FURTHER-STUDIES ON THE TOPOGRAPHY OF HUMAN PLATELET GLYCOPROTEIN-IIB - LOCALIZATION OF MONOCLONAL-ANTIBODY EPITOPES AND THE PUTATIVE GLYCOPROTEIN IIIA- AND FIBRINOGEN-BINDING REGIONS

Glycoprotein IIb (GPIIb) is a major glycoprotein of the human platelet plasma membrane, which together with glycoprotein IIIa (GPIIa) forms a Ca2+-dependent heterodimer, GPIIb/IIIa, which serves as the major fibrinogen receptor in activated platelets. The precise localization of the epitopes for six anti-GPIIb monoclonal antibodies (M1-M6) has been determined by a combination of enzymic and chemical cleavage procedures, peptide isolation, N-terminal sequence analysis, peptide synthesis and enzyme immunoassay. The following localizations were found: M1, beta-1-16-36, beta-2-4-24; M2, alpha-747-755; M-alpha-2, alpha-837-843; M3, alpha-849-857; M4, alpha-143-151; M5, alpha-550-558; M6, alpha-657-665. Besides considerations of the degree of exposure of these epitopes, several remarkable features are readily apparent. The earliest and main chymotryptic cleavage site of GPIIb in whole platelets is between alpha-cysteine-545 and alpha-phenylalanine-551. The epitope for M3 was located within the same sequence (alpha-842-857) as is the epitope for PMI-1 [Loftus, Plow, Frelinger, D'Souza, Dixon, Lacy, Sorge & Ginsberg (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7114-7118] in spite of the fact that the exposure of the latter in whole platelets is EDTA-dependent whereas that in the former is not. The epitope for M5 shares full homology with the 540-548 peptide stretch of the alpha-subunit of the vitronectin receptor, and this antibody cross-reacts with endothelial cells. The M6 epitope is located in the 25 kDa membrane-bound fragment of GPIIb, which is most resistant to chymotryptic digestion in whole platelets, contrary to what happens in isolated GPIIb in solution, where this epitope is destroyed at an early stage of chymotryptic digestion. This suggests that this region of GPIIb, somewhere between the epitope for M5 (alpha-550-558) and the epitope for M2 (alpha-747-755), may carry the surface of interaction of GPIIb with GPIIIa in the GPIIb/IIIa heterodimer. Finally, the sequence where the epitope for M6 has been located (alpha-657-667) was the only one found to be hydropathically complementary to the gamma-402-411 peptide of fibrinogen within the amino acid sequence of both GPIIb and GPIIIa. This complementariness, the EDTA- or thrombin-dependence of the exposure of the alpha-657-665 stretch in whole platelets to M6 and the ability of this antibody to inhibit platelet aggregation led us to postulate that this peptide stretch is a putative binding site for fibrinogen in the platelet receptor. The overlap between the M6 epitope and the putative binding site for the gamma-402-411 peptide sequence of fibrinogen would imply that the unmasking of the alpha-658-667 peptide stretch could be one of the structural changes in GPIIb/IIIa required for the induction of the fibrinogen receptor in activated platelets.