Platelets of COVID-19 patients are refractory due to thrombin receptor desensitizing in the bloodstream

Background : Coagulopathy is among the most alarming drivers of COVID-19-induced pathology. COVID-19 can result in blockade of the microvasculature in the lungs with microthrombi. While most of the studies concern COVID-19 impact on plasma coagulation, platelet dysfunction has been reported as well. However, the mechanism of platelet malfunctioning in COVID-19 has not been described yet. Aims : To determine the mechanism of COVID-19 induced platelet disfunction. Methods : 46 patients with confirmed COVID-19 (11 non-ICU, 26 ICU, and 9 ECMO) and 26 healthy volunteers were studied (independent ethics committee of NMRC PHOI No 3/2020). Citrated whole blood samples were diluted in Tyrode's buffer and activated by collagen and TRAP-6. Platelets of healthy donors were additionally washed and pre-treated by 0.5 nM of thrombin. Samples were analyzed using BC Navios flow cytometer. Additionally, light transmission aggregometry (AP-2110 SOLAR) of citrated platelet-rich plasma (PRP) with TRAP-6 and fucoidan as activators was conducted. Results : Platelet forward scattering parameter (FSC-A) for COVID-19 patients was significantly increased compared to healthy donors. This parameter reversibly correlated with mild thrombocytopenia observed in some patients. The amount of Annexin-V positive platelets was increased in all patients as well. Relative CD42b and CD62p binding upon activation were decreased, being statistically different in non-ICU and ICU patients. Both of the parameters also correlated to CRP concentration in patient blood plasma. Platelet aggregation was reduced as well. Altogether, platelets demonstrated refractoriness resembling desensitization of receptors. To prove this hypothesis, we performed thrombin pre-treatment of healthy donor platelets, which resulted in a phenotype resembling the COVID-19. Conclusions : Platelets of COVID-19 patients demonstrate refractoriness to activation through PAR1 receptor, probably because of a previous activation with thrombin in circulation. Together with their increased size and fraction of necrotic platelets, this suggests that in COVID-19 platelets encounter thrombin in circulation.

Platelets in covid-19: “innocent by-standers” or active participants?

One of the most dangerous features of the new coronavirus infection caused by the SARS-CoV-2 virus is the tendency of the hemostasis system of patients to excessive thrombus formation. Among the possible causes of this pathology, both the activation of vascular endothelial cells, leading to the exposure of tissue factor by these cells, and direct activation of the plasma hemostasis were named. Besides, there is a significant change in platelet responses to activation, which is not accompanied by significant thrombocytopenia. The mechanism of platelet dysfunction is rather controversial. On the one hand, there are suggestions that platelets can act as a direct “container” for the virus, thus spreading it throughout the body. On the other hand, the presence of viral RNA in platelets has been demonstrated in only one study, while other authors have obtained the opposite result. Another mechanism of the virus's direct effect on platelets is the penetration of the virus into megakaryocytes and the subsequent violation of thrombocytopoiesis. However, three of the four published works show that platelets from patients with SARS-CoV-2 are in an activated state (the so-called platelet pre-activation). This phenomenon can be caused by the direct influence of the virus and the effect of thromboinflammation in the lungs on platelet functions. Here we review the known data and possible causes of the platelet functionality changes observed in patients with SARS-CoV-2.

Results of multicenter monitoring of hemostasis parameters in patients with COVID-19

Materials and methods: a prospective non-randomized pilot multicenter study of the informativeness and clinical significance of hemostasis laboratory tests in 1210 patients with COVID-19 in disease course, including favorable and unfavorable outcomes, was performed. Hemostasis was assessed using clotting tests and D-dimer concentration, thromboelastography (TEG) and thrombodynamics (TD). Results: comparison of COVID-19 laboratory parameters and clinical picture showed that 75% of patients have pronounced activation of the plasma coagulation system upon admission to the hospital. Hypercoagulation is recorded in all tests, reaching a maximum in patients with subtotal (CT-3) and total (CT-4) lung lesion and/or resuscitation patients with a clinical picture of pulmonary embolism and unfavorable outcome. Low molecular weight heparins (LMWH) in a standard dosage leads to suppression of the initial hypercoagulable syndrome in more than half of the patients (from 75 to 31%). All patients without LMWH laboratory effect developed thrombotic complications. For clotting tests, insufficient sensitivity to changes in hemostasis against the background of LMWH was revealed. The D-dimer test effectively correlates with the severity and outcomes of COVID-19, but is not suitable for the control of heparin therapy, which is associated with the effect of lysis of existing blood clots and the lack of response to a decrease in the coagulation activity of patients. Methods of thromboelastography and thrombodynamics effectively record a decrease in the activity of the coagulation system and can be used to control heparin therapy. The correlation coefficient between the methods was 0,77. The dynamic indices of D-dimers, TEG and TD in severe patients and, especially, in patients with fatal outcomes revealed the greatest sensitivity to the control of heparin therapy in the Thrombodynamics test, which allows, along with thrombosis, to record hypercoagulable states and the risk of bleeding, which are the outcome of thrombohemorrhagic syndrome in patients with COVID-19. Материалы и методы исследования: проведено проспективное открытое нерандомизированное пилотное многоцентровое исследование информативности и клинической значимости лабораторных тестов гемостаза у 1210 пациентов с COVID-19 в динамике заболевания, включая благоприятные и неблагоприятные исходы. Оценку гемостаза проводили с использованием клоттинговых тестов и концентрации D-димера, тромбоэластографии (ТЭГ) и тромбодинамики (ТД). Результаты: при сопоставлении лабораторных показателей и клинической картины COVID-19 показано, что у 75% больных при поступлении в стационар наблюдается выраженная активация плазменной системы свертывания. Гиперкоагуляция фиксируется по всем тестам, достигая максимума у больных с субтотальным (КТ-3) и тотальным (КТ-4) поражением легких и/или реанимационных больных с клинической картиной тромбоэмболии легочной артерии и неблагоприятным исходом. Назначение низкомолекулярных гепаринов (НМГ) в стандартной дозировке приводит к подавлению исходного гиперкоагуляционного синдрома более чем у половины больных (с 75 до 31%). Все пациенты без лабораторного эффекта НМГ развили тромботические осложнения. Для клоттинговых тестов выявлена недостаточная чувствительность к изменениям гемостаза на фоне НМГ. Тест на D-димер эффективно коррелирует с тяжестью и исходами COVID-19, но непригоден для контроля гепаринотерапии, что связано с эффектом лизиса существующих тромбов и отсутствием ответа на снижение коагуляционной активности больных. Методы ТЭГ и ТД эффективно регистрируют снижение активности свертывающей системы и могут использоваться для контроля гепаринотерапии. Коэффициент корреляции между методами составил 0,77. Заключение: динамические индексы D-димеров, ТЭГ и ТД у тяжелых больных и особенно у пациентов с летальными исходами выявили наибольшую чувствительность к контролю гепаринотерапии у теста ТД, что позволяет наряду с тромбозами фиксировать гиперкоагуляционные состояния и риск кровотечений - исходы тромбогеморрагического синдрома у больных с COVID-19.