Fibrinogen and Antifibrinolytic Proteins: Interactions and Future Therapeutics

Thrombus formation remains a major cause of morbidity and mortality worldwide. Current antiplatelet and anticoagulant therapies have been effective at reducing vascular events, but at the expense of increased bleeding risk. Targeting proteins that interact with fibrinogen and which are involved in hypofibrinolysis represents a more specific approach for the development of effective and safe therapeutic agents. The antifibrinolytic proteins alpha-2 antiplasmin (α2AP), thrombin activatable fibrinolysis inhibitor (TAFI), complement C3 and plasminogen activator inhibitor-2 (PAI-2), can be incorporated into the fibrin clot by FXIIIa and affect fibrinolysis by different mechanisms. Therefore, these antifibrinolytic proteins are attractive targets for the development of novel therapeutics, both for the modulation of thrombosis risk, but also for potentially improving clot instability in bleeding disorders. This review summarises the main properties of fibrinogen-bound antifibrinolytic proteins, their effect on clot lysis and association with thrombotic or bleeding conditions. The role of these proteins in therapeutic strategies targeting the fibrinolytic system for thrombotic diseases or bleeding disorders is also discussed.

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Recent advances in understanding endogenous fibrinolysis: implications for molecular-based treatment of vascular disorders

Expert Reviews in Molecular Medicine ◽

10.1017/s1462399402004362 ◽

2002 ◽

Vol 4 (7) ◽

pp. 1-10 ◽

Cited By ~ 10

Author(s):

Peter F. Bodary ◽

Kevin J. Wickenheiser ◽

Daniel T. Eitzman

Keyword(s):

Animal Models ◽

Thrombus Formation ◽

Plasminogen Activator Inhibitor ◽

Coagulation Cascade ◽

Clot Lysis ◽

Plasmin Activity ◽

Plasminogen Activator Inhibitor 1 ◽

Fibrinolysis Inhibitor ◽

Activator Inhibitor

The fate of a forming thrombus is determined through the delicate balance between the coagulation cascade, favouring clot formation, and the fibrinolytic system, favouring clot lysis. These processes occur simultaneously, and enhancement of fibrinolysis has been shown to reduce occlusive thrombus formation in animal models. This review examines the roles of the major fibrinolytic factors involved in clot lysis. The regulation of plasmin activity by plasminogen activators, α-2-antiplasmin, plasminogen activator inhibitor 1, and thrombin-activatable fibrinolysis inhibitor, and their effects on thrombus formation in vivo are discussed. Since alterations in fibrinolytic capacity appear to affect thrombus formation in animal models, there is considerable interest in the pharmacological manipulation of fibrinolysis.

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Maturity onset diabetes of the young and fibrin-related thrombosis risk

Diabetes and Vascular Disease Research ◽

10.1177/1479164120963048 ◽

2020 ◽

Vol 17 (6) ◽

pp. 147916412096304

Author(s):

RC Sagar ◽

F Phoenix ◽

G Thanabalasingham ◽

K Naseem ◽

RA Ajjan ◽

...

Keyword(s):

Confocal Microscopy ◽

Plasminogen Activator Inhibitor ◽

Fibrin Clot ◽

Complement C3 ◽

Clot Lysis ◽

Maturity Onset Diabetes ◽

Plasminogen Activator Inhibitor 1 ◽

Onset Diabetes ◽

Increased Risk ◽

Fibrin Network

Background: Fibrin network characteristics determine predisposition to cardiovascular disease (CVD). Individuals with type 1 (T1DM) and type 2 diabetes mellitus (T2DM) have higher risk of CVD and display deranged fibrin network structure. Those with maturity onset diabetes of the young (MODY) may also be at increased risk but their fibrin clot properties have not been studied. Methods: Plasma clots properties from 13 individuals with HNF1A-MODY, 12 matched-individuals with T2DM and 12 with T1DM were studied using a validated turbidimetric assay and confocal microscopy. Plasma levels of fibrinogen, plasminogen activator inhibitor-1, complement C3 and C-reactive protein were also measured. Results: MODY clot maximum absorbance was 0.37 ± 0.03 AU, similar to T1DM (0.32 ± 0.03 AU; p = 0.26), but lower than T2DM (0.49 ± 0.03 AU; p = 0.02), with confocal microscopy confirming structural differences. Clot lysis time in MODY was similar to T1DM (456 ± 50 and 402 ± 20 s, respectively; p = 0.09) but shorter than T2DM (588 ± 58 s; p = 0.006). Comparing inflammatory/thrombotic proteins in HNF1A-MODY and T2DM, C3 levels were lower in MODY than T2DM (0.58 ± 0.09 and 0.80 ± 0.1 mg/ml, respectively; p < 0.01). Conclusions: HNF1A-MODY fibrin network alterations are at least as pronounced as in T1DM but less thrombotic than T2DM clots. Differences in fibrin clot characteristics comparing HNF1A-MODY and T2DM may, in part, relate to lower C3 levels.

