![Figure 1. Mechanisms and functional consequences of p38 activation in thrombin-stimulated platelets. (A) Thrombin-induced p38 phosphorylation is mediated by ADP and TxA2 secretion. Washed human platelets (4 × 108/mL) were preincubated for 5 minutes with either buffer alone, apyrase (0.1, 1, and 2 U/mL), AR-C (50 and 100 nM), MRS (100 μM), indomethacin (Ind, 5 μM), or AR-C (50 nM) and indomethacin (5 μM) together, and then stimulated with thrombin (0.005 U/mL) for 1 minute. Samples were then analyzed for P-selectin expression (fluorescence-activated cell sorter [FACS]), and phosphorylation of VASP (serine 239), p38, and Hsp27 (VASP-P, p38-P, Hsp27-P) on Western blots using phosphospecific antibodies. Data for P-selectin (mean ± SEM) are shown as fold increase with respect to the effect of thrombin alone designated as 1 (*significantly different at P < .05 compared with thrombin; n = 5). p38 inhibitors had no significant effect on thrombin-induced P-selectin expression (B-C), aggregation (D), and intracellular calcium mobilization (E). (B-C) Washed human platelets were preincubated with either DMSO, SNP (10 μM), p38 inhibitors SB203580 (20 μM) and SB202190 (20 μM), or control compound SB202474 (20 μM) for 5 minutes. Platelets were then stimulated with thrombin (0.005 U/mL [B] or 0.01 U/mL [C]). Data, expressed in arbitrary units for P-selectin, represent mean ± SEM for 4 independent experiments, with thrombin-stimulated samples designated as 1 (*significantly different at P < .05 compared with thrombin). Also shown are effects on VASP-P (Ser239), p38-P, and Hsp27-P obtained in at least 3 independent experiments. (D) Washed platelets were assayed for thrombin (0.005 U/mL)–induced aggregation in a turbidometric aggregometer. Platelets were preincubated for 5 minutes with RGDS (1 mM), SNP (10 μM), p38 inhibitors (SB203580, SB202190, 20 μM), a control (inactive) compound (SB202474, 20 μM), or DMSO, and then stimulated with thrombin. (E) Under similar experimental conditions, the effect of p38 inhibitors and the control compound on intracellular calcium was analyzed in platelets activated by 0.005 U/mL thrombin. Representative aggregation and calcium tracings from 3 independent experiments are shown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/109/2/10.1182_blood-2006-07-038158/4/zh80020706430001.jpeg?Expires=1735537157&Signature=Y261WV3lx9RzMZPNS7jOliF1L8W8uA6CZlFXKCTesFNdqdGNtx5kfFT2i4uZbmn-t-AiWaCEVZOqSAuS4xJLiPGfMunRMhiY9~bReyfT6RQ7eaz0yDeDJg4meODScDOQftkZAtrLvWtbgfj~GSu3kw1X0KZVRxEsoTyJIy11xoAIxlsFXLHVlVTZ546giEcMZTk6LAfnkURns23Q1RO-7CkE6ZW5BTx5lkKxre6VGz7GD2im61QAwNF7197q3gTYuTzntnf7WJJo4Bz-2B6Qjs7WbwEOjC6DBd06DOa2Kf5XoCqkMqNa0QZZkpf7riSkcVv8lLUd64yfXHN7fYJXuQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Mechanisms and functional consequences of p38 activation in thrombin-stimulated platelets. (A) Thrombin-induced p38 phosphorylation is mediated by ADP and TxA2 secretion. Washed human platelets (4 × 108/mL) were preincubated for 5 minutes with either buffer alone, apyrase (0.1, 1, and 2 U/mL), AR-C (50 and 100 nM), MRS (100 μM), indomethacin (Ind, 5 μM), or AR-C (50 nM) and indomethacin (5 μM) together, and then stimulated with thrombin (0.005 U/mL) for 1 minute. Samples were then analyzed for P-selectin expression (fluorescence-activated cell sorter [FACS]), and phosphorylation of VASP (serine 239), p38, and Hsp27 (VASP-P, p38-P, Hsp27-P) on Western blots using phosphospecific antibodies. Data for P-selectin (mean ± SEM) are shown as fold increase with respect to the effect of thrombin alone designated as 1 (*significantly different at P < .05 compared with thrombin; n = 5). p38 inhibitors had no significant effect on thrombin-induced P-selectin expression (B-C), aggregation (D), and intracellular calcium mobilization (E). (B-C) Washed human platelets were preincubated with either DMSO, SNP (10 μM), p38 inhibitors SB203580 (20 μM) and SB202190 (20 μM), or control compound SB202474 (20 μM) for 5 minutes. Platelets were then stimulated with thrombin (0.005 U/mL [B] or 0.01 U/mL [C]). Data, expressed in arbitrary units for P-selectin, represent mean ± SEM for 4 independent experiments, with thrombin-stimulated samples designated as 1 (*significantly different at P < .05 compared with thrombin). Also shown are effects on VASP-P (Ser239), p38-P, and Hsp27-P obtained in at least 3 independent experiments. (D) Washed platelets were assayed for thrombin (0.005 U/mL)–induced aggregation in a turbidometric aggregometer. Platelets were preincubated for 5 minutes with RGDS (1 mM), SNP (10 μM), p38 inhibitors (SB203580, SB202190, 20 μM), a control (inactive) compound (SB202474, 20 μM), or DMSO, and then stimulated with thrombin. (E) Under similar experimental conditions, the effect of p38 inhibitors and the control compound on intracellular calcium was analyzed in platelets activated by 0.005 U/mL thrombin. Representative aggregation and calcium tracings from 3 independent experiments are shown.
