Abstract
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.
