Platelet Procoagulant Activity Research Articles

Comprehensive investigation of platelet function in patients with cirrhosis

Cirrhosis presents with thrombocytopenia and possibly thrombocytopathy. Previous studies exploring platelet function gave conflicting results and most controversies are explained by the variety of methods employed for investigation. We sought to assess in-vitro the overall platelet function in cirrhosis.We investigated 34 patients by using the following tests. (i)Aggregometry. (ii)Measurement of the content of platelet granules. (iii)Cytometric platelet activation. (iv)Plasmatic markers of in-vivo platelet activation. (v)Platelet procoagulant activity by thrombin generation (TG) in platelet-rich plasma (PRP).TG measured in PRP for patients and controls was similar. Platelets from patients with cirrhosis showed reduction of aggregation and secretion of ATP. Similar results were observed for platelet activation parameters such as P-selectin expression and PAC-1 platelet binding. Plasma levels of βeta-thromboglobulin and soluble P-selectin, were increased in patients-vs-controls. In contrast, there were no patients-vs-controls differences for plasmatic platelet-factor-4. Results are consistent with a state of in-vivo platelet activation and decreased in-vitro aggregation. Since bleeding events following invasive procedures are uncommon in cirrhosis, we speculate that in-vitro aggregometry testing does not reflect the situation occurring in-vivo. Results of the study and pathophysiological considerations support the conclusion that platelet function in cirrhosis as determined by aggregometry, although somewhat impaired, may support the overall hemostatic potential, which is needed for most invasive interventions. These conclusions are in line with the recommendations of international guidelines, warning against indiscriminate use of prophylactic preprocedural administration of platelets before invasive procedures. Decision on platelet support should not be made based on in-vitro laboratory testing for platelet function.

Polyhexamethylene Guanidine Phosphate Enhanced Procoagulant Activity through Oxidative-Stress-Mediated Phosphatidylserine Exposure in Platelets.

Polyhexamethylene guanidine phosphate (PHMG-p) is a common biocidal disinfectant that is widely used in industry and household products. However, PHMG-p was misused as a humidifier disinfectant (HD) in South Korea, which had fatal health effects. Various health problems including cardiovascular diseases were observed in HD-exposed groups. However, the potential underlying mechanism of HD-associated cardiovascular diseases is poorly understood. Here, we examined the procoagulant activity of platelets caused by PHMG-p and clarified the underlying mechanism. PHMG-p enhanced phosphatidylserine (PS) exposure through alteration of phospholipid transporters, scramblase, and flippase. Intracellular calcium elevation, intracellular ATP depletion, and caspase-3 activation appeared to underlie phospholipid transporter dysregulation caused by PHMG-p, which was mediated by oxidative stress and mitochondrial dysfunction. Notably, antioxidant enzyme catalase and calcium chelator EGTA reversed PHMG-p-induced PS exposure and thrombin generation, confirming the contributive role of oxidative stress and intracellular calcium in the procoagulant effects of PHMG-p. These series of events led to procoagulant activation of platelets, which was revealed as enhanced thrombin generation. Collectively, PHMG-p triggered procoagulant activation of platelets, which may promote prothrombotic risks and cardiovascular diseases. These findings improve our understanding of HD-associated cardiovascular diseases.

A Mouse Model of the PAR4 Sequence Variant P310L Shows Decreased Platelet Reactivity and Thrombosis

