Treatment of platelets with a calmodulin inhibitor induces loss of GPVI from the platelet surface. Washed human platelets were resuspended in Tyrode buffer and treated with 150 μM W7. EDTA (10 mM) was included in some incubations. Platelet suspensions were incubated at room temperature for up to 4 hours, then mixed with 10 mM EDTA (final concentration), and centrifuged to isolate platelets from the incubation medium supernatant. Platelet pellets were lysed in buffer containing Triton X-100 and proteinase inhibitors and aliquots of platelet extracts or supernatants were electrophoresed on SDS/acrylamide gels, transferred to nitrocellulose, and probed with anti-GPVI monoclonal antibody, 6B12-3. Data are representative of at least 3 experiments with different donors.