Protease-Activated Receptor 3 Negatively Regulates Plasmin-Mediated Protease-Activated Receptor 4 Activation in Platelets.

Plasmin, a major extracellular protease, causes intracellular signals to mediate platelet aggregation. Previously, we reported that plasmin-mediated platelet aggregation predominantly occurs through proteolytic cleavage of protease-activated receptor 4 (Quinton et al J. Biol. Chem 2004). We showed that plasmin caused aggregation of mouse platelets more readily than human platelets. In this study, we investigated the mechanism of such a differential sensitivity of mouse platelets to plasmin, using transfected cell lines and platelets. In the platelet system, plasmin caused both human and mouse platelets to shape change and aggregate in a concentration-dependent manner with a different efficiency. Whereas 0.1 U/ml of plasmin causes full aggregation of mouse platelets, that dose only induces shape change in human platelets. In transfected COS7 cells, 1 U/ml plasmin caused a higher intracellular calcium mobilization through mouse PAR4 (mPAR4) than human PAR4 (hPAR4) activation. These results indicate that the mPAR4 is primarily more readily activated by plasmin than hPAR4, possibly due to differences in the primary sequence. Further, mouse and human platelets also differ in terms of the expression of PAR3. It is known that PAR3 acts as a co-receptor for thrombin-induced PAR4 activation. In order to evaluate the contribution of mouse PAR3 to plasmin-mediated mPAR4 activation, we co-expressed mPAR3 and mPAR4 in COS7 cells. Plasmin caused lower mobilization of intracellular calcium when mPAR3 and mPAR4 are co-expressed, compared to the expression of mPAR4 alone. These results indicate that mPAR3, instead of acting as a cofactor to potentiate mPAR4 activation, inhibits plasmin-mediated mPAR4 activation. Consistent with these results, PAR3 null mouse platelets also show a greater plasmin-induced calcium mobilization and aggregation compared to wild-type mouse platelets, which express PAR3 and PAR4. In conclusion, mouse platelets are more readily activated by plasmin than human platelets due to differences in the primary sequence of PAR4. In addition, mPAR3 acts as an inhibitory receptor to plasmin-mediated PAR4 activation, instead of acting as a co-receptor.