PAR4 Primarily Mediates Thrombin‐induced Morphological Changes in MEG‐01 Cells through PI3K/Akt and RhoA/ROCK Signaling Pathways

Platelet activation is critical in hemostasis and thrombosis. Thrombin most potently activates platelets via two protease-activated receptors (PARs): PAR1 and PAR4. While PAR1 activation results in a rapid and transient signal required for the initiation phase of platelet aggregation, PAR4 activation induces a sustained signal associated with later phases responsible for stable thrombus formation. The present study focused on the differential signaling pathways by PAR1 and PAR4 involved in thrombin-induced intracellular events in human megakaryoblastic leukemia cell line, MEG-01. Interestingly, we found that thrombin triggers not only Gq /PLC-mediated calcium signaling, but also G12/13 /RhoA/ROCK-mediated morphological changes in MEG-01 cells via PAR1 and PAR4, which showed close similarity to those observed in platelets. We developed a new image-based assay to quantify the morphological changes in living cells and extensively investigated on the underlying mechanism for thrombin-induced morphological changes in MEG-01 cells. Selective blockade of PAR1 and PAR4 by vorapaxar and BMS-986120, respectively, indicated that PAR4 activation primarily mediates thrombin-induced morphological changes in MEG-01 cells. Moreover, treatment of a set of kinase inhibitors showed that the PAR4-mediated morphological changes were predominantly mediated via PI3K/Akt and RhoA/ROCK signaling pathways. As in the platelets, PAR4-mediated signaling pathways are significant in thrombin-induced morphological changes in MEG-01 cells. Based on the close similarity to thrombin-induced signaling in platelets, MEG-01 cell line is a useful complementary in vitro model that can be used in research on platelets and thrombosis.