Activation of Piezo1 channels in compressed red blood cells augments platelet-driven contraction of blood clots.

BACKGROUND: Piezo1 is a mechanosensitive cationic channel that boosts intracellular [Ca2+]i. Compression of red blood cells (RBCs) during platelet-driven contraction of blood clots may cause the activation of Piezo1. OBJECTIVES: To establish relationships between Piezo1 activity and blood clot contraction. METHODS: Effects of a Piezo1 agonist, Yoda1, and antagonist, GsMTx-4, on clot contraction in vitro were studied in human blood containing physiological [Ca2+]. Clot contraction was induced by exogenous thrombin. Activation of Piezo1 was assessed by Ca2+ influx in RBCs and with other functional and morphologic features. RESULTS: Piezo1 channels in compressed RBCs are activated naturally during blood clot contraction and induce an upsurge in the intracellular [Ca2+]i, followed by phosphatidylserine exposure. Adding the Piezo1 agonist Yoda1 to whole blood increased the extent of clot contraction due to Ca2+-dependent volumetric shrinkage of RBCs and increased platelet contractility due to their hyperactivation by the enhanced generation of endogenous thrombin on activated RBCs. Addition of rivaroxaban, the inhibitor of thrombin formation, or elimination of Ca2+ from the extracellular space abrogated the stimulating effect of Yoda1 on clot contraction. The Piezo1 antagonist, GsMTx-4, caused a decrease in the extent of clot contraction relative to the control both in whole blood and in platelet-rich plasma. Activated Piezo1 in compressed and deformed RBCs amplified the platelet contractility as a positive feedback mechanism during clot contraction. CONCLUSION: The results obtained demonstrate that the Piezo1 channel expressed on RBCs comprises a mechanochemical modulator of blood clotting that may be considered a potential therapeutic target to correct hemostatic disorders.

A familial case of MYH9 gene mutation associated with multiple functional and structural platelet abnormalities.

Mutations in the MYH9 gene result in macrothrombocytopenia often associated with hemorrhages. Here, we studied the function and structure of platelets in three family members with a heterozygous mutation R1933X in the MYH9 gene, characteristic of closely related disorders known as the May-Hegglin anomaly and Sebastian syndrome. The examination included complete blood count, blood smear microscopy, platelet flow cytometry (expression of P-selectin and active integrin αIIbß3 before and after activation), the kinetics of platelet-driven contraction (retraction) of blood clots, as well as scanning/transmission electron microscopy of platelets. Despite severe thrombocytopenia ranging (36-86) × 109/l, none of the patients had hemorrhages at the time of examination, although they had a history of heavy menstruation, spontaneous ecchymosis, and postpartum hemorrhage. Flow cytometry showed background platelet activation, revealed by overexpression of P-selectin and active αIIbß3 integrin above normal levels. After TRAP-induced stimulation, the fractions of platelets expressing P-selectin in the proband and her sister were below normal response, indicating partial platelet refractoriness. The initiation of clot contraction was delayed. Electron microscopy revealed giant platelets with multiple filopodia and fusion of α-granules with dilated open canalicular system, containing filamentous and vesicular inclusions. The novel concept implies that the R1933X mutation in the MYH9 gene is associated not only with thrombocytopenia, but also with qualitative structural and functional defects in platelets. Platelet dysfunction includes impaired contractility, which can disrupt the compaction of hemostatic clots, making the clots weak and permeable, therefore predisposing patients with MYH9 gene mutations to the hemorrhagic phenotype.

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 αIIbß3 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.

Altered platelet and coagulation function in moderate-to-severe COVID-19.

To reveal if coagulopathies relate to the course of COVID-19, we examined 255 patients with moderate and severe COVID-19, receiving anticoagulants and immunosuppressive drugs. Coagulopathy manifested predominantly as hypercoagulability that correlated directly with systemic inflammation, disease severity, comorbidities, and mortality risk. The prolonged clotting tests in about » of cases were associated with high levels of C-reactive protein and antiphospholipid antibodies, which impeded coagulation in vitro. Contraction of blood clots was hindered in about ½ of patients, especially in severe and fatal cases, and correlated directly with prothrombotic parameters. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with platelet dysfunction. Clots with impaired contraction were porous, had a low content of compressed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin, suggesting that the uncompacted intravital clots are more obstructive but patients could also be prone to bleeding. The absence of consumption coagulopathy suggests the predominance of local and/or regional microthrombosis rather than disseminated intravascular coagulation. The results obtained (i) confirm the importance of hemostatic disorders in COVID-19 and their relation to systemic inflammation; (ii) justify monitoring of hemostasis, including the kinetics of blood clot contraction; (iii) substantiate the active prophylaxis of thrombotic complications in COVID-19.