Protease activated receptors 1 and 4 (PAR1, PAR4) on platelets mediate thrombin signaling and are vital for thrombus development. Hypercoagulation and thrombin generation are major risk factors for thrombosis. Sustained thrombin signaling through PAR4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single-nucleotide polymorphism (SNP, rs2227376) leads to an amino acid change in extracellular loop 3 (ECL3) of PAR4, which decreases PAR4 activation (PAR4-P310L). This SNP is also associated with a significantly lower risk for venous thromboembolism (VTE) in a GWAS meta-analysis. We aim to determine the mechanism by which PAR4-P310L (rs2227376) impacts platelet function and reduces the risk of VTE using mice with a homologous mutation (PAR4-P322L). Ex vivo platelet function was determined using flow cytometry and aggregometry. Arterial and venous thrombosis was examined using the ferric-chloride induced carotid artery injury and inferior vena cava (IVC) stasis models, respectively. Platelets from heterozygous (PAR4-322P/L) and homozygous (PAR4-322L/L) mice had decreased thrombin-mediated aggregation and decreased PAR4 reactivity compared to wild types. These mice also had a prolonged time to occlusion in arterial thrombosis, with 55% of PAR4-322P/L mice unable to develop stable thrombi within 30 minutes. In venous thrombosis, wild-type mice developed clotted IVCs that averaged 31 mg after 48 hours, while global PAR4-knockout mice averaged 21 mg (p<0.01), PAR4-322P/L 23 mg (p<0.01), and PAR4-322L/L 28 mg. Since platelet procoagulant activity is known to drive venous thrombosis, we used lactadherin to show that PAR4-P322L platelets express less phosphatidylserine in response to thrombin (5 nM) and convulxin (1 nM). Platelet-rich plasma from PAR4-P322L mice also generated less thrombin compared to wild-types, and whole blood from mice lacking PAR4 showed delayed clot formation in rotational thromboelastometry. Altogether, this suggests a direct impact of PAR4 signaling on platelet-mediated thrombin generation. Our new mouse model shows that the polymorphism in ECL3, PAR4-322L, decreases platelet reactivity and thrombosis. We also show PAR4-322L leads to less procoagulant platelets and decreases the endogenous thrombin potential. This decreased ability to generate thrombin offers a mechanism for PAR4's role in VTE. Our findings highlight a key role of PAR4 signaling in platelet activation and procoagulant activity in thrombosis.

Circulating Platelets in Thrombotic Antiphospholipid Syndrome Display High Procoagulant Activity and Surface Complement Deposition: Correlation with Thrombotic Risk

Background Antiphospholipid syndrome (APS) is characterized by circulating antiphospholipid antibodies (aPL) accompanied by arterial and/or venous thrombosis, and/or recurrent pregnancy loss. It commonly affects young women in whom it dramatically increases their risk of myocardial infarction, ischemic stroke, deep vein thrombosis and pulmonary embolism. Although the thrombotic risk associated with aPL is established, the underlying mechanisms are still incompletely defined and there are no definitive predictive biomarkers. As a result the management of APS involves indefinite anticoagulation. Increased activation of vascular cells, including platelets is considered to underlie the pathogenesis of APS, and may be mediated in some cases by complement. There is a considerable crosstalk between platelets and complement at various levels during thrombosis. First, activated complement proteins stimulate platelets through their cognate receptors on the platelet surface, and genetic deficiency of complement proteins in mice leads to decreased platelet responsiveness. Second, platelets secrete complement proteins into the medium upon activation. Third, agonist-stimulated platelets contribute to activation of both the classical and alternative pathways of the complement cascade. Hence, we sought to investigate the association between the complement-platelet axis and the prothrombotic pathology observed in APS. Methods Venous blood was drawn from patients with APS and healthy controls, and washed human platelets were prepared by differential centrifugation. Platelets either remained unstimulated or were treated with thrombin (0.1, 0.25, 0.5 U/ml) for 5 min and then labelled with FITC-PAC1, PE-anti-CD62P antibody, Alexa Fluor 488-anti-C4d conjugate, PE-Annexin V, Mitotracker Red, MitoSOX Red, Fluo4-AM, Rhod2-AM, Cell Event Green Caspase 3/7 detection reagent and FITC-IETD-FMK for 30 min in the dark, and then analyzed by flow cytometry to measure integrin activation, P-selectin expression (α-granule secretion), complement C4d binding, phosphatidylserine (PS) exposure, mitochondrial membrane potential, mitochondrial ROS, cytosolic calcium, mitochondrial calcium, caspase 3/7 activity (apoptosis) and caspase 8 activity (extrinsic apoptosis pathway), respectively. Platelet markers were correlated with complement binding and clinical characteristics of APS patients including number of thrombotic events and circulating levels of aPL. Results Thrombin-induced platelet integrin activation and P-selectin expression were identical in APS patients and healthy controls. However, platelets isolated from APS patients had significantly higher procoagulant activity (PS exposure) as well as complement C4d binding in both unstimulated and thrombin-stimulated states compared to healthy controls. There was a strong positive correlation between platelet PS exposure and C4d binding in APS patients, but not healthy individuals, suggesting a role for complement activation in formation of procoagulant platelets in APS. A subset of APS patients with high platelet procoagulant activity (PS exposure > mean + 2SD observed in healthy individuals) had lower mitochondrial membrane potential and increased mitochondrial calcium transients compared to healthy individuals, suggesting mitochondrial permeability transition pore (mPTP)-driven PS exposure. Another subset of APS patients with PS exposing platelets showed caspase activation indicating apoptosis. Platelet PS exposure and C4d binding correlated strongly with the number of thrombotic events as well as levels of circulating aPL, which identifies complement-induced procoagulant platelets as predictors and possible underlying mechanisms for thrombosis in APS. Conclusion Our preliminary studies suggest that PS-exposing platelets are generated in APS by multiple mechanisms including agonist-induced mPTP formation and apoptosis-associated caspase activation. Platelet PS exposure in APS is positively correlated with C4d binding, suggesting a role for complement activation in driving platelet procoagulant activity. Complement activation and PS exposure on platelets are strong predictors of thrombotic risk in APS and could serve as promising targets for novel antithrombotic agents in APS management.

