Autoimmune diseases, including systemic lupus erythematosus (SLE), have a high risk of thrombotic and hemorrhagic complications associated with altered platelet functionality. We studied platelets from the blood of SLE patients and their reactivity. The surface expression of phosphatidylserine, P-selectin, and active integrin αIIbβ3 were measured using flow cytometry before and after platelet stimulation. Soluble P-selectin was measured in plasma. The kinetics of platelet-driven clot contraction was studied, as well as scanning and transmission electron microscopy of unstimulated platelets. Elevated levels of membrane-associated phosphatidylserine and platelet-attached and soluble P-selectin correlated directly with the titers of IgG, anti-dsDNA-antibodies, and circulating immune complexes. Morphologically, platelets in SLE lost their resting discoid shape, formed membrane protrusions and aggregates, and had a rough plasma membrane. The signs of platelet activation were associated paradoxically with reduced reactivity to a physiological stimulus and impaired contractility that revealed platelet exhaustion and refractoriness. Platelet activation has multiple pro-coagulant effects, and the inability to fully contract (retract) blood clots can be either a hemorrhagic or pro-thrombotic mechanism related to altered clot permeability, sensitivity of clots to fibrinolysis, obstructiveness, and embologenicity. Therefore, chronic immune platelet activation followed by secondary platelet dysfunction comprise an understudied pathogenic mechanism that supports hemostatic disorders in autoimmune diseases, such as SLE.

Immunothrombosis can substantially affect the course and outcomes of severe infections and immune-mediated diseases. While inflammatory thrombi are neutrophil-rich, impact of neutrophils on clot contraction, a key modulator of thrombus stability and obstructiveness, was unknown. This study investigated how neutrophils and neutrophil extracellular traps (NETs) affect the rate and extent of platelet-driven clot contraction. Isolated human neutrophils were stimulated with phorbol-12-myristate-13-acetate (PMA) to induce NETosis, confirmed by fluorescence microscopy and scanning electron microscopy. Thrombin-induced clots, formed from whole blood or platelet-rich plasma, were supplemented with non-activated or PMA-activated neutrophils. Clot contraction kinetics and viscoelasticity were analyzed. PMA-activated neutrophils significantly enhanced the rate and final extent of clot contraction compared to controls. This promoting effect was abolished by deoxyribonuclease (DNAse) I, confirming that it was mediated by NETs embedded in the fibrin network. The factor Xa inhibitor rivaroxaban also abrogated this effect, indicating a role for NET-induced endogenous thrombin generation and platelet hyperactivation. Thromboelastography revealed that NETs made clots softer and more deformable. We conclude that activated neutrophils promote clot contraction via NETs embedded in the fibrin network, which enhance platelet contractility via endogenous thrombin production and increase clot deformability, suggesting that inflammatory thrombosis may require treatments addressing this enhanced contractility.

The spiny mouse (Acomys species) is capable of scarless wound regeneration through largely yet unknown mechanisms. To investigate whether this capacity is related to peculiarities of the hemostatic system, we studied the blood of Acomys cahirinus in comparison to Mus musculus (Balb/c) to reveal differences in blood composition and clotting in both males and females. In response to surgical manipulations, blood clots formed in wounds of Acomys comprised a stronger hemostatic seal with reduced surgical bleeding in comparison with Balb/c. Acomys demonstrated notably shorter tail bleeding times and elevated clottable fibrinogen levels. Histological analysis revealed that clots from Acomys blood had densely packed fibrin-rich clots with pronounced fibrin segregation from erythrocytes. Acomys exhibited superior plasma clot stiffness as revealed with thromboelastography. The latter two characteristics are likely due to hyperfibrinogenemia. Light transmission platelet aggregometry demonstrated that ADP-induced platelet aggregates in Acomys males are stable, unlike the aggregates formed in the plasma of Balb/c undergoing progressive disaggregation over time. There were no apparent distinctions in platelet contractility and baseline expression of phosphatidylserine. Hematological profiling revealed a reduced erythrocytes count but increased mean corpuscular volume and hemoglobin content in Acomys. These results demonstrate the distinctive hemostatic potential of Acomys cahirinus, which may contribute to their remarkable regenerative capacity.

Aim. To study the relationship of hemostatic disorders with inflammation and estimate their role in the course and outcomes of COVID-19. Materials and methods. We examined 215 consecutive patients with moderate and severe forms of acute COVID-19. The patients were on anticoagulants and immunosuppressive drugs. Hemostasis was assessed using the thrombodynamics assay, thromboelastography, fibrinogen and D-dimer levels, prothrombin time, and soluble fibrin-monomer complexes (ethanol gelation test). The hemostatic parameters were correlated with hematological and biochemical tests, including markers of inflammation (C-reactive protein, interleukins 6 and 8), as well as with the disease severity and outcomes. Results. Laboratory signs of coagulopathy were revealed in the vast majority of the cases. Despite the use of low-molecular-weight heparins in the prophylactic and therapeutic doses, coagulopathy in COVID-19 manifested predominantly as hypercoagulability that correlated directly with the systemic inflammation and metabolic changes due to liver and kidney dysfunction. A direct relationship was found between the grade of coagulopathy and the severity of COVID-19, including comorbidities and the mortality. The chronometric hypocoagulability observed in about 1/4 cases was associated with a high level of C-reactive protein, which may decelerate coagulation in vitro and thereby mask the true inflammatory thrombophilia. Persistent hyperfibrinogenemia and high D-dimer in the absence of consumption coagulopathy suggest the predominance of local and/or regional microthrombosis over disseminated intravascular coagulation. Conclusion. The results obtained substantiate the need for laboratory monitoring of hemostasis and active prophylaxis and treatment of thrombotic complications in COVID-19.