Inhibitor titres were defined by the dilution of inhibitor samples until 50% of the initial FVIII:C activity was neutralized. In all instances, incubation of samples with rhFVIII in the presence of VWF resulted in higher residual FVIII:C activity and lower apparent inhibitor titres compared with incubation with rhFVIII in the absence of VWF. The ratio of inhibitor titres with and without VWF was sevenfold lower in the presence of inhibitors from immunized VWFnullFVIIInull mouse plasma, fivefold lower in the presence of purified plasma PKC412 supplier IgG from human inhibitor patients, and sixfold lower

in the presence of cloned human monoclonal antibodies from inhibitor patients (Fig. 4). Thus, VWF has a protective effect on FVIII, reducing inactivation by inhibitors in both

mouse and human samples. This protective effect against inhibitor inactivation of FVIII was dose-dependent and similar irrespective of VWF source (rhVWF, plasma-derived human VWF, plasma-derived VWF from mouse) [31]. The protective effect of preformed complex of VWF and FVIII was then investigated by mixing up the experiments: rhVWF and rhFVIII were mixed together and allowed to form a non-covalent preformed complex. The mixture was then incubated with inhibitors from VWFnullFVIIInull mice. rhVWF was mixed together with inhibitors from VWFnullFVIIInull http://www.selleckchem.com/products/ink128.html mice, then incubated with rhFVIII. Control samples with no added VWF were assayed in parallel. The time dependence of the antigen-antibody reaction was assessed using the standard Bethesda assay method with assays performed immediately after the mixture and after a 2-hour incubation period. In all cases, FVIII levels were higher in the presence of VWF than in the absence of VWF, resulting in lower inhibitor titres (Fig. 5). Specifically, inhibitor titres were fivefold lower with preformed VWF/FVIII complex compared with when FVIII was MCE exposed to VWF and

antibodies at the same time. Simultaneous exposure of rhFVIII to VWF and inhibitory antibodies resulted in competition for binding to the FVIII molecule. The protective effect of VWF against inhibitory antibodies was more evident (50% better protection) when samples were assayed immediately after the mixture compared with after a 2-h incubation, highlighting the time dependency of VWF protection [31]. The effect of the presence of VWF in assay reagents on measured inhibitor titres was also explored. In this experiment, samples of immunized VWFnullFVIIInull mouse plasma with inhibitors ranging from 20 to 2000 BU mL−1 were incubated with rhFVIII in the presence and absence of VWF from plasma-derived and recombinant sources. The remaining FVIII activity was higher when VWF was present in the assay reagents, resulting in lower apparent inhibitor titres.