Platelet-Based Factor VIII Activity Is Inhibited By Anti-C2 Domain Antibodies That Don’t Significantly Inhibit Factor VIII Activity in a Standard Assay

The major complication of hemophilia A treatment is the development of inhibitory antibodies against factor VIII (fVIII), causing severe bleeding in spite of infused fVIII. Most antibodies are targeted to the A2 or the C2 domain of fVIII and the degree of inhibition by these antibodies measured in clinical fVIII assays fails to predict the severity of bleeding risk. We investigated whether the degree of inhibition of platelet dependent fVIII activity by a panel of monoclonal anti-C2 domain mAbs deviated from the degree of inhibition in a commercial one-stage assay.

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 PAR-1 and PAR-4, and factor XIa without other exogenous activators or vesicles. Results were compared to a commercial aPTT assay (Helena Laboratories, TX) and to results of a novel factor Xase assay developed to recapitulate the conditions in an APT. In the novel assay fresh, density-gradient purified platelets were activated by thrombin followed by inactivation with hirudin and incubated with fVIII and VWF, fIXa, and fX. In a second step, phospholipid vesicles, fVa and prothrombin were added to produce thrombin in proportion to the fXa generated in the first step. For comparison to the aPTT this assay was also done using purified sonicated phospholipid vesicles instead of activated platelets in the first step. The reaction was quenched with EDTA and thrombin generation was determined immediately in a thermosttated kinetics microtiter plate reader (Molecular Devices) at 25⁰C using the chromogenic substrate S-2238 (Diapharma). A panel of 10 defined, epitope mapped mAbs against the fVIIIC2 domain was chosen in order to survey the relationship between inhibition and binding of anti-C2 antibodies.

Results: The APT exhibited a log-linear relationship between fVIII and time to fibrin strand formation over a 4-log range, encompassing 0.01% - 100% plasma fVIII. Nine of 10 mAbs inhibited fVIII activity 89-96% while mAb F85 did not inhibit. There was excellent correlation for the degree of inhibition in the platelet-based factor Xase assay for 8 of 10 antibodies with F85 inhibiting the platelet Xase but not the APT and I109 inhibiting the platelet Xase assay less than the APT. In contrast, there was no correlation between inhibition of the aPTT-based assay and the platelet assay. Four mAbs did not inhibit the aPTT assay yet inhibited approximately 90% of platelet-based activity. The novel factor Xase assay, using phospholipid vesicles instead of activated platelets, showed inhibitory results that correlated to the degree of inhibition in the aPTT assay (which also utilizes purified phospholipids). Thus, the novel assay indicates that the degree of inhibition in both platelet-based and vesicle-based clotting assays is a consequence of the membrane type rather than unidentified clotting modifiers in plasma.

Conclusions: We have developed a novel factor Xase assay to measure the low levels of residual fVIII activity, at physiologic concentrations, in the presence of inhibitory antibodies. Antibody inhibition of fVIII activity correlated with inhibition measured in the aPTT and APT clotting assays,. Our results confirm that the anti-C2 inhibitory antibodies have substantially different inhibition on a platelet membrane than in an aPTT-based assay and that the differing degree of inhibition is primarily due to fVIII susceptibility to inhibition when functioning on platelet membranes vs. phospholipid vesicles. These results suggest that current inhibitor assays might underestimate the frequency of significant inhibitory antibodies.