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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Edward A. Doisy Department of Biochemistry and
Molecular Biology, St Louis University School of Medicine, MO.
Heparin is a commonly used anticoagulant drug. It functions
primarily by accelerating the antithrombin inhibition of coagulation proteinases, among which factor Xa and thrombin are believed to be the
most important targets. There are conflicting results as to whether
anticoagulant heparins can catalyze the antithrombin inhibition of
factor Xa in the prothrombinase complex (factor Va, negatively charged
membrane surfaces, and calcium ion), which is the physiologically
relevant form of the proteinase responsible for the activation of
prothrombin to thrombin during the blood coagulation process. In this
study, a novel assay system was developed to compare the catalytic
effect of different molecular-weight heparins in the antithrombin
inhibition of factor Xa, either in free form or assembled into the
prothrombinase complex during the process of prothrombin activation.
This assay takes advantage of the unique property of a recombinant
mutant antithrombin, which, similar to the wild-type antithrombin,
rapidly inhibits factor Xa, but not thrombin, in the presence of
heparin. A direct prothrombinase inhibition assay, monitoring thrombin
generation under near physiological concentrations of prothrombin and
antithrombin in the presence of therapeutic doses of low- and
high-molecular-weight heparins, indicates that factor Xa in the
prothrombinase complex is protected from inhibition by antithrombin
more than 1000 times, independent of the molecular size of heparin.
(Blood. 2001;97:2308-2313) The anticoagulant function of heparin is primarily
derived from its ability to accelerate the inhibition of the blood
coagulation proteinases by antithrombin.1-3 The mechanism
and the extent of this acceleration have been thoroughly investigated
for antithrombin inhibition of factor Xa and thrombin.4-15
It is not, however, known whether heparin is effective in catalyzing
the inhibition of factor Xa when the proteinase binds to factor Va on
negatively charged membrane surfaces in the presence of calcium (the
prothrombinase complex) to activate prothrombin to
thrombin.16-24 Results of several previous studies suggest
that the assembly of factor Xa into the prothrombinase complex is
accompanied by the protection of factor Xa from inhibition by the
antithrombin-heparin complex.18-21 Conflicting reports
exist about the extent to which factor Xa in the prothrombinase complex
is protected from inhibition by antithrombin in the presence of
different molecular weight heparins. These reports range from support
for nearly complete protection19,21 to less than 5- to
10-fold protection of factor Xa in prothrombinase from inhibition by
antithrombin in the presence of high- or low-molecular-weight high-affinity heparins.22-24 As expected, these results
have generated conflicting hypotheses within the scientific community
as to whether the therapeutic heparins are effective in catalyzing
prothrombinase inhibition by antithrombin, and if so, whether the
effect is dependent on the molecular size of heparin.
These conflicting results are believed to stem from the inability of
the existing assay systems to directly monitor the inhibition of
prothrombinase by antithrombin in the presence of prothrombin. This is
because both the enzyme and the product of the activator complex are
targets for rapid inhibition by antithrombin in the presence of
heparin.4,15 To overcome this problem, a mutant of human
antithrombin was prepared in which the reactive site loop of the
inhibitor from the P4-P4' site (nomenclature of Schechter and
Berger25) is replaced with the identical site of the
second factor Xa cleavage site
(Ile319-Asp-Gly-Arg-Ile-Val-Glu-Gly326) in
prothrombin. This mutant is fully characterized; it is folded properly,
has a normal affinity for heparin, and rapidly inhibits factor Xa, but
not thrombin, in the presence of heparin. Thus, the mutant was used to
evaluate, by a simple inhibition assay, the catalytic effects of
heparins of different representative molecular weights in antithrombin
inhibition of prothrombinase directly from the rate of thrombin
generation at near physiological concentrations of the reactants. The
results suggest that a physiological concentration of prothrombin
protects factor Xa in the prothrombinase complex by more than 3 orders
of magnitude from inhibition by the antithrombin-heparin complex
independent of the molecular size of heparin.
