FUNCTIONAL-CHARACTERIZATION OF GPIIB-SPECIFIC AND GPIIIA-SPECIFIC MONOCLONAL-ANTIBODIES - FURTHER EVIDENCE FOR THE EXISTENCE OF AGONIST-SPECIFIC ACTIVATED STATES OF THE PLATELET FIBRINOGEN RECEPTOR

In this work human platelet aggregation induced in vitro by ADP, colla gen, arachidonic acid and U-46619 (a thromboxane A(2) analogue) was us ed as a functional test to characterize 19 anti-GPIIb (M series) and a nti-GIIIa (P series) monoclonal antibodies whose epitope location is k nown for most of them. Additionally, now cytofluorimetry was applied t o study the epitope expression of these antibodies in resting, EDTA-tr eated and SFLLRN peptide (thrombin receptor agonist)-activated platele ts. Antibodies M6 (epitope located at GPIIbH 657-665), P23-7 (GIIIa 11 4-122) and P40 (GPIIIa 262-303) bind weakly to only 43%, 70% and 66%, respectively, of the resting platelet population. This binding was enh anced in EDTA-treated and in activated platelets. Platelet activation enhances the apparent binding of most of the other antibodies. Further evidence on the existence of agonist-specific activated states of GPI Ib/IIIa was provided by the agonist-dependent immunochemical inhibitio n in vitro of platelet aggregation by some of the anti-subunit antibod ies studied here. The most notable cases are those of P40 and M6, whic h at 140 nM inhibit most, the platelet aggregation induced by arachido nic acid and U-46619. On the other hand, three of the most strong and agonist-independent inhibitors, P37 (GPIIIa 101-109), P97 and P95-2 (G PIIIa N-terminal half) bind to resting platelets with high affinity (5 -8nM), compete with each other far binding to GPIIb-IIIa and their epi topes are located at the N-terminal domain of GPIIIa, where the recept or ligand binding site(s) have been found. Given that the formation of activated GPIIb-IIIa (GPIIb-IIIa()) is the first step at which the a nti-subunit antibodies can intervene as inhibitors and that agonist-sp ecific inhibitors should block only agonist-specific steps, while nons pecific inhibitors should block steps common to all the agonists, then our present work support the hypothesis that there are different agon ist-specific GPIIb-IIIa()s or, alternatively, different receptor envi ronments, that can be specifically blocked by some of the antibodies. These results add to earlier evidence on agonist-dependent ligand spec ificity and activated states found for this and other integrins. Final ly, the correlation between the in vitro inhibition of platelet aggreg ation and the antithrombotic activity in vivo is discussed for these a ntibodies.