key: cord-1031273-fnp6f81t authors: Hagisawa, Kohsuke; Kinoshita, Manabu; Takeoka, Shinji; Ishida, Osamu; Ichiki, Yayoi; Saitoh, Daizoh; Hotta, Morihiro; Takikawa, Masato; Torres Filho, Ivo P.; Morimoto, Yuji title: H12‐(ADP)‐liposomes for hemorrhagic shock in thrombocytopenia: Mesenteric artery injury model in rabbits date: 2022-02-15 journal: Res Pract Thromb Haemost DOI: 10.1002/rth2.12659 sha: 2f781e4b94bf01d149f00b5f40c2efd1a3377bd0 doc_id: 1031273 cord_uid: fnp6f81t BACKGROUND: Damage control resuscitation improves patient outcomes after severe hemorrhage and coagulopathy. However, effective hemostasis methods for these critical situations are lacking. OBJECTIVE: We evaluated the hemostatic efficacy of fibrinogen γ‐chain (HHLGGAKQAGDV, H12)‐coated, adenosine‐diphosphate (ADP)‐encapsulated liposomes (H12‐[ADP]‐liposomes) in thrombocytopenic rabbits with hemorrhagic shock. METHODS: Acute thrombocytopenia (80%) was induced in rabbits that also received mesenteric vessel injury, leading to hemorrhagic shock. Five minutes after injury, subjects received intravenous bolus injection with H12‐(ADP)‐liposomes (20 mg/kg), followed by isovolemic transfusion with stored red blood cells (RBCs)/platelet poor plasma (PPP) (RBC:PPP = 1:1 [vol/vol]), or lactated Ringer solution every 5 min to compensate blood loss. One group received H12‐(phosphate buffered saline [PBS]) liposomes followed by RBC/PPP. Additional groups were received isovolemic transfusion with RBC/platelet rich plasma (PRP) (RBC:PRP = 1:1 [vol/vol]), RBC/PPP, PPP alone, or lactated Ringer solution. RESULTS: Treatment with H12‐(ADP)‐liposomes followed by RBC/PPP transfusion and RBC/PRP transfusion effectively stopped bleeding in all thrombocytopenic rabbits. In contrast, three of 10 rabbits treated with RBC/PPP failed hemostasis, and no rabbits receiving lactated Ringer solution stopped bleeding or survived. Twenty‐four hours after hemorrhage, 80% of rabbits receiving H12‐(ADP)‐liposome followed by RBC/PPP transfusion survived and 70% of rabbits receiving RBC/PRP transfusion also survived, although RBC/PPP‐transfused rabbits showed 40% survival. Rabbits receiving H12‐(ADP)‐liposomes followed by lactated Ringer solution showed a transient hemostatic potential but failed to survive. H12‐(PBS)‐liposomes showed no beneficial effect on hemostasis. Neither the PPP group nor the lactated Ringer group survived. CONCLUSION: H12‐(ADP)‐liposome treatment followed by RBC/PPP may be effective in lethal hemorrhage after mesenteric vessel injury in coagulopathic rabbits. • An initial hemostatic agent is necessary for acute care surgery. • A new agent was developed for helping platelet aggregation. • This agent stopped bleeding after severe vessel injury in animals with altered coagulation. • This new treatment may be helpful for acute abdominal bleeding. Abdominal vascular injuries are one of the most lethal injuries in trauma patients, which mortality ranges from 20% to 60%. 1, 2 Patients with abdominal vascular injuries often exhibit the lethal vicious cycle of shock, consisting of acidosis, hypothermia, coagulopathy, and cardiac arrhythmias. 1 In such critical conditions, hemorrhage control and restoration of mesenteric tissue perfusion are crucial for life saving. 3 Mesenteric injury is found in approximately 5% of blunt trauma victims during laparotomy. 4 Prompt resuscitation is clinically important because it may cause significant blood loss or lead to bowel ischemia and necrosis, with eventually delayed rupture or ischemic strictures. 5, 6 Mesenteric arterial injury frequently complicates coagulopathy, increasing mortality to approximately 40%. 7, 8 Control of coagulopathy has been attempted by induction of damage control resuscitation (DCR) and massive transfusion protocol (MTP). Sorrentino et al. reported that refractory coagulopathy resulting from major abdominal vascular trauma decreased from 46% to 19% (during 1975-1980 vs 2004-2009 ) by adapting damage control surgery. 9 Moreover, institutional MTP may improve outcomes of patients with abdominal aortic injuries. Maciel et al. reported that introduction of MTP markedly increased patient overall survival from 14% to 47%. 10 Moore et al. proposed staged laparotomy for treating coagulopathy. 