SUBUNIT STRUCTURE AND FUNCTION OF PORCINE FACTOR XA-ACTIVATED FACTOR-VIII

Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by di fferent proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the Al and A2 domains, at Arg740 between the A2 and B domains, and at Arg1689 between the B and A3 domains to form an A1/A2/A3-C1-C2 hetero trimer. We now report a stable porcine fVIIIa(Xa) preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified su bunits of fVIIIa(Xa) revealed that factor Xa cleaves fVIII at Arg219 w ithin the Al domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVI IIa(Xa) preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIa(IIa) [Lollar, P., & P arker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIa(Xa),preparation contains five subunits, including fragm ents of the Al and A2 domains that remain noncovalently bound. Fluores cence anisotropy measurements indicated there was no difference in the affinity of fVIIIa(Xa) and fVIIIa(IIa) for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, t he fVIIIa(Xa), preparation bound dye-labeled factor IXa with 1:1 stoic hiometry, indicating that all fVIIIa(Xa) molecules in the preparation can bind factor IXa. However, fVIIIa(Xa) had 4-fold less procoagulant activity than fVIIIa(IIa). Kinetic analysis of fVIIIa cofactor activit y using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIa(IIa) relative to fVIIIa(Xa) within t he intrinsic fXase complex, rather than a difference in their stabilit ies.