title: Progress curve analysis of the kinetics with which blood coagulation factor XIa is inhibited by protease nexin-2.
abstract: Protease nexin-2 (PN-2), a soluble form of amyloid beta-protein precursor (APP) containing a Kunin protease inhibitor domain, has been shown to be a potent, reversible and competitive inhibitor of blood coagulation factor XIa (FXIa). We have analyzed progress curves of the hydrolysis of a sensitive fluorogenic substrate by FXIa in the presence of PN-2 to ascertain the kinetic rate constants governing the inhibition of FXIa by PN-2. The mechanism of this inhibition is best described as a slow equilibration between the free enzyme and inhibitor directly, without prior formation of a loosely-associated complex. The association rate constant (kon) and the dissociation rate constant (koff) were found to be 2.1 +/- 0.2 x 10(6) M-1 s-1 and 8.5 +/- 0.8 x 10(-4) s-1, respectively (n = 23). The inhibition constant calculated from these parameters (Ki) is 400 pM, in good agreement with previous reports. High molecular weight kininogen (HK) and Zn2+ ions exert opposite effects on the inhibition of FXIa by PN-2. HK protects FXIa from inactivation in a dose dependent and saturable manner (EC50 = 61 nM) whereas Zn2+ augments the ability of PN-2 to inhibit FXIa. When both Zn2+ ions and HK are present, only the accessory effect of Zn2+ is observed. PN-2 is known to be an abundant platelet alpha-granule protein (Van Nostrand et al., 1990a; Smith & Broze, 1992). We conducted sensitive measurements of FXIa activity in the presence of human platelets before and after their being activated with the thrombin receptor agonist peptide, SFLLRN-amide. We found that platelet activation, and ostensibly the release of PN-2, limits the lifetime of FXIa activity within the locus of activated platelets. As in the purified system, HK protects FXIa from inactivation and Zn2+ increases the inactivation of FXIa. However, when HK and Zn2+ are both present, it is the protective effect of HK which predominates and prolongs the lifetime of FXIa after platelet activation.
authors.affiliation: Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Scandura
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: W E
authors.initials: WE
authors.lastname: Van Nostrand
authors.forename: P N
authors.initials: PN
authors.lastname: Walsh
mesh_terms: D016564:Amyloid beta-Protein Precursor / Q000302:isolation & purification / Q000494:pharmacology*; D001665:Binding Sites; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D015949:Factor IXa / Q000037:antagonists & inhibitors*; D005347:Fibroblasts; D006801:Humans; D007700:Kinetics; D007704:Kininogens / Q000494:pharmacology; D008956:Models, Chemical; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
chemical_list: D016564:Amyloid beta-Protein Precursor; D007704:Kininogens; D015842:Serine Proteinase Inhibitors; D015949:Factor IXa; D014357:Trypsin; D015032:Zinc