(Circulation. 1995;92:962-967.)
© 1995 American Heart Association, Inc.
Articles |
Role of Thrombin Compared With Factor Xa in the Procoagulant Activity of Whole Blood Clots
From the Cardiovascular Division, Washington University School of Medicine, St Louis, Mo.
Correspondence to Paul R. Eisenberg, MD, MPH, Cardiovascular Division, Box 8086, Washington University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110.
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Abstract |
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Background Thrombi are known to induce activation of the coagulation system, which may be a mechanism for progression of thrombosis and its recurrence after thrombolysis. This study was designed to characterize the relative role of thrombin and activated factor X (factor Xa) as mediators of procoagulant activity of whole blood clots in blood and plasma.
Methods and Results Clots formed from human blood were incubated in recalcified (25 mmol/L CaCl2) citrated plasma or nonanticoagulated blood with increasing concentrations of recombinant desulfatohirudin (hirudin) to inhibit thrombin activity, recombinant tick anticoagulant peptide (TAP) or recombinant tissue factor pathway inhibitor (TFPI) to inhibit factor Xa, or heparin. Fibrinopeptide A (FPA) was assayed serially as an index of procoagulant (thrombin) activity. FPA generation was greatly accelerated by addition of clots to recalcified plasma (from 1251±211 ng/mL after 15 minutes without clot to 5916±1412 ng/mL with clot, n=7, P<.01) or whole blood (4803±761 ng/mL with clot compared with 546±233 without clot, n=5, P<.05) and was attenuated by inhibitors of thrombin (>90% inhibition of FPA with 0.05 µmol/L hirudin and 1.0 U/mL heparin) and factor Xa (>90% inhibition of FPA with 1.0 µmol/L TAP and 0.15 µmol/L TFPI) in a concentration-dependent manner. Preincubation of clots with tissue-type plasminogen activator sufficient to induce partial clot lysis increased the rate of thrombin-induced FPA generation by increasing the surface area of clot exposed to plasma. However, procoagulant activity induced by partially lysed clots was attenuated by lower concentrations of both thrombin and Xa inhibitors, presumably because access of the inhibitors to bound procoagulant molecules was facilitated. Comparable results were obtained with incubations in nonanticoagulated blood.
Conclusions These results indicate that factor Xa is primarily responsible for the procoagulant activity of clots in vitro and suggest a potential molecular mechanism for the observed efficacy of inhibitors of factor Xa in preventing recurrent thrombosis after coronary thrombolysis.
Key Words: thrombosis • thrombolysis • plasminogen activators • anticoagulants
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Thrombi are known to induce activation of the coagulation system,1 2 which may play a role in the recurrence of thrombosis after thrombolysis and the progression of thrombotic complications of atherosclerotic coronary vascular disease. Because a platelet-rich thrombus is the principal procoagulant surface exposed to blood after rupture of an atherosclerotic plaque or after coronary thrombolysis, the procoagulant activity of specific components of the thrombus will determine the progression of coronary thrombosis and the response to anticoagulant therapy.
Specific binding of thrombin to fibrin has generally been thought to be the primary mediator of thrombus-associated procoagulant activity, and it is known that fibrin-bound thrombin is resistant to antithrombin III–mediated inhibition.3 Thrombus-associated thrombin has been shown to induce fibrin formation3 and may activate coagulation cofactors V and VIII as well as platelets. The efficacy of the specific thrombin inhibitor hirudin in attenuating recurrent coronary reocclusion after thrombolysis in experimental and clinical studies is consistent with the critical role of thrombin in recurrent platelet-rich coronary thrombosis.4 5 6 However, we have recently shown that whole blood clots formed in vitro and thrombi recovered after arterial injury in vivo express sufficient factor Xa activity to induce marked activation of prothrombin.7 In addition, specific inhibitors of factor Xa have been shown to have efficacy at least equal to inhibition of thrombin in preventing recurrent thrombosis after thrombolysis in experimental animals.8 These results are consistent with the clinical observation that factor Xa–induced activation of prothrombin increases in patients treated with tissue-type plasminogen activator (TPA) for acute myocardial infarction.9 However, previous studies have not addressed the extent to which the procoagulant activity of arterial platelet-rich thrombi is attributable to the activity of bound thrombin compared with factor Xa. Furthermore, the relative efficacy of inhibitors of thrombin and factor Xa in attenuating thrombus-associated procoagulant activity has not been defined.
