© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Collagen–platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen1
aBiochemistry Department, Cambridge University, Cambridge, UK
bDepartment of Haematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
* Corresponding author. Tel.: +44-1223-766-089: fax: +44-1223-333-345.
Objective: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin 
2β1 in platelet activation by collagen. Methods: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. Results: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer 2β1-binding sequence in a repeat Gly-Pro-Pro structure supported 2β1-mediated platelet and HT 1080 cell adhesion and bound 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize 2β1, but was highly platelet activatory. Conclusions: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of 2β1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.
KEYWORDS Human platelets; Thrombosis; Collagens; Collagen-based peptides; Integrin 2β1; Glycoprotein VI
1 Amino acid sequences are defined using single-letter nomenclature (P*=Hyp); Ahx, 6-aminohexanoic acid; BSA, bovine serum albumin; CRP, collagen-related peptide; Fmoc, fluoren-9-ylmethoxycarbonyl; Gp, glycoprotein; mAb, monoclonal antibody; peptide-XL, crosslinked peptide; PRP, platelet-rich plasma.
2 Present address: Division of Hematology, Shizuoka General Hospital, 4-27-1 Kitaando, Shizuoka 420-0881, Japan.
3 Present address: Takashima General Hospital, 1667 Katsuno,Takashima-cho, Shiga-ken 520-1121, Japan.
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