title: Adhesive domains in the collagen III fragment alpha1(III)CB4 that support alpha2beta1- and von Willebrand factor-mediated platelet adhesion under flow conditions.
abstract: Seven overlapping peptides derived from the bovine alpha1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin alpha2beta1-recognition site has been assigned within this fragment to residues 522-528 of the collagen alpha1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both alpha2beta1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solid-phase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the alpha1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the alpha2 subunit of integrin alpha2beta1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the alpha2beta1-recognition site in this locality in alpha1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the alpha1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of alpha2beta1-, vWF- and rLAPP-binding sites all in close proximity in alpha1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.
authors.affiliation: The Postgraduate School of Biomembranes, Department of Haematology, University Hospital Utrecht, The Netherlands.
authors.forename: M W
authors.initials: MW
authors.lastname: Verkleij
authors.forename: M J
authors.initials: MJ
authors.lastname: IJsseldijk
authors.forename: G J
authors.initials: GJ
authors.lastname: Heijnen-Snyder
authors.forename: E G
authors.initials: EG
authors.lastname: Huizinga
authors.forename: L F
authors.initials: LF
authors.lastname: Morton
authors.forename: C G
authors.initials: CG
authors.lastname: Knight
authors.forename: J J
authors.initials: JJ
authors.lastname: Sixma
authors.forename: P G
authors.initials: PG
authors.lastname: de Groot
authors.forename: M J
authors.initials: MJ
authors.lastname: Barnes
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D003094:Collagen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D016023:Integrins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010973:Platelet Adhesiveness / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D038981:Receptors, Collagen; D011994:Recombinant Proteins / Q000378:metabolism; D012471:Salivary Proteins and Peptides / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D016023:Integrins; C073760:LAPP protein, Haementeria officinalis; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D038981:Receptors, Collagen; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; D014841:von Willebrand Factor; D003094:Collagen