Because the extent of local procoagulant activity is likely to be attributable, at least in part, to thrombus-associated procoagulants, the current study was designed to characterize the relative efficacy of inhibition of thrombin compared with factor Xa in attenuating thrombus-induced activation of the coagulation system. To accomplish this objective, we used whole blood clots prepared in vitro; these have been shown previously to exhibit procoagulant activity similar to that of thrombi recovered after arterial injury in vivo.7 The extent of attenuation of procoagulant activity then was characterized when the clots were incubated in recalcified citrated plasma or nonanticoagulated whole blood containing concentrations of antithrombin-dependent (heparin) and antithrombin-independent (hirudin) inhibitors of thrombin or inhibitors of factor Xa (recombinant tick anticoagulant peptide [TAP] or tissue factor pathway inhibitor [TFPI]) in the range of those documented as effective in vivo. The potential impact of fibrinolysis on the intensity of clot-associated procoagulant activity and its inhibition by the specific anticoagulants was assessed by preincubation of the clots with concentrations of TPA similar to those achieved during pharmacological fibrinolysis in vivo. We sought to determine the relative efficacy of specific inhibition of thrombin compared with that of factor Xa in attenuating the procoagulant activity of whole blood clots and the impact of fibrinolysis on the efficacy of specific inhibitors.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Formation of Whole Blood Clots
Clots were formed from human blood as described previously.10 Briefly, venous blood was collected, after discarding the first 3 mL, through a 19-gauge, winged infusion set (Terumo) into a polypropylene syringe without anticoagulants. One-milliliter aliquots were injected slowly into pieces of Tygon tubing (3.2-mm inside diameter, 4.8-mm outside diameter, 27 cm long; Fisher Scientific Products), and the ends were apposed to form a ring held together with latex tubing (4.8-mm inside diameter, 1.6-mm wall, 1 cm long; Fisher). The rings were rotated at 21 rpm and an angle of 80° for 90 minutes at 37°C on a tube rotator (Scientific Equipment Co) to simulate the flow of blood (approximately 30 mL/min). The resulting clots were removed from the tubing rings, rinsed vigorously with 0.9% saline, and placed in aliquots of plasma or whole blood as described below. The washing procedure was shown to remove both nonspecifically bound thrombin and trapped FPA as indicated by the lack of thrombin activity and the absence of measurable FPA in the last wash. We have shown previously that clots formed by these methods have an average diameter of 2 mm, a length of 5 to 10 mm, and weight of 5 to 10 mg (dry weight) and exhibit procoagulant activity identical to that of thrombi formed in vivo after electrical injury to the axillary artery in dogs.7
Incubations of Inhibitors of Thrombin and Factor Xa
With Clots in Plasma and Whole Blood
Citrated human plasma (1.5 mL,
American Red Cross) was added to
polypropylene tubes together with either 0.3 to 1.0 U/mL sodium heparin
(LyphoMed, Inc), 0.01 to 1.0 µmol/L recombinant desulfatohirudin
(hirudin, CGP39393, CIBA-GEIGY and GEN Therapeutica), 0.01 to 1.0
µmol/L recombinant TAP (Merck Sharp and Dohme), which specifically
inhibits factor Xa,11 or 0.015 to 0.15 µmol/L
recombinant TFPI (also known as lipoprotein-associated coagulation
inhibitor and extrinsic pathway inhibitor;
Monsanto), the physiological inhibitor
of the complex of tissue factor/VIIa/Xa that also inhibits free factor
Xa.12 Whole blood clots were added to one of a pair of
tubes with each concentration of inhibitor, and the plasma
was recalcified with 25 mmol/L CaCl2 (final concentration).
