Removal of the high-affinity thrombin-binding site by protease treatment. L2H/V cells were treated with mocarhagin, a protease that specifically removes the GP Ibα N-terminus by cleaving between residues 282 and 283. (A) Surface levels of GP Ibα and GP V were compared before and after treatment. GP Ibα was detected with AK2, a monoclonal antibody that binds the GP Ibα N-terminus, and GP V was detected with SW16. Mocarhagin treatment removed essentially all of the GP Ibα N-terminus from the cell surface, but had no effect on GP V levels. (B) Binding of thrombin at 1 nmol/L was evaluated and compared in mocarhagin-treated L2H/V cells v untreated cells and L2H cells. Removal of the GP Ibα N-terminus abolished high-affinity thrombin binding.