A human factor VIII inhibitor tolerating the endogenous FVIII with a unique A2 domain substitution in CRM + hemophilia A

Abstract number: P0648

Habart^* D., Novotny† M., Komrska‡ V., Dobrovolna^* M., Vorlova^* Z.

^*Institute of Haematology and Blood Transfusion, Czech Republic; ‡University Hospital Motol, Czech Republic †University of Uppsala, Sweden;

We report on a large kindred with mild/moderate hemophilia A in which factor VIII inhibitor developed. Studying of such rare inhibitors provided insight into the inhibitor pathophysilogy (Saenko et al. Haemophilia, 2002). The aim of our study was to characterize the structural basis of the factor VIII deficiency in this family and to determine the specificity and kinetics of the inhibitor. The FVIII activity and antigen were measured by the one stage assay and the ELISA. The FVIII inhibitor determinations were based on the Bethesda assay. A conformation sensitive gel electrophoresis and direct sequencing were used for mutation detection. The molecular modeling was performed by the SwissPdb program based on the 3D-model of FVIII. The HLA class II alleles at the loci DRB1, DRB3-5, and DQB1 were tested by PCR-SSC method. The levels of the FVIII:C 3–9% and the FVIII:Ag 35–55% constituted the diagnosis of cross-reactive material positive (CRM⁺) mild/moderate hemophilia A. In the FVIII gene a nucleotide change 1201C > G was detected, causing unique amino acid substitution Trp382Gly within the FVIII A2 domain. Molecular modeling predicted loss of an amino–aromatic interaction between the Trp382 and the backbone of Gln561. A conformation change of the loop 558–565, which is critical in modulating the FIXa activity in the Xase complex (Jenkins, Blood, 2002), might explain the CRM⁺ phenotype. The factor VIII inhibitor of 4–20 BU mL^-1 was detected in a member of the family, after failure of the substitution therapy. It was absent in five related patients, none of whom had the class II HLA alleles identical to the propositus. The inhibitor did not suppress the endogenous FVIII activity, suggesting tolerance to the endogenous FVIII. This hypothesis was verified by a modified Bethesda assay, using the post-DDAVP plasma from a related noninhibitory hemophiliac as a source of the 382Gly mutant FVIII. Plasma from the propositus inhibited the wild type but not the mutant FVIII. In agreement with this finding was a good response to the DDAVP in the propositus. Due to low titer of the inhibitor, the plasma dilution studies were inconclusive between the type I and II kinetics (Ling, Br J Haematol, 2001). We conclude that the inhibitor blocks the wild type and tolerates the endogenous FVIII. The endogenous FVIII is dysfunctional due to a unique amino acid substitution, Trp382Gly within the A2 domain, which is predicted to alter the cofactor activity of factor VIIIa to the factor IXa. Our results suggest that the inhibitor may block the interaction between the FVIII loop 558–565 and the factor IXa. Testing of the kinetics and the inhibitory epitope will be based on the isolated IgG.

Supported by: Grant NH/7615-3.