The tubes were placed in a 37°C water bath, and 250-µL aliquots of
plasma were removed at 5, 10, and 15 minutes after the addition of
calcium. To determine whether procoagulant activity of clots differed
in whole blood compared with plasma, a 19-gauge winged infusion set
(Terumo) was inserted into an antecubital vein of healthy volunteers
and 1 mL of whole blood was allowed to drip into polypropylene test
tubes containing the inhibitors. Clots were added, the
tubes were incubated at 37°C, and aliquots were removed serially as
for plasma. Aliquots of blood and plasma were transferred to tubes
containing 25 µL of a solution consisting of EDTA, aprotinin, and
D-phe-pro-arg-chloromethylketone (FPA
anticoagulant, Byck-Sangtek). Blood samples were centrifuged,
and the plasma was frozen at -70°C and assayed subsequently for FPA.
Studies involving acquisition of blood from human subjects were
approved by the Human Studies Committee at Washington University School
of Medicine.
Induction of Fibrinolysis
To determine whether fibrinolysis
altered the
procoagulant activity of clots, 2.5 µg/mL of TPA (Genentech, Inc) was
preincubated in citrated plasma or citrated whole blood with or without
added clot and inhibitors of thrombin or factor Xa at
37°C for 30 minutes. The plasma or blood then was recalcified, and
serial aliquots were obtained over 15 minutes for assay for FPA, as
described above.
Assessment of Clot Lysis
The extent of fibrinolysis induced
by incubation
of clots with TPA was assessed by addition of fibrinogen labeled with
125I to the whole blood before clots were formed.
Fibrinogen was labeled with 125I by the lactoperoxidase
method with use of Enzymobeads (Bio-Rad) according to the
manufacturer's instructions. Each clot contained approximately
300 000 cpm (1.6x106 cpm/µg fibrinogen). Several
of the clots were washed with saline, and the radioactivity was counted
for 1 minute in a gamma counter (NML 5010/5020 Organon Teknika). Other
clots were washed with saline and incubated in citrated plasma
containing 2.5 µg/mL of TPA for 30 minutes before counting the
radioactivity. Percent lysis over 30 minutes was calculated as the
difference in radioactivity in clots incubated with TPA and those not
incubated with TPA.
Mechanical Disruption of Clots
To determine whether
mechanical disruption of a clot simulating
pharmacological clot lysis potentiates procoagulant activity, clots
were minced with a scalpel into 1-mm3 pieces. The pieces
were incubated in recalcified plasma, and the aliquots were collected
for assay of FPA.
Assay for FPA
Assays for FPA were performed with use of a
radioimmunoassay
previously validated in our laboratory13 in which a
polyclonal antiserum (Byck-Sangtek) is used. Plasma samples were
treated with bentonite before analysis to remove fibrinogen and
large fibrinogen degradation products. We have previously shown
with reverse-phase high-pressure liquid chromatography
and carboxy terminal–specific antibodies that the assay is specific
for FPA.14 15 The lower limit of detection of FPA is
1.0
ng/mL and the linear range is 1.0 to 40.0 ng/mL. Intra-assay
variability is 5.7±0.7%.13 Samples with FPA levels
>40.0 ng/mL were diluted with pooled plasma containing no detectable
FPA.
Data Analysis
Values are expressed as mean±SEM. The
efficacy of specific
inhibitors in attenuating clot-induced procoagulant
activity was characterized by comparison of the concentrations of FPA
in blood or plasma incubated with clots in the presence of an
inhibitor to concentrations induced by incubation of clots
alone. Percent inhibition was defined as 1-(FPA with
inhibitor/FPA without inhibitor).
