(Circulation. 1995;92:485-491.)
© 1995 American Heart Association, Inc.
Articles |
Antithrombotic Effects of Orally Active Synthetic Antagonist of Activated Factor X in Nonhuman Primates
From the Division of Hematology and Oncology and Yerkes Regional Primate Research Center, Emory University School of Medicine, Atlanta, Ga, and Tokyo Research and Development Center, Daiichi Pharmaceutical Co, Ltd, Tokyo, Japan (S.K.).
Correspondence to Laurence A. Harker, MD, Division of Hematology and Oncology, Emory University School of Medicine, PO Drawer AR, Atlanta, GA 30322.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Since activated factor X (FXa) has a central role in hemostasis and thrombosis, it is an attractive target for antithrombotic strategies. Accordingly, we evaluated the relative antihemostatic and antithrombotic effects of an orally active amidinoaryl propanoic acid inhibitor of FXa, APAP, in baboons.
Methods and Results With a two-component thrombogenic device that
induced the concurrent formation of both arterial-type
platelet-rich and venous-type fibrin-rich thrombus when interposed
in chronic exteriorized arteriovenous (AV) femoral shunts flowing at 40
mL/min, thrombus formation was compared for oral versus parenteral APAP
by measurement of 111In-platelet deposition,
125I-fibrin accumulation, thrombotic obstruction of flow,
and circulating levels of blood biochemical markers of thrombosis. The
direct infusion of APAP (120 µg/min) into AV shunts proximal to
thrombogenic devices for 1 hour achieved local drug levels of 4.3±0.4
mg/L and substantially reduced the accumulation of platelets and
fibrin in the formation of venous-type fibrin-rich thrombus
(P<.01) but not in the formation of platelet-rich
arterial-type thrombus (P>.1). APAP was
subsequently removed from plasma with plasma clearance rates of
T50
of 6.3 minutes and T50ß of 99 minutes.
The oral administration of APAP (50 mg/kg) produced peak plasma levels
of 3.7±1.4 µg/mL at 30 minutes and gradually declining plasma
levels
over about 6 to 8 hours, with bioavailability estimated to be
approximately 5% to 12%. Oral APAP decreased platelet deposition
(P<.01) and fibrin accumulation (P<.05) in
venous-type thrombus but failed to decrease platelet or fibrin
accumulation in arterial-type thrombus (P>.1 in
both cases). Oral and infused APAP prolonged the activated partial
thromboplastin time and prevented thrombus-dependent elevations in
plasma fibrinopeptide A, thrombin–antithrombin III
complex, ß-thromboglobulin, and platelet factor 4
levels. Additionally, APAP produced dose-dependent inhibition of FXa
bound to thrombus on segments of vascular graft interposed in
exteriorized AV shunts for 15 minutes.
Conclusions An oral synthetic antagonist of FXa, APAP, inhibits the formation of venous-type fibrin-rich thrombus by inactivating bound and soluble FXa without impairing platelet hemostatic function.
Key Words: thrombosis • hemostasis • coagulation • anticoagulants • radioisotopes
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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The assembly of different macromolecular enzyme complexes by extrinsic and intrinsic pathways of coagulation both lead to cleavage of 52Arg in factor X (FX), thereby generating catalytic FX (FXa), which, together with the cofactor factor V, phospholipid surface, and ionic calcium, converts prothrombin to thrombin.1 Because of its pivotal role, FXa is an attractive target for antithrombotic therapy, particularly in view of the experimental evidence indicating that the inhibition of FXa, as opposed to the inactivation of thrombin, interrupts thrombus formation without impairing platelet hemostatic function.2 3
Natural polypeptidyl and synthetic peptidomimetic
antagonists of FXa exhibit selective anti-FXa activity in
vitro and produce antithrombotic effects in experimental animals when
administered parenterally, although none of these
compounds have been shown to produce significant anti-FXa activity in
vivo after oral
administration.4 5 6 7 8 9 10 11 12 13
However,
antithrombotic activity has recently been reported in rodents after
oral administration of the synthetic antagonist of FXa
(2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-amidino-2-naphthyl)
propanoic acid hydrochloride pentahydrate (APAP) (also designated
DX-9065a).14 15 16 17 18
This dibasic amidinoaryl propanoic acid
derivative (Fig 1
) inhibits FXa in purified systems with
a Ki of 41 nmol/L and three orders of magnitude
greater specificity for FXa than for thrombin or other coagulation
serine proteases.
