Binding of α-thrombin to surface-anchored platelet glycoprotein Ibα sulfotyrosines through a two-site mechanism involving exosite I

The involvement of exosite I in alpha-thrombin (FIIa) binding to platelet glycoprotein Ib alpha (GPIb alpha), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIb alpha amino terminal domain (GPIb alpha-N) fully sulfated on three tyrosine residues and solved the structure of its complex with FIIa. We found that sulfotyrosine (Tys) 278 enhances the interaction mainly by establishing contacts with exosite I. We then evaluated how substituting tyrosine with phenylalanine, which cannot be sulfated, affects FIIa binding to soluble or surface-immobilized GPIb alpha-N. Mutating Tyr(276), which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIb alpha-N; mutating Tyr(278) or Tyr(279), which mostly contact exosite I residues, reduced FIIa complexing in solution by 0-20% but affinity for immobilized GPIb alpha-N 2 to 6-fold, respectively. Moreover, three exosite I ligands-aptamer HD1, hirugen, and lepirudin-did not interfere with soluble FIIa complexing to GPIb alpha-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIb alpha-N and platelet GPIb nearly as much as aptamer HD22 and heparin, both exosite II ligands. Bound HD1 and hirugen alter Trp(148) orientation in a loop near exosite I preventing contacts with the sulfate oxygen atoms of Tys(279). These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIb alpha molecules. Through exosite engagement, GPIb alpha may influence FIIa-dependent processes relevant to hemostasis and thrombosis.