DETECTION OF AN EPITOPE-SPECIFIC FOR THE DISSOCIATED FORM OF GLYCOPROTEIN IIIA OF PLATELET MEMBRANE GLYCOPROTEIN IIB-IIIA COMPLEX AND ITS EXPRESSION ON THE SURFACE OF ADHERENT PLATELETS

Glycoproteins (GPs) IIb and IIIa form a Ca2+-dependent complex in plat elet membrane and change their conformation upon platelet activation a nd dissociation of the complex. A new anti-GPIIIa monoclonal antibody (mAb), CRC54, is described which could distinguish different conformat ional states of GPIIIa. This antibody (i) precipitated GPIIb-IIIa from platelet Triton X-100-lysate, (ii) recognized the GPIIIa band in West ern blotting of platelet SDS-lysate. and (iii) did not react with plat elets from a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa. Im munoblotting of chymotryptic digestion products of purified GPIIb-IIIa has shown that CRC54 epitope is located within residues 1-100 at the N-terminus of GPIIIa. CRC54 bound weakly to platelets in the presence of Ca2+ and Mg2+. 2.34+/-0.28 x 10(3) molecules per platelet at satura tion. The same level of binding was observed without any divalent cati ons in the medium. However, binding of CRC54 was increased by several times after treatment of platelets with EDTA, 10.04+/-0.28 x 10(3) mol ecules per platelet. Increase of CRC54 binding correlated with the dis sociation of GPIIb-IIIa complex which was followed by the decrease of the binding of another mAb, CRC64, directed against complex-specific e pitope of GPIIb-IIIa. Binding of CRC54 to platelets was changed neithe r by platelet activation in suspension with thrombin or ADP nor by the occupancy of GPIIb-IIIa ligand binding site with GRGDSR peptide. Howe ver, binding was significantly stimulated by platelet adhesion to poly styrene plastic. As measured using Cr-51-labelled platelets, binding o f I-125-CRC54 to adherent platelets in the presence of divalent cation s was about 4 times higher than to platelets in suspension, 8.68+/-0.4 8 x 10(3) per platelet. This increase was not due to the dissociation of GPIIb-IIIa since complex-specific antibody CRC64 still bound effect ively to the surface of adherent platelets. The data obtained indicate d that: (1) CRC54 recognized an epitope specific for the dissociated f orm of GPIIIa; (2) the CRC54-reactive epitope of GPIIIa is also expres sed on the surface of adherent platelets.