Shear induces GPVI shedding in washed platelets. Washed platelets (5 × 10⁸ platelets/mL) were left untreated (NT), or treated with either 0.5 μg/mL convulxin for 15 minutes, subjected to shear (10 000 seconds⁻¹) for various times, or subjected to different shear rates for 5 minutes, with and without 100μM GM6001 (A-E) or up to 2.5μM GI254023 (C). (A) Platelet pellets were lysed and analyzed by SDS-PAGE and immunoblotting using anti-GPVI cytoplasmic tail antibody (top panel) or anti-α_IIbβ₃ mAb CRC54 (bottom panel). (B) Supernatants were blotted with anti-GPVI mAb 12H1. (C) Platelet pellets were lysed and analyzed by SDS-PAGE and immunoblotting using anti-GPVI cytoplasmic tail antibody. Densitometry measurements (relative intensities) of each GPVI tail fragment in blot C were compared relative to the densitometry of the fragment detected in the 10 000-second⁻¹/5-minute sample. (D-E) Platelet pellets were lysed and analyzed by SDS-PAGE/Western blotting using (D) anti-GPV or (E) anti-GPIbα cytoplasmic tail antibodies.