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Binding of Factor VIII to von Willebrand Factor Is Enabled by
Cleavage of the von Willebrand Factor Propeptide and Enhanced by
Formation of Disulfide-Linked Multimers
Ana Victoria Bendetowicz,
Jill A. Morris,
Robert J. Wise,
Gary E. Gilbert, and
Randal J. Kaufman
From the Brockton/West Roxbury VAMC, West Roxbury, MA; the Brigham
and Women's Hospital, Boston, MA; the Harvard University Medical
School, Boston, MA; the Department of Biological Chemistry and the
Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI.
von Willebrand factor (vWF) is a multimeric adhesive glycoprotein
with one factor VIII binding site/subunit. Prior reports suggest that
posttranslational modifications of vWF, including formation of
N-terminal intersubunit disulfide bonds and subsequent cleavage of the
propeptide, influence availability and/or affinity of factor
VIII binding sites. We found that deletion of the vWF propeptide
produced a dimeric vWF molecule lacking N-terminal intersubunit
disulfide bonds. This molecule bound fluorescein-labeled factor VIII
with sixfold lower affinity than multimeric vWF in an equilibrium flow
cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide
in trans yielded multimeric vWF that displayed increased affinity for
factor VIII. Insertion of an alanine residue at the N-terminus of the
mature vWF subunit destroyed binding to factor VIII, indicating that
the native mature N-terminus is required for factor VIII binding. The
requirement for vWF propeptide cleavage was shown by (1) a point
mutation of the vWF propeptide cleavage site yielding pro-vWF that was
defective in factor VIII binding and (2) correlation between efficiency
of intracellular propeptide cleavage and factor VIII binding.
Furthermore, in a cell-free system, addition of the propeptide-cleaving
enzyme PACE/furin enabled factor VIII binding in parallel with
propeptide cleavage. Our results indicate that high-affinity factor
VIII binding sites are located on N-terminal disulfide-linked vWF
subunits from which the propeptide has been cleaved.
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 529-538
© 1998 by the American Society of Hematology.
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