A monoclonal antibody to platelet αIIbβ3 that inhibits protein disulfide isomerase binding and platelet aggregation.

abstractPlatelet integrin αIIbβ3 plays a pivotal role in hemostasis and thrombosis. Protein disulfide isomerase (PDI) binds to αIIbβ3 and mediates its activation. The binding region(s) on αIIbβ3 for PDI remains to be established. We identified a new anti-αIIbβ3 murine monoclonal antibody, R21C11, that inhibits PDI binding to platelets, ligand binding to αIIbβ3, and platelet aggregation. R21C11 inhibited recombinant PDI binding to platelets activated with the PAR1 thrombin receptor activating peptide SFLLRN (T6). Reciprocally, PDI partially reduced R21C11 binding to platelets. PDI was translocated to the platelet surface after activation and activated platelets showed higher PDI reductase activity. R211C11 decreased both the amount of PDI and the reductase activity. R21C11 partially inhibited both fibrinogen and PAC-1 binding to αIIbβ3 and platelet aggregation induced by ADP and T6. R21C11 bound slowly to unactivated platelets and more rapidly after T6 activation; eptifibatide, which induces the αIIbβ3 extended-open conformation, also increased the speed of R21C11 binding. Activation also increased total R21C11 binding. Cryogenic electron microscopy single-particle analysis of the R21C11 Fab-αIIbβ3 complex revealed that R21C11 binds to the β3 β-tail domain in both nearly bent and semiextended-closed conformations of αIIbβ3. R21C11 Fab clashed with the β3 β-I domain in the fully bent conformation, accounting for the slow binding rate of R21C11. These data are consistent with R21C11 inhibiting PDI binding by steric hindrance and the activation dependence of PDI binding. Taken together, these data suggest that the β-tail domain and/or neighboring area is the binding site for PDI on integrin αIIbβ3.