11 Although emergency laparotomy followed by gauze packing may be effective for intraabdominal bleeding, it cannot be usually performed in prehospital resuscitation. Hemostasis by tourniquet is quite effective for extremity bleeding in prehospital resuscitation but there are no effective tools for trunk hemorrhage. Resuscitative Endovascular Balloon Occlusion of the Aorta and junctional tourniquet may be potential tools for trunk bleeding but require skillful techniques. Although hemostasis by platelet transfusion may be effective in prehospital settings, it is logistically difficult because of the short half-life of platelet concentrates. The recent pandemics of the COVID-19 further limits the available blood supply for transfusions worldwide. 12 We have developed H12-(adenosine-diphosphate [ADP]) liposomes, which are effective for hemostasis against liver injury/ hemorrhage with acute thrombocytopenic coagulopathy and acute pulmonary hemorrhage caused by blunt injury. 13, 14 H12-(ADP)liposomes accumulate at bleeding sites through interaction with activated platelets via glycoprotein IIb/IIIa and augment platelet aggregation by releasing ADP at the bleeding site. H12-(ADP)liposomes remained intact in the blood circulation for up to 24 h after injection and accumulated at injured (bleeding) sites. 15 Thereafter, H12-(ADP)-liposomes released ADP and reinforced platelet aggregation with activated platelets within a few minutes. 16 The amount of ADP released from the liposome increased with decreasing lamellarity and with increasing membrane flexibility, owing to the conditions around the liposomes such as their compressed deformation by thrombus formation. 17 Released ADP was fully metabolized to allantoin and discharged to urine within 6 h. 15 H12-(ADP)-liposomes can be stored for at least 6 months at 4°C without shaking. 18 H12-(ADP)-liposomes may help hemostasis as an alternative to platelets in prehospital or urgent situations. 14, 19 Despite several trials, [20] [21] [22] an appropriate animal model of abdominal vascular injury complicated with severe acidosis and coagulopathy has not been provided. Therefore, we established a model of hemorrhagic shock with coagulopathy by vessel injury, based on our previous study. 23 Using this model, we evaluated the efficacy of H12-(ADP)-liposomes during initial DCR instead of platelet transfusion in thrombocytopenic rabbits with hemorrhagic shock caused by mesenteric vascular injury. In the current thrombocytopenic rabbits, platelet counts decreased to 50 ± 9 from 241 ± 35 (×10 3 /μl) of normal rabbits (approximately 80% thrombocytopenia). This study was conducted according to the guidelines of the Institutional Review Board for the Care of Animal Subjects of the National Defense Medical College and gained approval by the institutional review board (#16026). New Zealand White rabbits (2.5 ± 0.2 kg, male; Japan SLC, Hamamatsu, Japan) were used. The Conclusion: H12-(ADP)-liposome treatment followed by RBC/PPP may be effective in lethal hemorrhage after mesenteric vessel injury in coagulopathic rabbits. hemorrhagic shock, mesenteric artery, platelet transfusion, resuscitation, thrombocytopenia rabbits had free access to standard feed and water during a 7-day adaptation period before the experiment. A crossmatch test was applied between a donor and a transfused animal to eliminate transfusion of incompatible blood types. H12-(ADP)-liposomes were prepared as described previously. [8] [9] [10] [11] Briefly, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (1 g, 1.36 mmol), cholesterol (527 mg, 1.36 mmol), 1,5-dihexadecyl-N-s uccinyll-glutamate (189 mg, 272 μmol), 1,2-distearoyl-sn-glycero -3-phosphoethanolamine-N-[amino(polyethylene glycol)] (52 mg, 9 μmol), and H12-polyethylene glycol-Glu2C18 (47 mg, 9 μmol) (Nippon Fine Chemical Co Ltd, Osaka, Japan) were dissolved in t-BuOH and freeze-dried. The resulting mixed lipids were hydrated with phosphate buffered saline (PBS) containing 1 mM ADP using Durapore (pore size, 0.45, 0.20 μm; Millipore, Tokyo) to prepare H12-(ADP)-liposomes. After washing the liposomes with PBS followed by centrifugation (100,000g, 30 min, 4°C), the remaining ADP was removed using Sephadex G25 (GE Healthcare Japan, Tokyo). The diameter of H12-ADP-liposomes were 179 ± 53 nm. When the total lipid concentration was 20 mg/ml, the concentrations of ADP inside and outside of liposomes were 0.0732 and 0.0016 mg/ml, respectively. The encapsulation efficiency of ADP was estimated as 8.76% (Table S1 ). We also prepared H12-(PBS)-liposomes (without ADP), which skipped the process of containing ADP. Acute thrombocytopenia was achieved in rabbits essentially as described in our previous studies. 