Crosstalk of platelets with macrophages and fibroblasts aggravates inflammation, aortic wall stiffening, and osteopontin release in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a highly lethal disease with progressive dilatation of the abdominal aorta accompanied by degradation and remodelling of the vessel wall due to chronic inflammation. Platelets play an important role in cardiovascular diseases, but their role in AAA is poorly understood. The present study revealed that platelets play a crucial role in promoting AAA through modulation of inflammation and degradation of the extracellular matrix (ECM). They are responsible for the up-regulation of SPP1 (osteopontin, OPN) gene expression in macrophages and aortic tissue, which triggers inflammation and remodelling and also platelet adhesion and migration into the abdominal aortic wall and the intraluminal thrombus (ILT). Further, enhanced platelet activation and pro-coagulant activity result in elevated gene expression of various cytokines, Mmp9 and Col1a1 in macrophages and Il-6 and Mmp9 in fibroblasts. Enhanced platelet activation and pro-coagulant activity were also detected in AAA patients. Further, we detected platelets and OPN in the vessel wall and in the ILT of patients who underwent open repair of AAA. Platelet depletion in experimental murine AAA reduced inflammation and ECM remodelling, with reduced elastin fragmentation and aortic diameter expansion. Of note, OPN co-localized with platelets, suggesting a potential role of OPN for the recruitment of platelets into the ILT and the aortic wall. In conclusion, our data strongly support the potential relevance of anti-platelet therapy to reduce AAA progression and rupture in AAA patients.

PB0888 The Effect of Polyphenols on Platelet Procoagulant Activity and Fibrin Clot Structure

PB0888 The Effect of Polyphenols on Platelet Procoagulant Activity and Fibrin Clot Structure

PB0872 MicroRNA-223-3p is a Regulator of Platelet Procoagulant Activity

PB0872 MicroRNA-223-3p is a Regulator of Platelet Procoagulant Activity

PB1056 In situ Pulmonary Thrombosis is Regulated by Integrin αIIbβ3-Dependent Platelet Procoagulant Activity

PB1056 In situ Pulmonary Thrombosis is Regulated by Integrin αIIbβ3-Dependent Platelet Procoagulant Activity

Crucial roles of red blood cells and platelets in whole blood thrombin generation

AbstractRed blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI–induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.

