Treatment of platelets with RGD peptide does not block ligand-induced loss of GPVI from platelets. Washed human platelets (5 × 108/mL) were resuspended in Tyrode buffer alone or containing 100 μM RGD peptide and treated with either 0.5 μg/mL convulxin, 150 μM W7, or 1 U thrombin (Thr) for 90 minutes at room temperature. The treated platelets were then mixed with 10 mM EDTA and the platelets isolated by centrifugation. The platelet pellets were lysed in buffer containing Triton X-100 and proteinase inhibitors, and aliquots of the platelet extracts were electrophoresed on SDS/acrylamide gels, transferred to nitrocellulose, then probed with the anti-GPVI monoclonal antibody 6B12-3. This experiment is representative of at least 3 experiments with different donors.
