Agonist-induced PKD activation in platelets is dependent on classic PKC isoforms but independent of novel PKC isoforms. (A) Washed human platelets (2 × 10⁸/mL) were incubated with GFX (10μM), LY333531 (10μM), or vehicle (DMSO) for 10 minutes and subsequently stimulated with CRP (5 μg/mL) or thrombin (1 unit/mL) for 1 minute. (B) Washed human platelets (2 × 10⁸/mL) were incubated with various concentrations of LY333531 or vehicle (DMSO) for 10 minutes and subsequently stimulated with thrombin (1 unit/mL) for 1 minute under stirring conditions. Immunoblots were performed with P-PKD (Ser916), P-PKD (Ser744), or total PKD antibody. One representative experiment of 3 with identical results is shown. (C) Washed platelets (2 × 10⁸/mL) from WT, PKCα^−/−, PKCθ^−/−, or PKCδ^−/− mice, were incubated with various concentrations of CRP or thrombin for 1 minute. Immunoblots were performed with either P-PKD (Ser916) or total PKD antibody. One representative experiment (i) and densitometric analysis of 3 experiments (ii) are shown. For panel Cii, data shown are means ± SEM (n = 3).