Control of Platelet CLEC-2-Mediated Activation by Receptor Clustering and Tyrosine Kinase Signaling

Abstract

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.

Platelet calcium signaling by G-protein coupled and ITAM-linked receptors regulating anoctamin-6 and procoagulant activity

“…When stimulated by the snake venom rhodocytin, CLEC2 appeared to have a no more than limited role in platelet procoagulant activity [52]. This is in agreement with the reported moderate Ca 2+ rise induced by CLEC2 activation [53].…”

ASGR1 and Its Enigmatic Relative, CLEC10A

“…At high receptor occupancy, the receptor may aggregate to a greater extent, which would exclude CD45, the ubiquitous protein tyrosine phosphatase on immune cells, and allow the phosphorylated receptors to prolong the initiation signal. Clustering (dimerization) of the platelet receptor CLEC2 was required for phosphorylation by Syk/Src kinases of the hemITAM sequence (YITL) within the cytoplasmic domain and activation of downstream signaling, which involves PLCγ2 [177], a pathway similar to that for CLEC10A [89]. Ligand binding induced an increase in cytosolic Ca 2+ that reached a maximal level about 90 sec after adding ligand, which was dependent upon the fluidity/stability of the membrane [177].…”

“…Clustering (dimerization) of the platelet receptor CLEC2 was required for phosphorylation by Syk/Src kinases of the hemITAM sequence (YITL) within the cytoplasmic domain and activation of downstream signaling, which involves PLCγ2 [177], a pathway similar to that for CLEC10A [89]. Ligand binding induced an increase in cytosolic Ca 2+ that reached a maximal level about 90 sec after adding ligand, which was dependent upon the fluidity/stability of the membrane [177]. CLEC10A occurs on cell membranes as a trimer [178] and may form larger clusters with multivalent ligands.…”

In Silico Hemostasis Modeling and Prediction

“…The examples of successful models are usually not the stories of model development but rather stories of hypotheses tested and predictions performed; hypotheses that were too complex to be addressed with pure experiments. Roles of the negative feedback in the regulation of platelet activation via GPVI, 59 importance of spatial receptor distribution rather than copy numbers in the regulation of the response, 60 identification of the controlling stages in CLEC-2 signaling, 54 developing hypotheses for heterogeneity of integrin response in immune thrombocytopenia, 61 the role of mitochondrial calcium integration in procoagulant platelet formation, 50,62 importance of platelet surface-to-volume ratio and the number of mitochondria in programmed cell death of Wiskott-Aldrich syndrome platelets as a possible mechanism behind thrombocytopenia, 52 and interplay of protease-activated receptor 1 (PAR1) and PAR4 to form the optimal response of platelets to thrombin in time-and concentration-dependent manner 55 ; these are examples of how one might use a model of a signaling pathway to learn new information about the system.…”