CALCIUM-BINDING TO HUMAN PLATELET INTEGRIN GPIIB/IIIA AND TO ITS CONSTITUENT GLYCOPROTEINS - EFFECTS OF LIPIDS AND TEMPERATURE

Platelet plasma membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form a Ca2+-dependent heterodimer, GPIIb/IIIa, which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. Using equilibrium dialysis measurements, it was established that both GPIIb and GPIIIa in solution have low-affinity Ca2+-binding sites (K(d)0.2-0.3 mM), five in GPIIb and two in GPIIIa, and it was confirmed that only the alpha-chain of GPIIb (GPIIb-alpha) binds Ca2+. Furthermore, Ca2+ binding was found with two CNBr fragments of GPIIb, GPIIb-alpha-(1-285) and GPIIb-alpha-(314-489), which carry three out of the four putative Ca2+-binding sites. GPIIb/IIIa in solution has a single high-affinity Ca2+-binding site (K(d1) 80 +/- 30 nM at 21-degrees-C), whose degrees of saturation regulates the states of association of GPIIb and GPIIIa in the GPIIb/IIIa heterodimer at room temperature, and 3-4 medium-affinity Ca2+-binding sites (K(d2) 40 +/- 15-mu-M at 21-degrees-C). When GPIIb/IIIa was incorporated into liposomes, K(d1) decreased by an order of magnitude (9 +/- 3 nM at 21-degrees-C) and reached the dissociation constant estimated for the high-affinity Ca2+-binding sites at the platelet surface [Brass & Shattil (1982) J. Biol. Chem. 257, 1400-1405], whereas K(d2) remained unchanged. The high-affinity Ca2+-binding site of GPIIb/IIIa in solution at 4-degrees-C has almost the same affinity (K(d1) 65 +/- 20 nM) as at 21-degrees-C; however, at 37-degrees-C, either its affinity decreases enough so as to become experimentally indistinguishable from the medium-affinity Ca2+-binding sites determined at this temperature (number of binding sites 3.9 +/- 1.2 mol of Ca2+/mol of GP, K(d) 25 +/- 11-mu-M), or vanishes altogether. Studies on Ca2+-dependent dissociation of GPIIb/IIIa at 37-degrees-C in solution seem to support the former interpretation. Further work will be necessary to decide whether the dissociation of GPIIb/IIIa in the platelet membrane at 37-degrees-C is regulated by the degree of saturation of the high-affinity Ca2+-binding site, as occurs in solution. It is suggested that the high-affinity Ca2+-binding site could be related to the putative GPIIIa-binding region in GPIIb (residues 558-747 of the alpha chain).