Integrins are attractive therapeutic targets. Inhibition of integrin αIIbβ3 effectively blocks platelet aggregation. However, limitations with intravenous αIIbβ3 antagonists and failure of oral αIIbβ3 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 αIIbβ3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of αIIbβ3, (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic αIIbβ3 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 αIIbβ3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca2+-signaling. Blockade of the ADP receptor P2Y12 by AR-C69931MX and clopidogrel inhibits αIIbβ3 antagonist-induced platelet activation.Conclusion—
These findings can explain limitations of ligand-mimetic anti-αIIbβ3 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.