Increases in FPA induced by incubation of clots with plasma and blood were compared by ANOVA with a repeated-measures design (STATVIEW II, Abacus Concepts, Inc). Comparisons between inhibitors and different concentrations were analyzed by ANOVA and Scheffé's test. A value of P<.05 was considered significant.
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Results |
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Procoagulant Activity Induced by Whole Blood Clots
Incubation of whole blood or recalcified plasma at 37°C resulted in only minimal increases in FPA at 10 minutes (lag phase), with a subsequent more rapid increase in FPA between 10 and 15 minutes (Fig 1
). In contrast, incubation of nonanticoagulated whole
blood or recalcified plasma with whole blood clots resulted in marked
acceleration of the rate of increase in FPA over 15 minutes. The
increases in FPA observed in whole blood were not statistically
significant when compared with those in recalcified plasma; however,
four of six whole blood samples clotted by 15 minutes. The more
extensive clot-induced activation of the coagulation system in whole
blood probably reflects, in part, the contribution of
platelets.
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Relative Efficacy of Inhibition of Thrombin and Factor Xa on
Clot-Induced Procoagulant Activity in Recalcified
Plasma
The relative efficacy of specific inhibition of thrombin
compared
with factor Xa was tested with pharmacologically relevant
concentrations of heparin (antithrombin III–dependent thrombin and
factor Xa inhibition), hirudin (antithrombin III–independent thrombin
inhibition), TAP (factor Xa inhibitor), and TFPI (Xa and
tissue factor/VIIa/Xa inhibitor). Concentrations of FPA
induced by incubation of whole blood clots in the presence and absence
of different amounts of these inhibitors were compared. A
concentration of 1.0 U/mL of heparin inhibited 94% of the increase in
FPA induced by whole blood clots in recalcified plasma (Fig 2).
Concentrations of heparin similar to those achieved
by therapeutic anticoagulation (0.3 to 0.5 U/mL) attenuated increases
in FPA less markedly (73% with 0.3 U/mL and 83% with 0.5 U/mL of
heparin, P<.01 for all doses). Antithrombin
III–independent inhibition of thrombin activity with hirudin at
concentrations >0.01 µmol/L attenuated increases in FPA by
>90%.
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Inhibition of factor Xa alone with TAP or TFPI was also
effective for
inhibiting increases in FPA induced by addition of whole blood clots to
plasma (Fig 2
). TFPI exhibited nearly equivalent inhibition on
a molar
basis compared with hirudin, whereas the concentration of TAP required
was an order of magnitude higher.
Similar concentrations of inhibitors shown to be effective for inhibiting increases in FPA induced by whole blood clots added to recalcified citrated plasma were effective for attenuating increases in FPA at 15 minutes when clots were incubated in nonanticoagulated whole blood. Thus, 0.1 µmol/L hirudin inhibited increases in FPA by 87% (n=5) in blood compared with 96% (n=3) in recalcified plasma (P=NS), while 1.0 µmol/L TAP and 0.15 µmol/L TFPI inhibited increases in FPA by 87% and 95% (n=5), respectively, in blood compared with 95% and 97% (n=3) in plasma (P=NS).
Effect of Fibrinolysis on Procoagulant Activity
Induced by Clots and Its Inhibition
To characterize the effects of
fibrinolysis on
clot-associated procoagulant activity, whole blood clots were incubated
with 2.5 µg/mL TPA in either citrated plasma or blood for 30 minutes.
After preincubation with TPA, the plasma or citrated whole blood was
recalcified with 25 mmol/L CaCl2, and the change in
concentration of FPA was measured as described above to characterize
clot-induced activation of the coagulation system and elaboration of
thrombin activity. Incubation of whole blood clots with 2.5 µg/mL of
TPA induced 14.4±0.4% (n=6) clot lysis after 30 minutes, as
characterized by release of 125I-labeled fibrinogen into
the plasma. Partial fibrinolysis markedly potentiated
clot-associated procoagulant activity in recalcified citrated blood and
plasma (Fig 3). In plasma, concentrations of FPA
increased more rapidly and were somewhat higher than those observed in
blood; however, more rapid clotting occurred in blood than in plasma
(Fig 3).
