ИСТИНА

Background: Platelet activation relies on calcium signaling, however, platelets display heterogeneity in their calcium dynamics and functional responses upon stimulation. We analyze the heterogeneity of single platelet calcium responses to collagen, ADP and thrombin in healthy donors and hematological patients. Methods: Whole hirudinated blood of 9 Wiskott- Aldrich Syndrome, 3 immune thrombocytopenia and 7 MYH9-related thrombocytopenia patients and 18 healthy donors was collected in compliance with the Declaration of Helsinki. Fluorescence microscopy was used for characterization of platelet calcium responses. Aims: We aim to develop a diagnostic method for abnormal platelet activation detection relying on classification of single-platelet calcium profiles. Results: We distinguished four types of platelet calcium response: “no response” (i) with rare spiking, “spiking” (ii) with calcium oscillations with period T = 3-10 s, “clusters'' (iii) with several clusters of jammed together spikes, and “sustained high” (iiii) with a sustained high calcium level. The distribution of platelets of healthy donors shifted to response types that indicated higher calcium levels upon activation. Using bootstrap method, we obtained confidence intervals for mean fractions of healthy donors’ platelet populations within the groups. We provide a tool that tells if a patient has a normal platelet activation pattern by checking if fractions of his or her platelet population fit into these confidence intervals. Platelet calcium response profile of a patient with Wiskott-Aldrich syndrome has demonstrated weaker response to activation on neutral coating, while on collagen it demonstrated strong responses with 4%, 20%, 20% and 56% fractions of platelet population in groups (i), (ii), (iii), (iiii), respectively. Conclusions: The distribution of single platelets between response groups could be utilized as a diagnostic technique to determine the fractions of refractory platelets or hyper-active platelets within the platelet population. The reported study was funded by RFBR and the Royal Society of London (RS), project number 21-51-10005