title: Micromolar Ca2+ concentrations are essential for Mg2+-dependent binding of collagen by the integrin alpha 2beta 1 in human platelets.
abstract: Integrin receptor alpha(2)beta(1) requires micromolar Ca(2+) to bind to collagen and to the peptide GPC(GPP)(5)GFOGER(GPP)(5)GPC (denoted GFOGER-GPP, where O represents hydroxyproline), which contains the minimum recognition sequence for the collagen-binding alpha(2) I-domain (Knight, C. G., Morton, L. F., Peachey, A. R., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (2000) J. Biol. Chem. 275, 35-40). Platelet adhesion to these ligands is completely dependent on alpha(2)beta(1) in the presence of 2 mm Mg(2+). However, we show here that this interaction was abolished in the presence of 25 microm EGTA. Adhesion of Glanzmann's thrombasthenic platelets, which lack the fibrinogen receptor alpha(IIb)beta(3), was also inhibited by micromolar EGTA. Mg(2+)-dependent adhesion of platelets was restored by the addition of 10 microm Ca(2+), but millimolar Ca(2+) was inhibitory. Binding of isolated alpha(2)beta(1) to GFOGER-GPP was 70% inhibited by 50 microm EGTA but, as with intact platelets, was fully restored by the addition of micromolar Ca(2+). 2 mm Ca(2+) did not inhibit binding of isolated alpha(2)beta(1) to collagen or to GFOGER-GPP. Binding of recombinant alpha(2) I-domain was not inhibited by EGTA, nor did millimolar Ca(2+) inhibit binding. Our data suggest that high affinity Ca(2+) binding to alpha(2)beta(1), outside the I-domain, is essential for adhesion to collagen. This is the first demonstration of a Ca(2+) requirement in alpha(2)beta(1) function.
authors.affiliation: Department of Biochemistry, University of Cambridge, Building 0, Downing Site, Cambridge CB2 1QW, United Kingdom. djo21@hermes.cam
authors.forename: D J
authors.initials: DJ
authors.lastname: Onley
authors.forename: C G
authors.initials: CG
authors.lastname: Knight
authors.forename: D S
authors.initials: DS
authors.lastname: Tuckwell
authors.forename: M J
authors.initials: MJ
authors.lastname: Barnes
authors.forename: R W
authors.initials: RW
authors.lastname: Farndale
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000097:blood*; D002417:Cattle; D003094:Collagen / Q000097:blood*; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000097:blood* / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000097:blood* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000097:blood / Q000737:chemistry; D010973:Platelet Adhesiveness; D038981:Receptors, Collagen; D013915:Thrombasthenia / Q000097:blood*
chemical_list: D016023:Integrins; D010455:Peptides; D038981:Receptors, Collagen; D004533:Egtazic Acid; D003094:Collagen; D008274:Magnesium; D002118:Calcium