary artery disease and myocardial infarction (MI) have been reported in a number of studies within the last years. In the present study 29 male patients with an angiographically confirmed MI before the age of 45 and a control group consisting of 13 young males were studied in regard to their haemostatie and fibrlnolytic system, t-PA-activity, t-PAantigen and PAI-1 wece i n v e s t i g a t e d before and a f t e r a 20 minutes venous occlusion t e s t (V0). Since high serum l i p 0p r o t e i n (a) ( L p ( a ) ) -l e v e l s are known to be connected with an increased r i s k for a t h e r o s c l e r o s i s and MI, we f u r t h e rmore determined serum L p ( a ) -l e v e l s in p a t i e n t s and c o n t r o l s L p ( a ) -l e v e l s were s i g n i f t c a n t l y higher in the study group ~ed[an 21.8 mg/dl, range 1.6-90.5) in comparison to the c o n t r o l group (7.1, 1 . 0 -7 7 . 7 ) ( p < 0 . 0 1 ) . We derided the pat i e n t s into 2 subgroups: one c o n s i s t i n g of p a t i e n t s with a high r i s k for MI in terms of high Lp~a)-levels (Lp(a)> 20 mg/dl) and one with p a t i e n t s t h a t sho~ low serum Lp(a). In the p a t i e n t group with high t p ( a ) -l e v e l s s i g n i f i c a n t l y lower P A l -l -a c t i v i t y (med.3.1 U/ml, r. 0 . 5 -9 . 9 ) could be found, when compared to that with low t p ( a ) -l e v e l s (med. 6.6 U/ml, r. 1.2-23.0) (p 0.05). Plasma samples containing a LA demonstrated a shortening of clotting time with increasing amount of added PL. In contrast to the observations of other investigators different amounts of added PL were needed to minimize the clotting time of an individual plasma sample. Further increase of PL concentration led to an increase of clotting time. Independent from the used coagulation assay the calculated indexes in this group were significantly higher (p < 0.001) than those of normal or anticoagulated plasma. The test design seems to be a suitable confirmatory test for LA. Because of the variable PL concentration needed to minimize the clotting time, one particular index is probably not the best approximation for the in vitro activity of a LA. Zentrallabor der Medizinischen Universit~tsklinik Josef-Schneider-Straae 2, D-8700 W~rzburg Yos--Gly--Pro--Arg--NH ~ N CH3 This substrate is particularly useful for the determination of thrombin. Dissolved in 0.1 m HEPES buffer pH 7.0 it shows a X max of 522 nm in the UV-VIS spectrum. After cleavage of the tripeptide chain by thrombin the resulting N,N'-Dimethyl-4-aza-oxindol-azomethine dye has a k max of 559 nm. The substrate is also cleaved by other peptidases, but with significantly less specificity. In comparison, the molar extinction of the flee dye is 4.6 times higher than that of the uncleaved substrate. Because of the high specificity for thrombin on one hand and the significant difference between the extinctions of the dye and the substrate on the other hand, the new substrate is quite useful for global assays in spite of the relatively small k-shift of approx. 40 nm. We studied the properties of the new indicator in a photometric prothrombin time test. In addition it is possible to use the new substrate for reflectance read tests. Boehringer Hlgh dose SK in AMI lowers plasma ftbrlnogen to levels near zero whereby blood viscosity decreases. This favourable but short lasting effect may be prolonged by subsequent application of ancrod, In order to investigate this treatment reg&men 30 pat. with AMI were divided in 3 treatment groups: Gr. I heparin alone; n = 10, mean age 56.3 yrs; Gr. II high dose SK + heperin; n = 10, 55.4 yrs; Gr. III high dose SK followed by ancrod; n = 10, 56.7 yrs. A battery of haemorrheologlcal and haenmstasiological tests were performed before and 4,8,24 hrs, 8 and 15 days during treatment. While in Gr. I fibrinogen steadily increased until day 8, there was a steep fall of fibrinogen during SK-infusion in Gr. II with a subsequent exceeding increase until day 8. In Gr. III fibrinogen was lowered to values of about 1.2 g/1 during the 2 weeks of ancrod treatment with favourable effects on plasma viscosity, apparent whole blood viscosity and erythrocyte aggregaton index. Thrc~bin-antithrombin III complex (TAT) was increased before either treatment (7-18 pg/l), fell during heparin treatment to normal values, increased durlng SK infusion intermittently, but remained eievated for at Ieest 2 weeks during ancrod. The elevation of TAT during ancrod treatment correIated inverseIy with a decrease in AT III, plasminogen, and alpha-2-antiplasmin. Flbrin(ogen)degradation products (FDP) increased to about 300 pg/dl on day 1 and remeined elevated through ancrod treatment at leveIs of ~ 35 Mg/dl. FA was increased in gr.A and B due to a significant increase in tPA antigen concentration and e significant decrease in PAIi -and ~AP activity. These changes were significantly more pronounced in gr.C; thus in the latter group Fb-DP were significantly elevated. There were no significant differences between pts with and without portacaval shunt. 1985 until Oct.1989 . Until June 1988 86 Patients were treated with a standard dose of Urokinase(UK; 2,4 Mill. U/d) for a mean of 13 days. Within a dose finding multicenter study starting in July 1988 8 Patients were treated with rtPA 0,5 or 0,75 mg/kg/d and, starting March 1989 with rtPA 0,25 or 0,375 mg/kg/d. Therapy ge nerally lasted for 7 days. Furthermore 16 Patients under went thrombolytic therapy with ultra-high-dose-streptoki nase(UHSK) using 9 Mill. U/6h on 3 consecutive days. As cending phlebography was performed initially and after termination of the lysis. Complete clot dissolution without thrombotic residues of any hemodynamic relevance resulted in 35% of all evaluated pa tients: UK 27%, UHSK 75%, rtPA 43%. Lysis of more than 50% 0£ the o r i g i n a l clots was o b t a i n e d in 25% o f all c a s e s : UK 26%, UHSK 19%, r t P A 29%. Partial l y s i s to an e x t e n t l e s s t h a n 50% was r e a c h e d in 21%: UK 26%, UHSK 0%, r t P A 21%. 18% of all p a t i e n t s did not a c h i e v e a n y l y s i s at all: UK 21%, UHSK 6%, r t P A 7%. Complications d u r i n g t h e r a p y ( b l e e d i n g , p u l m o n a r y embolism, allergic r e a c t i o n s ) h a d to be noted in 12% (n=16) of 138 initially t r e a t e d p a t i e n t s . O f t h e s e t h e r a p y h a d to be t e r m i n a t e d p r e m a t u r e l y in 12 c a s e s (75%): UK 10/96 (10%), briSK 1/17 (6%), r t P A 1/15 (7%). Because of the affinity of coagulation factors to polyanions we investigated the effect of these procedures on coagulation parameters in the extracorporeal circuit. If ACD-plasma is treated by dextran sulfate adsorption, the activity of the coagulation factors V, IX, XI and XII is completely abolished, factor II is not affected. Factor VII activity decreases to 70Z, factor VIII to 23Z and factor X to 29Z, as compared to pre-treatment activities. Fibrinoge n is adsorbed at the beginning of the procedure 5inca the fibrinogen binding capacity of the column soon becomes saturated. Plasminogen decreases to 75Z and AT !II to 44Z of the original concentration. The HELP procedure completely depletes the activities of factors II, V, VIII and IX. Factor VII is reduced to 65Z, factor X to 12Z, factor XI to 40% and factor XII to 27Z of the pre-therapeutic activities. Fibrinogen and plasmlnogen precipitate completely; AT III concentration is reduced by 20%. All coagulation factor activities except of fibrinogen are restored within 24 to 48 hrs. No bleeding or thromboembolic complications in long-term treated patien%a using the HELP-system show that these acute raductiona do not lead to functional derangement of the coagul~ion System. Clinics, Robert-Koch-Str. 40, D-3400 G~ttingen, FRO Previously we have shown that the in vitro bleeding test (IVBT/Thrombostat 4000) is a very sensitive method for the detection of platelet function impairment (Blut 59." 188 (1989) ). Now we want to report on a significant increase of in vitro bleeding time andvolume in hemodilution (tab.), which was simulated using "reconstituted" blood with constant platelet concentratioru For hemodilution we either used platelet poor plasma (PPP), albumin, hydroxyethyl starch (HES), saline or dextran. The effect correlated with the grade of dilution. Al]m/min had the strongest influence followed by saline, dextran and HES. The primary hemostasis was changed least by PPP. We report on a 48 year old unmarried woman with a bleeding history due to an unclear thrombocytopathy. Her mother died from bleeding at the delivery of the 3rd child. The petient frequantlyhadhaematomas, petechiae, epistaxis and bleeding from the gums already in the childhood. Despite this, there have been no bleeding complications in connection with several operations. In 1960the patient had an acute rheumaticpolyarthritis which was treated with aspirin. In this context an Angio-Haemophilia (von Willebrandsyndrome) was diagnosed. This diagnosis was confirmed by a haemostasis laboratory of an university in 1972 and 1974. As the bleeding complications extended in the last years she received factor VIII concentrates and after a severe hepatitis platelet concentrates from random donors (in 1988 at least from 100 donors). In 1985 haemostasis laboratory of another university detected a cyclooxygenase deficiency and diagnosed an '~spirin-like-defect". In early 1 989 the patient was sent to our department in order to clarify the diagnosis and to find a therapeutical regimeru We could confirm the severe thrombopathy. The diagnosis couldn't be made definitively. . Independent from therapy the severity of thrombocytopathychanged considerably during clinical stay when tested by IVBT. In addition, only b y I V B T we easily were able to control the therapy: a good therapeutical effect of DDAVP (i.v. ~ i.n.) and HLA-selected platelets, no clear effect of FFP and no effect of phospholipids ( FibraccelR ). V. Kretschmer, D. W~, G. Dietrich Before routine use of the sensitive in vitro bleeding test (IVBT) the methodical evaluation of the influencing factors is necessary: We always changed one factor of a standard test (blood sample stored at room temperature for one hour, preincubation of blood, filter and CaCI 2 for 4 min, filter aperture 150 gm) determined bleeding time (BT), volume (BV) and compared the results with the standard test by Wilcoxon's rank test (p * .05, ** .01 ). 1 ) Blood samples should be examined between 30 and 180 min after drawing (tab: means for n = 10 Platelets internalize collagen fibers (CF). This process is accompanied by the formation of TXA 2 , the platelet shape change and the secretion of the granule contents. Computer assisted 3-D reconstruction from serial electron micrographs revealed that the internalized CF were closely associated with the contractile sphere in the platelet centre. Cytochalasin D (Cyto D) 0.1 HM suppressed the internalization of CF, the shape change, the formation of the contractile gel, the degranulation and the [lU'C]serotonin release but did not reduce the number of piatelets that adhered to CF. In contrast, Cyto D enhanced the degranulation and the [lU'C]serotonin release induced by U u,6619, although those plateiets remained discoid and showed their typical marginal bundle of microtubules and no contractile gel. PGE 1 1 IJM and IIoprost 0.5 nM inhibited the internalization and the phenomena of activation both with CF and U t1.6619. Aspirin neither affected the internalization of CF nor the shape change and degranuiation. However, aspirin suppressed the TXA2-mediated activation of the p/atelets that did not adhere to CF. The opposite effects which Cyto D exerts on CF-and U u,6619-induced platelet responses indicate that the internal contraction is essential for CF internalization and subsequent platelet activation but unneeded in U u,6619-induced platelet degranuiation. Cyto D prevents the internalization that follows the adhesion of CF. Internalization is independent of TXA 2 and can be inhibited by high intracellular cAMP. ( and conslsts of PC actlvator (Protac), PC-deficient plasma (substrate plasma) and a control plasma (about 60% PC). T~e sample is diluted with a specific diluent, then Protac is added and incubation takes place at 37°C for 5 minutes. Complete activation of PC is ensured within this time interval. The activafed sample is then added to substrate plasma: the prolongation that can be observed on the A~of the substrate Plasm a is related to the concentration (% activity) of PC in the sampl~ Calibration is automatically performed by the ACL-". Linearity has been ascertained in the activity range 10 ÷ 150%. Thanks to the sensivity of the AFIT reagent, a difference of about 100 seconds is obtained between 0~ and 100% activity in a typical calibration function prepared by dilution. The presence of polybrene in the diluent makes it possible to analyse samples under heparin thera[~y, avoiding interference up to I IU/ml heparin. We sho~the results of a c~parison -carried out on the ACL "'" -between Proclot "'" and a commercially available reagent, with samples collected from healthy donors and a variety, q~ patient~: under oral anticoagulant therapy; under heQarin therapy; with liver disease; with congenital or acquired PC deficiency. Instrumentation Laboratory GmbH, Klausnerring 4, D-8011Kirchheim-Heimstetten 133 110 exhibited by investigations of pletelet ~ perfused in glass models of vessel branchings