A Coagulation Mechanism for the Mild Bleeding Tendency in a Patient with Severe Hemophilia A Possessing an Arg1781His Mutation.

Abstract 2213

The severity and frequency of bleeding appears to be different in hemophilia A (HA) with similar factor (F)VIII activity, as a mild bleeding phenotype has been reported in 10–15% of individuals with severe HA. This heterogeneity in the clinical expression is presently not fully resolved, however. Severe HA patients with the point mutation Arg1781 to His (R1781H) in FVIII also represent a severe to mild/moderate clinical phenotype. We had a patient with mild phenotype (FVIII:C 0.9 IU/dl), presenting with this mutation. In this study, we investigated this mechanism using several global coagulation assays and purified assays with a recombinant FVIII mutant. The levels of all plasma coagulation, anti-coagulation, and fibrinolysis factors were within normal range, except for FVIII. Rotational thromboelastometry using patient whole blood revealed that the clot time and clot firmness time were significantly shortened compared to the control with similar level of FVIII:C, and were comparable to those observed at FVIII:C 5∼10 IU/dl. Thrombin and FXa generation tests using plasmas also demonstrated that the levels of peak thrombin in the former and the initial rate in the latter were comparable to those observed at FVIII:C 5∼10 IU/dl, respectively, supportive of significantly greater hemostatic potential, and potential effects upon formation of the tenase complex consisting of FVIIIa, FIXa, and FX on the phospholipid surface. The addition of excess amounts of FX to control plasma (FVIII:C 0.9 IU/dl) showed a similar waveform pattern in the R1781H patient sample, whilst the addition of FIXa did not show, might suggesting a significant alteration in the interaction of the R1781H patient FVIII(a) and FX. To clarify this mechanism, we prepared and stably expressed a recombinant, B-domainless FVIII mutant, R1781H. Thrombin generation tests using the control plasma with FVIII wild type revealed that the hemostatic function observed with R1781H FVIII (∼0.9 IU/dl) was comparable to that observed at FVIII:C ∼5 IU/dl, indicating the contribution of R1781H on the greater hemostatic potential of patients' plasma. Furthermore, a purified FXa generation assays showed that R1781H possessed an ∼1.9-fold decrease in K_m for FX compared to wild type (32.5±3.8 and 58.5±6.1 nM, respectively). This mutant did not affect the FVIIIa binding to either FIXa or phospholipid, however. These results demonstrated that the enhancement of binding affinity of FVIII R1781H for FX appeared to reflect the severity of the HA phenotype. This finding of the higher coagulant potential of the R1781H mutation may suggest utility as a new replacement therapy for HA patients. The addition of this mutant could yield a sufficient hemostatic effect at lower dose, and thus require less protein than a similar dose of native FVIII, as a ‘super-FVIII product’.