PMID: 7537313Apr 12, 1995Paper

An enzyme-linked immunosorbent assay to identify inhibitors of activation of platelet integrin alpha IIb beta 3

Journal of Immunological Methods
J Gao, S J Shattil

Abstract

The affinity of integrin alpha IIb beta 3 for adhesive ligands is tightly regulated by the platelet such that fibrinogen binding is observed only after platelet activation. Ligand binding is necessary for platelet aggregation, which contributes to vascular occlusion in pathological states. Therefore, we have developed an ELISA assay to screen for compounds that inhibit alpha IIb beta 3 activation. Washed platelets were incubated in microtitre wells with potential inhibitory compounds and stimulated with an agonist to activate alpha IIb beta 3. After the addition of biotin-PAC1, a fibrinogen-mimetic monoclonal antibody, the activation state of alpha IIb beta 3 was measured by sedimenting the platelets and quantitating the residual biotin-PAC1 in the cell-free supernatant in a streptavidin-based ELISA. This assay detected (1) specific PAC1 binding to activated platelets in response to a variety of agonists, and (2) dose-dependent inhibition of PAC1 binding by function-blocking anti-alpha IIb beta 3 monoclonal antibodies, by the tetrapeptide, RGDS, and by an alpha IIb beta 3-selective RGD peptidomimetic. Furthermore, the assay detected inhibition of PAC1 binding by intracellular inhibitors of platelet activation, including bisindo...Continue Reading

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