GPIb-IX–mediated cPLA2 activation in WT and LIMK1^−/− platelets, and the effect of MAPK inhibitors on LIMK activation. (A) Immunoblot analysis of WT and LIMK1^−/− platelets, stimulated in the presence or absence of 5 µg/mL VWF, 2 µg/mL botrocetin, or both, with antibodies recognizing phosphorylated cPLA2 Ser⁵⁰⁵(S⁵⁰⁵), total cPLA2, phosphorylated p38 ^{Thr180/Tyr182}(T¹⁸⁰/Y¹⁸²), or total p38. (B) Quantitative data from 4 experiments depicted in (A) using ImageJ (uncalibrated OD, mean ± SE). (C) Human platelets, preincubated with 25 μM SB203580, 3.5 μM U0126, or DMSO for 2 minutes, were stimulated with VWF (10 µg/mL) in the absence or presence of ristocetin (0.25 mg/mL) or stirred without stimulation in a platelet aggregometer at 37°C for 6 minutes. Lysates from these platelets were immunoblotted with antibodies against phosphorylated LIMK Thr^505/508 or against phosphorylated cPLA2 Ser⁵⁰⁵, and also with antibodies against LIMK1 and cPLA2 as loading controls. (D) Quantitative data for (C) expressed as a percentage of DMSO control.