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 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 alpha 2 beta 1 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 alpha 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 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 bound alpha 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 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. Recognition of alpha 2 beta 1 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.