Role of the B domain in proteolytic inactivation of activated coagulation factor VIII by activated protein C and activated factor X.
Hereditary deficiency of factor VIII (FVIII), haemophilia A, is treated by plasma-derived FVIII (pd-FVIII) or recombinant FVIII (rFVIII) infusions. B-domain-deleted FVIII (BDD-rFVIII), although generally safe and effective, was less effective than pd-FVIII in prophylaxis -- evidenced by a 2.5-fold higher bleeding incidence. Assessment of BDD-rFVIII activity in chromogenic and one-stage clotting assays gives up to 50% difference in activity values. As earlier studies demonstrated identical activation and cofactor activity of BDD-rFVIII and pd-FVIII, we decided to study susceptibility of thrombin-activated pd-FVIII, full-length rFVIII and BDD-rFVIII to proteolytic inactivation by activated protein C (APC) and activated factor X (FXa) in a purified system. Proteolysis was monitored by Western blot using monoclonal antibodies C5 and R8B12 specific for the A1 and A2 domains, respectively. Inactivation was monitored by measuring the residual cofactor activity of FVIII forms in a one-stage clotting assay. Proteolysis of A1 and A2 domains of activated BDD-rFVIII proceeded 11 or 13 times faster than that of pd-FVIII or full-length rFVIII. Inactivation of activated BDD-rFVIII was two to three times faster by APC and five to six times faster by FXa. We suggest that differences in proteolytic inactivation may contribute to differences between BDD-rFVIII and pd-FVIII in assaying and in clinical use.
Khrenov
Alexey V
AV
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. AKhrenov@som.umaryland.edu
Ananyeva
Natalya M
NM
Saenko
Evgueni L
EL
D015153:Blotting, Western; D005169:Factor VIII / Q000502:physiology*; D015944:Factor VIIIa / Q000378:metabolism*; D015951:Factor Xa / Q000494:pharmacology*; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology / Q000502:physiology*; D011486:Protein C / Q000494:pharmacology*; D013997:Time Factors
C451034:B-domain-deleted factor VIII; D010446:Peptide Fragments; D011486:Protein C; D015944:Factor VIIIa; D005169:Factor VIII; D015951:Factor Xa