Thrombin spatial distribution determines Protein C activation during hemostasis and thrombosis

Rebalancing of the hemostatic system by targeting endogenous anticoagulant pathways, like the Protein C system, is being tested as a means of improving hemostasis in patients with hemophilia. Recent intravital studies of hemostasis demonstrated that, in some vascular contexts, thrombin activity is sequestered to the extravascular compartment. These findings raise important questions about the context-dependent contribution of activated Protein C (aPC) to the hemostatic response since Protein C activation occurs on the surface of endothelial cells. Here, we used a combination of pharmacologic, genetic, imaging, and computational approaches to examine the relationships among thrombin spatial distribution, Protein C activation, and aPC anticoagulant function. We found that inhibition of aPC activity, either in mice harboring the Factor V-Leiden mutation or infused with an aPC blocking antibody, significantly enhanced fibrin formation and platelet activation in a microvascular injury model, consistent with aPC's role as an anticoagulant. In contrast, inhibition of aPC activity had no effect on hemostasis following penetrating injury of the mouse jugular vein. Computational studies showed that differences in blood velocity, injury size, and vessel geometry determine the localization of thrombin generation and, consequently, the extent of Protein C activation. Computational predictions were tested in vivo and showed that when thrombin generation occurred intravascularly, without penetration of the vessel wall, inhibition of aPC significantly increased fibrin formation in the jugular vein. Together, these studies show the importance of thrombin spatial distribution in determining Protein C activation during hemostasis and thrombosis.

Platelet Factor 4 (PF4) Modulates the Prothrombotic Nature of Neutrophil-Extracellular Traps (NETs): Therapeutic Implications of a NET-Stabilization Strategy

Sepsis is a dysregulated response to infection leading to life-threating organ damage. Although it remains one of the most common causes of mortality worldwide, it lacks targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens and prevent bacterial dissemination. However, when NETs are degraded by circulating nucleases they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger coagulation, induce complement activation, and cause oxidative tissue damage. We proposed a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4. Binding of PF4 enhances NET DNase-resistance, promotes NDP sequestration and increases bacterial capture, improving survival in murine sepsis. As NETs are considered prothrombotic, we were concerned that NET stabilization may increase the risk of clot formation. We therefore sought to determine the effect of PF4-NET stabilization on the thrombogenicity of NETs to learn if this strategy is safe for clinical application. To that end, we examined the effect of PF4 on the thrombotic potential of DNA and NET fragments at different states of nuclease digestion. High molecular weight (hmw) genomic DNA (hmwDNA, >50kbp) was isolated from human whole blood. hmwDNA was digested with restriction enzymes (EcoRI and AluI) for 15min to generate DNA fragments of ~4kbp and ~250bp, respectively. Neutrophils were also isolated from human blood and stimulated with 100 nM PMA to produce neutrophil-adherent NETs, which were cleaved from cell bodies by treatment with 4U/mL DNase I for 20 minutes, releasing NETs >50kbp (hmwNETs). Additional incubation of hmwNETs with DNase I yielded smaller NET fragments. We assessed in vitro activation of coagulation by DNA and NETs by measuring thrombin generation and fibrin formation in platelet-poor plasma using fluorogenic substrate and turbidity assays. Neutrophil-adherent NETs induced far less thrombin generation and fibrin formation in plasma than hmwDNA and hmwNETs. PF4 significantly increased lag time and reduced peak thrombin formation induced by both hmwDNA and hmwNETs. Binding of PF4 also delayed clot initiation time and reduced the rate of fibrin generation. Digestion of hmwDNA and hmwNETs to smaller fragments markedly enhanced thrombogenicity. We posited that shorter DNA fragments are more thrombogenic because they have a greater proportion of end-fragment DNA that exposes more single-stranded DNA. To test this hypothesis, we subjected hmwDNA and digested DNA to heat denaturation at 95°C and rapid cooling to generate single stranded DNA and found that this accelerated fibrin generation. Although the anti-thrombotic effect of PF4 was most pronounced with longer DNA and NET fragments, it continued to significantly reduce fibrin generation induced by shorter DNA fragments, perhaps by stabilizing the fragments to prevent exposure of single-stranded DNA. In conclusion, although prior studies have shown that NETs increase the risk of thrombosis in sepsis, we propose the counter-intuitive concept that PF4-stabilization decreases the risk of NET-mediated prothrombotic state by (1) inhibiting DNase cleavage of intact NETs and subsequent liberation of prothrombotic cfDNA from non-thrombogenic neutrophil-adherent NETs, and (2) preventing further digestion of circulating cfDNA into shorter and more prothrombotic fragments. Although NETs are a double-edged sword: capable of capturing pathogens but inducing host-tissue damage and thrombosis when degraded, treatment with PF4 tips the balance, limiting the capacity of NETs to induce fibrin generation and thrombosis, while enhancing their ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis and merits further investigation in translational studies. Disclosures No relevant conflicts of interest to declare.

