Effect of Src kinase inhibitors on 2MeSADP-induced cPLA₂ phosphorylation and TXA₂ generation. Washed and aspirin-treated human platelets were stimulated with 2MeSADP (100 nM), in the presence of fibrinogen (1 mg/mL) under stirring conditions for 60 seconds at 37°C (A). As per the requirement, platelets were preincubated with PP3 (10 μM), PP2 (10 μM), or PD173952 (25 μM) for 10 minutes at 37°C. The reaction was stopped using 3X sample loading buffer (with DTT). The lysates were subjected to Western blotting analysis and probed with phospho-cPLA₂ (Ser505) and PKCδ (lane loading control) (A). In the case of TXB₂ measurement (B), platelets were stimulated in the presence of Src kinase inhibitors or the inactive analog for 3.5 minutes at 37°C, following which the reactions were stopped and TXB₂ levels were analyzed as described in “Materials and methods.” The data are represented as the percent maximal TXB₂ generated by 2MeSADP in the absence of inhibitors. Each bar is the average of 3 experiments ± SEM from 3 different donors.