Effect of antiplatelet agents and tyrosine kinase inhibitors on oxLDL-mediated procoagulant platelet activity.

Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity exvivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Exvivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.

Small-Molecule Cyclophilin Inhibitors Potently Reduce Platelet Procoagulant Activity.

Procoagulant platelets are associated with an increased risk for thrombosis. Procoagulant platelet formation is mediated via Cyclophilin D (CypD) mediated opening of the mitochondrial permeability transition pore. Inhibiting CypD activity could therefore be an interesting approach to limiting thrombosis. In this study, we investigated the potential of two novel, non-immunosuppressive, non-peptidic small-molecule cyclophilin inhibitors (SMCypIs) to limit thrombosis in vitro, in comparison with the cyclophilin inhibitor and immunosuppressant Cyclosporin A (CsA). Both cyclophilin inhibitors significantly decreased procoagulant platelet formation upon dual-agonist stimulation, shown by a decreased phosphatidylserine (PS) exposure, as well as a reduction in the loss of mitochondrial membrane potential. Furthermore, the SMCypIs potently reduced procoagulant platelet-dependent clotting time, as well as fibrin formation under flow, comparable to CsA. No effect was observed on agonist-induced platelet activation measured by P-selectin expression, as well as CypA-mediated integrin αIIbβ3 activation. Importantly, whereas CsA increased Adenosine 5'-diphosphate (ADP)-induced platelet aggregation, this was unaffected in the presence of the SMCypIs. We here demonstrate specific cyclophilin inhibition does not affect normal platelet function, while a clear reduction in procoagulant platelets is observed. Reducing platelet procoagulant activity by inhibiting cyclophilins with SMCypIs forms a promising strategy to limit thrombosis.

Platelet pannexin-1 channels modulate neutrophil activation and migration but not the progression of abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is a common disease and highly lethal if untreated. The progressive dilatation of the abdominal aorta is accompanied by degradation and remodeling of the vessel wall due to chronic inflammation. Pannexins represent anion-selective channels and play a crucial role in non-vesicular ATP release to amplify paracrine signaling in cells. Thus, pannexins are involved in many (patho-) physiological processes. Recently, Panx1 channels were identified to be significantly involved in abdominal aortic aneurysm formation through endothelial derived Panx1 regulated inflammation and aortic remodeling. In platelets, Panx1 becomes activated following activation of glycoprotein (GP) VI. Since platelets play a role in cardiovascular diseases including abdominal aortic aneurysm, we analyzed the contribution of platelet Panx1 in the progression of abdominal aortic aneurysm. We detected enhanced Panx1 plasma levels in abdominal aortic aneurysm patients. In experimental abdominal aortic aneurysm using the pancreatic porcine elastase (PPE) mouse model, a major contribution of platelet Panx1 channels in platelet activation, pro-coagulant activity of platelets and platelet-mediated inflammation has been detected. In detail, platelets are important for the migration of neutrophils into the aortic wall induced by direct cell interaction and by activation of endothelial cells. Decreased platelet activation and inflammation did not affect ECM remodeling or wall thickness in platelet-specific Panx1 knock-out mice following PPE surgery. Thus, aortic diameter expansion at different time points after elastase infusion of the aortic wall was unaltered in platelet-specific Panx1 deficient mice suggesting that the modulation of inflammation alone does not affect abdominal aortic aneurysm formation and progression. In conclusion, our data strongly supports the role of platelets in inflammatory responses in abdominal aortic aneurysm via Panx1 channels and adds important knowledge about the significance of platelets in abdominal aortic aneurysm pathology important for the establishment of an anti-platelet therapy for abdominal aortic aneurysm patients.

SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet procoagulant activity.