Inhibition of Sars-Cov-2 Viral Replication and In Vivo Thrombus Formation By a Novel Plant Flavonoid

Plant-based flavonoids have been examined as inhibitors of β-coronavirus replication and as potential therapeutics for COVID19 based on their safety profile and widespread availability. SARS-CoV-2 viral replication is dependent on a cysteine protease known as 3CL protease, or main protease (Mpro), which cleaves the polyprotein translated from SARS-CoV-2 ssRNA into 11 functional proteins. This protease is highly conserved among β-coronaviruses and is intolerant of mutation. The main protein (Mpro) of SARS-CoV, SARS-CoV-2, and MERS has been identified as a target of flavonoids both by in silico and in vitro approaches. We have previously showed that select flavonoids inhibit protein disulfide isomerase (PDI), which is essential for normal thrombosis. These flavonoid PDI inhibitors block thrombus formation in vivo and have shown efficacy as antithrombotics in clinical studies. Given the substantial morbidity and mortality caused by COVID19-associated coagulopathy, we sought to identify a flavonoid that inhibits both SARS-CoV-2 Mpro and PDI, potentially blocking both viral replication and thrombus formation. While in silico studies identified many flavonoids as SARS-CoV-2 main protein (Mpro) inhibitors, no comprehensive in vitro testing of flavonoids against SARS-CoV-2 has previously been performed. We therefore evaluated 1,020 diverse flavonoids using high throughput screening for their ability to inhibit SARS-CoV-2 Mpro in a fluorescence-based Mpro substrate cleavage assay. This analysis identified four new flavonoid inhibitors of Mpro that had IC 50s ranging from 5-15 µM: amentoflavone, 3,8'-biapigenin, jaceidin triacetate, and pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). These compounds were equally or more potent than previously identified flavonoid inhibitors of SARS-CoV-2 Mpro, baicalein and myricetin. Structure activity relationships identified apigenin as an additional Mpro inhibitor. In a Vero-E6-based assay of SARS-CoV-2 replication, PGHG inhibited with an IC 50 = 4.9 µM. At 50 µM, apigenin showed 94±2.1% inhibition and baicalein 65±8.0% inhibition, while myricetin, amentoflavone, and 3,8'-biapigenin did not inhibit viral replication. Jaceidin triacetate was too toxic for further analysis. We next evaluated novel Mpro inhibitors for their ability to inhibit PDI. The most potent PDI inhibitor was PGHG, which blocked PDI reductase activity in an insulin turbidimetric assay with an IC 50 = 3.99±1.14 µM and in a di-eosin-GSSG assay with an IC 50 = 1.50±0.60 µM. When tested against isolated fragments of PDI, PGHG inhibited isolated a and a' fragments as well as ab, b'xa' and abb'x fragments, indicating that it acts on the a and a' domains of PDI. Since PDI is essential for thrombosis, we evaluated whether PGHG blocks platelet accumulation and fibrin formation following vascular injury. We infused mice with 25 mg/kg PGHG or vehicle and subsequently induced thrombus formation via laser-induced injury of an arteriole within the cremaster circulation. Infusion of PGHG resulted in a 82±6.2% inhibition of platelet accumulation and a 79±3.7% inhibition of fibrin formation. In contrast 25 mg/kg had no significant effect on tail bleeding in mice compared to vehicle control. Targeted therapies remain an important component of the armamentarium against COVID19. Our results show that a naturally occurring flavonoid, PGHG, found in Penthorum chinense Pursh , inhibits both SARS-CoV-2 replication and thrombosis without enhancing bleeding. This observation provides proof-of-principle for the development of plant-based flavonoid therapies for inhibition of β-coronaviruses and supports the further evaluation of PGHG for therapeutic use in COVID19. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Fibrin Network Formation and Lysis in Septic Shock Patients

Background: Septic shock patients are prone to altered fibrinolysis, which contributes to microthrombus formation, organ failure and mortality. However, characterisation of the individual patient’s fibrinolytic capacity remains a challenge due to a lack of global fibrinolysis biomarkers. We aimed to assess fibrinolysis in septic shock patients using a plasma-based fibrin clot formation and lysis (clot–lysis) assay and investigate the association between clot–lysis parameters and other haemostatic markers, organ dysfunction and mortality. Methods: This was a prospective cohort study including adult septic shock patients (n = 34). Clot–lysis was assessed using our plasma-based in-house assay. Platelet count, activated partial thromboplastin time (aPTT), international normalised ratio (INR), fibrinogen, fibrin D-dimer, antithrombin, thrombin generation, circulating fibrinolysis markers and organ dysfunction markers were analysed. Disseminated intravascular coagulation score, Sequential Organ Failure Assessment (SOFA) score and 30-day mortality were registered. Results: Three distinct clot–lysis profiles emerged in the patients: (1) severely decreased fibrin formation (flat clot–lysis curve), (2) normal fibrin formation and lysis and (3) pronounced lysis resistance. Patients with abnormal curves had lower platelet counts (p = 0.05), more prolonged aPTT (p = 0.04), higher lactate (p < 0.01) and a tendency towards higher SOFA scores (p = 0.09) than patients with normal clot–lysis curves. Fibrinogen and fibrin D-dimer were not associated with clot–lysis profile (p ≥ 0.37). Conclusion: Septic shock patients showed distinct and abnormal clot–lysis profiles that were associated with markers of coagulation and organ dysfunction. Our results provide important new insights into sepsis-related fibrinolysis disturbances and support the importance of assessing fibrinolytic capacity in septic shock.