and cvrves, platelet microthzx*abi d~/oped -provided their a~tasivity was e~nced -in those areas #here the platelets were bransp~ted towards the wall and touched its surface (~11~ H., Thcuatx~is Research, 8, 553-566, 1976 ). In the lxesent stt~, platelet ac~sivity to etdothelial monolayers and to glass surfaces was qualitatively sad quantitatively ex~m~wd. T~e effects of the antionagulonts beparin and citrate on the a~esion a~d ~#~re~atica fmctica were also on~ared. ~ne convective transport was s~lified and reduced to a rotationally symetrical stagnation point flow of plate/et rich plas~ PRP (Stagnation Point ~esio-~mgomter, ~). Non-stimulated platelets did ~ot a~tere to the e~otheli~. In order to evaluate the ontit~=~enic Woperties of the e~otheliuB as cum~xed to glass, the platelets were stimulated with ade~sin dil~c~hate in a concontratinn of 1 ~ which induced platelet acg~-ence. The time creese of the platelet deposition was recorded as an increase in the onmmt of delx~sited platelets per surface area and time tm/t. Tasae growth curves were calculated with an analooue ccqmter. By a~lyi~ the rescti~ kinetic equations of the tra,sport theory for the gro~:h of platelet ,~ (MO/ler-Mchnsaen H., Scholtes L., P~eataseologie: 2, 143-162, 1982) values were obtained for the growth rate constonts k~w and k~ -which describe, respectively, the a~saivity of the platelets to a surface and the aggregabil/ty of the plateleta to each other. Tes =ost imxr~mt results will be d/sct~ssed: ~e e~dothelium demmmtrated its intactneu as well as its antithrombogenic properties in remaining free of a~mring platelets not stimulated with A~. Q~mtificatica of its ~tithromboge~city -using ~P-stimalated platalets -deamstrated a ton-fold decrease in adhesion while aggregatica was only 2 times less with mMothelial surfaces as cca~L~d to glass surfaces. Four different methods for determination of soluble fibrin (SF) were compared, the SF-tPA-tast (COASET), SF-ELISA (Boehdnger Mennhelm), SF-erythrocytesagglutlnetlon-test (SF-EAT =FM-Test) and the SF-PS-turbldimetry. The latter Is based on the turbldimetry of the protamlne sulfate (PS) induced aggregation of soluble fibrin (SF), which can be detected without disturbing Influence by fibdnogen under defined test conditions. In the first part of the study, the fibdnogen-fibdn turnover was Induced In vitro by addition of thrombln or ancrod to sodium citrate plasma; after various time Intervals the action of the enzymes was stopped by hlrudln or antieamm, respec-tNaly. Apart from soluble flbdn flbrlnopaptld A (FPA) was determined In each eamp~e (n=96). Data obtained from the thrombln-and ancrod-lnduced flbdnogen turnover were evaluated separately, since different forms of soluble fibdn were sup~. The results of SF-EUSA, SF-tPA-test, SF-EAT. and FPA-EUSA correlated well with each other (r • 0.93). If only Increased values were coneldered, the SF-PS-turbldimetry correlated well with the results of the other SFmethods and with the FPA release (r • 0.91), but very low concentrations could not be detected. According to BRA~, an explanation for this may be the lack of aggregation at low soluble fibrin concentrations. The high correlation of these results Is remarkable, since the tested methods are based on differeot principles probably leading to different specificity. Subsequently these methods ware applied for determination of soluble fibrin In plasma samples from intensive care patients suffedng from DIC: In contrast to the in vitro studies the correlation was less satisfactory (r < 0.67). The SF-PSturbldlmetry yielded a sufficient seneltivlty, whereas the SF-EAT often failed. Heterogeneity of soluble flbdn (size, number of ollgomem and formation) and different sensitivity for flbdn(ogen) degradation products might be an explanation for weaker correlation In patients' plasma than In the In vitro studies. Haematologle, UnlveraltStskllnlken, 3400 G0ttlngen / FRG Patients with acute or prolonged coagulation disturbances as known in DIC were examined In several studies. In particular, the flbrln(ogen) derivatives proved to be very sensitive Indicators in diagnosing and monitoring of DIC. In acute pancreatitis, for example, the elevation of enzymes was followed by an Increase of soluble fibrin and subsequently of degradation products detectable by FbDP-, D55mg/dl: 93.6 ±26.5% vs. HDL<35mg/dl: 82.5 ±22.1%). DMI (r= -0.13"*) and systolic b.p. (r=-O.13 **) negatively correlated to EFA. The t-PA-activity on fibrin plates with added Ct-inactivator (n= 615, ~=92.5 ±19.1% of a normal pool) exhibited positive correlations to EFA (r= 0.50***), as well as to protein C (r=0.16***) and ATIII (r=0.27"**). (* =p14 IU/ml) however were 27-40~ higher in the HCF than the In LCF plasmas. PAI activities in the HCF samples were distinctly higher than in the LCF plasmas: PAI (HCF) B --19.6 +_ 0.8 AU/ml, VO --16.9 + 0.9 AU/ml vs. PAI (LCF) B = 13.4 + 1.0 AU/ml and VO = 10.2 + 1.2 AU/mI. From our results we conclude, that short centrlfugatlon at high centrifugal forces is suitable for preparation of acidified plasma for tPA activity determination. High tPA activities In plasma thereby may be better preserved from Inactivation. For PAI activity determination centrlfugstlon at 15,000xg Is unsuitable, because the higher PAI levels obtained here Indicate release of PAI activities from platlets and blood cells. Instltut f. Exp. H~matologle und Tranafuslonsmedlzln der Unlverslt&t Bonn, Slgrnund-Freud-Str. 25, 5300 Bonn I Previous studies have shown that l-alkyl-2(R)acetyl-glycero-3-phosphorylcholine (PAF) fails to induce platelet aggregation in platelet rich plasma of the rat (D. Namm, Thromb. Res. 25: 341, 1982 ; own results). The microembolization of labelled platelets in the lung was used as an in vivo model to evaluate the effect of PAF and inhibitory drugs. Following injection of 51Chromium-labelled homologous platelets into urethane-anesthetized rats the distribution was continously monitored using collimated crystal scintillation probes. Count rates and the ratio of two detectors (ci/c2) one placed above the thorax (cl), the other above the abdomen (c2) -were calculated and displayed by a microcomputer-based system. A bolus injection of PAF resulted in a rapid and dose-dependent sequestration of 51Chromiumlabelled platelets in the lung. This process was reversible and not redeemable by a second application of PAF indicating a desensitization. Nevertheless labelled platelets were still able to react to other platelet stimuli e.g. ADP. PAF-induced (5 ug/kg) pulmonary platelet accumulation could be completely overcome by prior injection of the specific PAF-antagonist WEB 2170 (BOEHRINGER INGELHEIM). Pretreatment of the animals with pentoxifylline and acetylsalicylic acid, administered successively (principle of HWA 5112) also exerted a significant inhibition° These results indicate that, in contrast to the situation in vitro, a PAF-specific activation of platelets is achievable in vivo. The detection of the multimeric structure of yon Willebrand factor is an important method of subtyping yon Willebrand's disease. We report an electrophore~c method, in which the yon Willebrand factor multimers are separated in a 1,0% agarose gel, using a discontinous buffersystem. The gel was lying on the water-repellent side of a ~el bond film and cooled at a temperature of 12vC. At the end of the separation the top side of the gel was covered with a nylonmembrane having a pore size of 0,2 ~m. The sandwich was with the nylon-membrane infront overturned on a carbon electrode, covered with filter papers, soaked in anode-buffer. After remove of the gel bond the sandwich was completed by bringing up filter papers, soaked in ~athode-buffer, and a second carbon electrode. The semi-dry blotting was performed by a constant current of 15mA for 17 hours. The visualization of von Willebramd factor multimers was carried out by use of a peroxidase conjugated rabbit antibody to human factor VIII vWf. The present method has a high degree of resolution and consumes, compared with conventional methods, using tank-blotting and immunochemical sandwich techniques, less buffer, antibody solutions and time. Problems, using radioactive marked antibodies, are avoided. Any change of hydrogen ion concentration could change the dissociation of proteins and possibly plasma and blood rheology. We measured therefore plasma and blood viscosity (capillary hose viscosimeter KSPV 4, RHEOMED for the plasma, WELLS-BROOKFIELD cone plate viscosimeter with shear rates of 230,4, 115.2, 46,08 and 23.04/'" for the blood) at different pH-values. We draw blood from three volunteers from an antecubital vein after venous stasis with a tourniquet for 5" and vigorous exercise with that arm. The initial pCO2 was between 54.1 and 75.3 mmHg, the pH between 7.232 and 7.06. Measurements were done immediately and then every 30" for 2 hours. The specimens were kept open, so that CO2 could leave the blood or plasma. At the last measurement the pCO2 was between 11.1 and 6.1 mmHg, the pH between 7,472 and 7.608. Plasma and blood viscosity remained statistically unchanged at all pH rp. pCO2-values. We conclude that alterations of the acid base balance do not influence blood or plasma rheology. Aim of this study was to find the best mathematical model fitting the curve that express the aggregation phenomenon when evaluated by means of impedance aggregometry on whole blood. Venous blood samples were collected from I00 healthy subjects (50 males and 50 females). Platelet aggregation was measured by a Chrono-Log aggregometer; collagen, at different concentration, was used as proaggregating agent. The mathematical analysis of the curves was performed by means of the least square method. The best model fitting the curves was the hyperbolic function Z=K*tm,where Z is the impedance and t the ti~le while K and m are the regression coefficients. The chi-square method and the Fisher's test confirmed the expressed hypothesis. The m parameter represents the per cent variation (positive, null or negative) of impedance for a per cent unitary variation of time. A normal distribution of the m values has been obtained from our samples. The normal range in our laboratory was 0.39+/-0.08. Values above or below this range could be respectively considered as expressing hyper or nypoaggregating situations. This simple but exact method could allow an easier clinical interpretation of platelet aggregation on whole blood. Aim of the present study was to investigate possible changes of platelet aggregability occurring during aging in healthy subjects. Platelet aggregation was evaluated in a group of 40 individuals (20 nlales, 20 females) whose age ranged from 50 to 5g years (mean 65.6 years).Criteria for inclusion were the absence of metabolic and/or cardio vascular disease, including hypertension, Furthermore, they were on no medications nor were they suffering from any acute or chronic illness. In the same way, 40 individuals watched for sew and smoking habits, whose age ranged from 40 to 59 years {wean 46.5 years),were enroled as control group. Platelet aggregability was evaluated by iaeans of impedance aggregometry using a whole blood Chrono-Log aggregometer; collagen at different concentration was used as proaggregating factor. Venous oIood sa~iples were collected without stasis in the fasting subject after 2 hours bed rest; blood was anticoagulated with sodium citrate 3.8%. Analysis of aggregation curves waS performed by means of calculation of a mathematical index (m-index), elaborated in our laboratory, that integrate the rate and percentage of aggregation. A significant increase of the m-index, ~aeaning a condition of relative iperaggregability, was present in the group of elder subjects compared to control group. B. Pilz, M. Steins, W. Stenzinger and J. van de Loo Autoimmune thrombecytopenia (AITP) frequently is associated with the detection of plateletreactive antibodies (pr-ab) in patients" sera. AITP as a clinical diagnosis can be supported by the finding of elevated values of pr-ab~ but negative results do not exclude AITP. We investigated blood samples taken from 47 patients (pts) with AITP for the presence of pr-ab using a competitive enzyme-immunoasssay (CELIA) according to Klefel et al. (Vex sang, 1987) . Platelet-specific antibodies (pa-ab) directed against membrane glycoprotelns (GP) were identified performing an antigen capture Elisa described by the same authors (Blood, 1987) . In 17 of 47 cases (36%) increased amounts of pr-ab, in 21 pts (45%) ps-ab and ~ in 12 cases (26%) both ps-ab and pr-ab were detected. Twelve of the 17 pts (71%) with elevated values of pr-ab were found to have IgG binding to GPIIb/IIIa complex and GPIb. However, in 9 of 21 pts with detectable anti-GP ab we failed to detect enlarged amounts of pr-ab. MOnster, Abtlg. A, H~mostaselabor, Domagkstr, 3, D-4400 MOnster It has been postulated that exidativly modified LDL may be involved in the generation of foam ceils from monocytes in the developing atherosclerotic lesion. Since fibrin(ogen) is also deposited in the plaque and mOnocytes are known to produce PCA in the presence of various stimulants, we investigated the effect of acetyl (Ac)-LDL and malondialdehyde (MDA)-LDL on monocyte PCA expression. Monocytes were isolated from peripheral blood of 29 healthy volunteers and cultured at 37=C in 5% CO=-air in a fully humidified atmosphere. At the end of the incubation period the cells were disrupted by freezing and homogenized. A factor VII and phospholipid dependent PCA was identified in the cell fragments, consistent with thromboplastic activity (TPA). Native LDL did not stimulate TPA in this cultures. Both modified LDLs displayed a kinetic-and dose-response stimulation of PCA, the maximum being seen after 24 h with 250 ug/ml modified LDL-protein. At this dose a 35fold TPA increase was observed whereas 2,5 ug/ml protein produced a 7-fold increse compared to native LDL. It is concluded, that this TPA formed in monocytes by modified LDL may contribute to the formation of fibrin in arteriosclerotic ~laques. generaily £s diagnosed using clinical criteria and routine laboratory tests. For the confirmation of diagnosis, determination of platelet -reactive immunoglobulins (Ig) is helpful, but not essential, because assays used to this end lack high specifity. Recently, enzyme-immunoassays (ELISA) for the detection of antibodies directed against platelet membrane glycoproteins (GP) have been developed, which may be more specific than standard tests mentioned above. Uslng the antigen capture ELISA described by Kiefel et al. (Blood, 1987) we analysed plasma from 53 patients (pta) for the presence of IgG directed against GP IIb/IIIa complex and GP Ib Anti-GP antibodies could be identified in 43% of pts (n=23). With regard to their GP-specifity antibodies were distributed among the pts as follows: Seven cases were found to have anti-GP IIb/IIIa, 8 pts had anti-GP Ib and in B cases both anti-GP IIb/IlIa and anti-GP Ib could be detected. MOnster, Abtlg. A, H~mostase-Labor, Domagkstr. 