Factor IXa protects factor VIIIa from activated protein C. Factor IXa inhibits activated protein C-catalyzed cleavage of factor VIIIa at Arg562.
Factor VIIIa is inactivated by both factor IXa and activated protein C. The latter protease rapidly attacked a site at Arg562 (A2 subunit), whereas both proteases slowly cleaved factor VIIIa at Arg336 (A1 subunit). Cofactor inactivation catalyzed by activated protein C was 8-fold faster than that catalyzed by factor IXa. Simultaneous reaction of factor VIIIa with the two enzymes resulted in a rate of inactivation intermediate to that observed for the individual proteases. Under these conditions, the activated protein C-catalyzed cleavage at Arg562 was inhibited such that cofactor inactivation resulted primarily from cleavage at Arg336. Substitution of factor IXa modified in its active site with 6-(dimethylamino)-2-naphthalenesulfonyl-glutamylglycylarginyl++ + chloromethyl ketone (DEGR-IXa) for the native enzyme yielded a similar rate of activated protein C-catalyzed cleavage at the A1 site, whereas cleavage at the A2 site was virtually eliminated. However, the inclusion of protein S resulted in a marked increase in cleavage at the A2 site that correlated with an increased rate of cofactor inactivation. Active site-modified activated protein C inhibited the factor IXa-dependent enhancement of factor VIIIa reconstitution from isolated subunits. In addition, the factor VIIIa-dependent fluorescence enhancement of DEGR-activated protein C was inhibited by EGR-IXa. These results indicate that factor IXa can reduce the rate of activated protein C-catalyzed cofactor inactivation by selectively blocking cleavage at the A2 domainal site, an effect reversed by protein S. One mechanism consistent with the reciprocal inhibitory effects of the proteases is that activated protein C and factor IXa occupy overlapping sites on the cofactor. Thus, factor IXa may protect factor VIIIa by preventing activated protein C binding.
Regan
L M
LM
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Lamphear
B J
BJ
Huggins
C F
CF
Walker
F J
FJ
Fay
P J
PJ
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D003619:Dansyl Compounds; D015949:Factor IXa / Q000494:pharmacology*; D015944:Factor VIIIa / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D011486:Protein C / Q000378:metabolism*; D017293:Protein S / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000590:Amino Acid Chloromethyl Ketones; D003619:Dansyl Compounds; D046911:Macromolecular Substances; D011486:Protein C; D017293:Protein S; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; D015944:Factor VIIIa; D001120:Arginine; D015949:Factor IXa