Structural Basis for the Platelet-Collagen Interaction: THE SMALLEST MOTIF WITHIN COLLAGEN THAT RECOGNIZES AND ACTIVATES PLATELET GLYCOPROTEIN VI CONTAINS TWO GLYCINE-PROLINE-HYDROXYPROLINE TRIPLETS

Peter A Smethurst,David J Onley,Gavin E Jarvis,Marie N O'Connor,C Graham Knight,Andrew B Herr,Willem H Ouwehand,Richard W Farndale

doi:10.1074/jbc.m606479200

Peter A Smethurst, David J Onley + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m606479200

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Abstract

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)n backbone. The adhesion of recombinant human Glycoprotein VI ectodo-main, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO.........GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)4 motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.

Highlights

Damage to the blood vessel endothelium results in the exposure of underlying extracellular matrix proteins, including the fibrillar collagens I and III, both abundant in that location
GPO triplets are abundant in collagens, forming ϳ10% of the fibrillar collagen COL domain, very few contiguous runs of GPO triplets occur; exceptions are the N-terminal domain of collagens I (␣1) and III (␣1), where four and three GPO triplets are present, respectively, and the C-terminal domain of collagen I ␣1, where five such triplets occur
The aim of this study was to determine the minimum arrangement of GPO triplets within Collagen-related peptide (CRP)

Summary

Introduction

Damage to the blood vessel endothelium results in the exposure of underlying extracellular matrix proteins, including the fibrillar collagens I and III, both abundant in that location. Each fibrillar collagen strand contains ϳ1000 amino acid residues arranged in the triple helical COL domain, comprised of (G-X-XЈ) repeats, where X is often proline and XЈ, hydroxyproline (O). Horii et al [13] recently described the crystal structure of the immunoglobulin (Ig)-like ligand-binding domain of GpVI and proposed a model for its interaction with collagen after docking with CRP-like structures. The authors show by analytical ultracentrifugation that monomeric CRP can interact with more than one copy of GpVI [13] This proposed binding mechanism is very different from that recently described for the complex of collagen with the Ig-fold-containing Staphylococcus aureus CNA protein [16]. The platelet reactivity of CRP has been shown to reside in the hydroxyproline content of its GPO triplets [17], but little is known of how they must be distributed within the collagen

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