title: Down-regulation of factor IXa in the factor Xase complex by protein Z-dependent protease inhibitor.
abstract: Protein Z-dependent protease inhibitor (ZPI) is a serpin inhibitor of coagulation factor (F) Xa dependent on protein Z, Ca2+, and phospholipids. In new studies, ZPI inhibited FIXa in the FXase complex. Since this observation could merely represent inhibition of the FXa product whose activity was measured, inhibition of FIXa was investigated five ways. 1) FXase incubation mixtures with/without ZPI/protein Z were diluted in EDTA; FXa activity was measured after reversal of its inhibition. 2) FXase incubation mixtures were immunoblotted for FXa product. 3) FX activation peptide region was 3H-labeled; release of 3H was used to measure FXase activity. 4) Activity was monitored in a FIXa-based clotting assay. 5) FIXa amidolytic activity was measured. In all cases, FIXa was inhibited by subphysiologic levels of ZPI. Unlike inhibition of FXa, inhibition of FIXa did not strictly require protein Z. Low concentrations of FVIIIa increased the efficiency of ZPI inhibition of FIXa; FVIIIa in molar excess was not protective of FIXa unless FIXa/FVIIIa interacted prior to ZPI exposure. Unusual time courses were observed for inhibition of both FIXa in the FXase complex and FXa in the prothrombinase complex. Activity loss stabilized in <100 s at a level dependent on ZPI concentration, suggesting equilibrium interactions rather than typical covalent serpin-protease interactions. Surface plasmon resonance binding experiments revealed binding and dissociation of ZPI/FIXa with Kd (app) of 9-12 nm, similar to the concentration of ZPI needed for 50% inhibition. ZPI may be an unusual physiologic regulator of both the intrinsic FXase and the prothrombinase complexes.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. heeb@scripps.edu
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Heeb
authors.forename: Katia M
authors.initials: KM
authors.lastname: Cabral
authors.forename: Lingjuan
authors.initials: L
authors.lastname: Ruan
mesh_terms: D001779:Blood Coagulation Factors / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015949:Factor IXa / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D009363:Neoplasm Proteins / Q000378:metabolism; D011994:Recombinant Proteins; D015843:Serpins / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism
chemical_list: D001779:Blood Coagulation Factors; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C416370:SERPINA10 protein, human; D015843:Serpins; D013925:Thromboplastin; D015949:Factor IXa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant