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Requirements of von Willebrand factor to protect factor VIII from
inactivation by activated protein C
SJ Koppelman, M van Hoeij, T Vink, H Lankhof, ME Schiphorst, C Damas, AJ Vlot, R Wise, BN Bouma and JJ Sixma
Department of Haematology, University Hospital Utrecht, The Netherlands.
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.
Volume 87,
Issue 6,
pp. 2292-2300,
03/15/1996
Copyright © 1996 by The American Society of Hematology
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