Platelet agonist–induced decrease of GP VI. Washed platelets were resting or stimulated with (A-I) collagen for 45 minutes at 37°C under static conditions. Whole platelet proteins were separated by SDS-PAGE under nonreducing conditions and immunoblotted with 9012.2 and C3a.19.5, followed by detection by ECL. The 98-kDa band corresponding to GP IIIa and the 63-kDa band corresponding to GP VI were optically scanned, and the density of the GP VI band relative to the GP IIIa band was calculated. This value was subtracted from the value found in control platelet lysate and expressed as the percent reduction from control. (A) Results are shown as percent reduction ± SEM, n = 5. Platelets were activated using (B) convulxin. (C) Platelets were resting (open bars) or stimulated with collagen 20 μg/mL (solid bars) for 30 seconds and 1, 5, 15, 25, and 45 minutes at 37°C under static conditions. Results are expressed as described above, n = 4 for each condition. (D-I) Washed platelets (3 × 10⁸/mL) were incubated at 37°C for 45 minutes in the presence of agonist, protease inhibitors were added, and the platelets were separated in an ultracentrifuge to remove microparticles. Supernatants from platelets activated in the presence of collagen and resting control lysate were loaded in equivalent amounts on SDS-PAGE under reducing conditions and electroblotted to membranes. Membranes were treated with polyclonal GP VI antibodies¹⁹  followed by anti–rabbit-HRP and detection by ECL. (D) The 63-kDa band corresponding to GP VI and the 57-kDa band corresponding to sGP VI were optically scanned, and the density of the sGP VI band relative to the GP VI band was calculated and expressed as percent released sGP VI. Results are shown as percent released ± SEM, n = 3. Platelets were activated using (E) mAb 9012.2 and (F) convulxin.