7, 9 Rabbits were anesthetized using intramuscular injections of ketamine (25 mg/kg) and xylazine (10 mg/kg). Anesthesia was maintained with intravenous injections of pentobarbital (15 mg/kg) every 30 min during the experiment. The adequacy of anesthesia was monitored by the loss of the ear pinch reflex. Anaesthetized rabbits were placed on a warming plate to maintain the body temperature at 37°C. Aseptic techniques were adopted for all surgical procedures. Surgical catheters (polyethylene indwelling needle 20 G; Terumo Co., Tokyo, Japan) were inserted into the femoral artery and vein in each rabbit. Thereafter, 25 ml/ kg of blood (sample 1) was withdrawn from the femoral artery, and the same volume of dextran 40 (Otsuka, Tokushima, Japan) was simultaneously transfused via the femoral vein. Forty minutes later, the next blood sample (25 ml/kg, sample 2) was withdrawn and the same volume of washed red blood cells (RBCs) prepared from sample 1 was transfused. This isovolemic blood exchange was repeated six times. The last transfusion of washed RBCs was performed without simultaneous blood withdrawal ( Figure S1A ). This blood exchange reduced platelet count to 50 ± 9 from 241 ± 35 (×10 3 /μl) of normal rabbits (approximately 80% thrombocytopenia). Using other thrombocytopenic rabbits, we examined an aggregating function of the residual platelets using the collagen test and ADP test. In addition, we performed the H12-(ADP)-liposome test (instead of ADP test) with and without ADP agonist (Multiplate Analyzer, Roche Diagnostics International AG, Rotkreuz, Switzerland) before and after thrombocytopenia and at 5 min after mesenteric bleeding in this model. At that test, we added 1.05 mg of H12-(ADP)-liposome for 3 ml of whole blood, according to the bolus injection dose of H12-(ADP)-liposomes (20 mg/ml/kg) in vivo study. As described previously, 7,9 blood samples withdrawn with a 10% volume of 3.8% (w/v) sodium citrate were centrifuged at 100 g for 15 min, and the supernatant was used as platelet rich plasma (PRP). The remaining sample was further centrifuged at 500 g for Figure S1B ). Donor rabbits were anesthetized as described previously. Thereafter, 50 ml/kg of blood was drawn from the femoral artery, and the same volume of normal saline was simultaneously transfused via the femoral vein, as described previously. 7 Until their euthanasia (performed using intravenous injections of pentobarbital 100 mg/kg), the animals donated approximately 30 ml of RBC concentrate. In brief, after removing PRP and PPP, the remaining RBCs were washed with acid citrate dextrose solution, finally adding the same volume of mannitol adenine phosphate solution (D-mannitol 1.457, Adenine 0.014, sodium dihydrogen phosphate 0.094, w/v%, Terumo Co, Tokyo, Japan). The allogeneic RBC concentrates were stored at 4°C in a refrigerator until use. After blood withdrawal and autologous RBC transfusion to make thrombocytopenia, rabbits underwent laparotomy and received mesenteric vessel injury at the jejunal mesentery approximately (Figure 1 ). The bleeding from the injured vessel was quantitatively evaluated by passing the injured ileum through a hole cut in a surgical glove. The exsanguinating blood was then collected in the glove and its volume precisely measured ( Figure S2A ). After abdominal closure, rabbit survivals were monitored for 24 h under ad libitum feeding with laboratory diet and water. Postoperative analgesia was performed with intramuscular injections of buprenorphine (0.02 mg/kg) twice immediately after wound closure and 12 h later. Blood cell counts were measured using a hematology analyzer (Erma PCE 170, Erma, Tokyo, Japan) at five time points: before the experiment, after completing the blood exchange, and 5, 15, and 30 min after mesenteric injury. To measure the plasma fibrinogen levels, AT III activity, PT, and aPTT, blood samples were collected in heparinized syringes and centrifuged at 500 × g at 4°C for 10 min, at four time points: before experiment, after completing the blood F I G U R E 1 Experimental design of resuscitation following mesenteric hemorrhage in rabbits with thrombocytopenic coagulopathy. Thrombocytopenia was induced in rabbits by repeated blood withdrawals and isovolemic transfusion of autologous washed RBCs. Thereafter, mesenteric injury was made in the jejunum, resulting in hemorrhagic shock due to mesenteric bleeding. At initial 5 min after bleeding, rabbits received H12-(ADP)-liposomes