FVIII inhibitors display FV?neutralizing activity in the prothrombin time assay

AbstractBackground

The coagulation factors (F)V and VIII are homologous proteins that support hemostasis through their regulation of FX activity. Hemophilia A (HA) patients have reduced FVIII activity and a prolonged bleeding time that is corrected through the administration of exogenous FVIII. Around one-third of severe HA patients develop FVIII neutralizing antibodies, known as “inhibitors,” which neutralize FVIII activity and preclude them from further FVIII therapy.

Objectives

We hypothesized that, based on the degree of homology between FV and FVIII (~40%), FVIII-neutralizing antibodies could cross react with FV. To test this hypothesis, a panel of recombinant, patient-derived, FVIII-neutralizing antibodies were screened for cross-reactivity against FV.

Methods

Factor V and FVIII activity was measured using one-stage clotting assays; structural analysis was carried out using a structural approach.

Results

We detected FV neutralizing activity with the anti-FVIII A2 domain antibody NB11B2. Because this antibody was derived from an HA inhibitor patient, FV-neutralizing activity was then evaluated in a number of HA inhibitor patient plasma samples; nine alloimmune samples had FV-neutralizing activity whereas no FV neutralizing activity was found in the two autoimmune samples available. We next examined the degree of surface homology between FV and FVIII and found that structural similarity could explain the cross reactivity of the anti-A2 antibody and likely accounts for the cross reactivity we observed in patient samples.

Conclusions

Although this novel observation is of interest, further work will be needed to determine whether FV neutralization in HA patient samples contributes to their bleeding diathesis.