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

Abstract

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.