期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2011
卷号：108
期号：21
页码：8628-8633
DOI：10.1073/pnas.1017042108
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The involvement of exosite I in -thrombin (FIIa) binding to platelet glycoprotein Ib (GPIb), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIb amino terminal domain (GPIb-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-N. Mutating Tyr276, which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIb-N; mutating Tyr278 or Tyr279, which mostly contact exosite I residues, reduced FIIa complexing in solution by 0-20% but affinity for immobilized GPIb-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-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIb-N and platelet GPIb nearly as much as aptamer HD22 and heparin, both exosite II ligands. Bound HD1 and hirugen alter Trp148 orientation in a loop near exosite I preventing contacts with the sulfate oxygen atoms of Tys279. These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIb molecules. Through exosite engagement, GPIb may influence FIIa-dependent processes relevant to hemostasis and thrombosis.
