Objective: Coronavirus disease 2019 (COVID-19) is associated with derangement in biomarkers of coagulation and endothelial function and has been likened to the coagulopathy of sepsis. However, clinical laboratory metrics suggest key differences in these pathologies. We sought to determine whether plasma coagulation and fibrinolytic potential in patients with COVID-19 differ compared with healthy donors and critically ill patients with sepsis. Approach and Results: We performed comparative studies on plasmas from a single-center, cross-sectional observational study of 99 hospitalized patients (46 with COVID-19 and 53 with sepsis) and 18 healthy donors. We measured biomarkers of endogenous coagulation and fibrinolytic activity by immunoassays, thrombin, and plasmin generation potential by fluorescence and fibrin formation and lysis by turbidity. Compared with healthy donors, patients with COVID-19 or sepsis both had elevated fibrinogen, d -dimer , soluble TM (thrombomodulin), and plasmin-antiplasmin complexes. Patients with COVID-19 had increased thrombin generation potential despite prophylactic anticoagulation, whereas patients with sepsis did not. Plasma from patients with COVID-19 also had increased endogenous plasmin potential, whereas patients with sepsis showed delayed plasmin generation. The collective perturbations in plasma thrombin and plasmin generation permitted enhanced fibrin formation in both COVID-19 and sepsis. Unexpectedly, the lag times to thrombin, plasmin, and fibrin formation were prolonged with increased disease severity in COVID-19, suggesting a loss of coagulation-initiating mechanisms accompanies severe COVID-19. Conclusions: Both COVID-19 and sepsis are associated with endogenous activation of coagulation and fibrinolysis, but these diseases differently impact plasma procoagulant and fibrinolytic potential. Dysregulation of procoagulant and fibrinolytic pathways may uniquely contribute to the pathophysiology of COVID-19 and sepsis.

Red blood cells (RBCs) demonstrate procoagulant properties in vitro, and elevated hematocrit is associated with reduced bleeding and increased thrombosis risk in humans. These observations suggest RBCs contribute to thrombus formation. However, effects of RBCs on thrombosis are difficult to assess because humans and mice with elevated hematocrit typically have coexisting pathologies. Using an experimental model of elevated hematocrit in healthy mice, we measured effects of hematocrit in 2 in vivo clot formation models. We also assessed thrombin generation, platelet-thrombus interactions, and platelet accumulation in thrombi ex vivo, in vitro and in silico. Compared with controls, mice with elevated hematocrit (RBC) formed thrombi at a faster rate and had a shortened vessel occlusion time. Thrombi in control and RBC mice did not differ in size or fibrin content, and there was no difference in levels of circulating thrombin-antithrombin complexes. In vitro, increasing the hematocrit increased thrombin generation in the absence of platelets; however, this effect was reduced in the presence of platelets. In silico, direct numerical simulations of whole blood predicted elevated hematocrit increases the frequency and duration of interactions between platelets and a thrombus. When human whole blood was perfused over collagen at arterial shear rates, elevating the hematocrit increased the rate of platelet deposition and thrombus growth. These data suggest RBCs promote arterial thrombosis by enhancing platelet accumulation at the site of vessel injury. Maintaining a normal hematocrit may reduce arterial thrombosis risk in humans.

The transglutaminase factor XIII (FXIII) stabilizes clots against mechanical and biochemical disruption and is essential for hemostasis. In vitro and in vivo models of venous thrombosis demonstrate that FXIII mediates clot size by promoting red blood cell (RBC) retention. However, the key source of FXIII and whether FXIII activity can be reduced to suppress thrombosis without imposing deleterious hemostatic consequences are 2 critical unresolved questions. FXIII is present in multiple compartments, including plasma (FXIII) as a heterotetramer of A and B subunits and platelets (FXIII) as an A homodimer. We determined the role of the FXIII compartment and level in clot contraction, composition, and size in vitro and using in vivo models of hemostasis and venous thrombosis. Reducing overall FXIII levels decreased whole blood clot weight but did not alter thrombin generation or contraction of platelet-rich plasma clots. In reconstituted platelet-rich plasma and whole blood clot contraction assays, FXIII, but not FXIII, produced high-molecular-weight fibrin crosslinks, promoted RBC retention, and increased clot weights. Genetically imposed reduction of FXIII delayed FXIII activation and fibrin crosslinking, suggesting FXIII levels mediate the kinetics of FXIII activation and activity and that the timing of these processes is a critical determinant of RBC retention during clot formation and contraction. A 50% reduction in FXIII produced significantly smaller venous thrombi but did not increase bleeding in tail transection or saphenous vein puncture models in vivo. Collectively, these findings suggest that partial FXIII reduction may be a therapeutic strategy for reducing venous thrombosis.

Tridegin is a potent and specific 66mer peptide inhibitor of coagulation factor XIIIa with six cysteines involved in three disulfide bonds. Three of the fifteen possible 3-disulfide-bonded isomers have been identified, which share a bridge between cysteines 19 and 25. We synthesized the three possible 2-disulfide-bonded analogs using a targeted protecting-group strategy to investigate the impact of the C 19 -C 25 bond on tridegin's folding, stability, and function. The FXIIIa inhibitory activity of the analogs was retained, which was shown by in vitro fluorogenic activity and whole blood clotting assays. Molecular dynamics simulations of wild type tridegin and the analogs as well as molecular docking studies with FXIIIa were performed to elucidate the impact of the C 19 -C 25 bond on conformational stability and binding mode. The strategy of

Obesity is a prevalent prothrombotic risk factor marked by enhanced fibrin formation and suppressed fibrinolysis. Fibrin both promotes thrombotic events and drives obesity pathophysiology, but a lack of essential analytical tools has left fibrinolytic mechanisms affected by obesity poorly defined. Using a plasmin-specific fluorogenic substrate, we developed a plasmin generation (PG) assay for mouse plasma that is sensitive to tissue plasminogen activator, α2-antiplasmin, active plasminogen activator inhibitor (PAI-1), and fibrin formation, but not fibrin crosslinking. Compared with plasmas from mice fed a control diet, plasmas from mice fed a high-fat diet (HFD) showed delayed PG and reduced PG velocity. Concurrent to impaired PG, HFD also enhanced thrombin generation (TG). The collective impact of abnormal TG and PG in HFD-fed mice produced normal fibrin formation kinetics but delayed fibrinolysis. Functional and proteomic analyses determined that delayed PG in HFD-fed mice was not due to altered levels of plasminogen, α2-antiplasmin, or fibrinogen. Changes in PG were also not explained by elevated PAI-1 because active PAI-1 concentrations required to inhibit the PG assay were 100-fold higher than circulating concentrations in mice. HFD-fed mice had increased circulating thrombomodulin, and inhibiting thrombomodulin or thrombin-activatable fibrinolysis inhibitor (TAFI) normalized PG, revealing a thrombomodulin- and TAFI-dependent antifibrinolytic mechanism. Integrating kinetic parameters to calculate the metric of TG/PG ratio revealed a quantifiable net shift toward a prothrombotic phenotype in HFD-fed mice. Integrating TG and PG measurements may define a prothrombotic risk factor in diet-induced obesity.