Background Activation of human platelets with thrombin proceeds via two protease-activated receptors (PARs), PAR1 and PAR4, that have identical main intracellular signaling responses. Although there is evidence that they have different cleavage/inactivation kinetics (and some secondary variations in signaling), the reason for such redundancy is not clear. Methods We developed a multicompartmental stochastic computational systems biology model of dual-receptor thrombin signaling in platelets to gain insight into the mechanisms and roles of PAR1 and PAR4 functioning. Experiments employing continuous flow cytometry of washed human platelets were used to validate the model and test its predictions. Activity of PAR receptors in donors was evaluated by mRNA measurement and by polymorphism sequencing. Results Although PAR1 activation produced rapid and short-lived response, signaling via PAR4 developed slowly and propagated in time. Response of the dual-receptor system was both rapid and prolonged in time. Inclusion of PAR1/PAR4 heterodimer formation promoted PAR4 signaling in the medium range of thrombin concentration (about 10 nm), with little contribution at high and low thrombin. Different dynamics and dose-dependence of procoagulant platelet formation in healthy donors was associated with individual variations in PAR1 and PAR4 activities and particularly by the Ala120Thr polymorphism in the F2RL3 gene encoding PAR4. Conclusions The dual-receptor combination is critical to produce a response combining three critical advantages: sensitivity to thrombin concentration, rapid onset and steady propagation; specific features of the protease-activated receptors do not allow combination of all three in a single receptor.

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With the progress of COVID-19 studies, it became evident that SARS-CoV-2 infection is often associated with thrombotic complications. The goal of our present study was to evaluate which component of clot formation process including endothelial function, platelets aggregation and plasma coagulation, as well as endogenous fibrinolysis in patients with COVID-19 correlates with the severity of the disease. We prospectively included 58 patients with COVID-19 and 47 healthy volunteers as a control group that we recruited before the pandemic started. It turns out that plasma coagulation with subsequent platelet aggregation, but not endothelial function, correlates with the severity of the COVID-19. IL-6 blockade may play a beneficial role in COVID-19 induced coagulopathy.

Platelet participation in hemostatic plug formation requires transition into an activated state (or, rather, variety of states) upon action of agonists like ADP, thromboxane A , collagen, thrombin, and others. The mechanisms of action for different agonists, their receptors and signaling pathways associated with them, as well as the mechanisms of platelet response inhibition are the subject of the present review. Collagen exposed upon vessel wall damage induced initial platelet attachment and start of thrombus formation, which involves numerous processes such as aggregation, activation of integrins, granule secretion and increase of intracellular Ca2+. Thrombin, ADP, thromboxane A , and ATP activated platelets that were not initially in contact with the wall and induce additional secretion of activating substances. Vascular endothelium and secretory organs also affect platelet activation, producing both positive (adrenaline) and negative (prostacyclin, nitric oxide) regulators, thereby determining the relation of activation and inhibition signals, which plays a significant role in the formation of platelet aggregate under normal and pathological conditions. The pathways of platelet signaling are still incompletely understood, and their exploration presents an important objective both for basic cell biology and for the development of new drugs, the methods of diagnostics and of treatment of hemostasis disorders.

Intracellular Ca2+ ions play an important role in the transmission and treatment of information that cells obtain from the ambient environment. Having received an external signal, a cell may increase the intracellular Ca2+ concentration within fractions of a second by a factor of several hundred. This phenomenon triggers activation of various cellular systems that generate a response to the external stimulus. In many cells under the effect of external signal the concentration of Ca2+ not only increases, but also starts oscillating. Both the frequency and amplitude of the oscillations are affected by the external signal strength. There are reasons to hypothesize that the conversion of the external signal into the oscillating intracellular signal has some important informational meaning. Methods to measure the dynamics of the intracellular Ca2+ concentration and mechanisms that generate the oscillations are reviewed, and hypotheses on how the cell decodes Ca2+ concentration oscillations are presented. Consideration is focused on the platelet, the cell that plays a key role in arresting hemorrhages. If a vessel is damaged, the platelet is rapidly activated. Identical platelets are divided in the process of arresting a hemorrhage into three populations with quite different missions. The platelet seems to somehow ‘interpret’ the set of external signals and uses the Ca2+ concentration oscillations to ‘choose’ the population to which it will belong. Owing to the platelet’s relative simplicity, one can expect that studies of that cell will shortly enable the decryption of the ‘code’ that drives Ca2+ concentration oscillations.

