Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens.

Emicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. However, details of the emicizumab-antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities (KD = 1.58, 1.52, 1.85, and 0.978 µM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens' epidermal growth factor (EGF)-like domains. We then performed KD-based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX-emicizumab-FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab's cofactor activity. The simulation also predicted that at 10.0-100 µg/ml of emicizumab-levels shown in a previous study to be clinically effective-the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The KD-based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab's cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions.

A putative inhibitory mechanism in the tenase complex responsible for loss of coagulation function in acquired haemophilia A patients with anti-C2 autoantibodies.

Acquired haemophilia A (AHA) is caused by the development of factor (F)VIII autoantibodies, demonstrating type 1 or type 2 inhibitory behaviour, and results in more serious haemorrhagic symptoms than in congenital severe HA. The reason(s) for this remains unknown, however. The global coagulation assays, thrombin generation tests and clot waveform analysis, demonstrated that coagulation parameters in patients with AHA-type 2 inhibitor were more significantly depressed than those in patients with moderate HA with similar FVIII activities. Thrombin and intrinsic FXa generation tests were significantly depressed in AHA-type 1 and AHA-type 2 compared to severe HA, and more defective in AHA-type 1 than in AHA-type 2. To investigate these inhibitory mechanism(s), anti-FVIII autoantibodies were purified from AHA plasmas. AHA-type 1 autoantibodies, containing an anti-C2 ESH4-epitope, blocked FVIII(a)-phospholipid binding, whilst AHA-type 2, containing an anti-C2 ESH8-epitope, inhibited thrombin-catalysed FVIII activation. The coagulation function in a reconstituted AHA-model containing exogenous ESH4 or ESH8 was more abnormal than in severe HA. The addition of anti-FIX antibody to FVIII-deficient plasma resulted in lower coagulation function than its absence. These results support the concept that global coagulation might be more suppressed in AHA than in severe HA due to the inhibition of FIXa-dependent FX activation by steric hindrance in the presence of FVIII-anti-C2 autoantibodies. Additionally, AHA-type 1 inhibitors prevented FVIIIa-phospholipid binding, essential for the tenase complex, whilst AHA-type 2 antibodies decreased FXa generation by inhibiting thrombin-catalysed FVIII activation. These two distinct mechanisms might, in part, contribute to and exacerbate the serious haemorrhagic symptoms in AHA.

Characterisation of an antibody specific for coagulation factor VIII that enhances factor VIII activity.

Many reports have identified factor (F)VIII inhibitory antibodies with epitopes located in all subunits of the FVIII molecule. Antibodies that promote FVIII activity do not appear to have been reported. We characterised, for the first time, a unique anti-FVIII monoclonal antibody, mAb216, that enhanced FVIII coagulant activity. The mAb216 shortened the activated partial thromboplastin time and specifically increased FVIII activity by approximately 1.5-fold dose-dependently. FXa generation and thrombin generation were similarly increased by approximately 1.4- and approximately 2.5-fold, respectively. An A2 epitope, not overlapping the common A2 epitope, was identified and the antibody was shown to enhance thrombin (and FXa)-catalysed activation of FVIII by modestly accelerating cleavage at Arg(372). The presence of mAb216 mediated an approximately 1.5-fold decrease in K(m) for the FVIII-thrombin interaction. Enhanced FVIII activity was evident to an equal degree, even the presence of anti-FVIII neutralising antibodies with epitopes in each subunit. In addition, mAb216 depressed the rates of heat-denatured loss of FVIII activity and FVIIIa decay by 2 to approximately 2.5-fold. We have developed an anti-A2, FVIII mAb216 that augmented procoagulant activity. This enhancing effect could be attributed to an increase in thrombin-induced activation of FVIII, mediated by cleavage at Arg(372) and a tighter interaction of thrombin with the A2 domain. The findings may cast new light on new principles for improving the treatment of haemophilia A patients.

[A spontaneous haemarthros in a postpartal woman: a case of acquired haemophilia A]. / Een spontane gewrichtsbloeding bij een vrouw post partum: een geval van verworven hemofilie A.

A woman aged 35 years presented with a haemarthros of the left elbow 6 months after her first parturition due to acquired haemophilia A. The bleeding episodes were mild and treated with recombinant activated factor VII. Because of the mild bleeding tendency and the chance of spontaneous remission it was decided to withhold immunosuppressive treatment. During the course of one year, the factor VIII level and the activated partial thromboplastin time (APTT) normalized and the autoantibodies disappeared. Four years after her first parturition, she had an uncomplicated pregnancy and the postpartum period was uneventful. Up to fourteen months postpartum, there are no signs of recurrence of the acquired haemophilia A. Acquired haemophilia A post partum usually presents in the first 3 months post partum. Frequently presenting symptoms are haemorrhages post partum, menorrhagia, soft-tissue haemorrhages and haemorrhages during surgical procedures. As a rule, the haemorrhages are slight. The titers of blocking antibodies are low in comparison with the titers in congenital haemophilia A.

Macrophages of human first trimester decidua express markers associated to alternative activation.

PROBLEM: Depending on the type of their activation, macrophages may promote TH1- or TH2-type of immune responses. To date, not much is known about the activation phenotype of decidua macrophages, which - together with NK cells - constitute the majority of bone marrow derived cells at this location. METHOD OF STUDY: The study was based on analysis of healthy first trimester decidua by immunohistochemistry and flow cytometry. We analyzed expression of markers characteristic for alternatively activated macrophages (Mphi2). RESULTS: The markers MS-1 (stabilin-1) and coagulation factor XIIIa were found expressed in the interior of decidua macrophages (DMphi). In contrast, indoleamine 2,3-dioxygenase (IDO), an enzyme induced in macrophages by IFNgamma, was not present in DMphi. CONCLUSIONS: First trimester DMphi display phenotypic markers associated to alternatively activated macrophages. In addition, absence of IDO indicates that DMphi are not under a predominant influence of IFNgamma.

Application of a new statistical approach to optimize the immunopurification of antihemophilia A factor.

Our aim was to optimize the immunopurification process of human factor VIII. This purification was performed using a mouse monoclonal anti-factor VIII light-chain antibody. Previous dissociation of the factor VIII-von Willebrand factor complex with CaCl2 led to a 50% increase of the factor VIII adsorption on the immunosorbent. The optimization of the elution step required the analysis of the effects of two parameters, pH and ionic strength, on four different responses: elution yield, concentration, specific activity and stability of factor VIII. For this purpose, a multifunctional method using Doehlert matrices for statistically designed experiments was applied. This methodology allowed us to obtain, with only seven experiments, a 60% increase of the elution yield and a two-fold increase of the specific activity of factor VIII.