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

摘要

The involvement of exosite I in a-thrombin (Flla) binding to platelet glycoprotein Iba (GPIbα), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIba amino terminal domain (GPIbα-N) fully sulfated on three tyrosine residues and solved the structure of its complex with Flla. 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 Flla binding to soluble or surface-immobilized GPIba-N. Mutating Tyr~(276), which mostly contacts exosite II residues, markedly reduced Flla interaction with both soluble and immobilized GPIba-N; mutating Tyr~(278） or Tyr~（279）, which mostly contact exosite I residues, reduced Flla complexing in solution by 0-20% but affinity for immobilized GPIba-N 2 to 6-fold, respectively. Moreover, three exosite I ligands—aptamer HD1, hiru-gen, and lepirudin—did not interfere with soluble Flla complexing to GPIba-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired Flla binding to immobilized GPIba-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 exo-sites of one Flla molecule independently interact with two immobilized GPIba molecules. Through exosite engagement, GPIba may influence Flla-dependent processes relevant to hemostasis and thrombosis.