3, D-4400 MOnster In a family living in Regensburg the mother and her two sons have a bleeding disorder with spontaneous hematomas, in the mother and the eider son a prolonged bleeding time of > 30 min. (Simplate I) was observed, while the second son had only a mild bleeding tendency (BT 15 min.) In the two patients with enhanced bleeding tendency, platelet counts were normal while the platelet size was maximally enlarged (mean volumes 21,5 f ] ) . Platelet adhesion to glass and human endothelial cell matrix (ECM) was strongly reduced. Collagen induced aggregation was t o t a l l y , ADP and epinephrine induced aggregation s l i g h t l y inhibited. Ristocetin induced aggregation was not affected. Surprisingly F V I I I R:Ag and Ristocetin-Cofactors in plas~'a w e r e s l i g h t l y d~creased. IF the platelet spreading test large spread platelet forms similar to other giant platelet thrombopathies were found. The "Thrombopathy Regensburg" d i f f e r s from the "May Hegg-] i n " anormaly by the missing of "DShle bodies", high specific platelet weight and inhibited aggregation and retention. In contrast to the Bernard Soulier-Syndrom, Ristocetin induced aggregation and glycoprotein I b content were normal. In a two dimensional electrophoreses (HR-2 D) in combination with flourography a defect of the glycoprotein I I I b molecule was detected. Further immunologic investigations and binding studies to thrombospondin are in progress. To evaluate the f i b r i n o l y t i c system as a possible pathogenetic factor of thrombotic disorders the a c t i v i t y of tissue-plasminogen activator (tPA) and its inhibitor (PAl-I) was measured before and after venous occlusion in 104 patients by functional assays. The results were compared with the tPA-and PAl-activity of 30 normal individuals. In healthy subjects a mean (± SEM) increase in tPA-act i v i t y from 1.53 ~ 0.89 IU/ml before to 19.86 C 11.31 IU/ ml after venous occlusion was found. PAl a c t i v i t y amounted to 12.05 C 5.41AU/ml before and 6.67 ~ 4.1AU/ml after venous stasis. In our thrombosis patients a highly significant lower tPAa c t i v i t y (11.68 ~ 10.02) was detected after venous occlusion (p•0.001). PAl-activity showed extreme variations after stasis but nevertheless was significantly higher (p<0.05) in patients with thrombotic disorders (11.96 ± 13.58 AU/ml). Although the patients differed in regard to concomitant treatment, 41 were on coumarine, 48 on heparin and 15 without a treatment at the time of blood sampling, no significant changes in tPA-or PAI-activity before and after venous stasis were detected among the three groups. In nearly 30% of our patients an impaired f i b r i n o l y t i c system was detected. Our results are in good agreement with those of a number of other investigators exceptthatwe could not confirm that cumarine treatment increases the f i b r i n o l y t i c system. We conclude that hypofibrinolysis is an important factor in the pathogenesis of thromboembolic disorders. Medizinische Klinik I I I der LMU-Universit~t MUnchen, Klinikum GroBhadern, D -8000 MUnchen 70. Plasmatic h y p e r c o a g u l a b i l i t y is defined as a status in patients, in which one or more serpin (serin protease inhibitor) complexes are elevated. This is the case in patients with a c t i v a t i o n of the intrinsic or extrinsic coagulation system. Both systems result in an activation of factor IX, so that its inactivated form is the first common s e r p i n c o m p l e x of the cascade. In patients under heparin treatment, sometimes it is the only indicator of plasmatic h y p e r c o a g u l a b i l i t y and frequently a unique elevated factor IX-AT complex can be seen in patients without heparin therapy. G e n e r a l l y it can be established, that in patients with activated coagulation but il~tact inhibitor potential (sometimes upholded by heparin), the serpin complex sequence is IXIAT > X, AT > TAT. In contrast to this, in patients with malignancies, shock or after serious operative treatment, the activation seems to be nearly u n r e s t r i c t e d by i n h i b itors with the sequence IXJAT < XIAT < TAT. We recommend to determine serpin complexes in those patients, which are known to be at high risk for thrombosis as well as in those patients, which are under heparin therapy for e s t a b l i s h i n g hypercoagulability. . While NO-compounds directly activate sGC, the nitrates need to be metabolized to release nitric oxide prior to activation of the enzyme. In vitro only NO-containing agents, but not nitrates, can stimulate sGC in platelets and potently inhibit aggregation. We have compared in a doubleblind study the effectiveness of each a single oral dose of molsldomlne (4mg), 5-1SMN (20rag) or placebo to inhibit platelet function ex vivo and to influence the fibrinolytic potential in twelve healthy volunteers. Before, 80 and 60 mln after drug intake plasma concentrations of tlssue-plasminogen-activator (t-PA) and the activity of plasminogenactivator-inhlbitor I (PAl-I) were determined. RESULTS: Platelet aggregation ex vivo was significantly inhibited after intake of molsidomine and to a minor degree following 6-ISMN. Plasma concentrations of t-PA were reduced by 10% after 5-1SMN and placebo, but increased by 6% after molsidomine. The activity of the inhibitor (PAl-I) was increased by 11% after 5-ISMN, while placebo and molsldomlne provoked decreases in PAl-activity by 6% and 13%, respectively. The overall fibrinolytic potential (calculated by the relation of t-PA/PAI) was absolutely unchanged after placebo, was decreased by 15% and 12% 30 and 60 min after 5-ISMN and was significantly increased by 7% (30 min) and by 45% (60 min, p<0.05) after intake of molsidomine. CONCLUSIOn: The present results suggest t h a t molsidomlne, but not organic nitrates, induces an increase in fibrlnolytic potential in vlvo. Since both drugs (molsidomine and 5-ISMN) have vasodilatory and p l a t e l e t i n h i b i t i n g properties ex vlvo, their different effects on fibrlnolytlc parameters may be an additional a d v a n t a g e of molsidomlne in the clinical practice. Serial blood samples from 8 patients were collected before, during and up to 30 min after PTCA via a heparin-eoated catheter placed into the coronary sinus. Platelets were identified in whole blood using a biotinylated anti-glycoprotein (GP) Ib monoclonal antibody (mAb) (I J-p3) and phycoerythrinstreptavidin. To quantitate the proportion of activated platelets, a panel of fluoresceinated mAbs specific for activated platelets was applied, including PACI for activated GP llb-IIIa complex, 9F9 for platelet-bound fibrinogen, anti-LIBS-i for ligand-induced binding sites on CP lib-Ilia, and S12 for CMP-140 (an m-granule membrane protein expressed on the platelet surface following secretion). Prior to PTCA, platelets from coronary sinus bIood samples bound minimal amounts of PACI, 9F9 and anti-LIBS-I. The baseline levels did not differ from those of normal subjects, indicating that activation-dependent epitopes were not expressed on the platelet surface. Angioplasty p:ocedure caused a transient platelet activation, as evidenced by a mean increase in binding of PACi (2 3 told) and anti-LIBS-i (3-4 fold) during and 15 min after termination of PTCA. The proportion of platelets positive for PACI or anti-LIBS-i increased from 2.0 ± 0.5% (SD) at baseline to 17 + 6% or 28 + 4%, respectively (p<0.O] in both cases). PTCA induced a lower increase in 9F9 binding and no increase in S12 binding, except in i patient. We conclude that using specific n~bs to evaluate platelet activation by FAFC, it is possible both to detect activated platelet subpopulations and to quantitate the level of functional changes in platelet membrane GP epitopes following PTCA. Research Institute of Scripps Clinic, La Jolla, CA, U.S.A. IM/4UNOFLUORESCENCE IN BERNARD-SOULIER SYNDROME AND GLANZMANN'S THROMBASTHENIA BY FLOW CYTOMETRIC ANALYSIS R.E. Scharf, A. Tomer, G.J. del goppo, and Z.M. Ruggeri We have studied platelet membrane glyeoproteins (GPs) in two patients with a lifelong bleeding tendency and in ten normal subjects using a fluorescence-activated flow cytometry technique (FAFC). To evaluate qualitative and quantitative abnormalities of platelet GPs, a panel of fluorescein isothiocyanate-eonjugated monoelonal antibodies (H~bs) was applied, including Tab for GP lib, Ab-15 for GP IIla, LJ-P4 for GP lib-Ilia complex, PACI for activated GP llb-llla complex, anti-LIBS-I for ligand-induced binding sites on GP lib-Ilia, and 3 mAbs (designated I~-P3, IJ-Ibl, iJ-Ibl0) that are directed against distinct epitopes of GP lb. In all individuals, FAFC analysis was performed in whole blood and platelet-rich plasma. Patient 1 showed no binding of LJ-P3, LJ-Ibl, and LJ-Ibl0, indicating the absence of normal GP Ib on the platelet surface. In contrast, membrane immunofluorescence of LJ-P4 was approximately 70% higher in this patient than in normal controls. Together with an increased forward light scatter, this finding indicated an increased number of the GP llb-IIia complex present on abnormally large platelets. Patient 2 displayed normal immunofluoreseence with LJ-P3 but no or negligible binding of mAbs directed to GP IIb (Tab; 12%), GP IIIa (Ab-15; 6%), or the OP IIb-IIIa complex (LJ-P4; 3%). Upon stimulation by ADP (i0 B #M), or phorbol myristate actate (2 #M), no significant increase in membrane immunofluorescence occurred in the patient's platelets using PACI, or anti-LIBS-i both directed to activation-dependent epitopes of the GP IIb-IIIa complex. This study demonstrates qualitative and quantitative abnormalities in platelet GP Ib and GP IIb-IIIa complex related to Bernard-Soulier syndrome (Patient i) and Glanzmann's thrombasthenia (Patient 2), respectively. We conclude that the FAFC technique provides a sensitive method to screen patients with platelet-related bleeding disorders and both to identify and quantitate abnormalities of platelet membrane GPs, using specific mAbs. In our studies we want to find evidence for our hypothesis that m e t a s t a t i c effects of colon tumour cells are not due to p l a t e l e t -c o l o n tumour cell aggregation. Breddin. Sulfated lactobionic acids have been found to produce antithrombotic effects in animal models of arterial and venous thrombosis after intravenous and subcutaneous administration. In order to study the pharmacodynamics of the anticoagulant effects of a sulfated lactobionic acid derivative (LW 10082), we utilized a primate model which has been previously used to investigate heparin and related glycosaminoglycans. Administration of 1-5 mg/kg dosages of LW 10082 produced a mild dose dependent effect on the APTT, TT and Heptest R. Although LW 10082 produced an inhibition of thrombin, the amidolytic antilla method was insensitive to detecting the pharmacodynamics of this agent. Modified plasma based (more sensitive) assays such as diluted APTT and thrombin generation assays showed better dose dependent activity. The bioavailability of LW 10082 was found to range between 25-50% depending upon the assay system. As determined by the APTT, bioavailability for 2.5 and 5.0 mg/kg dosages respectively, as measured by area under the curve was 22 54 #g/hr/ml, clearance was 2.0 and 1.6 ml/min/kg and voh~e of distribution was 0.8 and 0.9 L/kg. While additional studies are in progress, it appears that the plasmatic pharmacodynamic effects of this new antithrombotic agent, LW 10082, can be easily evaluated by the APTT, diluted APTT and modified thrombin generation tests. (TAT in normals: 60-~40 ~U/ml). 2. The CI-and CAH-patients showed only a slight tendency to elevated TATcomplexes but without significancy (AT III in CI-patients 65±21% of normals). 