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. Keywords: intracellular signalling/platelet receptors/tyrosine kinases/computational modelling AbbreviationsCLEC-2 -C-type lectin-like receptor 2; LEC -lymphatic endothelial cells; GPVI -glycoprotein VI; FcR -Fc receptor; ITAM -immune tyrosine activation motif; Syk -spleen tyrosine kinase; SFK -Src-family kinase; PI3K -phosphoinositide 3-kinase; PIP2 -phosphatidylinositol 4,5-bisphosphate; PIP3phosphatidylinositol 3,4,5-trisphosphate; LAT -linker adapter for T-Cells; PLCɣ2 -phospholipase Cɣ2; PGI2 -prostaglandin I2; mβCD -methyl β cyclodextrin; TIRF -total internal reflection; PTP1B -protein tyrosine phosphatase 1B; Btk -Bruton's tyrosine kinase, TxA2 -Thromboxane A2.

Development of a Simple Kinetic Mathematical Model of Aggregation of Particles or Clustering of Receptors

“…Other models for protein aggregation [23,32] also contain restrictions on their parameter values that are applicable to their particular case but are not compatible with other aggregation cases. Since the "2-equation model" is not detailed, it cannot give specific information about the system, but as it resembles general behavior, this model could be used as part of more complex systems such as thrombus formation models [62,63], or it could be introduced in the Kinase-Phosphatase Segregation model of TCR activation to more accurately account clustering process [64] or into the mathematical models of platelet activation [65,66].…”

Heterogeneity of Integrin αIIbβ3 Function in Pediatric Immune Thrombocytopenia Revealed by Continuous Flow Cytometry Analysis

“…In order to understand whether the increased calcium concentration in a quiescent state is linked to integrin activation, we developed a computational systems biology model of platelet calcium signaling and integrin activation (Figure 3a) based on our previously published models on platelet intracellular signaling [39][40][41][42]. The model had all compartments and major calcium signaling mechanisms.…”

“…However, at 15 times (100 µL LRP per 1530 µL Tyrode's) dilution, platelet reactivity appeared to be increased, yet this effect was not statistically significant (Figure S8d-f). Based on our additional experiments, this is based not on the increased plasma concentration, but on the increased platelet concentration, at which secondary platelet activation upon secretion becomes more pronounced [42,71,72]. To test the potential impact of the antiplatelet antibodies in ITP patient plasma, we have pre-incubated platelets with Tyrode's buffer, healthy donor platelet free plasma or ITP donor platelet free plasma under stirring conditions for 30 min.…”

“…A systems biology model of platelet calcium signaling was based on the ones developed previously [39,41,42]. In contrast to its predecessors, which were pure calcium signaling/homeostasis models, it contained an integrin activation module (Table S2).…”