![Fig. 2. Agonist-induced phosphorylation of MBS and the inactivation of myosin phosphatase in intact platelets. (A) Agonist-induced phosphorylation of MBS in intact platelets. [32P]Pi-labeled platelets were stimulated for the indicated time with either 1 μmol/L STA2, 0.1 U/mL thrombin, 1 μmol/L serotonin, or 1 μmol/L epinephrine. *An unknown protein of 120 kD. (B) Inhibition by HA1077 and Y-27632 of STA2-induced phosphorylation of MBS. [32P]Pi-labeled platelets incubated for 5 minutes with various concentrations of HA1077 (left panel) or Y-27632 (right panel) were stimulated for 30 seconds with 1 μmol/L STA2, without stirring. Results were expressed as the percentage of the value without the addition of compounds. Similar results were obtained in three other experiments, using different donor platelets. (C) Effects of HA1077 and Y-27632 on STA2-induced inactivation of myosin phosphatase in intact platelets. Human platelets incubated with saline (control; □), 20 μmol/L HA1077 (▪), or 10 μmol/L Y-27632 (▦) for 5 minutes were activated with 1 μmol/L STA2without stirring. MBS was immunoprecipitated with anti-MBS antibody and the activity of myosin phosphatase was determined immediately, as described in Materials and Methods. Results are expressed as the percentage of the value, without the addition of compounds. Data represent the mean ± SE of four experiments. *P < .05, **P < .01 (D) The inhibitory effect of receptor antagonists on agonist-induced phosphorylation of MBS in intact platelets. [32P]Pi-labeled platelets were pretreated with 0.1 μmol/L ONO-3708, 10 nmol/L M-1, 10 nmol/L yohimbine, 10 μmol/L Y-27632, or 20 μmol/L HA1077 for 5 minutes at 37°C and were activated with 1 μmol/L STA2, 1 μmol/L serotonin, or 1 μmol/L epinephrine for 30 seconds, without stirring. Similar results were obtained in three other experiments, using different donor platelets. (E) Inhibition by HA1077 and Y-27632 of thrombin-induced phosphorylation of MBS. [32P]Pi-labeled platelets incubated for 5 minutes with 20 μmol/L HA1077 or 10 μmol/L Y-27632 were stimulated for 30 seconds with 0.1 U/mL thrombin without stirring. Similar results were obtained in three other experiments, using different donor platelets.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/93/10/10.1182_blood.v93.10.3408.410k37_3408_3417/5/blod41037002ew.jpeg?Expires=1724235102&Signature=MDUzlyYldnGQjSbibz1IaM1nXtNhjhqEdSzoB4JmmxLu1FwFBSy0TK1ogW3EZyLQIeCCS6l70Cb2ZmkZ3UKsE7KVjWoX1iBLrq8EGlZxyG1AObLPZ6DBcTbu0lfAMt3YcsHJS04GGt1kt2lSulyEC2oUx6m9XAyB-mtjTJ7lNUjL4P1oMHBKonnc9Ajenzu2wwfBJYzMNauntYPmySKWyV9MfdAnF8cnKEMxyC85pJXgsGT8Ed1gLjFYyL0uYQBRc8LyAKx-vsVHcPAhMU~ohIV1-6EBx2acU02DlUTDuWhELHK1RQhjkc75xc809PKbg11RXVAOD8ycUIK8SrzQ4w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Agonist-induced phosphorylation of MBS and the inactivation of myosin phosphatase in intact platelets. (A) Agonist-induced phosphorylation of MBS in intact platelets. [32P]Pi-labeled platelets were stimulated for the indicated time with either 1 μmol/L STA2, 0.1 U/mL thrombin, 1 μmol/L serotonin, or 1 μmol/L epinephrine. *An unknown protein of 120 kD. (B) Inhibition by HA1077 and Y-27632 of STA2-induced phosphorylation of MBS. [32P]Pi-labeled platelets incubated for 5 minutes with various concentrations of HA1077 (left panel) or Y-27632 (right panel) were stimulated for 30 seconds with 1 μmol/L STA2, without stirring. Results were expressed as the percentage of the value without the addition of compounds. Similar results were obtained in three other experiments, using different donor platelets. (C) Effects of HA1077 and Y-27632 on STA2-induced inactivation of myosin phosphatase in intact platelets. Human platelets incubated with saline (control; □), 20 μmol/L HA1077 (▪), or 10 μmol/L Y-27632 (▦) for 5 minutes were activated with 1 μmol/L STA2without stirring. MBS was immunoprecipitated with anti-MBS antibody and the activity of myosin phosphatase was determined immediately, as described in Materials and Methods. Results are expressed as the percentage of the value, without the addition of compounds. Data represent the mean ± SE of four experiments. *P < .05, **P < .01 (D) The inhibitory effect of receptor antagonists on agonist-induced phosphorylation of MBS in intact platelets. [32P]Pi-labeled platelets were pretreated with 0.1 μmol/L ONO-3708, 10 nmol/L M-1, 10 nmol/L yohimbine, 10 μmol/L Y-27632, or 20 μmol/L HA1077 for 5 minutes at 37°C and were activated with 1 μmol/L STA2, 1 μmol/L serotonin, or 1 μmol/L epinephrine for 30 seconds, without stirring. Similar results were obtained in three other experiments, using different donor platelets. (E) Inhibition by HA1077 and Y-27632 of thrombin-induced phosphorylation of MBS. [32P]Pi-labeled platelets incubated for 5 minutes with 20 μmol/L HA1077 or 10 μmol/L Y-27632 were stimulated for 30 seconds with 0.1 U/mL thrombin without stirring. Similar results were obtained in three other experiments, using different donor platelets.
