Trypsin Causes Platelet Activation Independently of Known Protease-Activated Receptors

Abstract 2197

In platelets, Thrombin is the major agonist for PAR1 and PAR4, although PARs can be activated by other proteases. The physiological agonist of PAR3 has not been identified to date. To identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, G_q inhibitor YM-254890 or calcium chelator BAPTA-AM had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was completely abolished by ROCK inhibitor Y27632 or H1152, suggesting that trypsin-induced PAR4 null murine platelet shape change is mediated by G12/13, but not Gq pathway. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. To investigate whether Trypsin activated PAR3, we desensitized PAR4 in both WT and PAR3 null murine platelets. Surprisingly, trypsin caused similar shape change in PAR4-desensitized PAR3 null murine platelets as in PAR4 null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, a Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signaling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signaling pathway in platelets that is independent of PARs. This protease-induced novel signaling pathway regulates platelet shape change through SFKs and p160ROCK.