ИСТИНА

Background: Platelet surface receptor CLEC2 has a significant role in maintaining blood vascular integrity during inflammation. It shares signaling pathways with GPVI receptor. However, CLEC2 activation is notably delayed comparing to GPVI and the nature of this delay remains obscure. Different concepts of CLEC2 signaling have been proposed, for example Hughes et al. (Blood, 2015) believe that activation of Syk kinase lies upstream of SFK and Btk, while Manne et al. (J. Biol. Chem. 2015) assume the opposite. And whether CLEC2 activation affects platelet calcium signaling remains questionable. Aims: Systems biology analysis of intracellular signaling after ligation of CLEC2 in single platelets. Methods: A comprehensive 3D model of CLEC2 signaling was constructed based on experimental data and solved in VCell software (vcell.org). Fucoidan (Sigma 9072-19-9) was utilised as CLEC2 agonist in all experiments. For single platelet analysis Fura-2 loaded platelets were immobilised on fibrinogen and analised by TIRF microscopy. Alternatively, spreading of platelets on immobilized fucoidan was observed by the same technique. For model validation platelet activation by fucoidan was analised by aggregometry, flow cytometry and spectrofluorimetry. Results: The model describes diffusion of CLEC2 molecules to the lipid raft and recruitment of downstream proteins to the signalosome. Signaling scheme is described in fig 1. Changing cholesterol saturation of cell membrane affected diffusion speed of the CLEC2 molecules, thus slowing down formation of the CLEC2 cluster. The model predicted cytosolic calcium spiking, which was confirmed in experiments with single platelets. Conclusions: Slow activation of platelets by CLEC2 is due to the diffusion of it‘s oligomers to the signalosome, where a lipid raft prevents the cluster from the dissolving. CLEC2 activation leads to cytosolic calcium oscillations in single cells. Suggested scheme explains initial CLEC2 signaling stages and ties up representations of Hughes et al. and Manne et al.