We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal
272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two dimensional
crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified
human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease,
FVIII binding was seen only with the amino-terminal SP fragment III and not with the
carboxy-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked
FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed
to polystyrene beads. This binding was inhibited in a dose dependent manner by whole
vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited
by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced
and alkylated whole vWF. SP fragment II caused only minor inhibition. Trypsin cleavage
of SP fragment III produced a 35-kDa fragment containing the amino-terminal 272 amino
acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited
the binding of FVIII to vWF in a dose dependent manner. The other major fragment obtained
from this digestion was a two chain hetero-dimer composed of amino acid residues 273-511
and 674-728. This fragment did not inhibit FVIII binding. These studies demonstrate
that a major FVIII binding site resides within the first 272 amino acid residues of
vWF.
