Observation of calcium signaling in platelets - blood cells designed to be involved in stopping bleeding and forming blood clots - is an important part of fundamental research in hemostasis. However, such a study is possible only with the use of calcium fluorophores - small molecules that penetrate the platelet membrane due to their hydrophobic -AM part, which is then hydrolyzed by cytosol esterases. In this work, we consider the phenomenon of inhomogeneous loading of calcium fluorophores into platelets.

We used platelets from healthy adult donors loaded with various fluorescent probes (CalBryte590, DiOC6 (3), Fura Red, Fluo-4 and CellTracker Violet BMQC) and immobilized on antibodies to CD31 in parallel plane flow chambers. Total internal reflection fluorescence (TIRF) microscopy was used for observations.

We demonstrated that all studied probes are loaded heterogeneously, with 30% platelets being loaded with a probe 2-6 times higher than the population median value. Using the CalBryte590 probe as an example, we have shown that a decrease in the incubation temperature, the addition of Pluronic 127 to the incubation medium, or membrane cholesterol depletion significantly reduces the heterogeneity of the probe distribution in the population. By looking at platelet activation from the surface, we have shown that the probability of experiencing strong activation, as measured by the intensity of calcium oscillations, correlates with the amount of probe in the platelet.

Thus, we conclude that the type of fluorophore used and the conditions of its loading into platelets can significantly affect the results of experiments on the observation of calcium signaling in platelets.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder of autoimmune pathophysiology. The causes of ITP could be related to other pathology (viral, bacterial, or systemic), or ITP could develop without any apparent reason. While the immune system dysregulation mechanisms in ITP were described, its etiology remains unclear. Moreover, all existing treatment approaches are not specific for ITP, and its action is highly patient- specific. Here we describe recent findings in the origins and development of ITP and discuss novel experimental and theoretical approaches to diagnosing ITP and predicting therapy effects.

Small, non-nuclear cells, platelets, are primarily designed to form aggregates when blood vessels are damaged, stopping bleeding. To perform this function, platelets can implement several functional responses induced by various agonists and coordinated by a complex network of intracellular signaling triggered by a dozen of different receptors. This review, the second in a series, describes the known intracellular signaling pathways induced by platelet receptors in response to canonical and rare agonists. Particular focus will be on interaction points and “synergy” of platelet activation pathways and intermediate or “secondary” activation mediators that transmit a signal to functional manifestations.

It is known that in COVID-19, hypercoagulation and sometimes thrombocytopenia are related to disease severity. There is also controversial data on platelet participation in COVID-19 pathology. We aimed to determine the degree of platelet hyperactivation in COVID-19 patients. Whole blood flow cytometry with Annexin-V and lactadherin staining ("PS+ platelets") was utilized. Additionally, a stochastic mathematical model of platelet production and consumption was developed. Here we demonstrated that the percentage of PS+ platelets in COVID-19 patients was twofold that of healthy donors. There was a significant correlation between the amount of PS+ platelets and the percentage of lung damage in patients. No connection was found between platelet senescence and hospital therapy or patients' chronic diseases, except for chronic lung disease. Although no thrombocytopenia was observed in patients, the observed increase in platelet size (FSC-A parameter in flow cytometry) could indicate that platelet age is decreased in patients. The developed computational model of platelet turnover confirms the possibility of intense platelet consumption without noticeable changes in platelet count. We conclude that the observed platelet hyperactivation in COVID-19 could be caused by platelet activation in circulation, leading to platelet consumption without significant thrombocytopenia.

Store-operated calcium entry (SOCE) plays an important role in platelet function. It is generally assumed that the mechanism of SOCE relies on the direct interaction of STIM1 and ORAI1 proteins with specific STIM1:ORAI1 stoichiometry. However, in platelets, other pathways may take place. Here we aim to investigate the mechanisms of SOCE in platelets. We developed a lattice-based mathematical model that represented STIM1-ORAI1 interactions and applied it to both HEK cells, where SOCE mechanism is well established, and platelets. The model was able to describe STIM1-ORAI1 behavior in HEK cells successfully. We used the same parameters for protein interaction and applied them to platelets. As a result, we demonstrated that the number of STIM1 proteins on ER membrane could not assure the needed stoichiometry to proper SOCE in platelets.