Micromolar Ca2+ concentrations are essential for Mg2+-dependent binding of collagen by the integrin α2β1 in human platelets

Integrin receptor alpha(2)beta(1) requires micromolar Ca2+ to bind to collagen and to the peptide GPC(GPP)(5)GFO-GER(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 Mg2+. However, we show here that this interaction was abolished in the presence of 25 mu M EGTA. Adhesion of Glanzmann's thrombasthenic platelets, which lack the fibrinogen receptor alpha(IIb)beta(3), was also inhibited by micromolar EGTA. Mg2+-dependent adhesion of platelets was restored by the addition of 10 mu M Ca2+, but millimolar Ca2+ was inhibitory. Binding of isolated alpha(2)beta(1) to GFOGER-GPP was 70% inhibited by 50 mu M EGTA but, as with intact platelets, was fully restored by the addition of micromolar Ca2+. 2 mM Ca2+ did not inhibit binding of isolated alpha(2)beta(1) to collagen or to GFOGER-GPP, Binding of recombinant Lu, I-domain was not inhibited by EGTA, nor did millimolar Ca2+ inhibit binding. Our data suggest that high affinity Ca2+ binding to alpha(2)beta(1), outside the I-domain, is essential for adhesion to collagen, This is the first demonstration of a Ca2+ requirement in alpha(2)beta(1) function.