Inhibition by Y-27632, GF109203X, or both of STA₂-induced ATP secretion and phosphorylation of MLC₂₀ at Ser-19, MBS at Thr-696, and CPI in intact platelets.

Human platelets pretreated without or with 20 μM Y-27632, 5 μM GF109203X, or both for 3 minutes were stimulated with 0.5 μM STA₂ under conditions of nonstirring. Incubation was carried out for 30 seconds at 37°C to measure protein phosphorylation and for 2 minutes at 37°C to measure ATP release. Whole platelet lysates originating from 30 μL of platelet suspension were resolved by SDS-PAGE. This was followed by immunoblotting with anti–pThr695-MBS, anti–pThr38–CPI-17, and anti–pSer19-MLC₂₀ antibodies. MLC₂₀phosphorylation at Ser-19 was expressed as fold increase in the basal value, without the addition of inhibitors. Similar results were obtained in 3 other experiments using different donor platelets.