In systemic lupus erythematosus anti-dsDNA antibodies can promote thrombosis through direct platelet activation.

Systemic lupus erythematosus (SLE) is associated with a high risk of venous and arterial thrombosis, not necessarily associated with prothrombotic antiphospholipid antibodies (Abs). Alternatively, thrombosis may be due to an increased titer of anti-dsDNA Abs that presumably promote thrombosis via direct platelet activation. Here, we investigated effects of purified anti-dsDNA Abs from the blood of SLE patients, alone or in a complex with dsDNA, on isolated normal human platelets. We showed that anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes induced strong platelet activation assessed by enhanced P-selectin expression and dramatic morphological and ultrastructural changes. Electron microscopy revealed a significantly higher percentage of platelets that lost their discoid shape, formed multiple filopodia and had a shrunken body when treated with anti-dsDNA Abs or anti-dsDNA Ab/dsDNA complexes compared with control samples. In addition, these platelets activated with anti-dsDNA Ab/dsDNA complexes typically contained a reduced number of secretory α-granules that grouped in the middle and often merged into a solid electron dense area. Many activated platelets released plasma membrane-derived microvesicles and/or fell apart into subcellular cytoplasmic fragments. Confocal microscopy revealed that platelets treated with anti-dsDNA Ab/dsDNA complex had a heterogeneous distribution of septin2 compared with the homogeneous distribution in control platelets. Structural perturbations were concomitant with mitochondrial depolarization and a decreased content of platelet ATP, indicating energetic exhaustion. Most of the biochemical and morphological changes in platelets induced by anti-dsDNA Abs and anti-dsDNA Ab/dsDNA complexes were prevented by pre-treatment with a monoclonal mAb against FcγRIIA. The aggregate of data indicates that anti-dsDNA Abs alone or in a complex with dsDNA strongly affect platelets via the FcγRIIA receptor. The immune activation of platelets with antinuclear Abs may comprise a prothrombotic mechanism underlying a high risk of thrombotic complications in patients with SLE.

Differential sensitivity of various markers of platelet activation with adenosine diphosphate.

A number of techniques have been available to assess platelet activation, but their relative sensitivity is unknown and their usage is variable and not based on any rational criteria. Here, we compared the ability of several techniques based on morphological and biochemical markers to detect the first signs of ADP-induced platelet activation. Scanning electron microscopy of platelets was performed in parallel with flow cytometry to quantify the surface expression of P-selectin (marked by labeled anti-CD62P antibodies), active αIIbß3-intergrin (assessed by the binding of labeled fibrinogen) and phosphatidylserine (assessed by the binding of labeled Annexin V). When expressed as a fraction of activated platelets, shape changes were the most sensitive to a low ADP concentration compared to the biochemical markers in the following order of sensitivity: morphological changes>fibrinogen binding capacity>P-selectin expression> phosphatidylserine exposure. These results suggest the greater sensitivity of platelet microscopy and the importance of its combination with flow cytometry used to detect surface expression of the molecular markers of platelet activation.

Lytic Susceptibility, Structure, and Mechanical Properties of Fibrin in Systemic Lupus Erythematosus.