Aberrant stromal tissue factor and mycolactone-driven vascular permeability, exacerbated by IL-1β, orchestrate pathogenic fibrin formation in Buruli ulcer lesions

The neglected tropical disease Buruli ulcer, caused by Mycobacterium ulcerans infection, displays coagulative necrosis in affected skin tissues. We previously demonstrated that exposure to the M. ulcerans exotoxin mycolactone depletes the expression of thrombomodulin and impacts anticoagulation at the endothelial cell surface. Moreover, while widespread fibrin deposition is a common feature of BU lesions, the cause of this phenotype is not clear. Here, we performed sequential staining of serial tissue sections of BU patient biopsies and unbiased analysis of up to 908 individual non-necrotic vessels of eight BU lesions to investigate its origins. Most vessels showed evidence of endothelial dysfunction being thrombomodulin-negative, von Willebrand factor-negative and/or had endothelium that stained positively for tissue factor (TF). Primary haemostasis was rarely evident by platelet glycoprotein CD61 staining. Localisation of TF in these lesions was complex and aberrant, including diffuse staining of the stroma some distance from the basement membrane and TF-positive infiltrating cells (likely eosinophils). This pattern of abnormal TF staining was the only phenotype that was significantly associated with fibrin deposition, and its extent correlated significantly with the distance that fibrin deposition extended into the tissue. Hence, fibrin deposition in Buruli ulcer lesions is likely driven by the extrinsic pathway of coagulation. To understand how this could occur, we investigated whether clotting factors necessary for fibrin formation might gain access to the extravascular compartment due to loss of the vascular barrier. In vitro assays using primary vascular and lymphatic endothelial cells showed that mycolactone increased the permeability of monolayers to dextran within 24 hours. Moreover, co-incubation of cells with interleukin-1β exacerbated mycolactones effects, nearly doubling the permeability of the monolayer compared to each challenge alone. We propose that leaky vascular and lymphatic systems are important drivers of extravascular fibrin deposition, necrosis and oedema frequently seen in Buruli ulcer patients.

Neutrophil Extracellular Traps Participate in the Development of Gastric Cancer Associated Thrombosis

Background: Development of venous thromboembolism (VTE) is associated with high mortalities among gastric cancer (GC) patients. Neutrophil extracellular traps (NETs) have been reported to correlated with prothrombotic state in some diseases. We hypothesize that NETs participate in the development of GC-associated thrombosis.Methods: The level of NETs in blood and tissue samples of patients were analyzed by ELISA, flow cytometry and immunofluorescence (IF). NETs generation and hypercoagulation of platelets and endothelial cells (ECs) in vitro were observed by IF. NETs procoagulant activity (PCA) was performed by fibrin formation and thrombin-antithrombin complex (TAT) assays. Thrombosis in vivo was measured in murine model induced by flow stenosis in the inferior vena cave (IVC).Results: NETs were likely to form in blood and tissue samples of GC patients compared with healthy individuals. In vitro studies that GC cells and their conditioned medium (CM), but not gastric mucosal epithelial cell can stimulate NETs releasing from neutrophils. In addition, NETs induced hypercoagulable state of platelets by up-regulating the expression of phosphatidylserine (PS) and P-selectin on the cells. Furhter, NETs stimulated adhesion of normal platelets on glass surfaces. Similarly, NETs triggered the conversion of ECs to hypercoagulable phenotypes by down-regulating the expression of their intercellular tight junctions but up-regulating that of tissue factor (TF). Treatment of normal platelets or ECs with NETs augmented the level of plasma fibrin formation and TAT complex. Meanwhile, in the models of IVC stenosis, tumor-bearing mice showed stronger ability to form thrombi and NETs were abundantly accumulated in the thrombi compared with control mice. Notably, combination of DNase-1, activated protein C (APC) and Sivelestat markedly abolished the PCA of NETs.Conclusions: Our findings demonstrate that GC-induced NETs strongly increase the risks of VTE development both in vitro and in vivo. NETs are potential therapeutic targets in the prevention and treatment of VTE in GC patients.

Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin

Background Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. Methods Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. Results Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. Conclusion Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.