p38 and MEK inhibitors inhibit MLC phosphorylation and clot retraction. (A) Washed platelets at a concentration of 4 × 107/mL were preincubated with 500 μM aspirin and with 50 μM P2Y12 inhibitor, 2MeSAMP for 5 minutes at 37°C. Platelets were also pretreated with either 10 μM SB203580, 10 μM PD98059, or equal volume DMSO for 30 minutes at 25°C and added to polystyrene dishes coated with 100 μg/mL fibrinogen to induce adhesion and spreading at 37°C for 60 minutes, or kept in suspension. After washing to remove nonadherent platelets, adherent platelets or control platelets were solubilized as described in “Immunoblot detection of p38, ERK, and MLC phosphorylation in platelets.” Phosphorylation of p38, ERK, and MLC were analyzed by Western blot analysis using specific antibodies against phosphorylated p38, ERK, and MLC. A monoclonal antibody against tubulin was used to verify equal loading. (B,C) Platelet-rich plasma was anticoagulated with 0.38% sodium citrate and preincubated with (C) or without (B) 1 mM aspirin for 5 minutes at 25°C and with p38 and MEK inhibitor as indicated. Coagulation was induced with 0.2 U/mL thrombin, and clots were allowed to retract at 37°C and photographed. (D) Platelet-rich plasma was preincubated with either vehicle control, 10 μM SB203580, 3 μM U0126, or both inhibitors. Coagulation was induced with 0.2 U/mL thrombin, and clots were allowed to retract at 37°C and photographed. (E-G) Two-dimensional retraction of clots in panels B through D were measured using NIH ImageJ and expressed as percent retraction (mean ± SD). Statistical significance was determined using a Student t test.