or H12-(PBS)-liposomes intravenous bolus injection (20 mg/ml/kg), immediately followed by an isovolemic transfusion with allogenic stored-RBC/PPP The coagulation activity of whole blood was examined as previ- Blood gases and plasma lactate levels were measured at four time points: before experiment, after completing the blood exchange, and 15 and 30 min after mesenteric injury, using a blood gas analyzer (ABL 80, Radiometer, Copenhagen, Denmark). Numbers were reduced because we were unable to get blood samples from all rabbits. Two additional rabbits were prepared for H12-ADP-liposome + RBC/ PPP group. The mesenteric vessel specimens were obtained from the injury site at 1 h after injury. These specimens were prefixed with a fixative containing 4% paraformaldehyde and 0.5% glutaraldehyde Statistical analyses were performed using a software package (Stat View 4.02J, Abacus Concepts, Berkeley, CA). Survival rates were compared by Wilcoxon signed-rank test. Statistical evaluations between two groups were made using Student t test, and other statistical evaluations were performed using a one-way analysis of variance, followed by a Bonferroni post hoc test. Data are presented as means ± SD, with p < 0.05 considered to be statistically significant. After isovolemic blood exchange and platelet plasma apheresis, rabbits showed marked decreases in platelet counts (50 ± 8 × 10 3 /μl) and plasma fibrinogen levels (59 ± 23 mg/dl), indicating severe coagulopathy (PT and aPTT were out of measurable range). They also showed significant prolongation of CT and reduction of CR (Table 1) . RBC counts were 3.7 ± 0.6 × 10 6 /μl and hemoglobin (Hb) concentrations were 8 ± 1 g/dl in rabbits, which indicated mild anemia from loss of RBCs during the blood exchange. All rabbits showed a moderately reduced mean arterial pressure (MAP; 48 ± 7 mm Hg) at the end of blood exchange/platelet plasma apheresis (Table 1, Figure 2 ). They also showed lactic acidosis: pH declined to 7.15 ± 0.08, whereas plasma lactate levels were elevated to 7 ± 4 mmol/L (Table 1 ). Rabbits then received the mesenteric vessel injury. During the initial 5 min, their blood loss reached 16 ± 6 ml, which was approximately 11% of total blood volume (estimated as 54 ml/kg 24 ) At 5 min after the bleeding, MAP, Hb concentrations, and platelet counts further reduced to 28 ± 5 mmHg, 6 ± 1 g/dl, and 45 ± 11 × 10 3 /μl, respectively (Table 1, Figure 2 ). These parameters characterized coagulopathy as defined by Moore et al. 11 (MAP < 30 mmHg, Hb < 6 g/dl, platelet < 50 × 10 3 /μl, pH < 7.2). The area under the aggregation curve assessed by the collagen test or ADP test was not significantly changed between before experiment and after mesenteric vessel bleeding, which indicated that aggregating function of the residual platelets was well preserved in this model. H12-(ADP)-liposome test with ADP agonist showed similar results to the ADP test and H12-(ADP)-liposome test without ADP agonist showed no response ex vivo (Table S2 ). Consistently, total blood loss of rabbits treated with H12-(PBS)liposomes infusion followed by RBC/PPP was significantly larger than that of H12-(ADP)-liposomes followed by RBC/PPP. In all groups, clot amplitude reduced to 30%-40% levels compared with that of the previous experiment in a similar fashion (Table S5 ). Mesenteric bleeding caused lactic acidosis in rabbits. In brief, pH levels shifted to below 7.2 at either 15 or 30 min after injury in all groups (Table S6) (Table S6 ). The lesion near the surface of thrombus, adjacent to the injured vessel ( Figure 6A ) and the lesion inside of thrombus formation ( Figure 6B) were observed in toluidine blue-staining semithin section. In the former lesion, near the surface, platelets, erythrocytes, leukocytes, and fibrin were loosely contact with each other ( Figure 6C ). In contrast, inside of the thrombus (the latter lesion), platelets, erythrocytes, and leukocytes were densely contact with fibrin ( Figure 6D ). In both lesions, anhistous particles, which were approximately 200-400 nm in diameter (indicated by arrows), were observed around the platelets, erythrocytes, or fibrin deposits ( Figure 6E ,F). These anhistous particles were presumably considered H12-(ADP)-liposomes. 