ИСТИНА

Introduction. Platelet activation is triggered by interaction of different agonists with their receptors. Most physiological agonists, ADP and thrombin among them, bind to the G-protein-coupled receptors (GPCRs) leading to phospholipase Cβ (PLC) activation, inositol-3-phosphate (IP3) generation, and increase in cytoplasmic calcium concentration, which mediates a hierarchy of platelet responses. ADP activation of P2Y12 leads to cAMP decrease and protein kinase A (PKA) deactivation. Strong platelet activation with thrombin and/or GPVI agonists induces necrosis in a fraction of platelets, while weak activation with ADP or thromboxane A2 does not, though ADP can potentiate response to thrombin. The mechanisms of the interactions between the pathways and regulation of this signalling network are unclear. Aims. To develop a computational model of tetra-receptor (P2Y1-P2Y12-PAR1-PAR4) platelet signaling system and investigate platelet response to combination of thrombin and ADP. Methods. We developed a multicompartmental stochastic computational systems biology model of tetra-receptor thrombin-ADP signaling in platelets. Experiments employing continuous flow cytometry of washed, Fura Red-loaded and annexin-V labeled platelets were used to validate the model and test its predictions. Results. The model predicted cytosolic calcium oscillations after activation with ADP, thrombin or their combination, even for high levels of activation. Increase of activation level on any scale (addition of ADP to thrombin or increase in thrombin concentration) led to increase in the oscillation frequency without changes in amplitude. The predicted fraction of necrotic platelets could be increased by 50% by ADP addition to thrombin at 1-100 nM in line with experimental data. Computer modeling indicates that these effects of ADP are due to additional PLC activation by βγ-subunits of G-protein after their inhibition by PKA has been removed. Conclusions. We present a first platelet tetra-receptor (P2Y1-P2Y12-PAR1-PAR4) computational model that predicts that the intracellular signals from the four receptors are integrated at the level of PLC activation.