Effect of calcium on the stability of the platelet membrane glycoprotein IIb-IIIa complex.
Platelet membrane glycoproteins IIb and IIIa form a Ca2+-dependent heterodimer complex that contains binding sites for fibrinogen, von Willebrand factor, and fibronectin following platelet stimulation. We have studied the effect of Ca2+ on the stability of the IIb-IIIa complex using a IIb-IIIa complex-specific monoclonal antibody A2A9 to detect the presence of the complexes. Soluble IIb and IIIa interacted with A2A9-Sepharose only in the presence of Ca2+ with 50% IIb-IIIa binding requiring 0.4 microM Ca2+. In contrast, at 25 degrees C 125I-A2A9 binding to intact unstimulated platelets suspended in buffers containing EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was independent of the presence of Ca2+. However, the effect of Ca2+ chelators on 125I-A2A9 binding varied with temperature. At 37 degrees C, 125I-A2A9 binding to intact platelets became Ca2+-dependent with 50% binding requiring 0.4 microM Ca2+. This effect of temperature was not due to a change in platelet membrane fluidity because enrichment or depletion of platelet membrane cholesterol did not influence antibody binding. But, 125I-A2A9 binding to intact platelets at 25 degrees C did become Ca2+-dependent when the pH was increased above 7.4. Thus, at 1 nM Ca2+ and 25 degrees C, 50% antibody binding occurred at pH 9.0. Our studies demonstrate that Ca2+-dependent IIb-IIIa complexes are present on unstimulated platelets and that the Ca2+ binding sites responsible for the stability of these complexes are located on the external platelet surface. Our experiments also suggest that changes in platelet cytosolic Ca2+ do not regulate the formation of IIb-IIIa complexes.
L F
LF
Brass
S J
SJ
Shattil
T J
TJ
Kunicki
J S
JS
Bennett
D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000032:analysis; D002784:Cholesterol / Q000032:analysis; D004492:Edetic Acid / Q000494:pharmacology; D000939:Epitopes / Q000032:analysis; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007123:Immunoelectrophoresis, Two-Dimensional; D008563:Membrane Lipids / Q000032:analysis; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010980:Platelet Membrane Glycoproteins; D012995:Solubility; D013696:Temperature
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D002784:Cholesterol; D004492:Edetic Acid; D002118:Calcium