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.

Extent of intravital contraction of arterial and venous thrombi and pulmonary emboli.

Blood clots and thrombi undergo platelet-driven contraction/retraction followed by structural rearrangements. We have established quantitative relationships between the composition of blood clots and extent of contraction to determine intravital contraction of thrombi and emboli based on their content. The composition of human blood clots and thrombi was quantified using histology and scanning electron microscopy. Contracting blood clots were segregated into the gradually shrinking outer layer that contains a fibrin-platelet mesh and the expanding inner portion with compacted red blood cells (RBCs). At 10% contraction, biconcave RBCs were partially compressed into polyhedral RBCs, which became dominant at 20% contraction and higher. The polyhedral/biconcave RBC ratio and the extent of contraction displayed an exponential relationship, which was used to determine the extent of intravital contraction of ex vivo thrombi, ranging from 30% to 50%. In venous thrombi, the extent of contraction decreased gradually from the older (head) to the younger (body, tail) parts. In pulmonary emboli, the extent of contraction was significantly lower than in the venous head but was similar to the body and tail, suggesting that the emboli originate from the younger portion(s) of venous thrombi. The extent of contraction in arterial cerebral thrombi was significantly higher than in the younger parts of venous thrombi (body, tail) and pulmonary emboli but was indistinguishable from the older part (head). A novel tool, named the "contraction ruler," has been developed to use the composition of ex vivo thrombi to assess the extent of their intravital contraction, which contributes to the pathophysiology of thromboembolism.

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.

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.

Impaired contraction of blood clots precedes and predicts postoperative venous thromboembolism.

Deep vein thrombosis (DVT) is a common but unpredictable complication of surgical interventions. To reveal an association between the blood clot contraction (retraction) and the incidence of postoperative venous thrombosis, 78 patients with brain tumors that were operated on were studied, of which 23 (29%) were diagnosed with postoperative DVT. A clot contraction assay, along with other hemostatic and hematologic tests, was performed 1-3 days before the surgery and on the 1st day and 5-7th days after the surgery. On the 1st postoperative day, clot contraction was significantly suppressed in patients who subsequently developed DVT, compared to the patients without DVT. Importantly, this difference was observed at least 5 days before DVT had developed. The weakening of contraction on the 1st postoperative day was more pronounced in the DVT patients with malignant versus benign brain tumors, atherosclerosis, hypertension, as well as in patients receiving steroids before and during the operation. These results indicate that impaired clot contraction in the postoperative period is associated with imminent DVT, suggesting that it is a prothrombotic risk factor and promotional mechanism. The clot contraction assay has a predictive value in assessing the threat of postoperative thrombosis in patients with benign and malignant brain tumors.

Premorbid Hemostasis in Women with a History of Pregnancy Loss.

BACKGROUND: Congenital and acquired hemostatic disorders are among the pathogenic factors of pregnancy loss. Studying mechanistic relations between impaired hemostasis and fetal losses is important for the prognosis and prophylaxis of obstetric complications. OBJECTIVE: This article aims to establish latent hemostatic disorders in nonpregnant women as an important premorbid risk factor of pregnancy loss. METHODS AND RESULTS: Hemostasis was characterized using two relatively new in vitro assays, namely thrombodynamics (spatial clot growth) and kinetics of blood clot contraction, which together reflect the hemostatic or thrombotic potential. In addition, platelet functionality was assessed using flow cytometry. Our study included 50 women with a history of pregnancy loss and 30 parous women without previous obstetric complications. In patients with pregnancy loss, hypercoagulability was observed along with significant impairment of blood clot contraction associated with chronic platelet activation and dysfunction. Both hypercoagulability and defective clot contraction were significantly more pronounced in patients with a history of three or more miscarriages compared with patients with a history of one or two miscarriages. In addition, a significant inhibition of clot contraction was found in patients with miscarriage occurring after 10 weeks of gestation compared with those who lost a fetus earlier in pregnancy. CONCLUSION: These results indicate that chronic hypercoagulability and impaired clot contraction constitute a premorbid status in patients with pregnancy loss. The data confirm a significant pathogenic role of hemostatic disorders in pregnancy loss and suggest the predictive value of thrombodynamics and blood clot contraction assays in evaluating the risk of pregnancy loss.