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A long-acting PAI-1 inhibitor reduces thrombus formation

Thrombosis and Haemostasis ◽

10.1160/th16-11-0891 ◽

2017 ◽

Vol 117 (07) ◽

pp. 1338-1347 ◽

Cited By ~ 17

Author(s):

Shuangzhou Peng ◽

Guangpu Xue ◽

Lihu Gong ◽

Chao Fang ◽

Jingfei Chen ◽

...

Keyword(s):

Thrombus Formation ◽

Plasminogen Activator Inhibitor ◽

Fibrin Clot ◽

Computational Method ◽

Tissue Type ◽

Clot Lysis ◽

Plasminogen Activator Inhibitor 1 ◽

Long Acting ◽

Pai 1

SummaryPlasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC50 (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC50 value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.

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Altered fibrin clot structure and dysregulated fibrinolysis contribute to thrombosis risk in severe COVID-19

Blood Advances ◽

10.1182/bloodadvances.2021004816 ◽

2021 ◽

Author(s):

Malgorzata Wygrecka ◽

Anna Birnhuber ◽

Benjamin Seeliger ◽

Laura Michalick ◽

Oleg Pak ◽

...

Keyword(s):

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Fibrin Clot ◽

Clot Lysis ◽

Lag Phase ◽

Factor Xii ◽

Plasminogen Activator Inhibitor 1 ◽

Effect Analysis ◽

High Incidence ◽

Fibrinolysis Inhibitor

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.

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The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649570 ◽

1993 ◽

Vol 70 (02) ◽

pp. 301-306 ◽

Cited By ~ 51

Author(s):

Linda A Robbie ◽

Nuala A Booth ◽

Alison M Croll ◽

Bruce Bennett

Keyword(s):

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Fibrin Clot ◽

Clot Lysis ◽

Plasminogen Activator Inhibitor 1 ◽

Dependent Inhibition ◽

Activator Inhibitor ◽

Pai 1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

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Plasma fibrin clot properties in the G20210A prothrombin mutation carriers following venous thromboembolism: the effect of rivaroxaban

Thrombosis and Haemostasis ◽

10.1160/th17-01-0060 ◽

2017 ◽

Vol 117 (09) ◽

pp. 1739-1749 ◽

Cited By ~ 8

Author(s):

Agnieszka Janion-Sadowska ◽

Joanna Natorska ◽

Jakub Siudut ◽

Michal Zabczyk ◽

Andrzej Stanisz ◽

...

Keyword(s):

Venous Thromboembolism ◽

Fibrin Clot ◽

Clot Lysis ◽

Mutation Carriers ◽

Lysis Time ◽

Prothrombin Mutation ◽

Fibrinolysis Inhibitor ◽

Heterozygous Carriers ◽

Mutation Group ◽

Clot Structure

SummaryWe sought to investigate whether the G20210A prothrombin mutation modifies plasma fibrin clot properties in patients after venous thromboembolism (VTE) and how rivaroxaban treatment affects these alterations. We studied 34 prothrombin mutation heterozygous carriers and sex- and age-matched 34 non-carriers, all at least three months since the first VTE episode, before and during treatment with rivaroxaban. Clot permeability (Ks) and clot lysis time (CLT) with or without elimination of thrombin activatable fibrinolysis inhibitor (TAFI) were assessed at baseline, 2–6 hours (h) after and 20–25 h after intake of rivaroxaban (20 mg/day). At baseline, the prothrombin mutation group formed denser clots (Ks −12 %, p=0.0006) and had impaired fibrinolysis (CLT +14 %, p=0.004, and CLT-TAFI +13 %, p=0.03) compared with the no mutation group and were similar to those observed in 15 healthy unrelated prothrombin mutation carriers. The G20210A prothrombin mutation was the independent predictor for Ks and CLT before rivaroxaban intake. At 2–6 h after rivaroxaban intake, clot properties improved in both G20210A carriers and non-carriers (Ks +38 %, and +37 %, CLT −25 % and −25 %, CLT-TAFI −20 % and −24 %, respectively, all p<0.001), but those parameters were worse in the prothrombin mutation group (Ks −12.8 %, CLT +17 %, CLT-TAFI +13 %, all p<0.001). Rivaroxaban concentration correlated with fibrin clot properties. After 20–25 h since rivaroxaban intake most clot properties returned to baseline. Rivaroxaban-related differences in clot structure were confirmed by scanning electron microscopy images. In conclusion, rivaroxaban treatment, though improves fibrin clot properties, cannot abolish more prothrombotic fibrin clot phenotype observed in prothrombin mutation carriers following VTE.

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A fusion protein with improved thrombolytic effect and low bleeding risk

Thrombosis and Haemostasis ◽

10.1160/th09-04-0235 ◽

2009 ◽

Vol 102 (12) ◽

pp. 1194-1203 ◽

Cited By ~ 3

Author(s):

Lisheng Wang ◽

Qinglin Zhang ◽

Yide Qin ◽

Chutse Wu ◽

Xiudong Wang ◽

...

