ИСТИНА

Background: The serine protease thrombin is in the center of both plasma and cellular hemostasis. Human platelets possess two main receptors for thrombin, protease-activated receptors (PARs) PAR1 and PAR4. Both are capable of invoking all possible platelet activation responses and they have identical main intracellular signaling pathways. Although there is experimental evidence that they have different cleavage/inactivation kinetics (and some secondary variations in signaling), the reason for such redundancy is not clear: why there must be two discriminate receptors? Aims: Discrimination of the roles of PAR1 and PAR4 in platelet activation by thrombin. Methods: We developed a multicompartmental stochastic computational systems biology model of dual-receptor thrombin signaling in platelets. Experiments employing continuous flow cytometry of washed, Fura Red-loaded and annexin-V labeled platelets were used to validate model and test its predictions. Investigations were performed in accordance with the Declaration of Helsinki, and written informed consent was obtained from all donors. Results: The model described experimental data well; both receptors induced calcium spiking, with rapid and short-lived response from PAR1, while signaling by PAR4 developed slowly and propagated in time. Response of the dual-receptor system was both rapid and prolonged in time. PAR1 did not cause intracellular store depletion, while PAR4 did, which was followed by the activation of store-operated calcium entry. Different ratios of PAR1 and PAR4 led to different dynamics and dose-dependence of procoagulant platelet formation, which could explain the observed variability between donors. Conclusions: The dual-receptor combination is critical to produce a response combining three critical features: sensitivity to thrombin concentration, rapid onset and steady propagation; specific features of the protease-activated receptors do not allow combination of all three in a single receptor.