A murine monoclonal antibody to integrin αIIbβ3 that inhibits protein disulfide isomerase binding to platelets and platelet aggregation

Abstract L.W. and J.W. contributed equally Introduction: Platelet αIIbβ3 plays a pivotal role in hemostasis and thrombosis. Upon activation, it undergoes a change from a low-affinity, bent-closed, to a high-affinity, extended-open conformation, resulting in ligand binding and platelet aggregation. Protein disulfide isomerase (PDI) binds to αIIbβ3 and is proposed to regulate its activation, but the binding site on αIIbβ3 has not been identified. We now report the binding site of a new monoclonal antibody (mAb), R21C11, that binds to αIIbβ3, inhibits PDI binding to platelets, and appears to inhibit PDI binding by steric hindrance, suggesting that it is binding near or at the PDI binding site. Methods: Exogenous PDI binding: Alexa488-labeled PDI was added to washed platelets (WP), activated with the PAR1 thrombin receptor activating peptide SFLLRN (T6); binding was analyzed by flow cytometry (FC). Surface exposure of platelet endogenous PDI: WP were activated with T6 and PDI was detected with a rabbit anti-PDI antibody by FC. PDI reductase activity assay: WP were activated with T6 and a PAR4 activating peptide AYPGKF, washed, and mixed with Di-eosin-GSSG. Increased fluorescence, indicating reduction of the thiol in GSSG, was monitored (excitation 525 nm, emission 545 nm). Platelet aggregation: WP or platelet-rich plasma (PRP) was activated with T6 or ADP. mAb IV.3 was added to block binding of immunoglobulin Fc domain to FcγIIa receptor (FcγRIIa). mAb binding kinetics: WP were incubated with Alexa488-labeled mAbs and fluorescence was measured at multiple time points by FC. The structure of R21C11 Fab in complex with full-length αIIbβ3 purified from human platelets was determined by cryo-electron microscopy (cryo-EM) single particle analysis. Results: 1. Exogenous PDI binding: R21C11 at 40 µg/ml inhibited PDI binding to T6-activated platelets by 70 ± 22% (P=0.003; n=8) compared to background binding (binding in the presence of 10-fold excess unlabeled PDI). Reciprocally, PDI reduced 21C11 binding to platelets by 18 ± 7% (P=0.03; n=6) when added at 100-fold excess. 2. Endogenous PDI binding. Platelet surface PDI increased by 37±16% (P=0.02; n=4) with platelet activation. 40 µg/ml of R21C11 decreased the surface exposure of PDI by 40 ± 20% (P=0.006; n=5). Platelet activation also increased PDI reductase activity on platelets compared to unactivated platelets and R21C11 decreased the reductase activity by 27 ± 12% (P=0.02; n=4) calculated by initial slope. 3. Ligand binding: Initial studies demonstrated that intact R21C11 bound both to αIIbβ3 and FcγRIIa and so studies of fibrinogen and PAC1 binding to T6-activated platelets were conducted with R21C11 F(ab‘)2. R21C11 F(ab‘)2decreased the binding of both fibrinogen and PAC1 to platelets at 40 µg/ml. 4. Platelet aggregation: 40 µg/ml R21C11 inhibited the primary slope and the maximum extent of aggregation of WP induced by T6; it also reduced the aggregation of platelets in PRP induced by ADP. 5.The rate of R21C11 binding to WP was slow compared to the binding of mAb 7E3, which binds much slower than mAb 10E5. Platelet activation by T6 increased both the speed of R21C11 binding and the total amount bound. Eptifibatide, which induces αIIbβ3 to adopt the extended-open conformation, also increased the speed and amount of 21C11 bound to platelets. 6. R21C11 did not affect the binding of R21D10, the allosteric ligand-binding inhibitor mAb we recently reported that also inhibits PDI and fibrinogen binding. Combining the mAbs produced greater inhibition of fibrinogen binding than either mAb alone and greater inhibition of PDI binding than R21C11 alone at 40 µg/ml. 7. Cryo-EM single particle analyses of the R21C11 Fab-αIIbβ3 complex revealed that R21C11 binds to the β3 β-tail domain in both bent and semi-extended-closed conformations of αIIbβ3. Conclusions: mAb R21C11 binding to platelet αIIbβ3: 1. Inhibits exogenous PDI binding to platelets. 2. Is inhibited by exogenous PDI, suggesting that R21C11 inhibits PDI binding by steric hindrance. 3. Inhibits activation-induced endogenous PDI expression and platelet surface thiol-disulfide reductase activity. 4. Inhibits fibrinogen binding to αIIbβ3 and platelet aggregation 5. Is localized to an activation-dependent epitope on the β-tail domain of β3. These properties suggest that both exogenous and endogenous PDI bind to the disulfide-rich β3 β-tail domain, and that PDI's activity on disulfides in this region can contribute to activation of αIIbβ3.