An αIIbβ3 Monoclonal Antibody Traps a Semi-Extended Conformation and Allosterically Inhibits Large Ligand Binding

• A new monoclonal antibody to the αIIbβ3 I-EGF1 domain inhibits PDI binding, fibrinogen binding, and platelet aggregation.

• The antibody traps a semi-extended conformation of αIIbβ3 and inhibits fibrinogen binding via an allosteric mechanism.

Monoclonal antibodies (mAbs) have provided valuable information on the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation and binds to thrombin-activated αIIbβ3. Using human platelets as immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with a thrombin receptor-activating peptide (T6). R21D10 also partially inhibits T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and ADP-induced platelet aggregation. Mutual competition experiments show that R21D10 does not inhibit binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-β3 β-I domain) and immunoblot studies indicate that R21D10 binds to β3. Dissociation of αIIbβ3 by EDTA had minimal effect on R21D10 binding. Cryo-electron microscopy of the αIIbβ3-R21D10 Fab complex reveals that R21D10 binds to the β3 I-EGF1 domain and traps an intermediate conformation of αIIbβ3 with semi-extended leg domains. Binding of R21D10 produces a major structural change in the β3 I-EGF2 domain associated with a new interaction between the β3 I-EGF2 and the αIIb thigh domains, which may prevent the swing-out motion of the β3 hybrid domain required for high-affinity ligand binding and protect αIIbβ3 from EDTA-induced dissociation. R21D10 partially reverses the ligand binding priming effect of eptifibatide, suggesting that it can convert the swung-out conformation into the semi-extended conformation. We conclude that R21D10 inhibits ligand binding to αIIbβ3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.