The human antithrombin mutant in which P4-P4' residues of the
reactive site loop were substituted with the corresponding residues of
the second factor Xa cleavage site in prothrombin
(Ile319-Asp-Gly-Arg-Ile-Val-Glu-Gly326, named
HAT/Proth-2) was constructed by standard polymerase chain reaction
(PCR) mutagenesis methods and expressed in human 293 cells using
RSV-PL4 expression-purification vector system as previously described.26 This vector introduces a 12-residue epitope
for the monoclonal antibody, HPC4, to the N-terminus of the mutant protein for easy purification.26 The antithrombin mutant
was purified from cell culture supernatants to homogeneity by a
combination of immuno-affinity chromatography using an HPC4 antibody
linked to Affi-gel 10 (Bio-Rad, Hercules, CA) followed by
HiTrap-Heparin (Amersham/Pharmacia, Piscataway, NJ) ion exchange
chromatography with a gradient elution from 0.1 M to 3.0 M NaCl in 20 mM Tris-HCl, pH 7.4.
Human plasma-derived antithrombin, the active antithrombin-binding
pentasaccharide fragment of heparin and full-length high-affinity heparins containing the pentasaccharide with an average molecular mass
of approximately 8000 (approximately 26 saccharides) or approximately 21 000 (approximately 70-saccharides) were generous gifts from Dr
Steven Olson (University of Illinois, Chicago). Concentrations of
heparins were based on the antithrombin binding sites and were determined by stoichiometric titration of antithrombin with the polysaccharides, with monitoring of the interaction by changes in
protein fluorescence.4,27 Phospholipid vesicles containing 80% phosphatidylcholine and 20% phosphatidylserine (PC/PS) were prepared as described.28 Human coagulation factors, factor
Xa, factor Va, and prothrombin were purchased from Hematologic
Technologies (Essex Junction, VT). The chromogenic substrate,
Spectrozyme FXa (SpFXa), was purchased from American Diagnostica
(Greenwich, CT), and S2238 was purchased from Kabi
Pharmacia/Chromogenix (Franklin, OH). Unfractionated heparin from
porcine intestinal mucosa, sodium salt (169.2 USP U/mg), and Polybrene
were purchased from Sigma (St Louis, MO).
Kinetic methods
In the second assay, similar experimental conditions were used with the
exception that prothrombin was included in the reaction and the rate of
factor Xa inhibition by the mutant antithrombin-heparin complex was
directly measured from the inhibition of thrombin generation. In this
assay, either 1 nM human factor Xa alone or 0.2 pM in complex with 5 nM
human factor Va on 50 µM PC/PS vesicles in the same TBS buffer system
was used to initiate the activation of 1.5 µM human prothrombin in
the presence of 2.3 µM antithrombin in complex with catalytic levels
of heparin (1-1000 nM). At different time points (1-20 minutes), 10 µL aliquot of the reaction was removed and added to 90 µL
thrombin-specific chromogenic substrate, S2238, in TBS buffer
containing 20 mM EDTA and 1 mg/mL Polybrene (Sigma) (to stop both the
activation and the inhibition reactions immediately). The
concentration of thrombin generated at each time point in the absence
or presence of the inhibitor-heparin complex was determined from a
standard curve prepared from the cleavage rate of S2238 by known
concentrations of thrombin using a Vmax Kinetics Microplate
Reader (Molecular Devices) as described above. Thrombin generation in
the absence of heparin was normalized to 100, and the kobs
values were determined by computer fitting of the time-dependent
percentage inhibition of thrombin generation in the presence of varying
concentrations of heparin to a single exponential function. The
second-order association rate constants were obtained by dividing
kobs values to the concentrations of the
antithrombin-heparin complexes, as described above. Control experiments
showed that this assay was specific for the presence of both
antithrombin and heparin in the reaction because the linear rate of
thrombin generation (approximately 6 nM/min by the prothrombinase complex) was not changed by the presence of either antithrombin or
heparin alone for the 20-minute time-course analysis of the assays. In
all assays, thrombin generation was initiated by adding factor Xa,
either in free form or in complex with prothrombinase, to the reaction
wells containing antithrombin, heparin, and prothrombin. In a
modified form of this assay, the prothrombinase reaction was
initiated under the same experimental conditions described above, with
the exception that heparin was added to an ongoing prothrombinase
reaction after a 1-minute progression of the reaction.