Among complications of systemic lupus erythematosus (SLE), thrombotic events are relatively common and contribute significantly to the morbidity and mortality rates. An increased risk of thrombosis in various diseases has been shown to be associated with the lytic stability and mechanical stiffness of the fibrin clot determined by its structure. Here we studied alterations of the fibrin clot properties in relation to disease severity in SLE patients. Plasma clots from 28 SLE patients were characterized by the kinetics of formation and fibrinolytic dissolution (using dynamic turbidimetry), the network and fiber ultrastructure (scanning electron microscopy), viscoelasticity (shear rheometry), and the rate and degree of crosslinking (Western blotting) correlated with the disease activity, blood composition, and compared to clotting of pooled normal human plasma. Clots made from plasma of SLE patients were lysed faster with exogenous t-PA than control clots from normal plasma without a significant difference between those from active (SLEDAI>4) and inactive (SLEDAI<4) SLE patients. Clots from the blood of patients with active SLE were characterized by significantly slower onset, but faster rate of fibrin polymerization and a higher optical density due to thicker fibers compared to those from inactive SLE and control pooled normal plasma. The rheological parameters of the clots (storage and loss moduli) were significantly increased in the active SLE patients along with enhanced fibrin crosslinking and hyperfibrinogenemia. The structural and rheological alterations displayed a strong positive correlation with high fibrinogen levels and other laboratory markers of immune inflammation. In conclusion, changes in the blood composition associated with active systemic inflammation in SLE cause significant alterations in the lytic resistance of fibrin clots associated with changes in polymerization kinetics, viscoelastic properties, and structure. The formation of more rigid prothrombotic fibrin clots in the plasma of SLE patients is likely due to the inflammatory hyperfibrinogenemia and greater extent of crosslinking. However, the higher susceptibility of the SLE clots to fibrinolysis may be a protective and/or compensatory mechanism that reduces the risk of thrombotic complications and improves patient outcomes.

Platelet factor 4-containing immune complexes induce platelet activation followed by calpain-dependent platelet death.

Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy sometimes associated with thrombosis. The hallmark of HIT is antibodies to the heparin/platelet factor 4 (PF4) complex that cause thrombocytopenia and thrombosis through platelet activation. Despite the clinical importance, the molecular mechanisms and late consequences of immune platelet activation are not fully understood. Here, we studied immediate and delayed effects of the complexes formed by human PF4 and HIT-like monoclonal mouse anti-human-PF4/heparin IgG antibodies (named KKO) on isolated human platelets in vitro. Direct platelet-activating effect of the KKO/PF4 complexes was corroborated by the overexpression of phosphatidylserine (PS) and P-selectin on the platelet surface. The immune platelet activation was accompanied by a decrease of the mitochondrial transmembrane potential (ΔΨm), concurrent with a significant gradual reduction of the ATP content in platelets, indicating disruption of energy metabolism. A combination of PS expression and mitochondrial depolarization induced by the PF4-containing immune complexes observed in a substantial fraction of platelets was considered as a sign of ongoing platelet death, as opposed to a subpopulation of activated live platelets with PS on the plasma membrane but normal ΔΨm. Both activated and dying platelets treated with KKO/PF4 formed procoagulant extracellular microvesicles bearing PS on their surface. Scanning and transmission electron microscopy revealed dramatic morphological changes of KKO/PF4-treated platelets, including their fragmentation, another indicator of cell death. Most of the effects of KKO/PF4 were prevented by an anti-FcγRII monoclonal antibody IV.3. The adverse functional and structural changes in platelets induced by the KKO/PF4 complexes were associated with strong time-dependent activation of calpain, but only trace cleavage of caspase 3. The results indicate that the pathogenic PF4-containing HIT-like immune complexes induce direct prothrombotic platelet activation via FcγRIIA receptors followed by non-apoptotic calpain-dependent death of platelets, which can be an important mechanism of thrombocytopenia during HIT development.

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 αIIbß3 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.

Impaired contraction of blood clots as a novel prothrombotic mechanism in systemic lupus erythematosus.

