Abstract/Summary

Platelets are blood cells responsible for vascular integrity preservation. The activation of platelet receptor 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 modelling in combination with experimental approaches. We developed a stochastic multicompartmental computational model of CLEC-2 signalling. The model described platelet activation beginning with CLEC-2 receptor clustering, followed by Syk and SFK phosphorylation, determined by the cluster size. Active Syk mediated LAT protein phosphorylation and membrane signalosome formation, which resulted in the activation of Btk, PLC and PI3K, calcium and phosphoinositide signalling. The model parameters were assessed from published experimental data. Flow cytometry, TIRF 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 TIRF microscopy imaging. Our results suggest a refined picture of the platelet signal transduction network associated with CLEC-2. We show that the tyrosine kinases activation is not the only rate-limiting step in CLEC-2 induced activation of platelets. Translocation of receptor-agonist complexes to the signalling region and LAT-signalosome formation in this region are limiting CLEC-2-induced activation as well.