3. IXiAT-complexes were increased in all patients with liver diseases but without significancy according to an extended standard deviation. We suppose, that all patients with liver diseases show an activation of the coagulation, which can be b l o c k e d by serin protease inhibitors (serpins)like a n t i t h r o m b i n , a s long as it is about 50% of normals. In patients with considerable reduction of protein synthesis (AT below 50% P. Lenz, U. Becker, N. Heimburger, Thromb. Res. 50 (1988) 559 -573) . Here we present data which quantify the degree to w h i c h different types of PAI (PAI-I, PAI-2, PAI-3) contribute to the activity measured with our method. These differences reflect the kinetics of association between the inhibitors and urokinase which have been determined by other groups. When PAIs in the plasma sample are incubated with urokinase for 5 min, the only significant contribution to the inhibition of urokinase arises from PAI-I. By extending the incubation period to 30 min, PAI-2 can be determined in addition to PAI-I. The inhibition of urokinase by PAI-3 becomes apparent only in the presence of heparin. Thus it is possible to differentiate between the type of PAI p r e s e n t in plasma by varying the incubation period in the presence or absence of heparin. The interaction of plasminogen activator inhibitor-1 (PAl-l) with its binding protein vitronectin (VN) (JBC 263:15454-15461, 1988 ) was studied utilizing purified human components, and the possible contribution of VN in the extracellular matrix (ECM) of cultured human endothelial cells (EC) was addressed• While platelets and EC constitute two major sites of storage or production, respectively, of PAl-l, little is known about the distribution of VN at these sites. The high specific PAl-1 capacity in the ECM of cultured EC is suggestive for the stabilization of PAl-1 by matrix component(s). While VN was found associated in high molecular weight complexes together with plateletderived PAl-1 in the supernatant of stimulated platelets, only small amounts of VN were detectable in cultured EC. In contrast, substantial quantities of VN could be detected in the intact ECM of these cells. Direct binding of recombinant PAl-1 to the PAl-l-depleted ECM was inhibited mainly by antibodies against VN indicating that VN constitutes the primary binding component for PAl-1 in the ECM of EC. Binding studies of PAl-1 to immobilized VN further revealed that, unlike urokinase, heparin did not inhibit PAl-1 binding, although PAl-1 as well as biotinylated hepadn bound to the same 10-15 kDa fragments entailing the glycosaminoglycan binding domain of VN. These results provide evidence not only for the occurrence of both, VN and PAl-1 in platelet storage granules as well as in the ECM of EC, but also for a functional link between both components resulting in the stabilization of PAl-1 activity particularly at platelet-ECM sites of interaction. The extracellular VN-PAI.1 relationship may have significant implications for the regulation of plasminogen activation in general and the protection of the ECM against proteolysis. (VN) represents a multifunctional adhesive extra. cellular protein operative in the regulation of the hemostatic system. VN is found in plasma as intact molecule (M r 78,000) and in a partially proteolyzed form (M r 65,000); the ratio of both forms varies individually. The physiological significance of this distribution, however, is unclear. Monoclonal antibodies against VN were obtained from female balb/c mice immunized with a mixture of native and proteolyzed antigen. Positive clones were detected by ELISA type assay using highly purified human VN. One monoclonal (SP1C5) was selected by westernblot analysis for its reactivity exclusively with the intact VN molecule but not with the partially degraded po]ypeptide, indicating that SP1C5 may react with an epitope in the carboxy-terminal portion of VN, entailing the intact sensitive peptide bond (Arg-378, Ala-379). The reactivity of SP1C5 with different VN preparations correlated well with the concentration of proteolyzed VN in the respective samples. A direct correlation between decreasing reactivity of SP1C5 with timedependent proteolysis of intact VN by human plasmin or elastase but not by tPA was also found. Moreover, SP1C5 inhibited proteolysis of VN at the sensitive peptide bond. A sandwich ELISA was established using SP1C5 as tagging antibody and polyclonal anti-VN as detection antibodies. Drastic variances in the concentration of intact VN in normal individual plasma samples were found (30-100% of maximal reactivity), although the total VN concentration in these samples varied only minimally. The monoclonal SP1C5 may thus be helpful in monitoring VN-proteolysis by circulating proteases in plasma and to establish correlations with VN dys-function in the coagulation and fibrinolytic systems. A simple detection system is thereby provided which may also help to detect possible polymorphic forms of VN. The endothelial cell thrombin receptor thrombcmodulin (TM) is an inhibitor of coagulation (a) by serving as a cofactor for thrombin-induced protein C activation, (b) by directly affecting the procoagulant activity of thrombin and (c) by accelerating the inhibition of thrombin by antithrombin II1. We have recently proposed the presence of a dermatan sulfate-like glycosaminoglycan region in rabbit TM which may constitute a secondary binding site for thrombin, required for both activities (b),(c). In the present study the effects of TM on the inactivation of thrombin by another serine protease inhibitor, heparin cofactor II (HCll) whose activity is dependent on glycosaminoglycans, were investigated. In a dose-dependent fashion TM effectively protected thrombin against fast inactivation by HCII/dermatan sulfate. Similar effects were also found in this inhibition reaction if dermatan sulfate was omitted or was replaced by heparin, respectively. Removal of the secondary binding site of TM by chondroitin-ABC-lyase resulted in partial reduction of the thrcmbin protecting effect of TM. it is thus concluded that binding of TM to thrombin renders the enzyme resistant against inactivation by HCII. In another experimental set-up using intact cultured human endothelial cells no appreciable thrombin inhibition by HCII occurred on the surface of these cells. From these findings we conclude that under quiescent conditions neither in the fluid phase nor at the intact vessel wall thrombin inhibition by HCII is of major importance. Rather endothelial cell components may protect thrombin against inactivation by this protease inhibitor. MPG-Clinical Research Unit for Blood Coagui}ticn and Throm~ bosis, D-6300 Giessen (West Germany) and Lab. Hemostase, Hopital Purpan, Toulouse (France). BINDING OF FACTOR Vll/Vlla TO HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS. U. Reuning, K.T. Preissner and G. M011er-Berghaus The assembly of the extrinsic pathway activation complex on human umbilical vein endothelial cells (EC) was studied by binding assays with purified factor VII (FVlI) and factor Vlla (FVIla). Functionally, FVll(a)-binding was estimated by the rate of factor X (FX)-activation on the surface of endotoxin-stimulated and non-stimulated EC, respectively. FXa-formation occurred only on stimulated EC, was saturable within 90-120 min and could be inhibited by tissue factor (TF)-specific polyclonal antibodies, indicating the participation of EC-derived TF. Physically, binding of radiolabeled FVII/VIla occurred to stimulated as well as to unstimulated EC. Total bindina was calcium-, temperatureand time-dependent. Binding of 12~I-FVlI/FVIla was inhibited to about 50% by a 100-fold molar excess of unlabeled ligand(s). After reaching equlibrium within 4.5-6 h, reversibility of binding was demonstrated within 2 h by the displacement of 45-50% of bound radioligands. The interaction was characterized by a dissociation constant K D = 51.7 + 92.9 riM. Other vitamin K-dependent coagulation factors, in particular prothrombin, displaced binding of °I-FVll by 15-30% and binding of 1251-F'Vlla by 35-40%, whereas non-related proteins were not effective. These data suggest the involvement of an apparently TF-independent ECbinding site on both types of cells which appears not to be exclusively specific for FVlI/FVlla. The TF-mediated'binding was studied by adding 12OI-FVII/FVlla to EC in the presence of 5uM prothrombin as an unspecific competitor. Using this experimental approach bound radioactivity (about 60% of total) could be further suppressed by polyclonal TF-antibodies only on endotoxin-stimulated EC. The time course of specific FVII/ FVIlabinding, measured by determining the amount of dis-placable radioligands by TF.specific antibodies, reached saturation within 90-120 min, which was in good agreement with the kinetics of binding isothermes, measured via FXa-generation. Our results document that specific TF-dependent binding sites for FVII/ FVIla exist on perturbed EC and are required for effective FXactivation. MPG-Clinical Research Unit for Blood Coagulation and Thrombosis, D-6300 Giessen (West Germany). A new Thr~plastin prepared from rabbit brain ~as evaluated. The ~aterial gives a standard cdrve with more than 5 seconcls difference between 100 a~d 50 % and shows a good linearity up to the dilution 1:8 on the KC 10 coaguloceter. The material has a very high factor sensitivity as determined on mixtures of normal plas=a and deficient X pismaa. Both, inter and intra assay precisinn values of the new Thromboplasti~ show that the material is ver~ reproducable with c.v.'s in the 1 to 3 % range. With clinical samples of patients with liver disease, heparin therapy on oral anticoagulalinns or other diseases and also with normals a very good correlation between the new thromboplastin and a h~an placenta thromhuplastin was found in 195 samples. The correlation coafficleat was r = 0,97 and the equation of the regression line according to passlng/Bablok was y = 0,05 + 0,95 x for the comparison based on percent of ~ormal values. In a study on 102 normals and oral anticoagalated patients an ISI Value of 1,15 was found for the KC 10 using human thrc~beplast~ as a reference. ~ value is in accordance to In conclusion these data show that Thro~boplastda IS is a very sensitive thr~abeplastin with a low ISl value which is well suited for ~a~ostical and monitoring use. It shows ver~ similar performance cbaractsxistics ~ pati~t samples like huaan thromboplastin. Routine 02-atmosphere for cell cultivation ( lq5 mmHcjp0 ~ ) is hyperoxia and does not represent "tissue normoxia" (40 mmHg) of the vessel wall. Our previous results demonstrated the p02-induced modulation of the physiological behavior of hA/VSMC.We ilnvesticjated the proliferation and release of tissue-plasminocjen activator (tPA} and -inhibitor (PAl-l} from hSMC. HODS-Media-SMC from A.mammaria{Am) and V.saphena from patients with ACVB(n=120;35-71y)were cultivated under 20-1u~5 mmHcjp02 in our glas incubator unit in RPMI 1640medium. Therapeutic ASA-conc. (10-5M) was added on day 1,3, 5;cell counts and tPA-/PAi-l-ELISA-determinations in the supernatant on day 1,3,5,7. Increaslncj p02 from u,0 ~ 145 mmHg retards the proeration of hA/VSMC (p=0.0008}. Under 20 mmHcj p02 e.CJ.Am-SMC still show a 5-6fold higher proliferation than VsSMC. ASA retards the population doublincj time in VsSMC under ~,0 mmHcj (n=8;2.18 ~ 2.37 d.;p=0.02),however, not under lU,5 rnmHg (p =0.Sq), This is different to the heparin-action, hA/VSMC do re. llease tPA/PAI-1 during proliferation. There is a--7-fold increase of tPA on day 7 over day 1. There is no correlation between tPA-release and patient's acje (p=0.87;n=lU~). Increased proliferatlon decreases tPAI10~SMC (p=0.007;n=lq). Increased SMC-aging in culture increases tPA-release (p=0.024;n=15). ASA increases tPA-release in Vs/AmSMC ( n=9; p=0. 006). VsSMC release PAl-1 during proliferation which seems to be increased under tissue normoxia and ASA. ~i lSCUSSION: Tissue normoxia modulates the proliferation and brinolysis of hA/VSMC. This is important for studying the II>hysiolocjical behavior of hSMC in vitro and the influence of drugs, hA/VSMC do contribute to the endogenous vessel wall ~ibrinolysls which is modulated through the p02 and ASA. Factor XIII (F XIII) of the blood clotting cascade is a proenzyme which, after activation with thrombin, crosslinks proteins by a transamidation reaction, and has major functions in wound healing. The plasma form is built up by two different chains (a and b) from which the a-chain contains the active centre. F XIII Occurs also intracellularly in platelets, monocytes and placenta. In these cells only dimers of the a-chain are found. F XIII from placenta is well characterized and the amino acid sequence has been determined by Takahashi et al.(19~ For further characterization and understanding of the strUcture/function relationship x-ray diffraction analysis could be valuable. To enable such investigations we tried to obtain crystals with suitable quality.Previously Bohn etal, (1971) crystallized F XIII from placenta and platelets by dialysis against distilled water. Applying this method for F XIII from placenta we obtained small hexagonal and monoclinic crystals. To get larger crystals, we examined a nund0er of different crystallization conditions and found indeed some, yielding acceptable crystals from recombinant F XIII and F XIII of placenta origin. Precipitation with sodium acetate (9 -I0 % (w/v)) in Tris~buffered solution by vapour diffusion yields thin needles up to 1 .