THE FORMATION OF CA++-DEPENDENT COMPLEXES OF PLATELET MEMBRANE-GLYCOPROTEINS IIB AND IIIA IN SOLUTION AS DETERMINED BY CROSSED IMMUNOELECTROPHORESIS

Abstract

Triton X-100 soluble proteins from 125I-labeled human platelets were studied by crossed immunoelectrophoresis employing a multispecific rabbit antibody raised against whole normal platelets. Emphasis was placed upon an analysis of immunoprecipitates containing 125I-labeled major membrane glycoproteins and, in particular, a prominent immunoprecipitate containing a glycoprotein antigen(s) previously designated as protein 16. SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis of protein 16 precipitated by a monospecific alloantibody, IgG L..., confirmed the presence of both glycoproteins IIb and IIIa. 125I-IgG L..., at concentrations below that capable of precipitating protein 16 by itself, bound specifically to the precipitate containing protein 16 produced by the multispecific rabbit antibody. No other precipitates formed by the rabbit antibody contained either glycoprotein IIb or IIIa. When platelet proteins, incubated with optimum concentrations of EDTA or ethyleneglycol bis (B-aminoethylether) NN1-tetraacetic acid (EGTA), were electrophoresed against the rabbit antibody, previously unobserved immunoprecipitates that combined either free glycoprotein IIb or free glycoprotein IIIa were detected. Upon readdition of excess Ca2+, but not Mg2+, to the same protein samples, a single immunoprecipitate containing both glycoproteins was once again observed. Glycoproteins IIb and IIIa can form Ca2+-dependent complexes (protein 16) in Triton X-100 extracts of normal platelets. The potential significance of the reversible association of these glycoproteins to normal platelet function is discussed.