Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen

Objective: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificit y of this sequence for platelet activation; (3) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the r elative importance of glycoprotein VI and integrin alpha 2 beta 1 platelet activation by collagen. Methods: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platel ets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 s ubunit A-domain and to cause tyrosine phosphorylation of platelet proteins. Results: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or G ly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The pl atelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence fail ed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelet s. Platelet adhesion to this peptide was inhibited by intact anti-glycoprot ein VI antibody and its Fab fragment. The latter inhibited aggregation by t he peptide and Fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and b ound alpha 2 A-domain, but failed to activate platelets or to induce tyrosi ne phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp stru cture did not recognize alpha 2 beta 1, 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. Recogn ition of alpha 2 beta 1 is insufficient to cause activation. Interaction be tween 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 hi ghly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture. (C) 1999 Elsevier Science B.V. All rights reserved.