Platelets are blood cells responsible for vascular integrity preservation. The activation of platelet receptor C-type lectin-like receptor II-type (CLEC-2) could partially mediate the latter function. Although this receptor is considered to be of importance for hemostasis, the rate-limiting steps of CLEC-2-induced platelet activation are not clear. Here, we aimed to investigate CLEC-2-induced platelet signal transduction using computational modeling in combination with experimental approaches. We developed a stochastic multicompartmental computational model of CLEC-2 signaling. The model described platelet activation beginning with CLEC-2 receptor clustering, followed by Syk and Src family kinase phosphorylation, determined by the cluster size. Active Syk mediated linker adaptor for T cell protein phosphorylation and membrane signalosome formation, which resulted in the activation of Bruton's tyrosine kinase, phospholipase and phosphoinositide-3-kinase, calcium, and phosphoinositide signaling. The model parameters were assessed from published experimental data. Flow cytometry, total internal reflection fluorescence and confocal microscopy, and western blotting quantification of the protein phosphorylation were used for the assessment of the experimental dynamics of CLEC-2-induced platelet activation. Analysis of the model revealed that the CLEC-2 receptor clustering leading to the membrane-based signalosome formation is a critical element required for the accurate description of the experimental data. Both receptor clustering and signalosome formation are among the rate-limiting steps of CLEC-2-mediated platelet activation. In agreement with these predictions, the CLEC-2-induced platelet activation, but not activation mediated by G-protein-coupled receptors, was strongly dependent on temperature conditions and cholesterol depletion. Besides, the model predicted that CLEC-2-induced platelet activation results in cytosolic calcium spiking, which was confirmed by single-platelet total internal reflection fluorescence microscopy imaging. Our results suggest a refined picture of the platelet signal transduction network associated with CLEC-2. We show that tyrosine kinase activation is not the only rate-limiting step in CLEC-2-induced activation of platelets. Translocation of receptor-agonist complexes to the signaling region and linker adaptor for T cell signalosome formation in this region are limiting CLEC-2-induced activation as well.

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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.