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Background.Evaluation of the functional state of platelets in the composition of concentrates is necessary to improve the methods of their production, optimize storage conditions and terms, increase therapeutic efficacy, and reduce the risk of transfusion complications. Aim.Comprehensive study of the morphofunctional state of platelets during the storage of their concentrates. Material and methods.202samples of human platelet apheresis concentrates were studied for storage periods of up to 17days at a temperature of 2224C. The functional state of platelets was assessed using flow cytometry for the expression of P-selectin, active IIb3 integrin, and phosphatidylserines before and after biochemical stimulation. In addition, the mitochondrial potential of platelets, the concentration of intracellular adenosine triphosphate, contractile properties, induced aggregation, and morphological characteristics were studied according to scanning electron microscopy. Statistical analysis was performed by analysis of variance (ANOVA or KruskalWallis). Results.In non-activated platelets during storage, the spontaneous expression of P-selectin increased by an average of 7times, and phosphatidylserines by 3times. The induced expression of P-selectin, integrin IIb3 and phosphatidylserines was the highest on the day of receiving the concentrate and gradually decreased during storage to 1/2of the initial level. Platelet aggregation activity in response to collagen stimulation progressively decreased by an average of 100times, in response to the TRAP peptide by 1.5times. In contrast to the expression of activation markers, the ability of platelets to compress plasma clots in most samples changed slightly, within a few percent; in single samples, the contractile function dropped to zero by 47days of storage, which was combined with platelet hyperactivation and depletion. The mitochondrial potential of platelets and the content of adenosine triphosphate were relatively constant. Morphologically, discoid platelets predominated, however, starting from the 1st day of storage, spindle-shaped platelets, and from the 3rd day spherical forms and microaggregates of platelets accumulated. Conclusion.Platelets in the composition of concentrates initially have a high functional potential, which gradually decreases due to progressive spontaneous activation with a simultaneous decrease in reactivity and structural changes.

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 αIIbβ3 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 αIIbβ3 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.

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.

Aim. To establish the relationship of hematological disorders with the pathogenesis, course and outcomes of COVID-19. Methods. We examined 235 hospitalized patients with moderate and severe forms of acute COVID-19 receiving anticoagulants and immunosuppressive drugs. We studied the full blood cell counts and morphology along with the platelet function by flow cytometry in comparison with the clinical features and synthesis of inflammatory markers. To assess platelet contractility, blood clot contraction (retraction) kinetics was used in combination with scanning electron microscopy of platelets and blood clots. Results. Hemolytic anemia, neutrophilia and lymphopenia were associated with immature erythrocytes and leukocytes, indicating activation of hematopoiesis. Contraction of blood clots in COVID-19 was impaired, especially in severe and lethal cases, as well as in the presence of comorbidities, including myeloproliferative and coronary heart diseases and acute cerebrovascular disease. In male patients, the changes in clot contraction were more pronounced. Suppression of clot contraction correlated directly with anemia and coagulopathy, including a high D-dimer level, which confirms the pathogenetic significance of blood clot contraction in COVID-19. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with chronic platelet activation and secondary platelet dysfunction. The structure and cellular composition of blood clots depended on the extent of contraction;clots with impaired contraction were porous, had a low content of deformed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin. Conclusion. Blood cells undergoing both quantitative and qualitative changes are involved in the pathogenesis of COVID-19;the suppressed platelet-driven contraction of intravital blood clots may be a part of the prothrombotic mechanisms. © 2021 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/ licenses/by/4.0/.

To study the relationship of hemostatic disorders with inflammation and estimate their role in the course and outcomes of COVID-19.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.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.The results obtained substantiate the need for laboratory monitoring of hemostasis and active prophylaxis and treatment of thrombotic complications in COVID-19.Цель. Изучить связь гемостатических нарушений с воспалением и вклад коагулопатий в течение и исходы COVID-19. Материалы и методы. Обследованы 215 пациентов со среднетяжелой и тяжелой формами болезни на фоне антикоагулянтной и иммуносупрессивной терапии. Гемостаз оценивали по тестам тромбодинамики, тромбоэластографии, уровням фибриногена и D-димера, протромбиновому времени и растворимым комплексам фибрин-мономера. Показатели гемостаза сопоставляли с гемограммой и биохимией крови, включая маркеры воспаления (С-реактивный белок, интерлейкины 6 и 8), а также с клинической картиной. Результаты. Признаки коагулопатии выявлены у подавляющего большинства обследованных. Несмотря на применение низкомолекулярных гепаринов в профилактических и лечебных дозах, коагулопатия при COVID-19 протекает преимущественно по типу гипер- коагуляции, выраженность которой прямо коррелирует с системной воспалительной реакцией и метаболическими сдвигами вследствие дисфункции печени и почек. Обнаружена прямая связь между степенью гемостатических расстройств и тяжестью течения COVID-19, включая наличие сопутствующих заболеваний и вероятность неблагоприятного исхода. Примерно в 1/4 случаев обнаружена хронометрическая гипокоагуляция в сочетании с высоким уровнем С-реактивного белка в крови, который может тормозить свертывание in vitro и тем самым маскировать истинную тромбофилию при активном воспалении. Персистирующие высокие уровни фибриногена и D-димера при отсутствии признаков коагулопатии потребления свидетельствуют о преобладании локального и/или регионального микротромбоза над диффузным внутрисосудистым свертыванием крови. Заключение. Полученные результаты обосновывают необходимость лабораторного контроля системы гемостаза и активной профилактики тромботических осложнений, включая ограничение системной воспалительной реакции, при 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 ¼ 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.

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.

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.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex and largely unclear pathogenesis. Cellular phospholipid microvesicles released upon activation and/or death of a cell have been proposed to play a role in inflammatory autoimmune pathologies, including SLE. Here, circulating microvesicles of various cellular origins were marked with fluorescently labeled cell-specific antibodies and enumerated by flow cytometry in platelet-free plasma obtained from the heparinized blood of 29 SLE patients and 19 normal subjects. Significantly higher concentrations of endothelial-, monocyte-, and erythrocyte-derived microvesicles were found in the SLE patients compared to normal subjects with prevalence of microvesicles originating from endothelial cells. No significant difference was found for platelet-derived microvesicles. A correlation analysis of microvesicle counts with laboratory parameters and clinical features of SLE suggest differential implications of various cell-derived microvesicles in the pathogenesis of SLE. These data suggest that SLE is associated with functional alterations of endotheliocytes, monocytes, and erythrocytes followed by enhanced release of microvesicles that may contribute to inflammation and hypercoagulability.

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.