~m. Using ammonium sulfate (0,5 -0,9 % (w/v)) we received hexagonal and monoclinic crystals up to 0,5 mm long. Precipitation with arm~Onium acetate or ammonit~n sulfate (9,6 % (w/v)) in Tris-buffered solution by vapour diffusion in the hanging or laying drop leads to crystals with orthorhombic symmetzy. Single crystals with edges up to 0,25 ntm could be obtained. X-ray investigations of these crystals are ongoing to prove their quality for diffraction analysis. Research (1.8%), cerebral b l e e d i n g (0.9%), h a e m a t u r i a (0.4%), muscle bleeding (0.9%) and bleeding from tongue bite (5.4%) were less common. The risk of spontaneous b l e e d i n g is clearly dependent on the level of vWF. A f t e r surgery (159 surgical procedures) b l e e d i n g occurred in 64% of pts, The risk of b l e e d i n g is d e p e n d e n t on the s e v e r i t y of the d i s e a s e (vWF < 10%: 83%, vWF 10-30%, 75%, vWF 30-50 ~i b l~d i z~ The iirzt ~ajcr b ! ¢~d i~ event occurred at a m e d i a n age of 3.5 years in severe vWS (vWF <10%), at a median age of 7 years in i n t e r m e d i a t e cases (vWF 10-30%) and at a median age of 15 years in mild cases (vWF 30-50% Prewarmed aliquots ( 37°C, 70 ul) of platelet-rich plasma were directly added to medium (470 ul) composed of (in mH) Na+-propionate 140, HEPES 20, glucose 5, KCI i, MgCI 2 I, pH 6.7 at 37 ° C. The undissoelated acid permeates the cell membrane, acidifies the cytosol, and, thereby, activates the Na+/H + exchanger. The continuous uptake of Na + then causes cell swelling, due to the influx of osmotically obliged water. This addition was done in a euvette placed into an aggregometer. A rapid decrease in absorbance of such platelet suspensions was observed whose time course corresponded to a let order reaction. The observed changes directly reflect cell swelling via Na+/H + exchange : i) No change in absorbance occurred when platelets were incubated in K+-propionate medium, ii) Specific inhibitors of Na+/H + exchange completely inhibited the changes in absorbance iii) analysis of the external Na + requirement revealed that the changes in absorbance followed Lineweaver-Burk kinetics. These findings are identical to those obtained by electronic eel] sizing . While the rate constant of the change in ahsorbance was determined to be 21 x 10 -3 sec -I in normotension, this value was 30 x 10 -3 sec -I in essential hypertension. Max-Planck-lnstitut fur Biophysik, Kennedyallee 70, 6000 Frankfurt/M. 70 It was recently reported that platelets of patients with essential hypertension show an increased activity of the Na+/H + exchanger. The present study aimed at investigating i) whether this increased activity would result in an increased cytosolic pH (pH i) and ii) whether the increased activity is an epiphenomenon rather than a causative factor in essential hypertension. Platelets were loaded with the fluorescent pH i indicator BCECF. The platelets were suspended in HEPES buffer (pH 7.4) at 37~C and the cytosol was acidified by addition of increasing amounts of propionic acid. The recovery of pH i towards the initial values was recorded and the initial slopes of the fluorescence tracings were used to estimate the activity of Na+/H + exchange. The pH i of resting platelets in normotension (7.14 + 0.04, n=10) did not significantly differ from that in essential hypertension (7.16 + 0.04; n-8). In contrast, the initial rate of pH i recovery from an artificial acid load in essential hypertension was enhanced by a mean factor of 3.9 (range 2-5; n=8) as compared to platelets from normotensives. In contrast, platelets from patients with secondary hypertension (renal artery stenosis) had both a normal pH i and Na+/H + exchange activity. The following conclusions can be drawn: i) Overexpression of Na+/H + exchange activity in essential hypertension does not occur as a result of elevated blood pressure. In contrast, this phenomenon may constitute a causative factor in the pathogenesis of essential hypertension, ii) Overexpression of Na+/H + exchange activity does not result in an increased pH i. iii) The increased activity of the platelet Na+/H + exchanger may contribute to the hyperreactivity of platelets in essential hypertension. In a double-blind trial antithrombotic and adverse effects of a single daily injection of 1500 aP'I-F-U low molecular weight heparine plus 0.5 mg dihydroe rgotamina (LMWH/DHE) were co mpared to a twice daily in ection of 5000 IU unfractionated heparine plus 0.5 mg dihydreergotamine (UFH/DHE) in a total of 123 patients undergoing hip orknee surgery. Events of deep venous thrombosis were assessed by. means of the radiofibrinogen uptake test (RFU) and, whenever possible, verified by phlabography. Two patients with protein-C-deficlency are being followed up at our institution. Until 17 cases of this disorder have been described in the literature. A 5-year-old girl presented at the age or 8 months with ecchymosls and necrosis of the glutea[ area. Blood coagulation profile revealed protein-Cdeficiency (type I). Therapy with fresh-frozen plasma (FFP) and prothrombincomplex-concentrate (PPSB) was instituted which resulted in resolution of the necrosis. Transient skin bleedings remained unaffected. Upon administration of protein-C-concentrate every other day the child was free of symptoms for two years. Therapy had to be dlsconHnued because of severe anaphylactic reaction and administration of FFP (two times per week) was reinstituted. Again bleeding or skin and in additon severe retroocul~r hemorrhage was observed. Changing therapy to phenprocoumon-administration resulted in complete resolution of symptoms. A 6-year-old boy had had an intracranlal hemorrhage of two weeks, resulting in severe brain damage. Suspecting a diagnosis or dysfribrinogenemla FFP and cryoprecipitate was given, which prevented the child from further bleeding for two years. Unexpected profound necrosis of the skin with severe disseminated intravasal coagulation resolved again upon FFP administration but transient skin bleeding raised the suspicion of protein-C-deficiency (type I]]. e~eding was reduced by continuing this therapy. When FFP was replaced by PPSB after one year no further bleeding was observed. Nevertheless therapy had to be discontinued after two months because of deep venous thrombosis which lead to institution of phenprocoum-administration. Again complete resolution of symptoms could be achieved by this therapy. We conclude that phenprocoumon-administration was safe and effective in these two patients who had no further thromboembolic complications and no bleeding throughout treat,..ment period. Recombinant human erythropoietin (rh-EPO) has been shown to be effective in the treatment of renal anaemia. Additionally rh-EPO improves the haemostatic defect of uraemia. On the other hand, a hypertensinogen effect and an increased risk for thrombosis have been reported in haemodialysis HD) patients. 20 HD patients in Hombor~l were recruited for a multicenter, cebo-controlled study (MF 3981), aiming to assess the risk of rh-EPO. e investigated the effect of rh-EPO on micro-and macrocirculation, heemostasls and haemorheological paramsters. Initially, 10 patients received rh-EPO at a dose of 3 x 80 U/kg body weight per week which was subsequently ad usted according to the haematocrit, After 6 months the 10 patients receiving placebo were changed to rh-EPO therapy. Clinical and laboratory Investigations were done before, 1, 3, 6 and 12 months after the beginning of treatment. Low molecular weight heparins (LMWH) are more and more substituting uofractlonationatod heparin (UFH) as prophylactic agents against deep venous thrombosis. The use of LMWH appears to be more convenient, since due to an Increased haif-life and bioavailabilify only one injection per day is required. This, however, bears the risk of cumulation of anticoagulant effects during long term administration. We therefore investigated the effects of different LMWHs and UFH, when injected over a 5 day period in a randomized cross over study in 12 healthy subjects. Fragmin (KabWitrum, Munich), Fraxiparin (Sanofi, Munich), Embolex (without dihydroergotamine, Sandoz, Nemberg) and Liquemin (Roche, Grenzach-Wyhlen) were Injected subcutaneously using the recommended dosage at 8:00 for 5 days. Between the different drugs was a washout period of 14 d. Investigations on heemostasis were performed using standard methods and commercially available test systems. The anticoagulant effects (LMWHs calibrated against the WHO standard for LMWH) 3 h after the 5th injection (3 h after the I st injection in parentheses) are shown in the following Low molecular weight heparins (LMWH) were introduced as substitutes for uofractionated heparin (UFH) in order to decrease adverse effects of conventional prophylaxis of deep vein thrombosis. There are, however, controversial communications on the effect of UFH and LMWH on the fibrinolytic system. We therefore investigated the effects of different LMWHs and UFH, when injected over a 5 day period in a randomized cross ever study in 12 healthy subjects. Fragmin (KabWitrum, Munich), Fraxiparin (Sanofi, Munich), Embolex (without dihydroergotamine, Sandoz, NOmbcrg) and Uquemin (Roche, Grenzach-Wyhlen) were injected subcutaneously using the recommended daily dosage at 6:00 for 5 days. Between the different drugs was a washout period of 14 d. Investigations on heemostasis were performed using standard methods and commercially available test systems. No significant changes of plasminogen activator antigen and plasminogen activator inhibitor antigen (Biopool, Umea) were observed during successive administration. No significant changes of bleeding time (Duke), thromboxane B2 and 6-ksto-PGF1 (NEN, Dreielch) were found. Factor VIII antigen and von Willebrand factor antigen (Boehringer, Mannheim) remained unchanged. A significant increase of liver enzymes (ALAT > 30 U/I) was observed in 3 (of 12) subjects after Uquemin, and in one after Fraxiparin and Fragmln. Our data suggest no effects of UFH and LMWH on the fibrinolytic system as well as on the primary haemostasis. Elevation of liver enzymes may also occur after LMWH, however, may be less frequent when compared with UFH. Abteilung f0r Klinische H~mostaseologie und Transfusionsmedizin Universfl,~t des Saarlandes, D-6650 Homburg/Seer, F.R.G. Results of the TAT-ELISA in patients undergoing percutaneous transluminal coronary angloplasty (PTCA) and cardiopulmonary bypass surgery F.Mahlstedt, H.K6stering, G.Claus, J.U.Wieding; Medizinische Universit~tsklinik, 3400 G6ttingen The research conducted was focussed on investigating the reliability of the Thrombin-Antithrombin III-ELISA. Starting from the observation that the concentration of the TATcomplex in blood taken from an in-dwelling cannula (IDC) increased after 90 min, after a dose of 15 U of heparin per kg body weight had been injected, in vitro tests were carried out to examine the effect of the IDC and the time of passage through it. From this experiment it emerged that the results of the TAT-complex measured in blood taken from the IDC displayed higher readings than in blood obtained through cannulation with a butterfly and that it increased in proportion to the time of passage. In addition two groups of patients were examined. The first group consisted of patients who had to undergo a PTCA, whereas the second one were to undergo cardiopulmonary bypass surgery. It turned out that, after an injection of 15,000 U of heparin and after extracorporeal circulation, the TAT-complex increased, which suggests an increased activation of coagulation. Despite these results the question whether the TAT-test is a suitable one to be carried out routinely, still remains open because it is the method of taking blood samples which is of major importance, and falsely positive results are achieved very frequently. Therapy with vincristine and prednisolone caused a pronounced decrease in fibrinogen levels in 9 patients treated for lymphoid blast crisis (LBC) of chronic myeloid leukemia (CML). In two patients the initial decrease in white blood cell count (WBC) in response to therapy was accompanied by a marked decrease in fibrinogen levels to values below 80 ng/d], within 'he first three days of trealmu.L, a pronounced rise i,, b-Db,er ieveis~ the occurence of soluble fibrin in the circulation and a drop in platelet count to values below 20.000 cells/u]. Induction of disseminated intravascular coagulation DIC in these two patients caused profuse bleeding and necessitated substitution therapy with fibrinogen and platelet concentrates. In the remaining 7 patients no signs of DIC were detected after initiation of therapy, nevertheless 4 of them showed a moderate increase in D-Dimer levels. In these patients a well known side effect of steroid therapy, namely a decrease in fibrinogen levels accnrding to the half life of 96 hours was observed. Fibrinugen levels did not fall below 150 mg/dl and increased after dose reduction from 100 mg/day to 50 mg/day. We conclude from our results that "two types of disturbances in fibrinogen metabolism can be observed during vincristine/prednisolone therapy of LBC in CML: i) a decrease in fibrinogen levels according to its biological half life due to a steroid mediated impairment of liver synthesis and 2) a rapid fall in fibrinogen levels in the course of