GP VI proteolysis is inhibited by metalloproteinase inhibitors. Washed platelets (3 × 10⁸/mL) were pre-incubated with metalloproteinase inhibitors TAPI-1 or GM6001 for 10 minutes at 37°C prior to agonist stimulation. Platelets were activated for 45 minutes at 37°C with collagen, 10 μg/mL, in the absence or presence of GM6001 or the GM6001 negative control compound (A) or TAPI-1 (B). Platelet proteins were separated by SDS-PAGE under nonreducing conditions, immunoblotted with 9012.2 (GP VI) and C3a (IIIa), visualized by ECL, and expressed as percent reduction ± SEM described previously, n = 3-4. Platelet supernatants were prepared as described previously. Supernatants from platelets activated with convulxin, 10 ng/mL, in the presence and absence of GM6001 or the GM6001 negative control compound (C) or TAPI-1 (D) as well as resting control lysate were loaded in equivalent amounts on SDS-PAGE under reducing conditions and electroblotted to membranes. Membranes were treated with polyclonal GP VI antibodies,¹⁹  followed by anti–rabbit-HRP and detection by ECL Results are expressed as percent released sGP VI ± SEM, n = 3.