Factor VIII Inhibitors: Von Willebrand Factor Makes A Difference In Vitro and In Vivo
Authors: Shi Qizhen , Kuether Erin L. , Schroeder Jocelyn A. , Perry Crystal L. , Fahs Scot A. , Montgomery Robert R. 
Published: October 2019

Abstract
Abstract 709
The important association between von Willebrand factor (VWF) and factor VIII (FVIII) has been investigated for decades, but the effect of VWF on FVIII inhibitors is still controversial. Studies have demonstrated that some anti-FVIII inhibitory antibodies inhibit VWF-FVIII interaction, while others rely on the presence of VWF to inhibit FVIII activities. The influence of VWF on the Bethesda assay, which is routinely used in the clinic to determine the titer of FVIII-neutralizing inhibitors, is still uncertain because the plasma from hemophilia A patients with inhibitors contains normal levels of VWF. To explore the effect of VWF on the reactivity of FVIII inhibitors, we immunized VWF and FVIII double knockout (VWFnullFVIIInull) mice with recombinant human B-domain deleted FVIII (rhFVIII) to induce anti-FVIII inhibitory antibody development. Inhibitory plasma was collected and the titer of inhibitors was determined by Bethesda assay. Murine plasma-derived VWF (from FVIIInull mice) or recombinant human VWF (rhVWF) was used to study the influence of VWF on inhibitor inactivation of FVIII activity (FVIII:C). The remaining FVIII:C after inactivation was determined by chromogenic assay. When inhibitory plasma was incubated with rhFVIII in the presence of 1 U/ml VWF, the residual FVIII activity recovered was higher than in the absence of VWF, resulting in 6.82 ± 1.12 (n = 27) fold lower apparent inhibitor titers. This protective effect is VWF dose dependent. The source of VWF (plasma-derived murine VWF vs. rhVWF) did not affect its protection of FVIII from inhibitor inactivation and VWF does not affect FVIII:C measured in the chromogenic assay in the absence of inhibitors. Interestingly, we found that inhibitor inactivation of FVIII:C in the absence of VWF occurred much faster than in its presence. When the usual 2 hr. incubation at 37°C was omitted from the Bethesda assay, adding rhVWF to rFVIII before mixing with inhibitory plasma resulted in 67.29 ± 20.18 (n = 5) fold lower apparent inhibitor titers than without added VWF. In contrast, if VWF was added to inhibitory plasma first and then mixed with rhFVIII, the inhibitor titers were only 11.04 ± 3.56 (n = 5) fold lower than without added VWF. These results indicate that rhFVIII present in a preformed VWF-FVIII complex is protected from inhibitory antibody inactivation. Conversely, when VWF and inhibitory plasma are added to rhFVIII at the same time, the VWF and inhibitors appear to compete to bind to rhFVIII. Inhibitor titers were lower than in the absence of VWF, but the protective effect is not as efficient as when VWF and rhFVIII were already associated with one another before encountering inhibitors. To confirm the protective effect of VWF on FVIII from inhibitor inactivation, we infused FVIIInull or VWFnullFVIIInull mice with inhibitory plasma and rhFVIII followed by a tail clip survival test. When rhFVIII was infused into FVIIInull mice to 2% followed by inhibitory plasma infusion, all mice with inhibitor titer of 2.5 BU/ml (n = 4) survived tail clipping, and 2 of 4 survived with either 25 BU/ml or 250 BU/ml. If inhibitory plasma was infused first followed by rhFVIII infusion, then only 2 of 6 mice with inhibitor titers of 2.5 BU/ml survived tail clip challenge and none survived with 25 BU/ml and 250 BU/ml. In the first set of mice the infused FVIII was able to form a protective complex with endogenous VWF before encountering inhibitors, while in the second set FVIII is exposed to VWF and pre-infused inhibitory antibodies at the same time, a competitive binding that appears to reduce VWF's protective effect. In contrast, when rhFVIII was infused into VWFnullFVIIInull mice followed by inhibitory plasma infusion, no animals (n = 4 for each group) survived tail clipping with inhibitor titers of 2.5 BU/ml or higher. In summary, our studies demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo. While the role of VWF in stabilizing plasma FVIII in a milieu rich in proteases has been appreciated for decades, our results indicate that treatment utilizing products containing a complex of FVIII with VWF may be especially beneficial in hemophilia A patients with inhibitors.

Disclosures:
No relevant conflicts of interest to declare.