key: cord-1015600-ijhuusbk
authors: Wygrecka, Malgorzata; Birnhuber, Anna; Seeliger, Benjamin; Michalick, Laura; Pak, Oleg; Schultz, Astrid-Solveig; Schramm, Fabian; Zacharias, Martin; Gorkiewicz, Gregor; David, Sascha; Welte, Tobias; Schmidt, Julius J.; Weissmann, Norbert; Schermuly, Ralph T.; Barreto, Guillermo; Schaefer, Liliana; Markart, Philipp; Brack, Markus C.; Hippenstiel, Stefan; Kurth, Florian; Sander, Leif E.; Witzenrath, Martin; Kuebler, Wolfgang M.; Kwapiszewska, Grazyna; Preissner, Klaus T.
title: Altered fibrin clot structure and dysregulated fibrinolysis contribute to thrombosis risk in severe COVID-19
date: 2021-12-07
journal: Blood Adv
DOI: 10.1182/bloodadvances.2021004816
sha: 2e9c66bcbcd2409df7433b630cfcf74854cb128b
doc_id: 1015600
cord_uid: ijhuusbk

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. Here, we demonstrate altered levels of factor XII (FXII) and its activation products in critically ill COVID-19 patients in comparison to patients with severe acute respiratory distress syndrome due to influenza virus (ARDS-influenza). Compatible with this data, we report rapid consumption of FXII in COVID-19, but not in ARDS-influenza, plasma. Interestingly, the lag phase in fibrin formation, triggered by the FXII activator kaolin, was not prolonged in COVID-19 as opposed to ARDS-influenza. Using confocal and electron microscopy, we showed that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggers formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, clot lysis was markedly impaired in COVID-19 as opposed to ARDS-infleunza subjects. Dysregulatated fibrinolytic system, as evidenced by elevated levels of thrombin-activatable fibrinolysis inhibitor, tissue-plasminogen activator, and plasminogen activator inhibitor-1 in COVID-19 potentiated this effect. Analysis of lung tissue sections revealed wide-spread extra- and intra-vascular compact fibrin deposits in COVID-19 patients. Together, compact fibrin network structure and dysregulated fibrinolysis may collectively contribute to high incidence of thrombotic events in COVID-19.

Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is a corona virus that causes a multisystem disease emanating from the respiratory tract designated as a coronavirus disease (COVID)-19 [1] [2] [3] . Rapidly accumulating data suggests that a major underlying molecular mechanism in COVID-19-related morbidity and mortality is widespread endothelial injury associated with hyperactivation of the immune system, consequently leading to numerous haemostasis abnormalities [4] [5] [6] [7] [8] .The clinical relevance of these processes is highlighted by the association between abnormal levels of D-dimer and the 28-day mortality in patients with COVID-19 9-13 , and postmortem studies stressing the presence of micro-thrombi and capillarostasis in the lungs of affected subjects 14, 15 .

The high incidence of thrombotic events, in particular deep vein thrombosis and pulmonary embolism, in conjunction with mildly prolonged activated partial thromboplastin time (APTT) 16,17 , suggests a possible role of coagulation factor XII (FXII) in COVID-19 coagulopathy. FXII is a serine protease of the contact-phase system of blood coagulation which circulates in plasma as a single-chain zymogen 18 .

Following contact with anionic surfaces such as kaolin, but also extracellular RNA (eRNA) released from damaged cells 19 , neutrophil extracellular traps (NETs) 20 , or polyphosphates secreted from activated platelets 21 , FXII undergoes autoactivation to αFXIIa (herein referred to as FXIIa). FXIIa cleaves plasma prekallikrein (PK) to kallikrein (PKa), which in turn reciprocally activates FXII and amplifies FXIIa generation. As a consequence, the plasma kallikrein-kinin system is activated, leading to the release of the vasodilatory and vascular barrier disrupting peptide bradykinin from high molecular weight kininogen (HK) [22] [23] [24] [25] . Overall, activation of the contact-phase system may contribute to an increased production of thrombin and fibrin, although FXIIa/PKa-mediated conversion of plasminogen to plasmin may also affect fibrinolysis 26 .

A congenital deficiency of FXII in humans does not cause any bleeding complications, suggesting that FXII is dispensable for physiological haemostasis and fibrin formation 27 . However, the contact phase pathway may play an important role in thrombosis development when contact surfaces are exposed in scenarios such as trauma injury or bacterial and viral infections [27] [28] [29] . Indeed, numerous animal studies have confirmed a critical function of FXII in thrombus growth and stabilization under the mentioned conditions and provided the rationale for the development of new 4 FXIIa inhibitors, which ensure thrombo-protection in patients without causing a bleeding complications [27] [28] [29] [30] .

Given the high incidence of thromboembolic complications in severely ill COVID-19 patients 16,17 , we investigated the contribution of FXII to clot formation and its architecture in this patient cohort in comparison to patients infected with the influenza virus.

Additional methods are provided in the supplement.

Plasma samples from COVID-19 patients were obtained from the Charité-University (05/00). Blood was collected to the sodium citrate blood specimen collection tubes via standard venipuncture. All blood biospecimen were processed without a stabilizing reagent, at room temperature, within 5h of collection, and stored at -80°C.

All plasmas were used in analyses of circulating proteins as well as coagulation and fibrinolytic activity except when insufficient plasma from an individual subject was available. Baseline demographics and clinical characteristics of the donors and patients are shown in Table 1 .

Lung specimens were obtained from 10 ARDS patients (5 COVID-19, 5 influenza) and 5 donors by autopsy. Time from death to autopsy was matched. All Table 2 . 

Endogenous FXII was depleted from plasma using the goat anti-FXII antibody (cat.

no.: 206-0056; Zytomed Systems) covalently attached to magnetic beads (Thermo-Fisher Scientific). Afterwards, a hundred µl of plasma was supplemented with 30nM 6 exogenous FXII and the sample was incubated for 1h at 37°C. Aliquots were withdrawn after the indicated time points and analyzed by western blotting. In some experiments, plasma was preincubated with 12mg/mL CTI 30min prior to the addition of FXII.

The PKa-like activity assay and the activity of factor VIII (FVIII:C) were performed as described in 32 and 33 , respectively.

Statistical analysis was performed in R (version 4) using the ggpubr package 34, 35 .

Data are expressed as single data points with boxplot overlay indicating median and interquartile range, unless indicated otherwise. Multiple groups were compared by non-parametric Kruskal-Wallis test. Correlations were performed using Spearman's rank correlation coefficient.

For original data, please contact malgorzata.wygrecka@innere.med.uni-giessen.de

In the Berlin cohort, the plasma levels of FXII were decreased in severe COVID-19

patients as compared to donors ( Figure 1A , B; moderate: WHO severity score 3-4;

severe: WHO severity score 5-7). Disappearance of the FXII in plasma typically corresponds to its activation and conversion into the two chain FXIIa protein composed of the 50kDa heavy chain and 30kDa light chain. Detection of FXIIa in plasma is, however, hindered by its rapid inactivation and complex formation with C1 esterase inhibitor (C1INH). Thus to better monitor the presence of FXIIa in COVID-19 plasma, we monitored products of its activation, such as cleaved HK and PKa. As expected, disappearance of FXII in plasma was accompanied by HK cleavage, seen as diminished signal intensity of the intact HK band at 130kDa ( Figure 1C , D). A decrease in intact HK levels was associated with the appearance of cleaved HK fragments: the cleaved HK light chain band migrating at 55kDa and an additional 45kDa band representing a degradation product of the 55kDa cleaved HK light chain.

To further examine whether the reduction in intact levels of FXII and HK is a result of 7 the contact system activation, we measured the activity of plasma PKa. PKa-like activity was markedly elevated in severe COVID-19 patients in comparison to donors and patients with moderate SARS-CoV2 infection ( Figure 1E ). Furthermore, a strong negative correlation between the levels of intact HK/albumin ratio and the PKa-like activity in plasma of severe COVID-19 patients was observed ( Figure 1F ). Purified plasma proteins and deficient plasma samples were used to demonstrate the specificity of the bands shown in western blots ( Figure 1A , C; right panels).

To assess whether enhanced activation of FXII in critically ill COVID-19 patients between packing density of fibrin fibers and plasma fibrinogen concentration, with dense fibrin network in clots formed in plasma of patients exhibiting high fibrinogen levels ( Figure 2H ). A strong positive correlation between maximum turbidity values and fibrinogen concentration in plasma of COVID-19 patients was noted ( Figure 2J ).

As the architecture of fibrin clots may be influenced not only by fibrinogen but also FXIIa 36,37 , we next analyzed the impact of these two proteins on the clot structure in a purified system. As depicted in figure 3A high concentrations of fibrinogen increased peak turbidity values and this effect was potentiated by the addition of FXIIa.

