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Purification and characterization of factor VIII 372-Cys: a hypofunctional
cofactor from a patient with moderately severe hemophilia A
DP O'Brien, JK Pattinson and EG Tuddenham
Haemostasis Research Group, Clinical Research Centre, Harrow, Middlesex,
UK.
We have purified factor VIII from a patient with moderately severe
hemophilia A (FVIII, 4 U/dL; FVIII:Ag, 110 U/dL) and subjected the protein
to Western blot analysis after time course activation with thrombin. The
cross reacting material-positive (CRM+) FVIII has the normal distribution
of heavy and light chains before thrombin activation, and, after incubation
with the enzyme, appropriate cleavages are made at positions 740 and 1689.
However, the normal thrombin cleavage at position 372 in the heavy chain of
this molecule does not occur. This result is consistent with the
demonstration in the patient's leukocyte DNA of a C to T transition in
codon 372, leading to the substitution of a cysteine for an arginine
residue at the heavy chain internal cleavage site. The severely impaired
functional activity of this molecule confirms that the heavy chain of FVIII
must be proteolysed in order to effect full cofactor activation in vivo.
However, a threefold activation was detected when this protein was
incubated with thrombin. No evidence of thrombin-mediated cleavage at
position 336 in the heavy chain was detected, in contrast to the variant
recombinant B domainless-molecule, FVIII 372-Ile, described by Pittman and
Kaufman (Proc Natl Acad Sci USA 85:2429, 1988). Using gel permeation
studies of the FVIII/von Willebrand factor (vWF) complex before and after
thrombin activation, we have demonstrated that the 40 Kd A2 domain of wild
type FVIII dissociates from vWF after cleavage by the enzyme. In contrast,
incomplete dissociation was detected in the case of FVIII 372-Cys. We
conclude that the functional defect in FVIII 372-Cys is a consequence of
the resistance to proteolysis of the internal scissile bond in the heavy
chain.
Volume 75,
Issue 8,
pp. 1664-1672,
04/15/1990
Copyright © 1990 by The American Society of Hematology
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