Fibrin stimulation of platelets induces GPVI shedding. (A) Western blot for GPVI after stimulation of platelets in suspension by fibrinogen, fibrin, or fibrin in the presence of inhibitors. Washed platelets (500 × 109/L) were stimulated with fibrinogen (100 µg/mL) alone, thrombin (1 U/mL) in the presence of fibrinogen (polymerized fibrin), fibrin in the presence of Src and Syk inhibitors (dasatinib, 10 µM; PRT-060318, 10 µM), fibrin in the presence of GI254023 (2 µM, ADAM10 inhibitor) and GM6001 (25.7 µM, broad matrix metalloproteinase inhibitor), and monomeric fibrin in the presence of GPRP (10 mM) under stirring conditions for 1 h at 37°C in the presence of eptifibatide (9 µM) and CaCl2 (1 mM). GPRP was added with fibrinogen 3 minutes before thrombin stimulation to prevent fibrin polymerization. GI254023 and GM6001 were added 5 minutes before fibrinogen and thrombin stimulation. Membranes were blotted for GPVI and GPVI remnant after shedding. (B) Quantitation analysis of GPVI shedding induced by different forms of fibrin and fibrin-induced shedding in the presence of inhibitors. Percentage of GPVI represents levels calculated as a percentage of GPVI detected in unstimulated samples compared with GPVI levels after stimulation. Results are shown as mean ± SEM. A 1-way ANOVA was performed, with Tukey’s posttest, to compare fibrin-induced shedding in the presence of GPRP and the presence of inhibitors and fibrinogen alone to unstimulated samples. N.S., nonsignificant; n = 7 donors.