Accordingly, corn trypsin inhibitor (CTI), the inhibitor of FXIIa, reduced maximum turbidity of the clots generated by mixing fibrinogen and FXIIa ( Figure 3B ).

As sustained activation of FXII was described in COVID-19 38 , we next investigated the potential contribution of FXIIa to the regulation of fibrin clot structure in this patient group. To this end, FXII-depleted COVID-19 and ARDS-influenza plasma samples were recalcified in the absence or presence of exogenous FXII and the fibrin clots were visualized using the antibody against fibrinogen/fibrin. As depicted in figure   3C and D, FXII increased fibrin network density but not fibrin fiber diameter in COVID-19 plasma. Yet, no apparent effect of FXII on the clot architecture in ARDSinfluenza was observed ( Figure 3C, D) . Interestingly, the most prominent effect of FXII on fibrin network density was observed in COVID-19 plasma samples containing high levels of fibrinogen ( Figure 3D ). Together, these results imply that COVID-19 plasma contains FXII (auto)-activation cofactor(s) which trigger generation of FXIIa.

FXIIa then affects the fibrin clot structure in a thrombin-dependent and/or thrombinindependent manner. In line with this assumption, rapid decay of exogenous FXII in COVID-19 plasma was observed ( Figure 3E , F). CTI markedly delayed disappearance of FXII suggesting that auto-activation of FXIIa occurs in COVID-19

and ARDS-influenza plasma samples ( Figure 3E ). To demonstrate a direct, thrombinindependent, effect of FXIIa on the fibrin clot structure, we clotted hirudinpreincubated COVID-19 plasma with batroxobin in the presence of FXIIa and/or CTI and measured maximum turbidity. As shown in figure 3G , FXIIa increased maximum turbidity and this effect was diminished by CTI. These results are in line with the experiments performed in the purified system ( Figure 3A, B) . In sum, our results imply that FXIIa, in addition to its possible effect on thrombin generation, may also directly contribute to the fibrin network structure in COVID-19 plasma.

Elevated fibrin network density correlates with increased clot resistance to fibrinolysis Compact fibrin network density was previously found to impair clot fibrinolysis 39 .

Accordingly, we next evaluated the lysis resistance of fibrin clots in patient plasma, using an in vitro turbidimetric clot-lysis assay. Here, kaolin together with t-PA were added to plasma to initiate the intrinsic pathway of coagulation, followed by fibrindependent plasmin generation via t-PA-mediated activation of plasminogen in the same sample. While in donor plasma, the characteristic bell-shaped clot-lysis curve,

representing the complete fibrin clot dissolution, was observed, only partial clot-lysis was detected in ARDS-influenza samples, and clot-lysis was absent in COVID-19

samples over the entire time period of the experiment ( Figure 4A ). This observation is supported by the highest turbidity values at 60min in COVID-19 samples ( Figure 4B ).

Overall, clot lysis (as assessed by turbidity values at 60min) was observed in 84% of ARDS-influenza patients and only 30% of COVID-19 patients suggesting pronounced deregulation of the fibrinolytic system in SARS-CoV2-infected subjects in our cohort.

As expected, increasing amounts of fibrinogen and FXIIa prolonged clot lysis time,

with an additive effect being observed at the highest concentrations of both proteins ( Figure 4C ). The addition of CTI to the assay shortened clot lysis time ( Figure 4D ).

To test whether other components of the fibrinolytic system, such as t-PA, PAI-1 and TAFI, may be dysregulated in critically ill COVID-19 patients, we measured their levels by means of ELISA. The concentration of t-PA was elevated in COVID-19 as compared to donors and ARDS-influenza patients ( Figure 4E ). An increase of PAI-1 was also noted in plasma of ARDS-influenza and COVID-19 patients as opposed to

donors, yet, a significant difference between both patient groups was not detected ( Figure 4F ). Interestingly, TAFI was not only markedly elevated in both patient groups as compared to donors, but it was also higher in patients with COVID-19 as compared to ARDS-influenza ( Figure 4G ).