13, 17 In particular, dense coagulation clots appeared to squeeze anhistous particles by clot compression ( Figure 6F ). Management of abdominal vascular trauma has been standardized as According to several guidelines and papers, recombinant factor VIIa has been used for correcting coagulopathy in surgery or trauma, which were recommended under these conditions: platelet > 50 × 10 3 /μl, fibrinogen > 100 mg/dl, and HCT > 24%. [31] [32] [33] In the current study, H12-(ADP)-liposomes could be used in more severe conditions: platelet counts were 45 ± 11 × 10 3 /μl, fibrinogen level was 59 ± 23 mg/dl, and HCT was 20 ± 3% (Table 1) because H12-(ADP)-liposomes can directly promote platelet thrombi that is crucial for hemostasis in thrombocytopenic conditions. 13, 23 Consistent with the current findings, in our previous study H12-(PBS)-liposomes with PPP were applied for the liver penetrating hemorrhage in thrombocytopenic rabbits whose platelet counts decreased less than 50 × 10 9 /L, as low as in the current study. They had no additional platelet aggregation effect in comparison with PPP alone shown in similar ACT prolongation. Consequently, ADP loading is crucial for hemostasis in thrombocytopenia. 23 Previous studies indicated that ADP is the most appropriate agonist for enhancing platelet aggregation without adverse thrombotic event and it can be sufficiently encapsulated by liposomes. 34 Adding ADP also may help constriction of injured vessels via purinergic receptors on the endothelium and smooth muscle cells besides activation of platelets. 35, 36 H12-(ADP)-liposomes have several characteristics and advantages for application to DCR and emergency situations. Transcatheter arterial embolization 37, 38 or spasm-induced pharmacotherapy 39 based on invasive angiographical procedure can selectively achieve hemostasis. H12-(ADP)-liposomes could noninvasively achieve local hemostasis because they can accumulate at the bleeding site point. In addition, H12-(ADP)-liposomes are easy to store and provide for field care, owing to characteristics of artificial liposomes, which are suitable for initial treatment of urgent hemorrhage patients. As for the safety in a nonthrombocytopenic condition, we previously applied H12-(ADP)-liposomes for normal mice, whose platelet counts were kept > 400 × 10 9 /L. may enhance not only platelet aggregation but also its adhesion. In contrast, the H12-(ADP)-liposomes are designed to potentially F I G U R E 6 Transmission electron microscopic observation. A semithin section showed the loose coagulation clots (A) and dense coagulation clots (B) adjacent to the injured site. The clots had substantial involvement of platelets and fibrin around RBCs (C, D). Arrows indicate anhistous particles, which were presumably considered as H12-(ADP)-liposome (approximately 200-400 nm) 13, 17 (E, F). Although loose coagulation clots involved anhistous particles (E), dense coagulation clots squeezed anhistous particles by clot compression (F). Arrowheads indicate platelets. ADP, adenosine-diphosphate; RBC, red blood cell promote local activation of residual platelets proximal to injury as well as promote platelet aggregation. In this study, mesenteric bleeding was located on marginal branch defined as Fullen zone IV, 44 which was relatively mild trauma injury in comparison to the injury of proximal superior mesenteric artery. However, thrombocytopenic animals fell into robust hemorrhagic shock and the single bleeding site enabled to confirm the efficacy of H12-(ADP)-liposomes for hemostasis. As for mesenteric injured procedure, anatomical position was not distinctly similar in all rabbits because of their inherent vessel variability. In thrombocytopenic rabbits whose platelet counts decreased to less than 50 × 10 9 /L and as low as in the current study, H12-ADPliposmes showed similar hemostatic effect beyond 20 mg/kg of lipid concentration. 34 However, optimum liposome: platelet ratios should be elucidated for different platelet count conditions. Clinically, early platelet dysfunction may be one of the major reasons of trauma-induced coagulopathy in severely injured patients. 45 In our study, coagulopathy was mainly attributed to the hemodilution not to platelet dysfunction, and the current experimental model Resuscitation with H12-(ADP)-liposomes may be effective as platelet substitutes in lethal hemorrhage caused by mesenteric injury in rabbits with coagulopathy. The authors thank Prof. Hidenori Suzuki for supervising the transmission electron microscope observations and KNC Laboratories Co Ltd. for preparing the H12 peptides. The authors have no conflict of interest. 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