title: Subunit structure and function of porcine factor Xa-activated factor VIII.
abstract: Factor Xa and thrombin (factor IIa) activate factor VIII (fVIII) by different proteolytic pathways. Thrombin cleaves fVIII at Arg372 between the A1 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 heterotrimer. We now report a stable porcine fVIIIaXa preparation obtained by Mono S HPLC at pH 6. NH2-terminal sequence analysis of purified subunits of fVIIIaXa revealed that factor Xa cleaves fVIII at Arg219 within the A1 domain and at Arg490 within the A2 domain, as well as at Arg372, Arg740, and Arg1689. Analytical ultracentrifugation of the fVIIIaXa preparation yielded results consistent with a single, 148 kDa species, similar to previous results with fVIIIaIIa [Lollar, P., & Parker, C. G. (1989) Biochemistry 28, 666-674]. Thus, the major species in the fVIIIaXa preparation contains five subunits, including fragments of the A1 and A2 domains that remain noncovalently bound. Fluorescence anisotropy measurements indicated there was no difference in the affinity of fVIIIaXa and fVIIIaIIa for a fluorescent dye-labeled, active-site-blocked derivative of porcine factor IXa. Additionally, the fVIIIaXa preparation bound dye-labeled factor IXa with 1:1 stoichiometry, indicating that all fVIIIaXa molecules in the preparation can bind factor IXa. However, fVIIIaXa had 4-fold less procoagulant activity than fVIIIaIIa. Kinetic analysis of fVIIIa cofactor activity using purified factor IXa and factor X suggested this difference is due to greater activity of fVIIIaIIa relative to fVIIIaXa within the intrinsic fXase complex, rather than a difference in their stabilities.
authors.affiliation: Department of Medicine and Microchemical Facility, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Parker
authors.forename: J
authors.initials: J
authors.lastname: Pohl
authors.forename: M N
authors.initials: MN
authors.lastname: Blackburn
authors.forename: P
authors.initials: P
authors.lastname: Lollar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013552:Swine
chemical_list: D005169:Factor VIII; D015949:Factor IXa; D015951:Factor Xa