Thrombosis of the lung microvasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis that is endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting Spike-induced TMEM16 activation. As TMEM16F plays an important role in stimulating the procoagulant activity of platelets, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. Here we show that Spike, present either on the virion envelope or on the cell plasma membrane, promotes platelet activation, adhesion and spreading. Spike was active as a sole agonist or, even more effectively, by enhancing the function of known platelet activators. In particular, Spike-induced a marked procoagulant phenotype in platelets, by enhancing Ca2+ flux, phosphatidylserine externalization on the platelet outer cell membrane, and thrombin generation. Eventually, this increased thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine blocked this Spike-induced procoagulant response. These findings provide a pathogenic mechanism to explain lung thrombosis-associated with severe COVID-19 infection. We propose that Spike, present in SARS-CoV-2 virions or exposed on the surface of infected cells in the lungs, enhances the effects of inflammation and leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing Niclosamide for COVID-19 therapy.

Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure

BackgroundThe glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown. ObjectivesTo determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure. MethodsProcoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide. ResultsGPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired. ConclusionOur findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbβ3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.

The Btk inhibitor AB‐95‐LH34 potently inhibits atherosclerotic plaque–induced thrombus formation and platelet procoagulant activity

BackgroundNew antithrombotic therapies with less effect on bleeding are needed for coronary artery disease. The Btk inhibitor ibrutinib blocks atherosclerotic plaque–mediated thrombus formation. However, it is associated with increased bleeding, possibly due to non–Btk‐mediated effects. Btk‐deficient patients do not have bleeding issues, suggesting selective Btk inhibition as a promising antithrombotic strategy. ObjectivesTo compare the antithrombotic effects of the highly selective Btk inhibitor AB‐95‐LH34 (LH34) with ibrutinib. MethodsGlycoprotein VI and G‐protein coupled receptor‐mediated platelet function and signaling were analyzed in healthy human donor platelets by lumi‐aggregometry, flow adhesion, and western blot following 1 h treatment with inhibitors in vitro. ResultsLH34 showed similar inhibition of Btk‐Y223 phosphorylation as ibrutinib, but had no off‐target inhibition of Src‐Y418 phosphorylation. Similar dose‐dependent inhibition of aggregation to atherosclerotic plaque material was observed for both. However, in response to Horm collagen, which also binds integrin α2β1, LH34 exhibited less marked inhibition than ibrutinib. Both LH34 and ibrutinib inhibited platelet adhesion and aggregation to plaque material at arterial shear. Ibrutinib demonstrated the most potent effect, with complete blockade at high concentrations. Platelet activation (P‐selectin) and procoagulant activity (phosphatidylserine exposure) in thrombi were inhibited by LH34 and completely blocked by ibrutinib at high concentrations. Furthermore, plaque‐induced thrombin generation was reduced by higher concentrations of LH34 and ibrutinib. ConclusionsLH34 potently inhibits atherosclerotic plaque–induced thrombus formation and procoagulant platelet activity in vitro, with less off‐target inhibition of Src than ibrutinib, suggesting it is a promising antiplatelet therapy with the potential for reduced bleeding side effects.

Pathophysiological mechanisms of thrombosis in acute and long COVID-19.