Background/Introduction There are numerous reports regarding the direct endothelial damage by the SARS-CoV-2 that can lead to activation of both plasma hemostasis and platelet aggregation. However, the mechanism of interaction between endothelium and haemostasis in COVID-19 remains unclear. Purpose The aim of our study was to assess the relationship between each link of clot formation process (endothelial function, plasma coagulation, platelet aggregation) with the severity of the disease. Methods 58 COVID-19 patients were included in our study. Patients were divided into moderate (n=39) and severe (n=18) subgroups. All patients underwent a flow-mediated dilation (FMD) test, impedance aggregation, rotational thromboelastometry, thrombodynamics and von Willebrand factor antigen (vWF: Ag) quantification. All measurements were repeated on days 3 (point 2) and 9 (point 3) of hospitalization. Results COVID-19 patients demonstrated the enhanced plasma coagulation (clotting time, s 613,0 [480; 820], clot growth rate, μm/min 32,75 [29,3; 38,7]). At point 1 no significant difference in parameters of plasma coagulation between patients' subgroups was noted. At point 2 a significant decrease in the size (CS, μm 1278.0 [1216.5; 1356.5] vs 965.0 [659.8; 1098.0], p<0,01) and clot growth rate (μm/min 32,4 [29,2; 35,0] vs 17,7 [10,3; 24,4], p<0,01) under the influence of anticoagulants in the moderate subgroup compared with point 1 was observed. We didn't observe such phenomenon in severe subgroup. There was no significant difference in platelet aggregation between subgroups at point 1. During the course of the disease the patients in the moderate and severe subgroups demonstrated a significant increase in platelet aggregation induced by arachidonic acid and ADP (severe: AUC ARA 48,0 [25,0; 59,0] vs 77,5 [55,8; 92,7], p=0,04; AUC ADP 44,0 [41,0; 56,0] vs 58,0 [45,5; 69,0], p=0,04; moderate: AUC ARA 31,5 [19,8; 50,7] vs 56,0 [39,0; 76,0], p=0,01; AUC ADP 43,0 [20,0; 59,0] vs 56,6 [50,3; 70,5], p=0,04;), in moderate subgroup the significant increase in TRAP-induced aggregation was also noted (AUC TRAP 58,0 [41,0; 69,5] vs 76,0 [58,3; 81,5], p=0,048). There were no significant differences in the FMD-test results between the patient subgroups. FMD-test results were predominantly within the reference ranges (7,1 [4,0; 8,8]). Patients in the severe subgroup had significantly higher levels of vWF: Ag (228,0 [205,3; 240,7] vs 232,0 [226,0; 423,0], p=0,03). Conclusion SARS-CoV-2 infection was characterized by increased levels of vWF:Ag, that could represent the local endothelial damage, meanwhile there was no generalized endothelial dysfunction assessed via FMD-test in moderate to severe patients. At the same time the enhanced plasma coagulation in COVID-19 patients was observed. FUNDunding Acknowledgement Type of funding sources: None.

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For the first time, the influence of COVID-19 on blood microrheology was studied. For this, the method of filtering erythrocytes through filters with pores of 3.5 μm was used. Filterability was shown to significantly decrease with the increasing severity of the patient's condition, as well as with a decrease in the ratio of hemoglobin oxygen saturation to the oxygen fraction in the inhaled air (SpO2/FiO2). The filterability of ≤0.65, or its fast decrease during treatment, were indicators of a poor prognosis. Filterability increased significantly with an increase in erythrocyte count, hematocrit and blood concentrations of hemoglobin, albumin, and total protein. The effect of these parameters on the erythrocyte filterability is directly opposite to their effect on blood macrorheology, where they all increase blood viscosity, worsening the erythrocyte deformability. The erythrocyte filterability decreased with increasing oxygen supply rate, especially in patients on mechanical ventilation, apparently not due to the oxygen supplied, but to the deterioration of the patients' condition. Filterability significantly correlates with the C-reactive protein, which indicates that inflammation affects the blood microrheology in the capillaries. Thus, the filterability of erythrocytes is a good tool for studying the severity of the patient's condition and his prognosis in COVID-19.