Objective--Integrins are attractive therapeutic targets. Inhibition of integrin _IIb3 effectively blocks platelet aggregation. However, limitations with intravenous IIb₃ antagonists and failure of oral _IIb3 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 _IIb3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of _IIb3, (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic _IIb3 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 _IIb3 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 _IIb3 antagonist-induced platelet activation.

Conclusion--These findings can explain limitations of ligand-mimetic anti-_IIb3 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.