FACTOR-IXA PROTECTS FACTOR-VIIIA FROM ACTIVATED PROTEIN-C - FACTOR-IXA INHIBITS ACTIVATED PROTEIN-C-CATALYZED CLEAVAGE OF FACTOR-VIIIA AT ARG(562)

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 subun it). Cofactor inactivation catalyzed by activated protein C was 8-fold faster than that catalyzed by factor IXa. Simultaneous reaction of fa ctor VIIIa with the two enzymes resulted in a rate of inactivation int ermediate to that observed for the individual proteases. Under these c onditions, the activated protein C-catalyzed cleavage at Arg562 was in hibited such that cofactor inactivation resulted primarily from cleava ge at Arg336. Substitution of factor IXa modified in its active site w ith amino)-2-naphthalenesulfonyl-glutamylglycylarginyl chloromethyl ke tone (DEGR-IXa) for the native enzyme yielded a similar rate of activa ted protein C-catalyzed cleavage at the Al site, whereas cleavage at t he A2 site was virtually eliminated. However, the inclusion of protein S resulted in a marked increase in cleavage at the A2 site that corre lated with an increased rate of cofactor inactivation. Active site-mod ified activated protein C inhibited the factor IXa-dependent enhanceme nt 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 facto r IXa can reduce the rate of activated protein C-catalyzed cofactor in activation by selectively blocking cleavage at the A2 domainal site, a n effect reversed by protein S. One mechanism consistent with the reci procal 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 bindin g.