In vitro phosphorylation of platelet MBS and inactivation of myosin phosphatase by Rho-kinase. (A) Coimmunoprecipitation of Rho-kinase with platelet MBS. Immunoprecipitates with anti-MBS antibodies were immunoblotted with antibodies against MBS (left) and Rho-kinase (right). IP, immunoprecipitation antibodies used; IB, immunoblotting antibodies used; Ig, cross-reacted Ig. (B) In vitro phosphorylation of platelet MBS. MBS immunoprecipitates from platelet lysates were incubated without RhoA, with GDP-RhoA, and with GTPγS-RhoA for 30 seconds, as described in Materials and Methods. Protein phosphorylation was analyzed by SDS-PAGE, followed by autoradiography. (C) Inhibitory effect of HA1077 and Y-27632 on GTPγS-RhoA–dependent phosphorylation of platelet MBS. Different concentrations of HA1077 (left panel) or Y-27632 (right panel) were included in the reaction mixture, and MBS phosphorylation was analyzed, as described in Materials and Methods. Results were expressed as the percentage of the value, without the addition of compounds. The results are representative of three independent experiments. (D) Effects of HA1077 and Y-27632 on the activity of myosin phosphatase derived from MBS immunoprecipitates in the presence of GTPγS-RhoA. MBS immunoprecipitates were incubated with 20 μmol/L HA1077 or 10 μmol/L Y-27632 in the presence of GTPγS-RhoA for 30 seconds, and the activity of myosin phosphatase was determined immediately. The value shows the mean ± SE from three experiments. *P < .05 (E) Effects of various compounds on GTPγS-RhoA–dependent phosphorylation of platelet MBS. Immunoprecipitates with anti-MBS antibody from platelet lysates were incubated for 30 seconds with 10 nmol/L M-1, 10 nmol/L yohimbine, 100 nmol/L ONO-3708, 100 nmol/L KT5926, or 50 μmol/L W-7 in the presence of GTPγS-RhoA, as described in Materials and Methods. Protein phosphorylation was analyzed by SDS-PAGE, followed by autoradiography. Similar results were obtained in three other experiments.