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Title: Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX

Abstract

The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX. Initial rates of trichloroacetic acid-soluble /sup 3/H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl/sub 2/ (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of /sup 3/H preceded the appearance of Factor IXa activity, and the percentage of /sup 3/H released remained constant when the mole fraction of /sup 3/H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of /sup 3/H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include K/sub m/ (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (k/sub cat/) (7.7/s) is approximately 20-50 times higher than thatmore » reported by Zur and Nemerson for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania
OSTI Identifier:
6749869
Alternate Identifier(s):
OSTI ID: 6749869
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Clin. Invest.; (United States); Journal Volume: 73:5
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; THROMBOSIS; BIOCHEMICAL REACTION KINETICS; BIOCHEMISTRY; RADIOIMMUNOASSAY; TRACER TECHNIQUES; TRITIUM COMPOUNDS; CARDIOVASCULAR DISEASES; CHEMISTRY; DISEASES; ISOTOPE APPLICATIONS; KINETICS; LABELLED COMPOUNDS; RADIOASSAY; REACTION KINETICS; VASCULAR DISEASES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Walsh, P.N., Bradford, H., Sinha, D., Piperno, J.R., and Tuszynski, G.P.. Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX. United States: N. p., 1984. Web. doi:10.1172/JCI111343.
Walsh, P.N., Bradford, H., Sinha, D., Piperno, J.R., & Tuszynski, G.P.. Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX. United States. doi:10.1172/JCI111343.
Walsh, P.N., Bradford, H., Sinha, D., Piperno, J.R., and Tuszynski, G.P.. Tue . "Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX". United States. doi:10.1172/JCI111343.
@article{osti_6749869,
title = {Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX},
author = {Walsh, P.N. and Bradford, H. and Sinha, D. and Piperno, J.R. and Tuszynski, G.P.},
abstractNote = {The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX. Initial rates of trichloroacetic acid-soluble /sup 3/H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl/sub 2/ (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of /sup 3/H preceded the appearance of Factor IXa activity, and the percentage of /sup 3/H released remained constant when the mole fraction of /sup 3/H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of /sup 3/H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include K/sub m/ (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (k/sub cat/) (7.7/s) is approximately 20-50 times higher than that reported by Zur and Nemerson for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant.},
doi = {10.1172/JCI111343},
journal = {J. Clin. Invest.; (United States)},
number = ,
volume = 73:5,
place = {United States},
year = {Tue May 01 00:00:00 EDT 1984},
month = {Tue May 01 00:00:00 EDT 1984}
}
  • Since optimal rates of factor IX activation by factor XIa require the presence of calcium ions and the heavy chain of the enzyme as well as the active-site containing light chain, the authors have studied the effects of calcium ions and the heavy chain on the reaction kinetics. Whereas the amidolytic activities of factor XIa and of its active-site-containing light chain were almost indistinguishable, the two enzymes behaved quite differently when factor IX was the substrate. Factor XIa was 100-fold more potent in the presence of Ca/sup 2 +/ than in its absence. On the contrary, the presence or absencemore » of Ca/sup 2 +/ made very little difference in the case of the isolated light chain of factor XIa. Moreover, the enzymatic activity of the light chain was almost identical with that of intact factor XIa when Ca/sup 2 +/ was absent. Using an optimal concentration of Ca/sup 2 +/, they studied the activation in the presence of various concentrations of two monoclonal antibodies, one (5A4) directed against the light chain of faster XIa and the other (3C1) against its heavy chain. Analysis of 1/V vs. 1/S plots showed that whereas inhibition by 5F4 was noncompetitive, 3C1 neutralized the enzyme in a classical competitive fashion. They conclude that in the calcium-dependent activation of factor IX by factor XIa the heavy chain of the enzyme is involved in the binding of substrate and this is essential for optimal reaction rates.« less
  • Binding of /sup 125/I-Factor XIa to platelets required the presence of high molecular weight kininogen, was enhanced when platelets were stimulated with thrombin, and reached a plateau after 4-6 min of incubation at 37 degrees C. Factor XIa binding was specific: 50- to 100-fold molar excesses of unlabeled Factor XIa prevented binding, whereas Factor XI, prekallikrein, Factor XIIa, and prothrombin did not. When washed erythrocytes, added at concentrations calculated to provide an equivalent surface area to platelets, were incubated with Factor XIa, only a low level of nonspecific, nonsaturable binding was detected. Factor XIa binding to platelets was partially reversiblemore » and was saturable at concentrations of added Factor XIa of 0.2-0.4 microgram/ml (1.25-2.5 microM). The number of Factor XIa binding sites on activated platelets was estimated to be 225 per platelet (range, 110-450). We conclude that specific, high affinity, saturable binding sites for Factor XIa are present on activated platelets, are distinct from those previously demonstrated for Factor XI, and require the presence of high molecular weight kininogen.« less
  • The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a wall shear rate of 57, 300, or 1130 sec{sup {minus}1}. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2{endash}-to 3{endash}fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminatedmore » by the lipoprotein-associated coagulation inhibitor. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.« less
  • Factor IX is a blood clotting protein that contains three regions, including a {gamma}-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF0like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel {beta}-sheets, and preliminary distance geometry calculations indicate thatmore » the protein has two domains, separated by Trp{sup 28}, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the {beta}-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.« less
  • Whether the factor VII/tissue factor complex that forms in tissue factor-dependent blood coagulation must be activated to factor VIIa/tissue factor before it can activate its substrates, factor X and IX, has been a difficult question to answer because the substrates, once activated, back-activate factor VII. The earlier studies suggested that human factor VII/tissue factor cannot activate factor IX. Studies have now been extended to the activation of factor X. Reaction mixtures were made with purified factor VII, X, and tissue factor; in some experiments antithrombin III and heparin were added to prevent back-activation of factor VII. Factor X was activatedmore » at similar rates in reaction mixtures containing either VII or factor VIIa after an initial 30-sec lag with factor VII. In reaction mixtures with factor VII a linear activation of factor X was established several minutes before cleavage of {sup 125}I-labeled factor VII to the two-chain activated molecule was demonstrable on gel profiles. These data suggest that factor VII/tissue factor cannot activate measurable amounts of factor X over several minutes. Overall, the results support the hypothesis that a rapid preferential activation of factor VII bound to tissue factor by trace amounts of factor Xa is a key early step in tissue factor-dependent blood coagulation.« less