Diffusional and chemical control in the tyrosine kinase network of platelet CLEC-2 signalling

Abstract

C-type lectin-like receptor 2 (CLEC-2) is platelet membrane glycoprotein implicated in maintenance of blood vessel integrity and development of lymphatics. Organization and regulation of tyrosine kinase signalling network associated with CLEC-2 is poorly understood. Here we aimed to investigate CLEC-2 signal transduction using computational systems biology methods in combination with experimental approaches. We developed a 3D/stochastic multicompartmental computational model of CLEC-2 signalling, which was built around membrane-associated signalosome formation. The model predicted that both Syk and Src-family kinases phosphorylate CLEC-2, and that CLEC-2 ligation induces cytosolic calcium spiking. This was experimentally confirmed using flow cytometry and TIRF microscopy, respectively. Sensitivity analysis suggested that the CLEC-2 translocation to the signalosome region is one of the rate-limiting steps in the signal transduction process. In agreement with this prediction, CLEC-2-induced platelet activation was strongly temperature-dependent (unlike that mediated by G-protein coupled receptors) and was delayed by lipid raft disruption. Our results suggest a revised picture of the CLEC-2 signal transduction network functioning that emphasizes the crucial role of lipid raft structural rearrangement followed by tyrosine kinase feedback interplay.