Rho/Rho-kinase downstream of G(12/13) plays an important role in the regulation of calcium-independent platelet shape change. We have previously shown that proline-rich tyrosine kinase 2 (Pyk2) is activated downstream of G(12/13) pathways. In this study, we evaluated the role of Pyk2 in G(12/13)-induced platelet shape change. We used low concentrations of YFLLRNP, a heptapeptide binding to protease-activated receptor 1 (PAR1), or PAR4-activating peptide AYPGKF in the presence of G alpha q inhibitor YM254890 to selectively stimulate G(12/13) pathways. We found that G(12/13)-induced platelet shape change was completely inhibited in the presence of Pyk2 inhibitors AG17 and TAT-Pyk2-CT, suggesting an important role of Pyk2 in platelet shape change. In addition, AYPGKF-induced shape change in Gq -/- platelets was completely inhibited in the presence of AG17 or RhoA/p160(ROCK) inhibitor Y27632, confirming the role of Pyk2 in RhoA-dependent shape change. Furthermore, AYPGKF-induced platelet aggregation and dense granule secretion were inhibited by blocking Pyk2 or RhoA. Finally, G(12/13)-induced myosin phosphatase target subunit 1 (MYPT1) phosphorylation was inhibited by AG17, confirming that Pyk2 regulates RhoA/p160(ROCK) activation in platelets. These results demonstrate that Pyk2 downstream of G(12/13) pathways regulates platelet shape change as well as platelet aggregation and dense granule secretion through the regulation of RhoA/p160(ROCK). (C) 2020 Elsevier Inc. All rights reserved.