A mutant of human antithrombin (HAT/Proth-2) was prepared in which
the reactive site loop of the inhibitor from the P4-P4' site was
replaced with the identical site of the factor Xa cleavage site
(Ile319-Asp-Gly-Arg-Ile-Val-Glu-Gly326) in
prothrombin. The mutant was purified to homogeneity by a combination of
an HPC4 immuno-affinity and a HiTrap-Heparin (Amersham/Pharmacia) ion
exchange column chromatography, as described in "Materials and
methods." Similar to the wild-type antithrombin, the mutant was
eluted from the HiTrap-Heparin column at approximately 0.8 M NaCl (data
not shown). Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE)
analysis, under nonreducing conditions, suggested that the mutant had a
similar relative molecular mass as the plasma-derived antithrombin
(Figure 1). Similar to plasma
antithrombin, the mutant formed stable complexes with factor Xa in both
the absence and the presence of heparin (Figure 1A). However, unlike
plasma antithrombin, the mutant did not form a stable complex with
thrombin in either the absence or the presence of low-molecular-weight
heparin (Figure 1B). In the presence of a full-length (approximately
70-saccharides) high-affinity heparin, a small fraction of the mutant
antithrombin appeared to form a stable complex with thrombin
(Figure 1B, lane 9). The lack of stable mutant antithrombin-thrombin
complexes was not due to thrombin recognizing the mutant as a substrate because no cleavage product was detected in either the absence or the
presence of heparin (Figure 1B, lanes 6-9). Consistent with these data,
kinetic analysis suggested that the mutant inhibited factor Xa only
approximately 5 to 10 times more slowly than the wild-type antithrombin
in the absence and the presence of heparin and the pentasaccharide
fragment of heparin, but it did not react with thrombin at a detectable
rate in either the absence or the presence of pentasaccharide. Its
reactivity with thrombin was impaired by approximately 5 orders of
magnitude in the presence of a full-length (approximately
70-saccharides) high-affinity heparin (Table
1). Similar to the wild-type
antithrombin, the mutant bound to high-affinity heparins with a
Kd of approximately 20 nM, as determined from
changes in intrinsic protein fluorescence on binding to
heparin27 (data not shown). Because the heparin-catalyzed proteinase inhibition by the mutant was minimally affected for factor
Xa but virtually abolished for thrombin, the mutant was used to
evaluate the catalytic effect of heparins of various molecular sizes in
antithrombin inhibition of prothrombinase during prothrombin activation
directly from the inhibition of thrombin generation.
First, the assay system was validated by studying the kinetics of
factor Xa or prothrombinase inhibition by the mutant antithrombin in
the presence of heparins by an amidolytic activity assay that monitored
hydrolysis of SpFXa and compared the results with those obtained for
the wild-type antithrombin under identical conditions. Thus, in Figure
2, the pentasaccharide-mediated wild-type
antithrombin inhibition of factor Xa was measured in either the absence
or the presence of factor Va on PC/PS vesicles from remaining
amidolytic activities toward cleavage of the chromogenic substrate
SpFXa (first assay), as described in "Materials and methods." In
this assay, second-order rate constants of approximately
6 × 105 M
Next, the catalytic effects of longer chain heparins (approximately 26 and approximately 70-saccharides high-affinity heparins or
unfractionated [UFH] heparin) in antithrombin inhibition of factor Xa
and prothrombinase were evaluated in both assay systems (Figure
4, shown for H70 only).
Comparisons of the rate constants derived from the first assay
(monitoring the cleavage of SpFXa) for these studies suggested that
binding of factor Xa to factor Va on PC/PS vesicles in the absence of
prothrombin confers only an approximately 2-fold (H26),
approximately 6-fold (H70), or approximately 12-fold (UFH)
protection against inhibition by the mutant of antithrombin (Table 2).
