Treatment of platelets with GPVI agonists induces loss of GPVI from the platelet surface. (A) Washed human platelets (5 × 10⁸/mL) were resuspended in Tyrode buffer and treated with either 20 μg/mL CRP or collagen or 0.5 μg/mL convulxin (Cvx) for up to 60 minutes, or 1 U thrombin (Thr) for 60 minutes. EDTA (10 mM), where indicated, was included in some incubations. Platelet suspensions were incubated at room temperature, then mixed with 10 mM EDTA (final concentration) and centrifuged to isolate platelets from incubation medium supernatants. Platelet pellets were lysed in 1% Triton X-100 and aliquots of platelet extracts or supernatants were electrophoresed on SDS/acrylamide gels, transferred to nitrocellulose, then probed with anti-PECAM-1 antibody WM59 or anti-GPVI monoclonal antibody 6B12-3. An aliquot of resting platelet lysate is included on the left hand side of supernatant fractions for molecular weight comparison. Data are representative of at least 3 experiments with different donors. Two separate preparations each of collagen and CRP were tested and no difference was observed between each preparation of GPVI agonist for production of GPVI fragment. (B) Washed platelets were treated with 0.5 μg/mL convulxin or a combination of 2.5 μg/mL botrocetin (Botr0) plus 10 μg/mL von Willebrand factor (VWF) for 60 minutes. Incubations were then treated and analyzed as described for panel A.