Inactivation of factor VIII by factor IXa.
Factor VIII (FVIII) is the nonproteolytic cofactor for FIXa in the tenase complex of blood coagulation. FVIII is proteolytically activated by thrombin and FXa in vitro to form a heterotrimer with full procoagulant activity. Activated protein C inactivates thrombin-activated FVIII through cleavage adjacent to position Arg 336 in the cofactor. We have investigated the interaction of FIXa and FVIII and subjected FVIII polypeptides to N-terminal amino acid sequence analysis. Contrary to previous reports, we were unable to demonstrate the activation of FVIII by FIXa. Incubation of these two proteins at equimolar or close to equimolar concentrations resulted in the inactivation of FVIII, coincident with cleavage of the FVIII heavy chain adjacent to Arg 336 and the light chain adjacent to Arg 1719. These cleavages were detected in the presence or absence of thrombin, indicating that FIXa does not stabilize thrombin-activated FVIIIa. APC cleaved FVIII at the same position in the heavy chain, and simultaneous incubation of FVIII, APC, and FIXa did not result in stabilization of the cofactor. We conclude that FIXa does not play a role in the stabilization or activation of FVIII.
O'Brien
D P
DP
Haemostasis Research Group, Clinical Research Centre, Harrow, Middlesex, England.
Johnson
D
D
Byfield
P
P
Tuddenham
E G
EG
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001780:Blood Coagulation Tests; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D004591:Electrophoresis, Polyacrylamide Gel; D015949:Factor IXa / Q000378:metabolism*; D005169:Factor VIII / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D010743:Phospholipids; D011994:Recombinant Proteins; D005169:Factor VIII; D015949:Factor IXa; D002118:Calcium