Purification and characterization of factor VIII 1,689-Cys: a nonfunctional
cofactor occurring in a patient with severe hemophilia A
DP O'Brien and EG Tuddenham
Haemostasis Research Group, Clinical Research Centre, Middlesex, England.
We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL
VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after
thrombin activation by Western blotting with monoclonal antibodies (MoAbs).
Normal or patient citrated plasma was ultracentrifuged,
cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void
volume fractions were reduced and subjected to ion exchange chromatography
yielding material of specific activity approximately 1,000 U/mg protein
(VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is
fully activatable by thrombin with proteolytic fragmentation as previously
described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII
1,689-Cys has the normal distribution of factor VIII light and heavy chains
prior to thrombin activation. After exposure to thrombin the heavy chain
polypeptides were fully proteolysed but the light chain was totally
resistant to cleavage. This is consistent with the demonstration in the
patient's leucocyte DNA of a C to T transition in codon 1,689 converting
Arg to Cys at the light chain thrombin cleavage site as previously
described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light
chain of Factor VIII 1,689-Cys is not released from von Willebrand factor
(vWF) by thrombin, but this is not the sole cause of the functional defect
since the protein purified free of vWF has no coagulant activity. We
conclude that the functional defect in factor VIII 1,689-Cys is a
consequence of failure to release the acidic peptide from the light chain
upon thrombin activation.
Volume 73,
Issue 8,
pp. 2117-2122,
06/01/1989
Copyright © 1989 by The American Society of Hematology
