Stimulation of platelets with GPCR agonists do not induce GPVI shedding. (A) Western blot for GPVI after platelet stimulation by GPVI agonists, thrombin, or A23187. Washed platelets (500 × 109/L) were stimulated with collagen (30 µg/mL), CRP (30 µg/mL), thrombin (1 U/mL), and calcium ionophore (A23187: 10 µM), a positive control for GPVI shedding, in suspension under stirring conditions for 1 h at 37°C, in the presence of eptifibatide (9 µM) and CaCl2 (1 mM). Representative figure of data from at least 12 individual donors. Membranes were blotted with an anti-GPVI antibody for GPVI (60-65 kDa) and the GPVI remnant band (10-17 kDa) observed after shedding. (B) Western blot for GPVI after platelet stimulation with GPCR agonists, PAR-1 peptide (SFLLRN: 100 µM), PAR-4 peptide (AYPGKF: 100 µM), U46619 (10 µM), U46619 (10 µM) +ADP (10 µM), and ADP (10 µM) in the presence of eptifibatide (9 µM) and CaCl2 (1 mM) under the same conditions as before. Membranes were blotted with an anti-GPVI antibody for GPVI, as stated earlier. (C) Quantitation analysis of GPVI shedding after platelet stimulation with various GPVI and GPCR agonists. GPVI shedding represented as percentage of intact GPVI remaining compared with unstimulated GPVI levels. Results are shown as mean ± standard error of the mean. A 1-way ANOVA with Tukey’s posttest was performed to compare shedding with unstimulated platelets. n = 8+ donors.
