FXa induces GPVI shedding in washed platelets. Washed platelets resuspended at 5 × 10⁸/mL in Tyrode buffer had no treatment (NT) or were mixed with (A) 0.1 μg/mL CVX, 10 μg/mL purified FX or FXa (10 μg/mL purified FX plus a 1:1 molar ratio of RVV to generate FXa) or (B) 5mM NEM or 10 μg/mL purified FXa. GM6001 (100μM) was added to platelets before addition of other factors where indicated. Platelet lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-GPVI cytoplasmic tail IgG or anti-GPV cytoplasmic tail IgG (B bottom panel), and visualized using chemiluminescence. A vertical line indicates a repositioned gel. Washed platelets (10⁷/mL in Tyrode buffer) were pretreated with 10μM PP1 or PP2, 20μM BAPTA-AM, 1 μg/mL rivaroxaban (RIVA), or 10μM PAR1 inhibitor SCH79797 and then mixed with 10 μg/mL purified FXa for up to 1 hour as indicated. Some samples included 100μM GM6001. Control samples were activated with 10μM phorbol myristate acetate (PMA). Surface levels of (C) P-selectin (anti–P-selectin IgG) and (D) active α_IIbβ₃ (OG-fibrinogen binding) were assessed by flow cytometry, and (E) sGPVI levels in supernatant fractions were assessed by ELISA as described in “Measuring sGPVI.”