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

Abstract

Glycoproteins (GPs) IIb and IIIa form a Ca(2+)-dependent complex in platelet membrane and change their conformation upon platelet activation and dissociation of the complex. A new anti-GPIIIa monoclonal antibody (mAb), CRC54, is described which could distinguish different conformational states of GPIIIa. This antibody (i) precipitated GPIIb-IIIa from platelet Triton X-100-lysate, (ii) recognized the GPIIIa band in Western blotting of platelet SDS-lysate, and (iii) did not react with platelets from a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa. Immunoblotting 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 saturation. The same level of binding was observed without any divalent cations 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) molecules per platelet. Increase of CRC54 binding correlated with the dissociation of GPIIb-IIIa complex which was followed by the decrease of the binding of another mAb, CRC64, directed against complex-specific epitope of GPIIb-IIIa. Binding of CRC54 to platelets was changed neither by platelet activation in suspension with thrombin or ADP nor by the occupancy of GPIIb-IIIa ligand binding site with GRGDSR peptide. However, binding was significantly stimulated by platelet adhesion to polystyrene plastic. As measured using 51Cr-labelled platelets, binding of 125I-CRC54 to adherent platelets in the presence of divalent cations was about 4 times higher than to platelets in suspension, 8.68 +/- 0.48 x 10(3) per platelet. This increase was not due to the dissociation of GPIIb-IIIa since complex-specific antibody CRC64 still bound effectively to the surface of adherent platelets. The data obtained indicated that: (1) CRC54 recognized an epitope specific for the dissociated form of GPIIIa; (2) the CRC54-reactive epitope of GPIIIa is also expressed on the surface of adherent platelets.

Stimulation of platelet glycoprotein IIb‐IIIa (αIIbβ3‐integrin) functional activity by a monoclonal antibody to the N‐terminal region of glycoprotein IIIa

“…2B). Much lower concentrations and a routine turbidometric method were used in the initial study of CRC54 and that is why the aggregation response was not detected in a previous paper [8]. Fab fragments of CRC54 were also able to stimulate platelet aggregation ( Fig.…”

“…According to its ability to blot GPIIb-IIIa chymotryptic fragments the CRC54 epitope was mapped within the first 100 N-terminal residues of GPIIIa [8]. In this study we used previously described conformation-dependent monABs to test if CRC54 epitope could overlap or be sterically close to other conformation-dependent epitopes of GPIIb-IIIa.…”

“…MonAB CRC54 is directed against GPIIIa and has been partially described previously [8]. Antibody pl-80 [13] and antibody PMI-1 [4,14] are directed against LIBS epitopes on GPIIb, antibodies LIBSI, LIBS2, and LIBS6 against LIBS epitopes on GPIIIa [5], and antibody pl-55 recognizes the activated conformation of GPIIbIlia [10].…”

“…Labeling of monABs, Fab fragments and fibrinogen (Calbiochem, USA) with 1251 and binding experiments were performed essentially as described elsewhere [8,19]. Washed platelets (109/ml) were preincubated without any additions or in the presence of RGDS, RGES (both peptides were generously provided by Dr. G. Bespalova, Cardiology Research Center, Moscow, Russia), CRC54 or EDTA for 30 min at 37°C and with thrombin (Sigma, St. Louis, MO) for 5 min at 22°C.…”

Evidence that thrombocytopenia observed in humans treated with orally bioavailable glycoprotein IIb/IIIa antagonists is immune mediated

“…EDTA treatment of platelets at 37°C has been reported to dissociate the GP IIb/IIIa complex, resulting in the dissociation of conformational sensitive antibodies. 40,[41][42][43] After the initial incubation with whole blood in the absence ( Figure 1B, no drug/EDTA) or presence of L-739758 (drug/EDTA), EDTA was added for 15 minutes at room temperature, and PPP was prepared. DDABs were quantitatively recovered from the platelet surface by EDTA treatment, suggesting that conformational and calcium-sensitive GP IIb/IIIa epitopes are the molecular target.…”