Accelerated Spatial Fibrin Growth and Impaired Contraction of Blood Clots in Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease associated with thrombotic complications. To elucidate pathogenic mechanisms, hemostatic disorders in RA were correlated with other laboratory and clinical manifestations. Hemostasis was assessed using relatively new complementary tests, the spatial growth of a plasma clot (Thrombodynamics assay), and contraction of whole blood clots. Platelet functionality was assessed with flow cytometry that quantified the expression of P-selectin and the fibrinogen-binding capacity of platelets before and after activation with a thrombin receptor-activating peptide. Parameters of fibrin clot growth and the kinetics of contraction of blood clots were significantly altered in patients with RA compared to the control group. In Thrombodynamics measurements, an increase in the clot growth rate, size, and optical density of plasma clots altogether indicated chronic hypercoagulability. The rate and extent of blood clot contraction in patients with RA was significantly reduced and associated with platelet dysfunction revealed by an impaired response to activation. Changes in the parameters of clot growth and contraction correlated with the laboratory signs of systemic inflammation, including hyperfibrinogenemia. These results confirm the pathogenic role of hemostatic disorders in RA and support the validity of fibrin clot growth and the blood clot contraction assay as indicators of a (pro)thrombotic state

Chronic Immune Platelet Activation Is Followed by Platelet Refractoriness and Impaired Contractility

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

Effects of Hyperhomocysteinemia on the Platelet-Driven Contraction of Blood Clots

Hyperhomocysteinemia (HHcy) is associated with thrombosis, but the mechanistic links between them are not understood. We studied effects of homocysteine (Hcy) on clot contraction in vitro and in a rat model of HHcy. Incubation of blood with exogenous Hcy for 1 min enhanced clot contraction, while 15-min incubation led to a dose-dependent suppression of contraction. These effects were likely due to direct Hcy-induced platelet activation followed by exhaustion, as revealed by an increase in fibrinogen-binding capacity and P-selectin expression determined by flow cytometry. In the blood of rats with HHcy, clot contraction was enhanced at moderately elevated Hcy levels (10–50 μM), while at higher Hcy levels (>50 μM), the onset of clot contraction was delayed. HHcy was associated with thrombocytosis combined with a reduced erythrocyte count and hypofibrinogenemia. These data suggest that in HHcy, platelets get activated directly and indirectly, leading to enhanced clot contraction that is facilitated by the reduced content and resilience of fibrin and erythrocytes in the clot. The excessive platelet activation can lead to exhaustion and impaired contractility, which makes clots larger and more obstructive. In conclusion, HHcy modulates blood clot contraction, which may comprise an underappreciated pro- or antithrombotic mechanism

Combined computational modeling and experimental study of the biomechanical mechanisms of platelet-driven contraction of fibrin clots

While blood clot formation has been relatively well studied, little is known about the mechanisms underlying the subsequent structural and mechanical clot remodeling called contraction or retraction. Impairment of the clot contraction process is associated with both life-threatening bleeding and thrombotic conditions, such as ischemic stroke, venous thromboembolism, and others. Recently, blood clot contraction was observed to be hindered in patients with COVID-19. A three-dimensional multiscale computational model is developed and used to quantify biomechanical mechanisms of the kinetics of clot contraction driven by platelet-fibrin pulling interactions. These results provide important biological insights into contraction of platelet filopodia, the mechanically active thin protrusions of the plasma membrane, described previously as performing mostly a sensory function. The biomechanical mechanisms and modeling approach described can potentially apply to studying other systems in which cells are embedded in a filamentous network and exert forces on the extracellular matrix modulated by the substrate stiffness

Combined computational modeling and experimental study of the biomechanical mechanisms of platelet-driven contraction of fibrin clots

Abstract While blood clot formation has been relatively well studied, little is known about the mechanisms underlying the subsequent structural and mechanical clot remodeling called contraction or retraction. Impairment of the clot contraction process is associated with both life-threatening bleeding and thrombotic conditions, such as ischemic stroke, venous thromboembolism, and others. Recently, blood clot contraction was observed to be hindered in patients with COVID-19. A three-dimensional multiscale computational model is developed and used to quantify biomechanical mechanisms of the kinetics of clot contraction driven by platelet-fibrin pulling interactions. These results provide important biological insights into contraction of platelet filopodia, the mechanically active thin protrusions of the plasma membrane, described previously as performing mostly a sensory function. The biomechanical mechanisms and modeling approach described can potentially apply to studying other systems in which cells are embedded in a filamentous network and exert forces on the extracellular matrix modulated by the substrate stiffness

Fatal dysfunction and disintegration of thrombin-stimulated platelets.

Platelets play a key role in the formation of hemostatic clots and obstructive thrombi as well as in other biological processes. In response to physiological stimulants, including thrombin, platelets change shape, express adhesive molecules, aggregate, and secrete bioactive substances, but their subsequent fate is largely unknown. Here we examined late-stage structural, metabolic, and functional consequences of thrombin-induced platelet activation. Using a combination of confocal microscopy, scanning and transmission electron microscopy, flow cytometry, biochemical and biomechanical measurements, we showed that thrombin-induced activation is followed by time-dependent platelet dysfunction and disintegration. After ~30 minutes of incubation with thrombin, unlike with collagen or ADP, human platelets disintegrated into cellular fragments containing organelles, such as mitochondria, glycogen granules, and vacuoles. This platelet fragmentation was preceded by Ca2+ influx, integrin αIIbβ3 activation and phosphatidylserine exposure (activation phase), followed by mitochondrial depolarization, generation of reactive oxygen species, metabolic ATP depletion and impairment of platelet contractility along with dramatic cytoskeletal rearrangements, concomitant with platelet disintegration (death phase). Coincidentally with the platelet fragmentation, thrombin caused calpain activation but not activation of caspases 3 and 7. Our findings indicate that the late functional and structural damage of thrombin-activated platelets comprise a calpain-dependent platelet death pathway that shares some similarities with the programmed death of nucleated cells, but is unique to platelets, therefore representing a special form of cellular destruction. Fragmentation of activated platelets suggests that there is an underappreciated pathway of enhanced elimination of platelets from the circulation in (pro)thrombotic conditions once these cells have performed their functions