However, these compounds were virtually ineffective in catalyzing the
inhibition of prothrombinase during prothrombin activation even if
their concentrations were increased by up to 2 orders of magnitude
(Table 3, Figure 4B shown for H70 only). In a slightly
modified form of this assay, when heparins were added to an ongoing
prothrombinase reaction after a 1-minute progression of the reaction,
similarly no inhibition of thrombin generation was observed with
pentasaccharide (1000 nM), and approximately 0.5% to 1% (per minute)
inhibition of thrombin generation was observed with the longer chain,
high-affinity heparins (100-200 nM). Whether this minor inhibition of
thrombin generation by the antithrombin mutant in the presence of the
longer chain heparins was due to the inhibition of free factor Xa that
might have been in dissociable equilibrium with factor Xa in the
prothrombinase complex or due to the inhibition of prothrombinase is
unknown. Based on such results, k2(app) values of less than
10 M
Assembly of factor Xa into the prothrombinase complex is required for the generation of thrombin during the blood coagulation process.16,17 Complex formation not only enhances the rate of thrombin generation approximately 3 × 105-fold, it is also accompanied by protection of factor Xa from inhibition by antithrombin.16,17 Because factor Xa is an important target for heparin during anticoagulant therapy, knowing the extent to which prothrombinase complex formation protects the proteinase from heparin-catalyzed inhibition by antithrombin is crucial for treating patients with thrombosis. However, attempts to quantitatively address this question have resulted in inconsistent and conflicting data in the literature.19,21-24 This problem is believed to arise primarily from the inherent complexity of the assay systems needed to measure the rate of prothrombinase inhibition by antithrombin. This is because factor Xa and thrombin, the proteinase and the product of prothrombinase, are both inhibited at near diffusion-limited rates by the antithrombin-heparin complex.4,15,30 Thus, in some previous studies, prothrombin had to be excluded from the assays to measure the factor Va-mediated protective effect against inhibition by the antithrombin-heparin complex by a simple amidolytic activity assay.23,24 Results of the current study suggest that the inhibition rates obtained from such assays do not represent the heparin-catalyzed inhibition of a fully assembled prothrombinase complex in the presence of prothrombin. In one previous study, the heparin-catalyzed inhibition of prothrombinase was studied in the presence of prothrombin. However, complicated mathematical models had to be developed for data analysis because the rate of prothrombinase inhibition had to be calculated from the initial rate of thrombin generation, which itself is concomitantly and rapidly inhibited by the inhibitor-heparin complex.22 In this previous study, the prothrombinase complex formation resulted in an approximately 5-fold protection against inhibition by the heparin-antithrombin complex. Results of current direct prothrombinase inhibition assays do not support the reliability of these previous findings. Another factor that might have contributed to the discrepancies in the literature is that results of some of the previous studies mentioned above have been analyzed based on the traditional view that a heparin-induced conformational change in the reactive site loop of antithrombin is solely responsible for the cofactor effect of heparin in factor Xa inhibition. Thus, heparin-catalyzed prothrombinase inhibition by antithrombin was measured in the presence of Ca++ and compared to that of free-factor Xa inhibition in the absence of Ca++. However, recently the existence of a binding site for heparin on the proteinase domain of factor Xa in a region homologous to heparin binding exosite 2 of thrombin was reported.15,29 It was demonstrated that heparin binding to this site accelerates factor Xa inhibition by antithrombin several hundred-fold more if the Gla-domain of the proteinase is neutralized by physiological concentrations of Ca++.15,29 This increased acceleration was mediated by a bridging mechanism based on the bell-shaped dependence of the additional reaction rate enhancement on heparin concentration.15,29,30 It is likely that in some of these previous studies, the additional template cofactor effect of high-molecular-weight heparins has been wrongly attributed to the ability of longer chain heparins to overcome the protective effect of prothrombinase complex formation against inhibition by antithrombin. The mechanistic basis for resistance of prothrombinase to inhibition by the antithrombin-heparin complexes in the presence of prothrombin was not investigated in detail. It is known, however, that the assembly of factor Xa into the prothrombinase complex dramatically lowers the Km of prothrombin (less than 1 µM) for binding to prothrombinase.31 In contrast, antithrombin, free or in complex with low-molecular-weight heparins, has a very high dissociation constant (approximately 200 µM) for binding to factor Xa.32 It follows, therefore, that in the presence of prothrombin, antithrombin may not effectively compete with the substrate prothrombin for binding to the catalytic center of factor Xa in the prothrombinase complex. Although binding of full-length heparin chains to an exosite on factor Xa can significantly improve the affinity of factor Xa for binding to antithrombin in the presence of physiological Ca++,15,29,30 previous kinetic data nevertheless suggest that the heparin-binding exosite of factor Xa overlaps with the binding site for factor Va, prothrombin, or both in prothrombinase.29 Thus, therapeutic doses of heparin cannot bind to this site to accelerate the prothrombinase inhibition by antithrombin because the affinity of factor Xa for factor Va-prothrombin is several orders of magnitude higher than that for the polysaccharide, and very high concentrations of heparin would be required to dissociate factor Xa from the prothrombinase complex.29,31,33 In summary, the results of this study suggest that prothrombin confers more than 3 orders of magnitude of protection for prothrombinase against inhibition by antithrombin in complex with either low- or high-molecular-weight heparins. The clinical ramifications of this observation are that the high-affinity pentasaccharide and other similarly low-molecular-weight heparins possessing predominantly anti-factor Xa activity may only be effective anticoagulant drugs for prophylactic purposes. Once prothrombinase is assembled on injured endothelium or on activated platelets, it is resistant to heparin-catalyzed inhibition by antithrombin in the presence of plasma concentrations of prothrombin. Thus, for the management of ongoing thrombosis, heparin therapy may have to be directed to the inhibition of thrombin with appropriate molecular-size heparins.
