Anti-heavy-chain monoclonal antibodies directed to the acidic regions of the factor VIII molecule inhibit the binding of factor VIII to phospholipids and von Willebrand factor.

Recent studies have shown that inhibitors develop against acidic regions of the FVIII molecule, which contain important functional sites. However, their mechanisms of inhibition are not well understood. In this study, two anti-human FVIII mouse monoclonal antibodies (MAbs), directed towards the exposed acidic regions of the FVIII molecule, were developed, characterised and their mechanisms of inhibition investigated. The two MAbs, F7B4 and F26F6, had inhibitory titres of 32 and 944 BU/mg respectively, had high affinities for the FVIII molecule (K(D) approximately nM range) and recognised sequences V(357)-F(360) on the acidic a1 region and E(724)-L(731) on the acidic a2 region of the FVIII heavy-chain (HC), respectively. F7B4 inhibited the rate of FXa generation by activated FVIII, whilst both antibodies inhibited FVIII activation by thrombin and blocked thrombin cleavage of FVIII. Furthermore, F7B4 and F26F6 inhibited FVIII binding to (a) phospholipids (IC(50): 77 nM and 40 nM respectively), and (b) VWF (IC(50): 93 nM and 267 nM respectively), despite both having HC specificity. Experiments with F(ab')(2) fragments confirmed the above findings. Taken together these data represent novel findings in that anti-acidic HC antibodies can inhibit FVIII function by a variety of mechanisms, in particular by interfering with the binding of FVIII to phospholipids & VWF.

Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.

Factor VIII (FVIII) is assayed by one-stage and two-stage clotting methods and by chromogenic methods, although the chromogenic method has largely replaced the two-stage clotting assay. Clinical plasma samples are assayed mostly by one-stage assays, but most manufacturers of concentrates use the chromogenic method, which is more precise and is the reference method of the European Pharmacopoeia and the International Society on Thrombosis and Haemostasis (ISTH). For most plasma-derived concentrates, assays against the World Health Organization (WHO) concentrate standard give similar results with the one-stage and chromogenic methods, but for products produced by the "method M" monoclonal antibody process, the one-stage potency is 25 to 30% higher than the chromogenic potency. For full-length recombinant products assayed against a plasma-derived concentrate standard, one-stage potencies are about 10% lower than chromogenic potencies, but for the B-domain deleted recombinant product ReFacto, the discrepancy is larger-from 20 to 50%. These discrepancies emphasize the need for an international methodology for labeling of concentrates. In ex vivo assays of hemophilic plasmas after infusion of concentrates, large discrepancies are found among laboratories and with different assay methods when a plasma standard is used. In most studies, the chromogenic potencies are higher than the one-stage potencies, and the discrepancy is highest for recombinant products. This discrepancy can be largely eliminated by the use of concentrate standards, diluted in FVIII-deficient plasma, to assay postinfusion plasma samples.