DIC most likely induced by the release of procoagulants from detoriating blast cells, leading to severe bleeding. Therefore blood coagulation parameters should carefully be monitored in such clinical settings. Human and bovine platelets were fixed by impact freezing and freeze-substitution in resting state and after activation. Electron micrographs of serial sections were digitized by a Macintosh II computer (Apple Inc.), and threedimensional reconstruction was done with regard to the surface shape, the distribution of internal organelles, the contractile sphere, and the distribution of microtubules. The program allows presentation of different selections of the components of the models under various aspects. Transparent reconstruction of some surfaces give insight into internal organizations. The cryo-preparation acts very rapid. Conformational changes of platelets therefore most likely are the result of activation, and not to be regarded as artifacts due to the slow process of chemical fixation. Both platelet types have a discoid shape in the resting state. In both types activation causes the formation of a contractile sphere. By this way cellular compartments are redistributed and centralized. The dense tubular system forms a branched membrane complex with narrow lumina which occasionally enlarge into distinct vesicles. AS bovine platelets have no surface-connected canalicular system, they perform no shape change in the way human platelets do. Nevertheless, they exhibit membrane compartments that have contact to the cell surface and result from fusion of gr@nule membranes with the cell surface when secretion has occurred. The computer models give evidence of the complex morphological reorganization that occurs when platelets transform from the resting into the activated state. Endotoxins induce a variety of functional alterations in cultured endothelial cells such as expression of tissue thromboplastin, downregutation of thrombomodulin, and increased plasminogen activator inhibitor 1 synthesis. In order to gain more insight into the expression of surface protein expression, we immunized mice with human umbilical vein endothelial cells (HUVEC), which had been pretreated with endotoxin for 24 hours, and raised monoclonal antibodies directed against endotoxin-treated HUVEC. Four monoclonals were isolated which showed markedly increased binding to endotoxin-treated HUVEC. One of these monoclonals, designated EC-HTC7, has been characterized. EC-H7C7-binding to HUVEC increased after 4 h treatment with endotoxin (from S. enteritides), reached a maximum at 8 hours and remained constant up to at least 24 hours. The expression of the antigen was dose-dependent As little as 1 ng/ml of endotoxin induced an increase of binding of EC-H7C7 to HUVEC, maximal binding was observed at 30 ng/ml. The antibody showed no binding to human fibroblasts before or after stimulation with endotoxin. The epitope recognized by this antibody was not induced by treating the cells with thrombin or phorbol esters which are known to induce the expression of tissue thromboplastin, indicating that it does not react with tissue thromboplastin. The antibodies raised against endotoxin-activated HUVEC most likely recognize so far unknown epitopes on the surface of endothelial cells, and may help to characterize the surface alterations induced by endotoxin. showed a significantly enhanced HR, particularly in severe PAD end diabetic macro-angiopath~;mainly due to:codsfderabIy blevated RBC aggregation and increased plasma viscosity. Both haemorheological parameters closely correlated with fibrinogen concentration. SPA proved to be activated in the same patient groups. These changes may promote thrombogeneeis, especially in areas characterized by e pronounced passive movement of blood towards the vascular wall. Additionally, they indicate an increased risk of blood flow limitation in the disturbed microcirculation. Therefore special treatment to improve diminished blood fluidity and to inhibit raised plstelet activity is indicated. (n=2) were tested for presence of antibody to hepatitis C using a qualitative enzyme-linked immunosorbent assay provided by Ortho Diagnostic Systems. Patients were between 1 and 12 years old and had exclusively receive~ virus-inactivated concentrates (wet heat: 60 C, 1Oh; steam heat: 1Oh, 1200 mbar (factor VIII), 10h,1200mbar,1h,1375mbar (factor IX); UV/betapropiolactone) for management of bleeding episodes. Anti-HCV-antibodies were detected in 1/25 patients. The patient positive for antibody to HCV was a 6 year old boy with moderate hemophilia A, who to our knowledge had only received several 1000 units of a wetheated product. There had never been any clinical or biochemical evidence of hepatitis. Further investigations are necessary to exclude with certainty other possible sources of infection with HCV in this given patient. We conclude that virus inactivation procedures applied in the preparation of presently used concentrates are effective in reducing the transmission of HCV. Nevertheless we feel that HCV-infected plasma has to be excluded from the manufacturing process by means of an anti-HCV donor screening. The multimeric glycoprotein von Willebrand Factor (vWF) fulfills two biological functions: it promotes platelet adhesion to exposed subendothelium and acts as a carrier protein for coagulation Factor VIII (FVIII). The present investigation was concerned with the carrier function of vWF for FVIII, specifically the vWF component of FVIII/vWF complex was studied. Native, plasmaderived FVIII/vWF complex was analyzed under nondenaturating conditions by ultracentrifugation using a 3-30% linear sucrose density gradient. vWF multimer analysis of the starting material and vWF antigen containing fractions demonstrated a clear separation of the differently sized vWF multimers. The bulk of FVIII activity was found in the fractions composed of vWF dimer only. In all those fractions that not contained vWF dimer, FVIII activity was not measured. These data indicate that the binding protein for FVIII is the vWF-dimer. Moreover, ligand blot analysis of highly purified FVIII and electropboretically separated vWF-multimers showed exclusively binding of the FVIII molecule to the VWF dimer. Thus, the present data give clear evidence that the FVIII/vWF complex is formed between intact FVIII molecule and the dimeric form of vWF only. Klinische Forschungsgruppe fur Blutgerinnung und Thrombose der Max-Plantk-Gesellschaft, 6300 Giessen, und Behringwerke Marburg*, 3500 Marburg. D. Diamantis, B. P6tzsch, M. H%rtgen*, K. Schwemmle* and G. M~l l e r -B e r g h a u s von Willebrand Factor (vWF) is s y n t h e s i z e d by endothelial cells (EC) and megakaryocytes. Elevated vWF p l a s m a levels have been shown in different diseases a f f e c t i n g the v a s c u l a r e n d o t h e l i u m and have been interpretated to be a sign of EC damage. However, there are m a n y questions c o n c e r n i n g the m e c h a n i s m of vWF increase in these p a t i e n t s and the information one can get from increased vWF levels. These questions were a d d r e s s e d in a clinical trial on patients u n d e r g o i n g isolated extremity perfusion. During this procedure, the leg e x h i b i t i n g the malignant t u m o r is connected to an extracorporeal c i r c u l a t i o n and p e r f u s e d for 1 hour with a n t i -c a n c e r agents. EC damage during the perfusion p r o c e d u r e was d e m o n s t r a t e d by light and electron microscopy. Three days after perfusion had been performed, maximal v W F antigen and r i s t o c e t i n cofactor activities values were observed, while a maximal d e c r e a s e of platelet count w i t h elevated PF4 values was seen I hour after operation. The time interval between maximal p l a t e l e t c o n s u m p t i o n and vWF antigen peak d e m o n s t r a t e d the EC origin of the measured vWF. Furthermore, the time interval between EC injury and m a x i m a l vWF plasma level revealed de novo synthesis of vWF. From these results we c o n c l u d e that isolated limb p e r f u s i o n with a n t i -c a n c e r agents is a useful model for s t u d y i n g EC damage in vivo. Klinische F o r s c h u n g s g r u p p e ffir B l u t g e r i n n u n g und Thrombose der M a x -P l a n c k -G e s e l l s c h a f t und Zentrum f~r Chirurgie* der Justus Liebig U n i v e r s i t~t Giessen, 6300 Giessen. Recently, we demonstrated that plasma-derived VN specifically binds to human cultured endothelial cells and thereby promotes attachment and spreading of these cells to polystyrene petri dishes (Blood 71: 1581; 1988 ). In the present study we investigated whether VN may also promote the attachment of human endothelial and mesothelial cells to expanded polytetrafluorethylen (ePTFE) and polyurethane. ePTFE and polyurethane were passively precoated overnight with a solution of 25 ug/ml vitronectin, 25 ug/ml fibronectin and 0,2% (w/v) bovine serum albumin. Mesothelial cells derived from human omentum and endothelial cells isolated from human umbilical veins were seeded at a density of 2,4 x 105 cells/cm 2 and the extent of cell attachment was determined by light and scanning electron microscopy and by counting the cells after trypsinization. After 90 min more than 80% of the surface of ePTFE was covered with endothelial or mesothelial cells in the fibronectin and vitronectin coated wells, but the cells were more homogeneously distributed over the graft surface in the vitronectin coated wells. A monolayer of cells could not be achieved neither with fibronectin nor with vitronectin precoating on ePTFE after this time interval. In contrast to ePTFE, cells seeded on polyurethane pracoated with vitronectin and fibronectin formed a monolayer of ceils after this time interval. These data demonstrate that vitronectin can be used as a precoating substance for endothelial celt seeding on ePTFE and polyurethane. Thrombosis, D-6300 Giessen. In the last years interactions of plasminogen-activators with the coagulation system have been reported. During fibrinolytic therapy a rise of FPA and thrombin-antithrombin complex (TAT) has been observed. We investigated the in vitro effect of several plasminogen activators in high concentrations on citrated normal plasmas. The reagents used in final concentrations of 100.000 ul ml plasma were rt-PA (Actilyse Thomae, Biberach = 1 mg), prourokinase Sandoz/N~rnberg, Urokinase Deutsche Kabil-M~nchen, Streptokinase Behringwerke Marburg. rt-PA, prourokinase and urokinase had a low thrombinlike proteolyric activitiy when tested on the chromogenic substrate S-2ZSa, I00.000 u/ ml corresponding to 0.4-5 ulml, When added to plasma an effect was even noted for concentrations of 10.000 ulml for rt-PA and puk. With urokinase the effect was lesser visible. Streptokinase had no effect. At the same time a rise of FPA could be observed from about 10 ng/ml to 2000-3000 ng/ml for rt-PA, PUK and less with 1000 nglml for urokinase. Streptokinase induced a rise only about 50 ng/ml. At the same time the high concentrations of plasminogen activator, prourokinase and urokinase led to a loss of antithrombin activity of 30-50 % in plasma as well as in AT concentrate (Kybernin, Behringwerke) with a small rise of TAT.-This effect was again not noted with SK. -We conclude that several kind of plasminogen activators especially those with an affinity for fibrin have besides their fibrinolytic activity an additional but Lesser effect on the coagulation system. This work was supported by Stiftung Sandoz. In der Gerinnungsanalytik ~berwiegen zur Zeit noch auf koagulometrischer Basis arbeitende Ger~te wie beispielsweise der KC 10 oder KC 40 (Amelung). Jedoch i s t die methodische Umsteiiung auf nephelometrische Bestimmungen und Automation bereits in voliem Gange, seit Ger~te wie der MLA 900/1000 yon Baxter oder der ACL 300 yon I i kommerziell verfOgbar sind. Bei dieser Vergleichsuntersuchung wurde der MLA 900/1000 hinsichtlich der Vergleichbarkeit Pr~zision und Handhabung dem KC 10 gegenUberges t e i i t . Der MLA 900/1000 ~berzeugt auch in Bezug auf seine Pr~zision und gute Korrelationsm~glichkeit, insbesondere bei Quick, PTT und mit Ein-schr~nkung auch Fibrinogenbestimmung nach CIauss. Die Handhabarkeit i s t gut und f~hrt im Vergleich zum KC 10 zu einer Arbeitserleichterung. Probleme ergeben sich jedoch bei der Uberwachung yon Beparintherapien mit Hilfe der PTZ sowie bei der vom Hersteller propagierten Quick-derived Fibrinogen Bestimmung. Hierbei i s t , jedenfalls zur Zeit, die erforderliche Standardisierbarkeit noch nicht e r f U l l t . V o r t e i l h a f t i s t beim MLA 900/1000 zus~tzlich die MOglichkeit zur DurchfUhrung yon Bestimmungen mit chromogenen Substraten, beispielsweise bei der AT I l l -und Plasminogenbestimmung. Derzeit werden analoge Vergleichsuntersuchungen mit dem ACL 300 R yon I i durchgefUhrt, deren Ergebnisse zum Zei%punkt der Veranstaltung ebenfalls v o r l i egen werden. Z e n t r a l i n s t i t u t for Blutgerinnungswesen und Transfusionsmedizin der Heinrich-Heine-Universit~t DUsseldorf, Moorenstr. 