Inhibitory effects by a PKC inhibitor GF109203X on PMA- or STA₂-induced phosphorylation of MBS at Thr-696, CPI, and MLC₂₀ phosphorylation at Ser-19 in intact platelets.

Human platelets pretreated with various concentrations of GF109203X for 2 minutes were stimulated with 100 nM PMA (A) or 0.5 μM STA₂ (B) for 30 seconds at 37°C under conditions of nonstirring, and 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. Inhibitory effects by GF109203X on agonist-induced phosphorylation of CPI (▴), MBS at Thr-696 (■), and MLC₂₀ at Ser-19 (●) were expressed as a percentage of the phosphorylation level of platelets induced by each agonist. Inhibition of PMA-induced MBS phosphorylation by GF109203X was expressed as a percentage of the phosphorylation level found in STA₂-stimulated platelets without GF109203X. Values represent the average of 3 experiments (SD < 10%).