COVID-19 patients have a high incidence of thrombosis, and thromboembolic complications are associated with severe COVID-19 and high mortality. COVID-19 disease is associated with a hyper-inflammatory response (cytokine storm) mediated by the immune system. However, the role of the inflammatory response in thrombosis remains incompletely understood. In this review, we investigate the crosstalk between inflammation and thrombosis in the context of COVID-19, focusing on the contributions of inflammation to the pathogenesis of thrombosis, and propose combined use of anti-inflammatory and anticoagulant therapeutics. Under inflammatory conditions, the interactions between neutrophils and platelets, platelet activation, monocyte tissue factor expression, microparticle release, and phosphatidylserine (PS) externalization as well as complement activation are collectively involved in immune-thrombosis. Inflammation results in the activation and apoptosis of blood cells, leading to microparticle release and PS externalization on blood cells and microparticles, which significantly enhances the catalytic efficiency of the tenase and prothrombinase complexes, and promotes thrombin-mediated fibrin generation and local blood clot formation. Given the risk of thrombosis in the COVID-19, the importance of antithrombotic therapies has been generally recognized, but certain deficiencies and treatment gaps in remain. Antiplatelet drugs are not in combination with anticoagulant treatments, thus fail to dampen platelet procoagulant activity. Current treatments also do not propose an optimal time for anticoagulation. The efficacy of anticoagulant treatments depends on the time of therapy initiation. The best time for antithrombotic therapy is as early as possible after diagnosis, ideally in the early stage of the disease. We also elaborate on the possible mechanisms of long COVID thromboembolic complications, including persistent inflammation, endothelial injury and dysfunction, and coagulation abnormalities. The above-mentioned contents provide therapeutic strategies for COVID-19 patients and further improve patient outcomes.

Novel Snake Venom Derived Hemocoagulase Reverses Anticoagulant Effect of Factor Xa Inhibitors, Restores Hemostatic Clot Formation, and Limits Bleeding in Vitro and In Vivo

Background: Direct oral anticoagulants (DOACs) such as rivaroxaban and apixaban are factor Xa (FXa) inhibitors widely used to treat thromboembolism and prevent stroke. Despite improvements in patient outcomes compared to vitamin K antagonists, major bleeding still occurs and there is an urgent unmet need for reversal agents in the event of major bleeding or when emergency surgery is needed. Slounase is a snake venom derived batroxobin (a thrombin-like enzyme) containing FXa that converts fibrinogen to fibrin in a manner distinctly different from thrombin. We recently reported slounase enhances platelet-fibrin clot formation in vivo in heparin-anticoagulated mice suggesting slounase may bypass coagulation to restore hemostasis and prevent bleeding. Here, we investigate if slounase reverses the anticoagulant effect of FXa inhibitors and restores hemostatic clot formation as a novel bypassing agent. Methods: In this study we used in vitro thromboelastography (TEG) to test the effect of slounase on whole blood from healthy human donors spiked with a range of apixaban or rivaroxaban doses. Clot formation kinetics were recorded for 60 minutes and the parameters reaction time and maximum amplitude (MA) were analyzed. The effect of rivaroxaban and apixaban on hemostasis in vivo was determined in wild type (WT) mice pretreated with apixaban (30mg/kg) or rivaroxaban (10mg/kg) via oral gavage using intravital microscopy laser-induced cremaster arteriole thrombosis model. WT mice pretreated with apixaban or rivaroxaban were further treated with slounase (1U/kg) and reversal of anticoagulant effect of FXa inhibition was determined in vivo under intravital microscopy. The dynamics of platelet recruitment and fibrin formation within the growing clot were analyzed for change in fluorescent intensity over the course of thrombus formation in response to vascular injury. Additionally, mice were pretreated with FXa inhibitors via oral gavage and then intravenously dosed slounase (1U/kg) 10 minutes prior to tail bleeding assay to test the effect on bleeding time and blood loss. Results: A high dose of apixaban and rivaroxaban spiked in human whole blood caused full inhibition of the TEG parameters and an inability to form a stable thrombus in vitro. However, slounase treatment dose dependently restored the parameters of clot formation into the range of control. The in vivo laser-induced cremaster arteriole model was used to test slounase as an intervention to apixaban and rivaroxaban pretreatment. The severe hemostatic defect was determined by limited platelet accumulation and fibrin formation at the site of vascular injury in both groups. Additionally, platelet adherence was transient and fibrin formation was unstable in mice treated with rivaroxaban and apixaban. Intravenous intervention with slounase (1U/kg) increased platelet accumulation and fibrin formation at the site of vascular injury. Slounase treatment reversed the anticoagulant effect of FXa inhibitors in vivo and restored the hemostatic clot formation in response to vascular injury in mice treated with FXa inhibitors. In the in vivo tail bleeding assay WT mice pretreated with FXa inhibitors bled continually until intervention via cauterization. Slounase pretreatment significantly decreased blood loss and bleeding time into the range of WT controls. Conclusions: Slounase is a potential bypassing agent for reversal of anticoagulation by DOACs rivaroxaban and apixaban. Using TEG, our data demonstrated the anticoagulant effect of FXa inhibitors in vitro and the reversal of anticoagulation by slounase. Slounase dose dependently restored clot formation in human whole blood pretreated with an inhibitory dose of FXa inhibitors. Slounase intervention improved hemostasis in vivo by increasing platelet accumulation and fibrin formation at the site of vascular injury. Additionally, slounase treatment decreased bleeding and blood loss in an in vivo bleeding assay demonstrating its potential to quickly reverse effects of DOACs and prevent bleeding episodes. This study further demonstrates slounase is a novel bypassing agent that promotes platelet procoagulant activity and converts fibrinogen to fibrin independent of thrombin activity.