5, 4000 DUsseldorf F VII-deficiency (hypoproconvertinemia), an autosomal recessive defect in the extrinsic pathway, was first described by Alexander et al. in 1951. A severe congenital F VII-deficiency is rare (1:400 OOO), a partial defect is found more often. The F VII-activity of homozygotes is below 10%, the F VIIactivity of heterozygotes ranges between 20 and 75% of the normal values. There is no close correlation between the hemorrhagic diath#sis and the F VII-activity: 10% of all homozygotes do no~ suffer from bleedings, whereas 10% of all heterozygotes show hemorrhagic diathesis. A case of a three-year-old girl with reduced F VII-acti-vity (14.5-17%) and no hemorrhagic diathesis so far is reported. Because of a preoperative screening coagulation test for tonsillectomy, a F VII-deficiency was diagnosed. Although the child had shown no bleeding tendency so far, the child received PPSB; after therapy F VII-activity raised, however the PTT was now prolonged (78"'). Finally after therapy with a F VII-concentrate alone a rise of F VIE-activity and prothrombine dime could be achieved without change of the PTT. It is postulated that the change of the PTT was caused by the heparin which is contained in the PPSB. Because PPSB contains less F VII-activity than a F VII-concentrate alone, the patient was given a high dosage of PPSB (20 U/ kg/12 hra) -which means he received more heparin than in a F VII-concentrate. We conclude that for a patient with an isolated F Vlldeficiency undergoing surgery a therapy with F VII-concentrate should be the therapy of the first choice. Significant difference between PC activity and antigen was found in 23 patients with proteinuria exceeding Ig/24 h and varying degree of renal insufficiency. PCI, ap-macroglobulin, urea and uremic toxins were investigated as a possible cause for this difference. Results showed a significant higher PCI in patients than in controis (p 0.001), but only a very weak inverse correlation to PC-activity. Elevated PCI due to enhanced liver synthesis in nephrotic syndrome seems unlikely since there was no correlation between PCI and cholinesterase. Neither uremic toxins nor urea nor a~-macroglobuIin were responsible for reduced PC-activity compared to the antigen. It is suggested that PCI may play an important role in exerting differences between functional and immunological PC. If elevated PCI participates on thrombogenesis in patients with high grade proteinuria with our data could not be shown. Phenprocoumon was administered to 10 healthy volunteers at a dosage of 15 and 12 mg in the first two days. Four of them received 1 mg vitamin K after 3 days. Bloc, d samples were drawn every 12 hours up to 7th day. Factor VII and protein C reached minimal levels of 10 to 15% during the third day. Concentrations (In mean) of thrombln-antithromblnitl-complexes (TAT) and flbrlnobeptld A (FPA) were markedly increased during the first two days whereas soluble flbdn remained within the normal range. Especially the prothrombin fragments (F1,2) proved to be very sensitive in the diagnostics of hypercoagulabllity. Concentrations were elevated during th~ first two days and subsequently fell to subnormal values Indicating the efficacy of the anticoagulant therapy. Two days after the last phenprocoumon administration, coagulation factors and protein C tended to normalize but In some courses TAT and FPA levels were increased again. This study provides Insights into the activation of the coagulation system : TAT and F1 +2 proved to be sensitive indicators of thrombln generation, which, however, due to the Inhibitory function of antithrombinlll, does not necessarily lead to an increased flbrlnogan-flbrln turnover with FPA release and formation of soluble fibrin. The measurement of TAT and In particular of F1 +2 may demonstrata a latent hypercoagulabllity even before fibrinogan-tumover is increased. Additionally, these results elucidates the inhibitory capacity of the protein C complex. Consequently protein C as well as antithrombinlll should be measured (using functional assays) before onset of phenprocoumon therapy, in order to diagnose the Inhibitory potential of the patient's coagulation system and to prevent coumadn necrosis or other thromboambolic events. Furthermore, at initial dosages of mote than 9 mg phenprocoumon per day an additional anticoagulant therapy with heparin should be considered. Aspects Of Preoperative Care Zieger The use of low molecular weight heparin (LMWH, Fragmin, KabWitrum) in comparison with unfractionatad beparin (UFH, Braun, Melsungen) was investigated in a cross over study in 27 haemodialysis (HD) patients over 12 month. The patients (15 female, 12 male) had a mean age of 65 years and were on HD for 5,7 years. 5 patients died during the study and one was transplanted. LMWH was given initially at a 60% dose of previous UFH requirement and subsequently adjusted according to AP'I-r and thrombin time (l-r) values. Several assays were performed prior to, 30 rain and after HD including: AP13-, "IT, anti-Xa, anti-Ila (Boehringer, Mannbeim), t-PA and PAl-1 (Biopool, Umea), von Willebrand factor antigen (Boehringer, Mannheim) and factor VIII:C antigen.A mean heparin dose of 4958 U (UFH) vs. 4216 U (LMWH) was given per dialysis. Mean APTT (Boehdnger, Mannbeim) values before HD were 33.8 s and 33.9 s, and after HD were 49.9 and 46.3 s for UFH and LMWH, respectively. Significant correlations between chromogenic substrata anti-Xa assay (Ka-bWitrum) and Heptest (Haemachem, St.Louis) were observed during UFH (r=0.71) and LMWH (r=0.72). The correlation between APTT and anti-Xawas r= 0.62 for UFH, but only r= 0.27 for LMWH. Factor vIn:CAg decreased during LMWH treatment (425_+ 151 to 317_+ 260 U/dl), but remained constant during UFH (313_+122 and 292_+144 U/dl). Mean platelet count showed no differences during LMWH (228_+84x 1000/ul) or UFH (220+89 x 1000/ul) treatment. Transfusion requirements were 1.59 _+ 2.62 U and 1.6 -+ 1.44 U red cells during UFH and LMWH treatment, respectively. Shunt thromboses were observed in 1 (UFH) and 3 (LMWH) patients. Our data confirm earlier findings that LMWH may be used as an anticoagulant for HD, however, dose finding, drug monitoring and possible advantages have to be investigated and confirmed in further studies.Abteilung for Klinische H~mostaseologie und Transfusionsmedizin und Abteilung for Dialyse und Nephrologie der Universitt=t des Saarlandes, D-6650 Homburg/Saar, F.R.G. a-2-Macroglobulin (a2M) is a proteinase inhibitor of broad specificity and one of the major plasma proteins in man. Monocytes interact with azM-proteinase complexes internalising and degrading them. Since activated leukocytes (PMN) release large amounts of reactive oxidants the goal of this study was to determine whether these agents are able to inactivate the inhibitor. Mimicking the leukocyte attack in normal human plasma by N-chleramines we observed an oxidant concentration dependent inactivating effect on purified and plasma azM. The oxidant concentration for complete inactivation of the inhibitor was found to be comparable w i t h the "lethal" dose for human a-2-antiplasmin and antithrombin I I I . The inactivation of a2M by oxidants is the result of a specific oxidative damage since the corresponding serine proteases as well as CI-inhibitor were chloramine resistant under the conditions used. I t might be speculated that the oxidants attack the bait region of the trap molecule a2M. According to our results, the amount of oxidants released by I x 10 ~ activated P M N would be sufficient to destroy azM activity of about I Nl of human plasma. Consequently, in local areas of inflammation activated leukocytes may well be able to create microcompartments of uncontrolled protease activity by generation of reactive oxidants.Oxidants seem to alter enzyme/ inhibitor balances in favour of the enzyme.Univ. Department of Hematology, Section of Hemostasis and Thrombosis, General Hospital "Virgen del Roche", University of Seville, E-41013 Seville, Spain Shape change (1), internalization of surface-bound iigands (2) and the exocytosJs of secretory organelles (3) characterize stimulated platelets. These reactions involve membrane rearrangements and the action of the contractile cytoskeleton and appear to run off simultaneously within the first seconds of activation. To study the earliest structural dynamic processes during activation the platelets were stimulated for 10 up to 60 seconds with thrombin or collagen and than chemically fixed or -for demonstration of membrane fusion -rapidly frozen (impact freezing) and freezesubstituted. Using electron micregraphs from serial sections the structural alterations were reconstructed. (1) Within the first seconds of piatelet stimulation the membranes of the "surface connected system" were evaginated for the purpose of surface enlargement required to change shape. (2) Simultaneously, the plateJets started to internalize surface areas with bound ligands. The plasmalemmaJ invaginations were observed attached to the contractile get and thus they followed the centripetally moving constriction. This could be demonstrated for several surface bound ligands as cationized ferritin, fibrin as weJI as collagen fibers. The internalization of fibrillar ligands by platelets in this mode caused the retraction of fibers. (3) Within the first seconds after stimulation the membranes of the secretory organel]es form appositions to the plasmalemma or to membranes of the SCS and seemed to maintain this position during shape change and interna] contraction. The exocytosis started after formation of a fusion pore and was then continued by compound exocytosis of secretory granules. The observed membrane rearrangement may explain how piatelets are able to carry out their different actions (shape change, surface-ligand interaction, release reaction and retraction) within a short period of time. Thereby, a retrieval of membranes and integrated components seems to occur. In recent years the knowdlege on the regulation of the fibrinolytic system has substantially increased. Several clinical conditions have been associated with alterations of the fibrinolytic system in plasma. However, it is difficult to draw definite conclusions from the laboratory data obtained as the plasma levels of several components of the fibrinolytic system, such as tissue-plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAl-l), are influenced by diurnal variation, age, exercise, stress, nutrition. In order to study the fibrinolytic system in plasma, it is necessary to standardize influencial factors. We designed a highly standardized procedure for the investigation of the fibrinolytic system in plasma. Ten healthy volunteers were investigated three times, with a minimal interval of 7 days between each examination. Blood was withdrawn under fasting conditions in the morning between 7.30 and 9.00 a.m., after a resting period of 20 minutes, before and after 10 minutes of venous occlusion (VO). In order to evaluate the day to day variation, the coefficient of variation (CV) was estimated for each parameter before and after VO. The mean CV for the eug!obulin lysis time was 9.5% before VO and 9.3% after VO. The CVs for tPA were 14.4% and 27.0% before and after VO, respectively. PAl-1 antigen showed a CV of 10.5% before and 19.0% after VO, and the CV for PAl-capacity were 5.0% before and 33.0% after VO. There was a significant correlation between PAl-1 antigen and PAl.capacity before and after VO (r=0.64, p<0.0001, and r=0.72, p<0.0001). These data indicate that the day to day variations in parameters of the fibrinolytic system can be kept within a low range, provided a highly standardized procedure is chosen for blood withdrawal MPG-Clinical Research Unit for Blood Coagulation and Thrombosis, D-6300 Giessen, and Department of Internal Medicine II, Hassenklinik, D-6330 Wetzlar. The effects of the 10w molecular weight heparin Fragmin R (LMW) were compared to standard heparin (SH) in 2 long,term studies In heemodielysis patients, LMW heparin showed a significantly lower Upctyti¢ activity than SH heparln. A repeated stimulation of lipolysis during long-term therapy with standard heparin can lead to an exhaustion of the lipolytic activity end can thereby contribute to the formation end progression of hypertriglyceridemla. This could be shown in 5 healthy persons: • 4-hour therapy with SH heparin in comparative doses te haemodialysis patients lead to a significant reduction of the stimulation of the Ilpolysie for 24 hours. On the other hand • 4hour treatment with LMW heparin did not lead to an exhaustion of the lipolyric activity. In the first study 70 newly accepted patients receiving haemo. dialysis were treated randomly with either LMW or SH heparln for 12 month. A significant rise In trlglycerldea levels (TG) as well as in the VLDL-fraction occurred in the SH heperin group, Especially extremely high TG values (>450 mg/d!) were found in 6 patients after 12 month in the SH group but not In the LMW group, in the LMW group there was no rise in TG. Cholesterol, LDL and HDL did not change in either group. To check whether established hypertriglycerldaemia in HD patients is reversible, 218 paUents with triglycerid values ever 350 rag% were treated randomly with either SH or LMW hepartn for 12 month, in the LMW heparin group there was a significant reduction of the elevated TG values, not however in the SH heparin group, The clinical significance of these favourable results effects on triglycerides lies in their participation in the development of atherosclerotic vascular changes In hemodlalysis patients. Especially the stherogenic potency of triglyceride-rlch remnant particles in hemodialysis patients had been pro-Yen. Organ complications of P. falciparum malaria have been linked to hemostat!