title: Simple collagen-like peptides support platelet adhesion under static but not under flow conditions: interaction via alpha2 beta1 and von Willebrand factor with specific sequences in native collagen is a requirement to resist shear forces.
abstract: The aim of this study was to define the need for specific collagen sequences and the role of their conformation in platelet adhesion to collagen under both static and flow conditions. We recently reported that simple triple-helical collagen-related peptides (CRPs), GCP*(GPP*)10GCP*G and GKP*(GPP*)10GKP*G (single-letter amino acid code, P* = hydroxyproline; Morton et al, Biochem J 306:337, 1995) were potent stimulators of platelet activation and were able to support the adhesion of gel-filtered platelets examined under static conditions. The present study investigated whether these same peptides were able to support platelet adhesion under more physiologic conditions by examining static adhesion with platelet-rich plasma (PRP) and adhesion under flow conditions. In the static adhesion assay, we observed 20% surface coverage with platelet aggregates. In marked contrast, there was a total lack of adhesion under flow conditions examined at shear rates of 50 and 300 s-1. Thus, the interaction of platelets with the CRPs is a low-affinity interaction unable on its own to withstand shear forces. However, the addition of CRPs to whole blood, in the presence of 200 micromol/L D-arginyl-glycyl-L-aspartyl-L-tryptophan (dRGDW) to prevent platelet aggregation, caused an inhibition of about 50% of platelet adhesion to collagens I and III under flow. These results suggest that the collagen triple helix per se, as defined by these simple collagen sequences, plays an important contributory role in the overall process of adhesion to collagen under flow. The monoclonal antibody (MoAb) 176D7, directed against the alpha2 subunit of the integrin alpha2 beta1, was found to inhibit static platelet adhesion to monomeric but not fibrillar collagens I and III. However, under flow conditions, anti-alpha2 MoAbs (176D7 anf 6F1) inhibited adhesion to both monomeric and fibrillar collagens, indicating that alpha2 beta1 is essential for adhesion to collagen under flow, independent of collagen conformation, whether monomeric or polymeric. To obtain further insight into the nature of the different adhesive properties of CRPs and native collagen, we investigated the relative importance of von Willebrand factor (vWF) and the integrin alpha2 beta1 in platelet adhesion to collagen types I and III, using the same shear rate (300 s-1) as used when testing CRPs under flow conditions. Our results, together with recent data of others, support a two-step mechanism of platelet interaction with collagen under flow conditions. The first step involves adhesion via both the indirect interaction of platelet glycoprotein (GP) Ib with collagen mediated by vWF binding to specific vWF-recognition sites in collagen and the direct interaction between platelet alpha2 beta1 and specific alpha2 beta1-recognition sites in collagen. This suffices to hold platelets at the collagen surface. The second step occurs via another collagen receptor (thought to be GPVI) that binds to simple collagen sequences, required essentially to delineate the collagen triple helix. Recognition of the triple helix leads to strengthening of attachment and platelet activation.
authors.affiliation: Postgradual School of Biomembranes, Department of Haematology, University Hospital Utrecht, Utrecht, The Netherlands.
authors.forename: M W
authors.initials: MW
authors.lastname: Verkleij
authors.forename: L F
authors.initials: LF
authors.lastname: Morton
authors.forename: C G
authors.initials: CG
authors.lastname: Knight
authors.forename: P G
authors.initials: PG
authors.lastname: de Groot
authors.forename: M J
authors.initials: MJ
authors.lastname: Barnes
authors.forename: J J
authors.initials: JJ
authors.lastname: Sixma
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D003094:Collagen / Q000737:chemistry / Q000276:immunology / Q000502:physiology* / Q000648:ultrastructure; D018056:Hemorheology; D006801:Humans; D016023:Integrins / Q000378:metabolism / Q000502:physiology; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D010973:Platelet Adhesiveness / Q000187:drug effects* / Q000502:physiology; D011487:Protein Conformation; D038981:Receptors, Collagen; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D016023:Integrins; D010446:Peptide Fragments; D038981:Receptors, Collagen; D014841:von Willebrand Factor; D003094:Collagen