Addition of membrane-permeable CBPS causes dissociation of the Sema4D–calmodulin complex and cleavage of Sema4D. (A) Platelets were incubated with 10 μΜ FITC-myr-CBPS, FITC-CBPS, FITC-myr-vCBPS, or FITC-vCBPS for 15 minutes, fixed with formaldehyde, and analyzed by flow cytometry. (B) Platelets were treated with 10 μΜ FITC-myr-CBPS, FITC-CBPS, FITC-myr-vCBPS, or FITC-vCBPS peptide for 30 minutes, allowed to spread on immobilized fibrinogen for 60 minutes, and examined by differential interference contrast (DIC) and confocal fluorescence microscopy. The images are representative of at least 3 independent experiments. (C) Platelets (5 × 10⁸/mL) were incubated with 5.0 μΜ CBPS or vCBPS for 30 seconds, lysed, and immunoprecipitated with anti-Sema4D N-terminal antibody (1E8G9). Proteins were immunoblotted with an anti-calmodulin antibody and an anti-Sema4D C-terminal antibody (n = 3). (D) Gel-filtered platelets were incubated with 50 μΜ CBPS. Soluble 120-kDa fragment was detected with an N-terminal antibody. Full-length 150-kDa Sema4D in the platelet pellets was detected by using the Sema4D C-terminal antibody. (E) Gel-filtered platelets were incubated with CBPS or vCBPS for 60 minutes at 37°C. (F,G) Gel-filtered platelets were incubated with CBPS, vCBPS, or W7 for 60 minutes at 37°C. Platelet pellets and supernatants were separated and blotted with GPVI antibody (6B-12) or GPIbα antibody (SZ2). *P < .05; ***P < .001 compared with control (n = 3). N.S., not significant (n = 3).