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To determine whether increased exposure of procoagulants as a consequence of partial clot lysis accounted for increases in clot-associated procoagulant activity, whole blood clots were extensively minced and incubated in recalcified citrated plasma, and concentrations of FPA were measured by assay. All of the samples incubated with minced clots were clotted by 15 minutes, and in clots preincubated with TPA, FPA increased to 25 145±7064 ng/mL. In contrast, FPA increased to 4583±726 ng/mL in recalcified plasma incubated with clots not exposed to TPA (P<.01 compared with clots preincubated with TPA). Thus, increases in procoagulant activity appeared to be attributable, at least in part, to increased exposure of clot-associated procoagulants to plasma.
To characterize
the efficacy of thrombin and Xa inhibition on
attenuating clot-associated procoagulant activity after partial
fibrinolysis, heparin, hirudin, TAP, and TFPI were
added to recalcified citrated plasma preincubated with whole blood
clots in TPA for 30 minutes, as described above. Procoagulant activity
was markedly attenuated by each of the inhibitors after
fibrinolysis at concentrations lower than those
required to inhibit procoagulant activity induced by clots that had not
been preincubated with TPA (Fig 4). Thus,
fibrinolysis induced by preincubation with TPA
potentiated the inhibition of procoagulant activity achieved by
addition of specific inhibitors of thrombin and factor Xa.
At the concentrations tested in whole blood, there was a trend toward
greater inhibition of clot-associated procoagulant activity with
hirudin and TFPI after partial fibrinolysis (Fig 5).
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To verify the dependence of clot-associated procoagulant activity on serine proteases, whole blood clots were incubated with D-phe-L-pro-L-arg-chloromethylketone (PPACK) in recalcified citrated plasma. After 15 minutes, FPA concentration was increased minimally with concentrations of 1, 5, and 10 µmol/L PPACK (68, 51, and 54 ng/mL, respectively, n=2).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Procoagulant activity of residual thrombi, which can promote rethrombosis and limit the efficacy of fibrinolytic agents, has been attributed to preexisting thrombin bound to fibrin that is reexposed during fibrinolysis.3 Our results showing concentration-dependent and nearly equivalent attenuation of FPA generation with inhibitors of either thrombin or factor Xa in recalcified plasma or blood incubated with whole blood clots (Fig 2
)
indicate that the procoagulant activity of clots is dependent on de
novo activation of prothrombin mediated by factor Xa/Va and not
primarily preexisting clot-bound thrombin. Complete inhibition of clot
procoagulant activity by incubation in plasma-containing PPACK verified
its dependence on serine proteases. Results in vitro are
consistent with those in experimental animals and preliminary
results in patients showing marked attenuation of thrombotic
reocclusion after coronary fibrinolysis by
conjunctive administration of either direct inhibitors of
thrombin such as hirudin,4 5 6
Hirulog,16 and
argatroban17 18 or inhibitors of prothrombin
activation including TAP,8 TFPI,19 and low
molecular weight heparin.20 Further studies will be
necessary to determine the relative importance of factor Xa compared
with clot-associated thrombin in the progression of thrombosis in vivo.
The presence of clot-associated factor Xa implies that inhibition of
factor Xa in combination with inhibition of thrombin during
fibrinolysis may be most efficacious for suppression of
procoagulant activity and may therefore facilitate prompt and
persistent coronary recanalization.
Generation of FPA in recalcified plasma and blood containing clot was
increased more than fourfold by preincubation of the clots with TPA
(Fig 3). Potentiation of procoagulant activity was not mediated
by
either plasmin or TPA-induced cleavage of FPA from fibrinogen; it was
not observed in plasma containing TPA but no added clot (data not
shown). Results of experiments showing similar potentiation when clots
were minced before incubation with plasma in the absence of TPA suggest
that increased procoagulant activity attributable to incubation with
TPA is due to the increased surface area of clots induced by partial
fibrinolysis.
