Studies of Protein Disulfide Isomerase (PDI) Binding to Platelets and Production of a Murine Monoclonal Antibody (mAb) to Integrin αIIbβ3 That Inhibits PDI Binding and Platelet Aggregation

L.W. and J.W. contributed equally

Introduction: The platelet integrin αIIbβ3 plays a pivotal role in both hemostasis and thrombosis, and is an established target for antiplatelet therapy. Upon activation, αIIbβ3 undergoes a conformational change from a low-affinity, bent-closed conformation to a high-affinity, extended-open conformation, resulting in the binding of fibrinogen and von Willebrand factor and platelet aggregation. αIIbβ3 activation is regulated by protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide bond formation, reduction, and isomerization. One of us (LW) previously showed that PDI binds to thrombin-activated platelets, but the αIIb or β3 conformation(s) and domain(s) to which PDI binds is unknown.

Methods: PDI was produced in E. coli and purified and labeled with Alexa-488. Human platelets and Alexa-488-PDI were stimulated with a variety of agonists either in the absence or presence of unlabeled PDI (background control) and then analyzed by flow cytometry. For monoclonal antibody (mAb) production, BALB/cAnN mice were immunized with human platelets and their spleen cells isolated and fused with a non-secretory myeloma cell line. Hybridomas were isolated by colony picking in semi-solid media and culture supernatants were assayed for their ability to inhibit PDI binding to platelets activated with the PAR1 thrombin receptor activating peptide SFLLRN (T6). One mAb that inhibited PDI binding (21D10) was assessed for its effects on platelet aggregation. The epitope of 21D10 on αIIbβ3 was explored by comparing the binding properties of 21D10 to that of previously characterized anti-αIIbβ3 mAbs. Using cryo-electron microscopy (cryo-EM) single particle analysis, we determined the structure of the Fab fragment of 21D10 in complex with full-length αIIbβ3 purified from human platelets.

Results: T6, the PAR4 thrombin receptor activating peptide AYPGKF, and collagen-related peptide induced PDI binding to washed platelets [mean fluorescence intensity (MFI) of 547 ± 21 compared with 229 ± 12 for background control, p < 0.001; 576 ± 111 vs 215 ± 27 for background control, p < 0.05; and 934 ± 99 vs 167 ± 14 for background control, p < 0.001, respectively]. The reducing agent dithiothreitol (DTT) also enhanced PDI binding (MFI of 595 ± 147 vs 178 ± 24 for background control, p < 0.05).

mAb 21D10 and its Fab fragment (40 μg/ml) inhibited PDI binding to platelets [MFI of 357 ± 25 vs 567 ± 60 for buffer control, p < 0.01 (figure); and 286 ± 38 vs 507 ± 86 for buffer control; p < 0.05)], fibrinogen binding (6,493 ± 815 vs 15,058 ± 1,935 for buffer control, p < 0.001; 1,714 ± 276 vs 14,917 ± 2,129 for buffer control, p < 0.0001), and maximum platelet aggregation (mean ± SD of 32 ± 8 vs 67 ± 2% for buffer control, p < 0.01; and 30 ± 8 vs 69 ± 3% for buffer control; p < 0.01). For comparison, the PDI inhibitor rutin at 45 μM produced similar inhibition.

21D10 did not inhibit the binding of mAbs 10E5 or 7E3, both of which inhibit fibrinogen binding to αIIbβ3, to platelets, and vice versa. EDTA treatment of platelets at either 22°C, pH 7.4, or 37°C, pH 8.0, had minimal effect on 21D10 binding, while both conditions inhibited 7E3 binding and the latter inhibited 10E5 binding. 21D10 reacted with the nonreduced but not reduced β3 subunit by immunoblotting. With chymotrypsin digested platelets, both 21D10 and the mAb 7H2, which binds to the PSI domain of β3, bound to bands extending down to 66 kDa, but 21D10 also bound to the bands below 66 kDa, whereas 7H2 did not. The cryo-EM structure of the 21D10 Fab-αIIbβ3 complex revealed that 21D10 binds to two different αIIbβ3 conformations that differ in the orientation of the leg domains (in one the legs are nearly bent and in the other the legs are partially extended). In both conformations, 21D10 binds to the PSI, I-EGF-1 and I-EGF-2 domains of β3 (figure).

Conclusion: We have shown that in addition to thrombin, T6, a PAR4 activating peptide, collagen-related peptide, and DTT can induce PDI binding to platelets. We produced a mAb to human platelets (21D10) that can inhibit T6-induced PDI binding to platelets. Immunoblotting and cryo-EM analysis shows that 21D10 binds to the PSI, I-EGF1, and I-EGF2 regions of β3. If 21D10 prevents PDI binding by steric hindrance, it is likely that PDI binds to β3 at a site distant from the RGD ligand binding pocket. The functional effects of 21D10 may reflect its inhibition of PDI to platelets, but it may also reflect inhibition of other related proteins, in particular, ERp57.