The murine monoclonal antibody (mAb) PT25-2 induces aIIbβ3 to bind ligand and initiate platelet aggregation. The underlying mechanism is unclear, because previous mutagenesis studies suggested that PT25-2 binds to the aIIb b propeller, a site distant from the Arg-Gly-Asp-binding pocket. To elucidate the mechanism, we studied the aIIbb3-PT25-2 Fab complex by negative-stain and cryo-electron microscopy (EM). We found that PT25-2 binding results in aIIbb3 partially exposing multiple ligand-induced binding site epitopes and adopting extended conformations without swing-out of the b3 hybrid domain. The cryo-EM structure showed PT25-2 binding to the aIIb residues identified by mutagenesis but also to 2 additional regions. Overlay of the cryo-EM structure with the bent aIIbb3 crystal structure showed that binding of PT25-2 creates clashes with the aIIb calf-1/calf-2 domains, suggesting that PT25-2 selectively binds to partially or fully extended receptor conformations and prevents a return to its bent conformation. Kinetic studies of the binding of PT25-2 compared with mAbs 10E5 and 7E3 support this hypothesis. We conclude that PT25-2 induces aIIbb3 ligand binding by binding to extended conformations and by preventing the interactions between the aIIb and b3 leg domains and subsequently the bI and b3 leg domains required for the bent-closed conformation.
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