Of interest, and contrary to a previous report,21 we found
that inhibition of the procoagulant activity associated with whole
blood clots was facilitated by partial fibrinolysis
(Figs 4 and 5). This may have resulted from the
release of factor Xa
and/or thrombin from the clot, making them more accessible to
inhibitors, or from the more efficient penetration of
inhibitors into the clot after partial
fibrinolysis.
Concentrations of hirudin, TAP, and TFPI found to suppress procoagulant
activity of clots in vitro were analogous to those shown to reduce
reocclusion after fibrinolysis in
vivo.4 5 8 19 Comparable
results were obtained with
inhibitors in either plasma or whole blood. The
concentrations of hirudin and TFPI required to inhibit the procoagulant
activity of clots were quite similar (Fig 2), consistent with
the one-to-one stoichiometry of prothrombin activation to thrombin
by factor Xa. The concentrations of TAP required to achieve comparable
inhibition of thrombin generation were nearly 10-fold higher, possibly
because of the slow kinetics of binding of TAP to factor
Xa.9 Although the physiological action
of TFPI in vivo is believed to be the inhibition of factor Xa mediated
by the complex of tissue factor and factor VIIa,22 our
data with whole blood clots that presumably lack tissue factor show a
marked, direct inhibitory effect of TFPI on factor Xa
activity, confirming an earlier report.12 It is unlikely
that TFPI inhibits the activity of the factor IXa/VIIIa complex because
TFPI does not bind or interact with factor IXa in purified systems
(personal communication, Dr J. Miletich). Thus, in vivo,
pharmacological concentrations of TFPI may significantly attenuate both
the activity of Xa and its generation by the tissue factor/VIIa
complex.
Clinical Implications
Pharmacological fibrinolysis has become
an
established intervention after acute myocardial
infarction.23 However, its success is often limited by
failure to achieve adequate coronary
recanalization in as many as 50% of treated
patients and by early reocclusion in 8% to 15% of recanalized vessels
despite administration of heparin.24 Furthermore, early
withdrawal of heparin after fibrinolysis is frequently
associated with recurrent ischemia, which is indicative of
persistent thrombosis.25 Impaired
fibrinolysis and persistent thrombosis appear to result
from competing procoagulant activity, attributable in part to the free
plasmin generated by fibrinolytic agents that can activate
prothrombin as well as factors V and
XII.14 15 26 27 28
Procoagulant activity has been attributed also to thrombin bound to
fibrin that not only induces continued activation of fibrinogen but
activation of platelets and factors V and VIII, which increases the
activity of factor IXa/VIIIa and factor Xa/Va complexes.29
The limited efficacy of heparin observed both clinically and in
experimental animals appears to reflect relative protection of
clot-bound thrombin and factor Xa to inhibition by
heparin–antithrombin III. The results of this in vitro study show that
thrombin elaboration induced by incubation of clots with recalcified
plasma or whole blood is due to clot-associated factor Xa activity.
Thus, in vivo inhibition of thrombin alone may not be effective once
antithrombin agents are discontinued because clot-associated Xa
activity may not be inhibited. Accordingly, inhibitors of
factor Xa alone or a combination of direct potent
inhibitors of thrombin and factor Xa may be particularly
effective for facilitating fibrinolysis and warrant
testing in vivo.
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Acknowledgments |
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This study was supported in part by NHLBI SCOR in Coronary and Vascular Diseases HL-17646. We acknowledge the invaluable technical assistance of Mary Jane Eichenseer, Debbie Tinker, John Girotto, Evan Wasserman, and Brad Reznick. We thank CIBA-GEIGY for providing recombinant hirudin, Merck Sharp and Dohme for providing recombinant TAP, and Monsanto for providing recombinant TFPI. We also thank Ava Ysaguirre and Barbara Donnelly for typing the manuscript.
Received August 15, 1994; revision received February 2, 1995; accepted February 20, 1995.
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