A Mechanistic Model for Paradoxical Platelet Activation by Ligand-Mimetic α IIb β 3 (GPIIb/IIIa) Antagonists

Abstract

Objective— Integrins are attractive therapeutic targets. Inhibition of integrin α IIb β 3 effectively blocks platelet aggregation. However, limitations with intravenous α IIb β 3 antagonists and failure of oral α IIb β 3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. Methods and Results— Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic α IIb β 3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of α IIb β 3 , (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic α IIb β 3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic α IIb β 3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca 2+ -signaling. Blockade of the ADP receptor P2Y 12 by AR-C69931MX and clopidogrel inhibits α IIb β 3 antagonist-induced platelet activation. Conclusion— These findings can explain limitations of ligand-mimetic anti-α IIb β 3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.