Shakhnoza M. Saliakhutdinova

Background: Blood clot contraction/retraction depends on the force-generating actomyosin and on the platelet integrin αIIbβ3, which transmits intracellular forces to fibrin. Before clotting, fibrinogen binds to activated integrin αIIbβ3, mediating platelet aggregation. The relationship between platelet aggregation and subsequent platelet-driven clot contraction remains unclear. Methods: We investigated the effects of platelet aggregation on clot contraction by selectively blocking the αIIbβ3-fibrinogen binding using the RGDW peptide. The ability of RGDW to disrupt αIIbβ3-fibrinogen binding was assessed by platelet aggregometry. The time-course of clot contraction was monitored optically in whole blood or platelet-rich plasma and modeled mathematically. Clot stiffness was assessed using Thromboelastography. The effect of the RGDW peptide on the structure of PRP-clots was examined using scanning electron microscopy. Results: The RGDW peptide dose-dependently inhibited TRAP-induced platelet aggregation. Both in whole blood and in plasma, the peptide dose-dependently prolonged the lag-period and slowed the rate without affecting the final extent of contraction. Thromboelastography showed that RGDW dose-dependently increased maximum clot stiffness in blood. Scanning electron microscopy revealed that RGDW treatment resulted in formation of smaller fibrin agglomerates surrounding non-aggregated platelets. A theoretical model allowed us to decipher mechanisms underlying the kinetic effects of RGDW. Conclusion: Blocking the binding of integrin αIIbβ3 to fibrinogen and preventing platelet aggregation delays and slows subsequent clot contraction without affecting the final degree of shrinkage. These findings indicate a modulatory role of fibrinogen-mediated platelet aggregation in clot contraction and highlight the unforeseen effects of selective inhibitors of platelet aggregation on the contraction of blood clots and thrombi.

Introduction. Immunothrombosis significantly exacerbates the course and outcomes of inflammatory diseases. Immune thrombi contain leukocytes, primarily neutrophils, that modulate their properties such as resistance to lysis, mechanical stability (embologenicity), and others. We have previously shown that activated neutrophils enhance clot contraction (retraction); however, the pathways and pathogenetic consequences of the influence of inflammatory cells on clot retraction remain unknown. Aim: to establish the mechanisms underlying the stimulatory effect of activated neutrophils on the rate and extent of blood clot retraction mediated by platelet contraction. Materials and methods. Isolated human neutrophils were activated with phorbol 12-myristate 13-acetate (PMA). Thrombin-induced clots were formed in whole blood or platelet-rich plasma with the addition of non-activated or PMA-activated neutrophils forming neutrophil extracellular traps (NETs). Clot retraction kinetics was tracked optically, and the viscoelastic properties of clots were measured using thromboelastography. Results. PMA-activated neutrophils significantly increased the rate and the final extent of clot retraction. These effects were associated with the presence of NETs detected in the clots and their degradation which eliminated the stimulatory effect of neutrophils. The experimentally identified mechanisms of how NETs influence blood clot retraction included the hyperstimulation of platelets by endogenous thrombin and/or alterations in the mechanical properties of clots. Specifically, rivaroxaban, a factor Xa inhibitor, neutralized the stimulatory effect of NETs on clot and plasma retraction// on clot retraction both in whole blood and platelet-reach plasma, indicating the important role of active thrombin generation and secondary platelet hyperactivation in the process of retraction. Furthermore, according to the results of thromboelastography, in the presence of NETs clots were less stiff and, consequently, more susceptible to compressive deformation. Conclusion. PMA-activated neutrophils within blood clots form NETs that incorporate into a fibrin network and enhance clot retraction via two independent mechanisms: the generation of endogenous thrombin causing platelet hyperactivation, and an increase in clot susceptibility to mechanical compression. Understanding these mechanisms may facilitate the development of novel treatments for immunothrombosis based on modulation of NETs formation, as well as on their biological and mechanical properties that influence the retraction of inflammatory blood clots and thrombi.

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.