The aim of this work was to examine a possible role of clot contraction/retraction in thrombotic complications of systemic lupus erythematosus (SLE). Using a novel automated method, we investigated kinetics of clot contraction in the blood of 51 SLE patients and 60 healthy donors. The functionality of platelets in the SLE patients was assessed using flow cytometry by expression of P-selectin and fibrinogen-binding capacity. The rate and degree of clot contraction were significantly reduced in SLE patients compared with healthy subjects, especially in the patients with higher blood levels of anti-dsDNA antibodies. The reduced platelet contractility correlated with partial refractoriness of platelets isolated from the blood of SLE patients to stimulation induced by the thrombin receptor activating peptide. To test if the anti-dsDNA autoantibodies cause continuous platelet activation, followed by exhaustion and dysfunction of the cells, we added purified exogenous anti-dsDNA autoantibodies from SLE patients to normal blood before clotting. In support of this hypothesis, the antibodies first enhanced clot contraction and then suppressed it in a time-dependent manner. Importantly, a direct correlation of clot contraction parameters with the disease severity suggests that the reduced compactness of intravascular clots and thrombi could be a pathogenic factor in SLE that may exaggerate the impaired blood flow at the site of thrombosis. In conclusion, autoantibodies in SLE can affect platelet contractility, resulting in reduced ability of clots and thrombi to shrink in volume, which increases vessel obstruction and may aggravate the course and outcomes of thrombotic complications in SLE.

Reduced Contraction of Blood Clots in Venous Thromboembolism Is a Potential Thrombogenic and Embologenic Mechanism.

Contraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin that can potentially modulate the obstructiveness and integrity of thrombi. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in the blood of 55 patients with VTE. In addition, we studied the ultrastructure of ex vivo venous thrombi as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction in vivo. The extent and rate of contraction were reduced by twofold in clots from the blood of VTE patients compared with healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared with that of those with isolated venous thrombosis, suggesting that less compacted thrombi are prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness. Morphologically, 75% of platelets from VTE patients were spontaneously activated (with filopodia) compared with only 21% from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared with healthy individuals. The results obtained suggest that the impaired contraction of thrombi is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi.

Activated Monocytes Enhance Platelet-Driven Contraction of Blood Clots via Tissue Factor Expression.

Platelet-driven reduction in blood clot volume (clot contraction or retraction) has been implicated to play a role in hemostasis and thrombosis. Although these processes are often linked with inflammation, the role of inflammatory cells in contraction of blood clots and thrombi has not been investigated. The aim of this work was to study the influence of activated monocytes on clot contraction. The effects of monocytes were evaluated using a quantitative optical tracking methodology to follow volume changes in a blood clot formed in vitro. When a physiologically relevant number of isolated human monocytes pre-activated with phorbol-12-myristate-13-acetate (PMA) were added back into whole blood, the extent and rate of clot contraction were increased compared to addition of non-activated cells. Inhibition of tissue factor expression or its inactivation on the surface of PMA-treated monocytes reduced the extent and rate of clot contraction back to control levels with non-activated monocytes. On the contrary, addition of tissue factor enhanced clot contraction, mimicking the effects of tissue factor expressed on the activated monocytes. These data suggest that the inflammatory cells through their expression of tissue factor can directly affect hemostasis and thrombosis by modulating the size and density of intra- and extravascular clots and thrombi.

Contraction of Blood Clots Is Impaired in Acute Ischemic Stroke.

OBJECTIVE: Obstructive thrombi or thrombotic emboli are the pathogenic basis of ischemic stroke. In vitro blood clots and in vivo thrombi can undergo platelet-driven contraction (retraction), resulting in volume shrinkage. Clot contraction can potentially reduce vessel occlusion and improve blood flow past emboli or thrombi. The aim of this work was to examine a potential pathogenic role of clot contraction in ischemic stroke. APPROACH AND RESULTS: We used a novel automated method that enabled us to quantify time of initiation and extent and rate of clot contraction in vitro. The main finding is that clot contraction from the blood of stroke patients was reduced compared with healthy subjects. Reduced clot contraction correlated with a lower platelet count and their dysfunction, higher levels of fibrinogen and hematocrit, leukocytosis, and other changes in blood composition that may affect platelet function and properties of blood clots. Platelets from stroke patents were spontaneously activated and displayed reduced responsiveness to additional stimulation. Clinical correlations with respect to severity and stroke pathogenesis suggest that the impaired clot contraction has the potential to be a pathogenic factor in ischemic stroke. CONCLUSIONS: The changeable ability of clots and thrombi to shrink in volume may be a novel unappreciated mechanism that aggravates or alleviates the course and outcomes of ischemic stroke. The clinical importance of clot or thrombus transformations in vivo and the diagnostic and prognostic value of this blood test for clot contraction need further exploration.

Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)γRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcγRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcγRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcγRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk.