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

Alessandro Zarpellon, Reha Celikel, James R. Roberts, Richard A. McClintock, G. Loredana Mendolicchio, Kevin L. Moore, Hua Jing, Kottayil I. Varughese, Zaverio M. Ruggeri

Research output: Contribution to journalArticlepeer-review

Abstract

The involvement of exosite I in α-thrombin (FIIa) binding to platelet glycoprotein Ibα (GPIbα), which could influence interactions with other substrates, remains undefined. To address the problem, we generated the GPIbα amino terminal domain (GPIbα-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 GPIbα-N. Mutating Tyr276, which mostly contacts exosite II residues, markedly reduced FIIa interaction with both soluble and immobilized GPIbα-N; mutating Tyr278 or Tyr 279, which mostly contact exosite I residues, reduced FIIa complexing in solution by 0-20% but affinity for immobilized GPIbα-N 2 to 6-fold, respectively. Moreover, three exosite I ligands - aptamer HD1, hirugen, and lepirudin - did not interfere with soluble FIIa complexing to GPIbα-N, excluding that their binding caused allosteric effects influencing the interaction; nonetheless, all impaired FIIa binding to immobilized GPIbα-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 Tys 279. These results support a mechanism in which binding occurs when the two exosites of one FIIa molecule independently interact with two immobilized GPIbα molecules. Through exosite engagement, GPIbα may influence FIIa-dependent processes relevant to hemostasis and thrombosis.

Original languageEnglish
Pages (from-to)8628-8633
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number21
DOIs
Publication statusPublished - May 24 2011

Keywords

  • Platelet activation
  • Platelet aggregation
  • Protease-activated receptor
  • Tyrosine sulfation
  • Tyrosylprotein sulfotransferase-2

ASJC Scopus subject areas

  • General

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