ИСТИНА

Background: Under physiological conditions, platelets are stimulated by several agonists simultaneously. Moreover, receptor network can be important even when not all of them are occupied by ligands. Aims: We developed a computational systems biology model of platelet signaling encompassing receptors PAR1, PAR4, P2Y12, P2Y1 and pathways to integrin activation and procoagulant activity in order to investigate the interplay of Gq- and Gi-dependent signaling. Methods: Kinetics of fibrinogen and annexin V binding to Fura Redloaded platelets were investigated by continuous flow cytometry, confocal and total internal reflection fluorescence microscopy. The model comprised set of ordinary differential equations integrated in COPASI software (www.copasi.org). Results: The model included about one hundred reactions governing signal transduction after ligation of G-protein coupled receptors and leading to cytosolic calcium increase and protein kinase A (PKA) inactivation. PKA and calcium then influenced CalDAGGEFI activation and subsequent Rap1b-GTP formation and integrin activation. The validation of the model was conducted using experiments on cytosolic calcium, fibrinogen and annexin V binding dynamics after platelet stimulation with a combination of ADP and thrombin or PARactivating peptides. The model predicted that ADP would increase the frequency of calcium oscillations, the levels of fibrinogen binding and procoagulant activity, and P2Y12 antagonists would decrease them even when platelets are stimulated via PAR1 only. The predictions were confirmed experimentally. | 1251 Conclusions: Here we presented the first comprehensive model describing platelet intracellular signaling after simultaneous activation by ADP and thrombin. PKA is a crucial controlling element in platelet response even when stimulating signal comes though PAR receptors and does not contain ADP.