BACKGROUND: Immunothrombosis, or inflammatory thrombosis, largely determines the course and outcomes of severe infectious and autoimmune diseases. Inflammatory thrombi are rich in neutrophils; however, how inflammatory cells affect the contraction (retraction) of a blood clot, an important process that influences the course and outcomes of hemostatic disorders, remains unknown. AIM: This study aimed to assess the effect of activated neutrophils and neutrophil extracellular traps on the rate and degree of blood clot retraction mediated by activated platelet contraction. METHODS: Isolated human neutrophils were activated with phorbol 12-myristate 13-acetate to induce the formation of neutrophil extracellular traps, which were visualized using immunofluorescence (citrullinated histone H3 and DNA) and scanning electron microscopy. Thrombin-induced clots were formed for three sample groups from whole blood (n = 5 for each group) or platelet-rich plasma (n = 4 or n = 5 for each group): (1) without added neutrophils (control), (2) with nonactivated neutrophils, and (3) with activated neutrophils. Clot-retraction kinetics were recorded optically. The final degree of retraction was the difference between the initial and final clot size, expressed as a percentage of the initial value. Statistical analysis was performed using the Student’s t-test, one-way analysis of variance with a Sidak post hoc test, and the Friedman test. The significance level was set at 95%. RESULTS: Phorbol 12-myristate 13-acetate induced the activation of neutrophils and formation of neutrophil extracellular traps, which merged into the fibrin structure. When added to whole blood or platelet-rich plasma, activated neutrophils significantly (p 0.05) increased the final degree of clot retraction (50% ± 5% in blood and 86% ± 3% in platelet-rich plasma) compared with nonactivated neutrophils (45% ± 3% and 80% ± 1%, respectively). The kinetic parameters of retraction demonstrated accelerated contraction in the presence of activated neutrophils (greater mean velocity in platelet-rich plasma: 0.070%/s ± 0.003%/s vs. 0.065%/s ± 0.001%/s; p = 0.03). The stimulatory effect of activated neutrophils on clot retraction disappeared following neutrophil extracellular trap degradation with deoxyribonuclease I, in whole blood (degree of retraction 44% ± 5% vs. 54% ± 2%; p = 0.02) and platelet-rich plasma (82% ± 3% vs. 87% ± 2%; p = 0.03), indicating a key role of neutrophil extracellular traps within clots. CONCLUSION: Activated neutrophils accelerate and enhance blood clot retraction. This effect is mediated by the formation of neutrophil extracellular traps that merge into the fibrin network.

Immunothrombosis can affect substantially 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 activated 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 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 into 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.

Background. COVID-19 is a predominantly respiratory disease caused by the SARS-CoV-2 virus that has spread as a pandemic since 2020; characterized by high mortality and unexplored long-term outcomes. Aim. To describe the morphological changes in the lungs and other organs in severe forms of COVID-19. Material and methods. A macro- and microscopic examination of the lungs, kidneys, liver, brain and heart of 45 patients who died from complications of COVID-19 was carried out. Histological preparations were stained with hematoxylin and eosin, by the Picro-Mallory stain, as well as with fluorescent antibodies and dyes to visualize neutrophil extracellular traps. Results. The leading pathomorphological manifestations of COVID-19 were inflammation and microthrombosis in the lungs. At the same time, diffuse alveolar damage often developed as a manifestation of acute respiratory distress syndrome in an adult. Diffuse leukocyte infiltration of the lungs was combined with the formation of neutrophil extracellular traps in the lumens of the blood vessels. Diffuse microthrombi were found in most lung tissue samples (87%), while macroscopic thrombi were much less common (16%). In single cases, the cause of death was thromboembolism of the branches of the pulmonary artery, originating from deep vein thrombi of the lower extremities or the right atrial appendage. In the tissues of the liver, heart, kidneys and brain, moderate alterative (degenerative and necrobiotic) microscopic changes were observed, as well as morphological signs of concomitant chronic diseases. Conclusion. COVID-19 is characterized by pathogenically interrelated morphological manifestations of inflammation and primary pulmonary (micro)thrombosis in the form of accumulation of leukocytes both in blood clots and in the alveoli, while regional pulmonary immunothrombosis is associated with diffuse damage to the alveoli; in the heart, brain, liver and kidneys, predominantly dystrophic and necrobiotic changes occur, which are typical of secondary injuries and/or concomitant pathology.