I would like to thank Dr Steven Olson for critical reading, Dr Akash Mathur for assistance with Figure 1, and Audrey Rezaie for proofreading the manuscript.
Submitted September 21, 2000; accepted December 11, 2000.
Supported by grant R01 HL 62565 from the National Heart, Lung, and Blood Institute of the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Alireza R. Rezaie, Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, 1402 S Grand Blvd, St Louis, MO 63104; e-mail: rezaiear{at}slu.edu.
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© 2001 by The American Society of Hematology.
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N. Brufatto and M. E. Nesheim Analysis of the Kinetics of Prothrombin Activation and Evidence That Two Equilibrating Forms of Prothrombinase Are Involved in the Process J. Biol. Chem., February 21, 2003; 278(9): 6755 - 6764. [Abstract] [Full Text] [PDF]
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D. M. Monroe, M. Hoffman, and H. R. Roberts Platelets and Thrombin Generation Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1381 - 1389. [Abstract] [Full Text] [PDF]
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 A. K.C. Chan, J. Rak, L. Berry, P. Liao, M. Vlasin, J. Weitz, and P. Klement Antithrombin-Heparin Covalent Complex: A Possible Alternative to Heparin for Arterial Thrombosis Prevention Circulation, July 9, 2002; 106(2): 261 - 265. [Abstract] [Full Text] [PDF]
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N. S. Quinsey, J. C. Whisstock, B. Le Bonniec, V. Louvain, S. P. Bottomley, and R. N. Pike Molecular Determinants of the Mechanism Underlying Acceleration of the Interaction between Antithrombin and Factor Xa by Heparin Pentasaccharide J. Biol. Chem., May 3, 2002; 277(18): 15971 - 15978. [Abstract] [Full Text] [PDF]
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A. R. Rezaie Partial Activation of Antithrombin without Heparin through Deletion of a Unique Sequence on the Reactive Site Loop of the Serpin J. Biol. Chem., January 4, 2002; 277(2): 1235 - 1239. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
and 
forms). Lane
2, human plasma antithrombin (HAT). Lanes 3 to 5, plasma antithrombin
incubated with factor Xa in the absence of heparin, in the presence of
pentasaccharide, or in the presence of approximately 70-saccharide
heparin, respectively. Lane 6, mutant antithrombin. Lanes 7 to 9, mutant antithrombin incubated with factor Xa in the absence of heparin,
in the presence of pentasaccharide, or in the presence of approximately
70-saccharide heparin, respectively. MW, molecular weight marker. (B)
Identical to panel A, except that thrombin (IIa) was used to initiate
the reactions.
1 second
using either the chromogenic substrate
SpFXa (Figure 
) or the presence
of 50 µM PC/PS minus (
) or plus 5 nM factor Va (
) in TBS buffer
containing 1 mg/mL BSA, 0.1% PEG 8000, and 2.5 mM Ca++. At
the indicated time points, aliquots of the reaction were removed to 500 µM SpFXa containing 1 mg/mL Polybrene (Sigma), and the remaining
enzyme activity was determined by an amidolytic activity assay using
Spectrozyme Fxa (American Diagnostica). Solid lines are nonlinear
regression fit of data to a pseudo first-order rate equation, as
described in "Materials and methods."
