Effect of CBPS on platelet activation and ADAM17 activity. (A) Detection of P-selectin surface expression by flow cytometry in platelets treated with CBPS or vCBPS. Gel-filtered platelets were incubated with PMA (10 μM), CBPS (50 μΜ), or vCBPS (50 μΜ) at 37°C for 15 minutes. Platelets were fixed with formaldehyde and stained with PE-conjugated mouse anti-human CD62P antibody. Data are representative of at least 3 independent experiments using different donors. (B) A fluorogenic substrate was used to detect ADAM17 activity on platelets incubated with 50 µM CBPS or vCBPS for 30 minutes at 37°C. Fluorescence (320/405 nm) was quantified, and all values were corrected for background fluorescence. *P < .05 compared with control (n ≥ 3). N.S., not significant. (C) Gel-filtered platelets were preincubated with GM6001 (100 μΜ) or TAPI-2 (10 μΜ), incubated with CBPS (50 μΜ) or PMA (10 μΜ), and then immunoblotted for full length and soluble Sema4D. **P < .01 (n = 3).