Requirements of von Willebrand factor to protect factor VIII from inactivation by activated protein C

Abstract

The interaction of factor VIII with von Willebrand factor (vWF) was investigated on a quantitative and qualitative level. Binding characteristics were determined using a solid phase binding assay and protection of factor VIII by vWF from inactivation by activated protein C (aPC) was studied using three different assays. Deletion mutants of vWF, a 31-kD N-terminal monomeric tryptic fragment of vWF that contained the factor VIII binding site (T31) and multimers of vWF of different size were compared with vWF purified from plasma. We found that deletion of the A1, A2, or A3 domain of vWF had neither an effect on the binding characteristics nor on the protective effect of vWF on factor VIII. Furthermore, no differences in binding of factor VIII were found between multimers of vWF with different size. Also, the protective effect on factor VIII of vWF was not related to the size of the multimers of vWF. A 20-fold lower binding affinity was observed for the interaction of T31 with factor VIII, and T31 did not protect factor VIII from inactivation by aPC in a fluid-phase assay. Comparable results were found for a mutant of vWF that is monomeric at the N- terminus (vWF-dPRO). The lack of multimerization at the N-terminus may explain the decreased affinity of T31 and vWF-dPRO for factor VIII. Because of this decreased affinity, only a small fraction of factor VIII was bound to T31 and to vWF-dPRO. We hypothesized that this fraction was protected from inactivation by aPC but that this protection was not observed due to the presence of an excess of unbound factor VIII in the fluid phase. Therefore, vWF, T31, and vWF-dPRO were immobilized to separate bound factor VIII from unbound factor VIII in the fluid phase. Subsequently, the protective effect of these forms of vWF on bound factor VIII was studied. In this approach, all forms of vWF were able to protect factor VIII against inactivation by aPC completely. We conclude, in contrast with earlier work, that there is no discrepancy between binding of factor VIII to vWF and protection of factor VIII by vWF from inactivation by aPC. The protective effect of T31 was not recognized in previous studies due to its low affinity for factor VIII. The absence of multimerization observed for T31 and vWF- dPRO may explain the low affinity for factor VIII. No other domains than the binding site located at the D′ domain were found to be involved in the protection of factor VIII from inactivation by aPC.

Evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease

“…We conclude from this observation that the half-life of FVIII in these dogs was independent of the survival of all of the multimeric vWF species in the circulation. The ®nding that LMW-rvWF induced an increase in endogenous FVIII levels is consistent with in vitro observations that multimers of different sizes have equivalent af®nities for FVIII [6].…”

A qualitative and quantitative analysis of von Willebrand factor contained in a very high‐purity plasma‐derived FVIII concentrate

“…Moreover, Koppelman et al. [40] showed that all molecular forms of VWF were able to protect FVIII against inactivation by activated Protein C.…”

Development of a novel automated screening method for detection of FVIII Inhibitors

“…Factor VIII (FVIII) clotting factor circulates as an inactive plasma protein tightly associated with von Willebrand factor . In response to blood vessel injury, FVIII is cleaved between its A1 and A2 domains, resulting in an unstable heterotrimeric factor VIIIa molecule which interacts with Factor IX in a subsequent, well‐characterized series of reactions that result in blood clot formation .…”