To demonstrate the in vivo relevance of our findings, we stained for fibrin the autopsy lung tissue sections from SARS-CoV2-and influenza-infected ARDS patients as well as subjects who died due to no respiratory causes. Notably, time from death to autopsy was matched for all groups examined. As demonstrated in figure 5A , intraand extra-vascular fibrin aggregates were observed in both severe COVID-19 and ARDS-influenza patients. However, in contrast to ARDS-influenza subjects, in the lungs of COVID-19 patients the deposits of fibrin appeared to be more widespread and evenly present not only in vascular but also alveolar spaces. In ARDS-influenza patients, fibrin deposit were predominantly observed in alveolar spaces and present in selected regions of the lung ( Figure 5A, C) . Overall, in COVID-19 lungs fibrin clots were more compact and homogeneous whereas in ARDS-influenza lungs they were widespread and characterized by regions of high and low fibrin fiber density ( Figure   5B , D). In order to provide mechanistic insights into the reported hypercoagulable state of severe COVID-19 patients, we compared changes in the contact phase system activation and fibrinolysis between COVID-19 patients, individuals with ARDSinfluenza, and donors (healthy subjects). While some critical parameters such as fibrinogen, t-PA, and TAFI were significantly increased, FXII levels were reduced in severe COVID-19, and the process of fibrin formation and the resulting fibrin clot structure and lysis were substantially different between patient cohorts. Histological data provided evidence for widespread, compact fibrin deposition in the lungs of patients with COVID-19 as opposed to those with ARDS-influenza. 11 In particular, the levels of FXII were decreased in severe COVID-19 patients as compared to ARDS-influenza and donors and FXII-activation products, such as cleaved HK and PKa-like activity, were altered in patients with SARS-CoV2 infection.

This scenario very likely reflects FXII consumption due to its auto-activation on negatively charged surfaces and its reciprocal activation by PKa. Decreased FXII levels in COVID-19 plasma are also in accordance with moderately elevated APTT values reported in other studies 43, 44 . The exacerbated consumption of FXII in severe COVID-19 is further supported by our in vitro studies, in which the supplementation of COVID-19 plasma with exogenous FXII resulted in its rapid activation, presumably due to the presence of FXII auto-activation cofactors or increased PKa activity. Increased levels of fibrinogen and elevated thrombin generation potential were found to contribute to the formation of stable clots that are composed of a dense network of the thin fibrin strands 36, 55 . Accordingly, clots generated in COVID-19 plasma exhibited higher packing density and were more resistant to lysis as compared to clots formed in ARDS-influenza plasma. Further experiments with COVID-19 plasma revealed that next to fibrinogen also FXIIa may regulate clot compactness. While, increased fibrinogen levels can independently promote thrombus formation and stability 55 , the role of FXII in this process seems to be more complex and dependent on the presence of cofactors, which enable FXII (auto)-activation. FXIIa may then regulate fibrin network density in a thrombin-dependent and a thrombin-independent manner.

While, the ability of FXIIa to convert factor XI (FXI) to FXIa and thereby to promote thrombin generation is well documented 56, 57 , elucidation of the mechanism of a direct effect of FXIIa on fibrin clot architecture requires further research. FXIIa binds with high affinity to fibrinogen/fibrin 36 , whether this interaction facilitates fibrin fiber crosslinking or incorporation of other components into a clot is speculative at the movement. Though, high turbidity of fibrin clots observed in COVID-19 may speak for the intercalation of, e.g. NET components, into the fibrin network. Cell free-DNA and histones (both NET components) were found to promote more opaque and fibrinolysis resistant clots 58, 59 . In addition, cell free-DNA was reported to bind to fibrinogen, fibrin, FXII, FXIIa, HK, FIXa, FXIa, fibronectin, and vWF 58,60-62 . Thus, its 13 intercalation into fibrin network could not only accelerate FXII auto-activation but also serve as a platform that brings plasma-proteins and fibrin fibers together resulting in the formation of turbid and fibrinolysis resistant clots.

The persistent vessel occlusion, seen in critically ill COVID-19 patients, seems to be reinforced by markedly increased plasma levels of TAFI and moderately increased amounts of PAI-1. Elevated levels of t-PA try to counterbalance this prothrombotic environment, however, are not sufficient to compensate for increased procoagulant activity in patients with COVID-19. In conclusion, pathological events described in COVID-19 create milieu favoring activation of FXII. In combination with high levels of fibrinogen, FXIIa may contribute 14 to compact, lysis resistant clot formation in a thrombin-dependent and a thrombinindependent manner. This prothrombogenic microenvirement is further promoted by dysregulated fibrinolysis. Our study advances understanding of the common and divergent aspects related to clot formation and lysis during ARDS-influenza and COVID-19.

We thank E. Bieniek for her excellent technical assistance. We also thanks A. Seipp Competing interests: None declared. 

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