Platelet Membranes and Activation Pathway Determine Inhibitory Activity of Anti-Factor VIII C2 Domain Antibodies

Abstract

Inhibitory antibodies against factor VIII (fVIII) are the major complication of hemophilia A treatment, causing severe bleeding in spite of infused fVIII. Most antibodies are directed against the A2 or the C2 domain of fVIII and the degree of inhibition in clinical fVIII assays does not predict the severity of bleeding risk. We recently discovered that fVIII binds to a macromolecular complex including low molecular weight fibrin and the αIIbβ3integrin on membranes of thrombin-stimulated platelets rather than exposed phosphatidylserine (PS). Because clinical fVIII assays utilize PS-rich vesicles to support fVIII activity we asked whether the degree of inhibition of platelet-dependent activity by anti-C2 domain mAb’s differs from inhibition on PS-rich phospholipid vesicles.Methods: Platelet dependent fVIII activity was measured in a defined activated platelet time (APT) assay containing fresh, density gradient-purified human platelets and fVIII deficient plasma supplemented with fVIII. Coagulation was triggered by simultaneous addition of calcium, thrombin receptor activation peptides for PAR-1 and PAR-4, and either factor XIa (intrinsic pathway) or factor fXa (extrinsic pathway) without standard exogenous activators or vesicles. aPTT assays were performed on the same plasma-preparations using a commercial preparation of activator and phospholipid vesicles. Assays were performed using BBL fibrometers, electromechanical devices. A panel of 12 defined, epitope-mapped mAb’s against the fVIII-C2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies. mAb’s at 10 µg/ml were pre-incubated with fVIII for 60 min in the absence of VWF before mixing the fVIII-antibody mixture with plasma.Results: Platelets from nine donors supported a similar, near log-linear relationship between fVIII concentration and fibrin strand formation spanning at least 10,000-fold (0.0001 - 1 unit/ml). Time to fibrin strand formation was inversely related to platelet concentration over a range of at least 1 x 106 - 2 x 108/ml. Factor XIa and factor Xa concentrations of 20 pM and 0.01 pM, respectively were chosen emulating the intrinsic and extrinsic pathways.Four mAb’s inhibited fVIII activity <10% in the aPTT assay, four others by 50-80%, with the final four in the 90-99% range. fVIII activity in the intrinsic APT had little correlation to inhibition in the aPTT assay with four mAb’s causing less inhibition of the APT and the remaining 8 antibodies having 5-10 fold more inhibition. No mAb caused inhibition that was within 2-fold for the aPTT and APT assays.For the extrinsic APT, triggered by fXa, all antibodies produced greater than 99% inhibition of fVIII activity with 10 antibodies having 2-10 fold greater inhibition than with the intrinsic pathway.Conclusions: These results show that both platelet membranes and the pathway to fVIII activation determine the degree of inhibition by anti-C2 antibodies. The inhibition of platelet-dependent fVIII activity has little correlation to inhibition of the aPTT fVIII activity. Further, anti-C2 antibodies show greater inhibition of platelet-based activity when the fVIII activation pathway is via factor Xa vs. factor XIa. Additional studies will be needed to determine whether residual fVIII activity from the intrinsic APT or extrinsic APT better correlates to bleeding risk and whether the basis for activation pathway-dependent inhibition correlates to different proteolytic cleavage sites for factor VIII. DisclosuresMeeks:Genentech: Membership on an entity’s Board of Directors or advisory committees; Grifols: Membership on an entity’s Board of Directors or advisory committees; CSL Behring: Membership on an entity’s Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity’s Board of Directors or advisory committees; Biogen: Membership on an entity’s Board of Directors or advisory committees; Shire: Membership on an entity’s Board of Directors or advisory committees. Novakovic:Harvard Medical School: Patents & Royalties: Methods and Assays for Factor VIII Activity. Gilbert:Baxalta/Shire: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Harvard Medical School: Patents & Royalties: Methods and Assays for Factor VIII Activity; Grifols: Membership on an entity’s Board of Directors or advisory committees; Bayer: Membership on an entity’s Board of Directors or advisory committees, Research Funding.