Keyword(s):

Fusion Protein ◽

Anticoagulant Activity ◽

Bleeding Risk ◽

Fibrin Clot ◽

Clot Lysis ◽

Recognition Sequence ◽

Linker Peptide ◽

C Terminus ◽

N Terminus

SummaryTo resolve the therapeutic dilemma between efficacy of thrombolysis and bleeding risk associated with the use of a combination of thrombolytic and anticoagulant treatments, we created a fusion protein. Staphylokinase was fused to the N-terminus of hirudin using thrombin recognition sequence as linker peptide, resulting in a fusion protein STH.We hypothesised that STH would be cleaved by thrombin at the thrombus site, releasing staphylokinase and hirudin to perform bifunctionally, and attenuating bleeding risk. SDS-PAGE andWestern blot analyses indicated that the linker peptide could be specially recognised and cleaved by thrombin. Amidolytic and thromboelastogram assays showed that the N-terminus of hirudin in STH was blocked by staphylokinase and linker peptide, impeding hirudin’s anticoagulant activity. Once cleaved, STH displayed 35.7% of the anticoagulant activity of equimolar hirudin and exhibited anticoagulant effects in the fibrin clot lysis assay.Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of hirudin retained its high affinity for thrombin. Moreover, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Thus, STH may have the capacity to perform bifunctionally and release anticoagulant activity in a thrombus-targeted manner in vivo, which may reduce the bleeding risk that often accompanies high thrombolytic efficacy in the treatment of thromboembolic diseases.

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Altered fibrin clot structure contributes to thrombosis risk in severe COVID-19

10.1101/2021.09.17.460777 ◽

2021 ◽

Author(s):

Malgorzata Wygrecka ◽

Anna Birnhuber ◽

Benjamin Seeliger ◽

Laura Michalick ◽

Oleg Pak ◽

...

Keyword(s):

Thrombus Formation ◽

Fibrin Clot ◽

Clot Lysis ◽

Factor Xii ◽

Tissue Sections ◽

Thrombosis Risk ◽

Activation Rate ◽

Activation Products ◽

Kaolin Clotting Time ◽

Clot Structure

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 two independent cohorts of critically ill COVID-19 patients in comparison to patients suffering from 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 kaolin clotting time was not prolonged in COVID-19 as compared to ARDS-influenza. Using confocal and electron microscopy, we show 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, we observed clot lysis in 30% of COVID-19 patients and 84% of ARDS influenza subjects. Analysis of lung tissue sections revealed wide-spread extra- and intra-vascular compact fibrin deposits in COVID-19. Together, our results indicate that elevated fibrinogen levels and increased FXII activation rate promote thrombosis and thrombolysis resistance via enhanced thrombus formation and stability in COVID-19.

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Herpesviruses enhance fibrin clot lysis

Thrombosis and Haemostasis ◽

10.1160/th11-08-0601 ◽

2012 ◽

Vol 107 (04) ◽

pp. 760-768 ◽

Cited By ~ 5

Author(s):

Edwin S. Gershom ◽

Amanda L. Vanden Hoek ◽

Scott C. Meixner ◽

Michael R. Sutherland ◽

Edward L.G. Pryzdial

Keyword(s):

Vascular Disease ◽

Plasminogen Activator ◽

Disease Risk ◽

Plasminogen Activator Inhibitor ◽

Aminocaproic Acid ◽

Fibrin Clot ◽

Clot Lysis ◽

Cell Infection ◽

Proteolytic Activation

SummaryThe incorporation of virus- and host-derived procoagulant factors initiates clotting directly on the surface of herpesviruses, which is an explanation for their correlation to vascular disease. The virus exploits the resulting thrombin to enhance infection by modulating the host cell through protease activated receptor (PAR) 1 signalling. Prior reports demonstrated that at least one herpesvirus expresses surface annexin A2 (A2), a cofactor for tissue plasminogen activator (tPA)-dependent activation of plasminogen to plasmin. Since plasmin is both a fibrinolytic protease and PAR agonist, we investigated whether herpesviruses enhance fibrinolysis and the effect of plasmin on cell infection. Herpes simplex virus types 1 (HSV1) and 2, and cytomegalovirus (CMV) purified from various cell lines each accelerated the proteolytic activation of plasminogen to plasmin by tPA. Ligand blots identified A2 as one of several plasminogen binding partners associated with the virus when compared to an A2-deficient virus. This was confirmed with inhibitory A2-antibodies. However, A2 was not required for virus-enhanced plasmin generation. HSV1, HSV2 and CMV accelerated tPA-dependent fibrin clot lysis by up to 2.8-fold. Modest plasmin generation and fibrinolysis was detected independent of exogenous tPA, which was inhibited by plasminogen activator inhibitor type-1 and ε-aminocaproic acid; however, the molecular basis remains speculative. Up to a ∼6-fold enhancement of infection was provided by plasmin-mediated cell infection. Inhibitory antibodies revealed that plasmin increased HSV1 infection through a mechanism involving PAR2. Thus, virus-enhanced fibrinolysis may help explain the paradox of the highly procoagulant in vitro herpesvirus surface eliciting only relatively weak independent vascular disease risk.

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