Effects of platelets activated by different agonists on fibrin formation and thrombin generation

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.

Extracellular Mitochondria Activate Microglia and Contribute to Neuroinflammation in Traumatic Brain Injury.

Traumatic brain injury (TBI)-induced neuroinflammation is closely associated with poor outcomes and high mortality in affected patients, with unmet needs for effective clinical interventions. A series of causal and disseminating factors have been identified to cause TBI-induced neuroinflammation. Among these are cellular microvesicles released from injured cerebral cells, endothelial cells, and platelets. In previous studies, we have put forward that cellular microvesicles can be released from injured brains that induce consumptive coagulopathy. Extracellular mitochondria accounted for 55.2% of these microvesicles and induced a redox-dependent platelet procoagulant activity that contributes to traumatic brain injury-induced coagulopathy and inflammation. These lead to the hypothesis that metabolically active extracellular mitochondria contribute to the neuroinflammation in traumatic brain injury, independent of their procoagulant activity. Here, we found that these extracellular mitochondria induced polarization of microglial M1-type pro-inflammatory phenotype, aggravating neuroinflammation, and mediated cerebral edema in a ROS-dependent manner. In addition, the effect of ROS can be alleviated by ROS inhibitor N-ethylmaleimide (NEM) in vitro experiments. These results revealed a novel pro-inflammatory activity of extracellular mitochondria that may contribute to traumatic brain injury-associated neuroinflammation.

CD44–fibrinogen binding promotes bleeding in acute promyelocytic leukemia by in situ fibrin(ogen) deposition

AbstractEarly hemorrhagic death is still the main obstacle for the successful treatment of acute promyelocytic leukemia (APL). However, the mechanisms underlying hemostatic perturbations in APL have not been fully elucidated. Here, we report that CD44 on the membrane of APL blasts and NB4 cells ligated bound fibrinogen, resulting in in situ deposition of fibrin and abnormal fibrin distribution. Clots formed by leukemic cells in response to CD44 and fibrinogen interaction exhibited low permeability and resistance to fibrinolysis. Using flow cytometry and confocal microscopy, we found that CD44 was also involved in platelet and leukemic cell adhesion. CD44 bound activated platelets but not resting platelets through interaction with P-selectin. APL cell-coated fibrinogen-activated platelets directly induce enhanced procoagulant activity of platelets. In vivo studies revealed that CD44 knockdown shortened bleeding time, increased the level of fibrinogen, and elevated the number of platelets by approximately twofold in an APL mouse model. Moreover, CD44 expression on leukemic cells in an APL mouse model was not only associated with bleeding complications but was also related to the wound-healing process and the survival time of APL mice. Collectively, our results suggest that CD44 may be a potential intervention target for preventing bleeding complications in APL.