¢ alterations including fibrin formation, Therefore, we analyzed coagulation factor XIII (F,XIII) plasma levels in 45 patients with P, falolparum malaria before, during and after antiparasitic treatment. 22 of these patients had organ complications (complicated malaria ). Antigen concentrations ( subunits a and s ) were measured by Laurell electrophoresis while plasma activity was determined functionally as F,XIII dependent incorporation of 125-I-putrescin into casein. Subunit a and s antigen concentrations and activity levels correlated closely to each other (r =0.80; pC 0.001).F,XIII levels (activity 74%, subunit a 77%; subunit s 90% ) were somewhat decreased In untreated P. falciparum malaria and increased (p,¢ 0,05 ) during antiparasitic treatment. 14 of 22 patients with organ complications, but none of 23 patients with uncomplicated malaria had F.XIII activity and subunit a levels below 50% or subunit s levels below 75% (p'~0,001). Low F.XIII activity and antigen (a and s ) levels correlated (p 4: 0.001 ) to high parasitemJa ( -0.66, ~ 0.SY, and -0.53, resp.), but not to thrombin-antithrombin-lU (TAT) plasma levels.The results show that P. faleiparum malaria can be associated with a reversible decrease in F.Xltl antigen and activity levels. Low F.XIII antigen and activity levels tend to be associated with organ complications (complicated malaria ), Since both subunlts (a and s) are involved, and since no correlation was found between F.XIII and TAT levels, it could be suspected that -in addition to hemostatic alterations -unspecific proteolysis might play a role In complicated P. falciparum malaria. of an acquired vWd. The s i g n i f i c a n c e of these findings will be discussed. Therefore, we measured plasma levels of thrombin-antithrombin-Ill (TAT) complexes, tibdn monomeres ( F M ) and plasminogen activator inhibitor ( P A l ) activity in order to test whether fibrin metabolism is affected during Illness. TAT complexes were measured by sandwich ELISA, FM via their stimulatory effect on the activity of tissue plasminogen activator, and PAl activity (PAl 1 + 2 ) was determined functionally as the inhibitory effect of the sample versus a defined quantity of uroklnase.AI! three parameters were elevated in untreated P. falciparum malaria (TAT 8.0 ng/ml; FM 8.6 nmol/I; PAl 5.1 U/ml ). In comparison to pretreatment levels, they decreased during antiparasitic therapy ( p < 0 . 0 0 1 ) . FM correlated to TAT ( r = + 0.40; p.~ 0.01), and PAl ( r = + 0 . 3 7 ; p < 0 . 0 5 ) , while all three parameters correlated to parasitemia (TAT: r = + 0.44; FM: r =+0.48; PAl: r = + 0.48; p < 0 . 0 1 ) and were higher in complicated than in uncomplicated malaria (TAT" p~ 0.05; FM: p < 0.01; PAl: p < 0.001).These results show that P. falciparum malaria is associated with signs of increased fibrin formation in viva while the elevated PAl activity levels act against fibrinolysis. In eleven healthy young subjects the effects of a short exhaustive bicycle exercise were examined on thrombin-antithrombin III komplex, tissue-plasminogen activator, complement fragments C3a and C4a and histamin in the blood. The analyses were carried out thirty minutes before and immediately before exercise, immediately post-exercise and thirty and sixty minutes later. As evaluated from the percentage change of haemoglobin and haematocrit, the plasma volume did not change significantly after the exercise test. Immediately postexercise thrombin -antithrombin III, tissueplasminogen activator, complement fragments C3a and C4a and histamin were all significantly elevated (p < 0.01) compared with the pre-exercise values. Thirty and sixty minutes later the values normalized and significant differences with the pre-exercise values could no longer be measured.The present results support the concept of an activation in vivo of coagulation, fibrinolysis as well as of the complement system after short maximal exercise. Institut der Universit~t -GH -Paderborn, Warburger Str. i00, 4790 Paderborn. ASSOCIATED WITH ORTHOTOPIC LIVER TRANSPLANTATION (OLT) G.Himmelreich, H. Rices, B.Kierzek, B. Lefebre. P. Neuhaus. D. Huhn. OLT is frequently complicated by bleeding, especially after graft reperfusion. One of the causes may be hyperfibrinolysis.Activity and antigen of tissue-type plasminogen activator (tPA; tPA-Ag), activities of plasminogen activator inhibitor (PAI) and "intrinsic plasminogen activators" (iPA), urokinase-type plasminogen activator antigen (uPA-Ag) as well as thrombelastography (TEG) and further parameters of plasmatic coagulation were monitored in the course of 9 consecutive OLTs (table). Our data confirm previous results that indica£e hyperfibrinolysis developping during the anhepatic stage (a.st.) and peaking in the early reperfusion (rep.) period mostly due to tPA increase. In addition intrinsic fibrinolytic activity may be involved. We believe that antifibrinolytic therapy may be indicated in OLT. A protocol was designed to detect and quantitate tumor cell-derived ur0kiname-type plamin0gen activator (uPA) in solution by ELISA, and Western blot, and in tumor cells by immun0histnchemist~. Son0cl0nal antibody (m0AB) #394 was used to locate uFA in breast cancer cells in f0rmalin-fixed paraffinembedded tissue specimens (APA~2 method) in the cytoplasm and on the plasma membrane. MoAB #394 in combination with the bi0tinylated meAB #377 was applied to quantitate various forms of nrokinase by FLISA. As verified by Western bi0t analysis, m0AB #377 and #394 bind to the pr0enzyme form (pr0-uPA), the high and 10w molecular weight form of uPA (HMW-uPA, LMW-uPA). MOAB #377 is directed to an epit0pe on the A-chain of uPA whereas #394 detects an epit0pe on the B-chain. The ELISA detects the pr0enz~e form of ur0kinase {pr0-uPA), pr0te01ytically degraded enzymaticslly active and inactive uPA, and uPA c0mplexed with the inhibit0rs PAI-I and PAI-2. The detection limit is < 40 pg uPA / ml. The assay was used for quantitati0n of uPA in urine, plasma, and tumor cell extracts. Himmelreich, B. Kierzek, G. Blumhardt,P. Neuhaus, H. Riess.OLT is frequently complicated by bleeding, especially after graft reperfusion. Hyperfibrinolysis has been recognized to develop during the anhepatic phase and to get worse in the early per±ode of graft liver reperfusion. We analysed early samples of graft liver perfusate (GLP) in 9 consecutive OLTs. Tissue-type plasminogen activator activity (10.2±5.8 U/ml; mean±SD) and antigen (25.4±13.1 U/ml)) as well as "intrinsic fibrinolytic activity " (1.1±0.6 U/ml) and urokinase-type plasmingen activator antigen (2.0±2.2 U/ml) were clearly elevated whereas plasminogen activator inhibitor was less increased (7.1±9.6 U/ml) above the normal ranges.This was reflected by signs of overt hyperfibrinolysis in thrombelastography. The extent of fibrinolytic activity detected in the GLPs correlated with the increase in fibrinolytic activity observed in the patient between the late anhepatic and early reperfusion periods. Our observation may be helpfull for the timing and dosing of antifibrinolytic therapy in OLT.Medizinische Klinik und Poliklinik sowie Chirurgische Klinik und Poliklinik, Universit~tsklinikum Rudolf Virchow, Freie Universit&t, Spandauer Damm 130, D-1000 Berlin 19. Turbidimetric measurements of platelet function lack a reference with which different instruments and procedures could be standardized. As a consequence, quantitative results strongly depend on the type of the aggregometer, the mode of its adjustment and cannot be directly compared between different laboratories. We present a method of a standardization that refers to the platelet concentration. It is based on a linear relationship between the signal measured and the platelet concentration. We developed a computer-assisted universal aggregometer in which the range is adjusted with a dispersion of definite t u r b i d i t y and the platelet-suspending medium, the transmission is transformed into absorbance and the still remaining deviations from a line are corrected by software-assisted calibration.The calibration can be stored for different platelet preparations and even different cell species, e.g. neutrophils, and is reused for different samples and preparations. With that calibration, any phenomenon of piatelet activation which is turbidimetrically monitored can be referred to an actual (in aggregation measurements) or pseudo (in shape change or secretion measurements) percentual change in platelet concentration. This procedure also strongly increase the sensitivity and accuracy with which small changes in the t u r b i d i t y during weak aggregation, shape change or alpha-granule secretion can be reliably measured. The computer software provides on line monitoring of the signals from 4 aggregometer channels and calculates the final results. The instrument works with platelet concentrations between 0.5 and 8 x 108/rnl. An universal interface can also be connected to the analogous exit of any conventional aggregometer. ( Fla.,1984) . Un~l now a viral transmission was easily detected by screening tests for HBV or HIV, but NANB hepafifis could only he excluded by surrogate tests. Now we have the possibility to prove a NANBV transmission by detection of anfi HCV with the second generation "ortho-HCV" enzyme linked immunceorbent assay (ELISA) developed by Ortho Diagnosfi~, New Jersey. A further criterion for safety is the Inactivation of parvovirus B19, because this virus is designated as one of the most thermoresistaet viruses in blood products. Anti HCV tests and anti B 19 tests were conducted in three groups of *virgin" patients, who were exclusively treated with defined concentrates: Group 1) 49 haemophilia A patients, who were treated since 1980 with a pasteurized factor Viii concentrate, heated in aqueous solufion at 60 o C for 10 hours (Heamate ® HS, Behringwerke Marburg). Group 2) 5 haemophilia A petJents, focmer high respoeder pafients, who had received high doses of factor VIII concentrates (Haemete ® HS) and vapor We have repeatedly demonstrated the preventive effect of the specific thrombin inhibitor hirudin on DIC in various animal models. In this context, special interest was devoted to the influence of hirudin on the clinically relevant endotoxin-induced localized or generalized microthrombosis. The generalized form known as Shwartzman reaction (GSR) was induced by endotoxin infusion (2.5 mg/kg x h)in young pigs. The localized Shwartzman reaction (LSR) was induced by intradermal application of endotoxin in rabbits, followed by intravenous provocative injection of endotoxin on the next day. To detect f i b [~ deposits the animais were pretreated with ~ ~I-fibrinogen and the accumulation of radioactivity was measured with a gamma c o u~ ter. When hirudin (0.05 mg/kg x h)waa given,the endotoxin-induced consumption of clotting factors which is typical of GSR was less pronounced and the number of fibrin deposits in the lungs, kidneys, liver and spleen was diminished. The increase in the right ventricular pressure was delayed. The animals survived the otherwise lethal endotoxin infusion. Compared to hirudin, heparin (0.5, i or 2 mg/kg x h) could not prevent the endotoxin-induced changes and the an9 mals died during the endotoxin infusion. Hirudin prevented also the development of the LSR. Fibrin accumulation in the skin area and the haemorrhagic-necrotic reaction were suppressed by hirudin (0.2 mg/kg s.c.) 30 min prior to the provocative endotoxin injection.Institut for Pharmakologie und Toxikologie der Medizinischen Akademie Erfurt, Nordh~user Str. DDR-5010 Erfurt HEMOSTATIC FACTORS AS INDICATORS OF POSTTRAUMATIC ORGAN FAILURE M. J~chum , D. Nast-Kolb 2, W. Schramm 3, M. Spannagl , Ch.Waydhas 2, H. Fritz I Severe polytraumatic events are often deteriorated by multiple organ failure (MOF) in the later posttraumatic course. Therefore, the availability of easily measurable plasma parameters predicting a forthcoming organ complication as early as possible is urgently needed. In this respect we evaluated the predictive value of the hemostatic parameters prothrombin, antithrombin III, plasminogen, O62-antiplasmin , prokallikrein and C1-inactivafor [either separately or summarized in the PFI-index according to AASEN) in a prospective study on 69 polytraumized patients (Injury Severity Score > 30 points). In addition, we studied factor VIII antigen as well as tissue plasminogen activator (t-PA), t-PA inhibitor I and D-dimers. During the observation period (usually 14 days) 11 patients developed lethal MOF, 29 patients overcame reversible organ failure and 29 patients did not show signs of organ dysfunction. At the time of admission to the hospital (mean value: 49 min post trauma) AT III, prothrombin, D-dimems and t-PA indicated the development of later organ failure with specificities and positive predictive values above 60 %. All other parameters did not behave as clinically relevant prognostic factors in this respect. Moreover, the data of AT III and ~2-antiplasmin at the time of admission allowed a clear differentiation between patients who died and those who overcame the traumatic events. From the 4th posttraumatic day onwards also the PFIindex indicated the later outcome with a sensitivity and a specificity value above 60 %.1~t. Klin.Chemie u.Kli~.Biochemie in der 2Chirurg. Klinik Innenstadt, ~Med. Klinik Innenstadt, Universit[t M~nchen