Effects of HA1077 and Y-27632 on agonist-induced ATP secretion of human platelets and MLC phosphorylation at Ser¹⁹ in intact platelets. (A) Effects of HA1077 and Y-27632 on agonist-induced ATP secretion. Human platelets incubated for 3 minutes in the aggregometer with various concentrations of HA1077 (left panel) or Y-27632 (right panel) were stimulated with 0.1U/mL thrombin (•), 0.05 U/mL thrombin (○), 1 μmol/L STA₂(▪), or a mixture of 1 μmol/L serotonin and 1 μmol/L epinephrine (▴) for 30 seconds, without stirring. Control levels of ATP secretion induced by 0.1 U/mL thrombin, 0.05 U/mL thrombin, STA₂, and a mixture of serotonin and epinephrine were 354 ± 23.1, 184 ± 15.5, 173 ± 13.1, and 39.6 ± 2.58 pmol/10⁸ platelets (n = 3), respectively. The values represent the average of three experiments (SD <10%). (B) Effects of HA1077 and Y-27632 on agonist-induced MLC phosphorylation at Ser¹⁹ in intact platelets. Washed platelets preincubated with various concentrations of HA1077 (left panel) or Y-27632 (right panel) were stimulated with 0.1 U/mL thrombin (•), 0.05 U/mL thrombin (○), 1 μmol/L STA₂ (▪), or a mixture of 1 μmol/L serotonin and 1 μmol/L epinephrine (▴), without stirring. The extent of 20-kD MLC phosphorylation was expressed as the percentage of 20-kD MLC in the monophosphorylated form. The values represent the average of three experiments (SD <10%)