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In well-characterized quantitative thrombosis models in baboons, the antithrombotic efficacy of APAP has been compared for oral versus infusional administration by measurement of arterial-type platelet-rich and venous-type fibrin-rich thrombus formation, changes in circulating biochemical markers of thrombosis, and thrombus-bound FXa and assessment of the antihemostatic effects of this FXa antagonist.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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Animal Studies
These studies were carried out in 10 normal male baboons (Papio anubis) weighing 9 to 12 kg that had been quarantined and observed to be disease-free for at least 3 months. All studies were approved by the Institutional Animal Care and Use Committee and were in compliance with procedures and methods outlined by the National Institutes of Health (Guide for the Care and Use of Laboratory Animals, National Institutes of Health, Bethesda, Md, NIH publication 86-23) as well as the Animal Welfare Act and related university policies.
Blood counts and hematocrits were measured on whole blood collected in EDTA (2 g/L) with a J.T. Baker model 9000 whole-blood analyzer.19 20 21 Template bleeding time measurements were performed on the shaved volar surface of the forearm as described.19 20 21
Exteriorized femoral arteriovenous (AV) access shunts were surgically implanted for interposition of thrombogenic devices, drug infusions, and blood sampling.19 20 21 These AV shunts do not detectably activate platelets or fibrinogen.19 20 21 Thrombogenic devices were incorporated into the AV shunts of awake animals. Blood flow through the shunt was maintained at 40 mL/min with a clamp placed distal to the device and was measured continuously with an ultrasonic flowmeter (model 201, Transonics Systems).
Models of Thrombosis
Thrombus was formed by interposing
thrombogenic devices in
exteriorized AV shunts and exposing the devices to native
nonanticoagulated blood flowing at 40 mL/min for 1 hour. The
two-component device19 20 21 consisted of
a proximal tubular
segment (2 cm longx4.0 mm ID) of knitted Dacron vascular graft
(Bioknit, C.R. Bard, Inc) followed by a distal region of expanded
diameter composed of polytetrafluoroethylene tubing (2 cm longx9.3 mm
ID) exhibiting disturbed and static flow. The segments of vascular
graft induced the formation of platelet-rich thrombus, simulating
arterial thrombosis, while the expanded chamber produced
fibrin-rich thrombus resembling venous thrombosis.19
Before studies were begun, autologous baboon platelets were labeled with 1 mCi 111In-tropolone.22 Labeling efficiencies averaged about 90%. After at least 1 hour was allowed for the reinfused cells to distribute within the vasculature, thrombogenic devices were incorporated into the shunt system and exposed to native flowing blood for 1 hour. The accumulation of 111In-labeled platelets within each device region was measured continuously with a gamma scintillation camera (General Electric 400T MaxiCamera). Data were stored at 5-minute intervals and analyzed with a computer-assisted image processing system interfaced with the camera (Medical Data Systems A3, Medtronic Inc) as reported previously.19 20 21 Images of the vascular graft and distal expansion segment were taken in 128x128 byte mode with a 15% energy window and analyzed with 2-cm-long (10-pixel) regions of interest for each component composing the device. The total number of deposited platelets in each component was calculated by dividing the deposited platelet radioactivity by the whole-blood 111In-platelet activity and multiplying by the circulating platelet count.19 20 21
Homologous baboon fibrinogen was purified and labeled with 125I as described.19 20 21 The labeled fibrinogen preparation was >90% clottable. Ten minutes before thrombus formation was initiated, 5 µCi of 125I-fibrinogen was injected intravenously. After exposure to blood for 1 hour, the thrombogenic device was thoroughly rinsed with isotonic saline and cut into regions corresponding to the Dacron graft segments and expanded flow region. After at least 30 days was allowed for the 111In to decay, the device components were counted for 125I-activity with a gamma counter. Total fibrin accumulation was calculated by dividing the deposited 125I-radioactivity by the clottable fibrinogen radioactivity and multiplying by the circulating fibrinogen concentration as measured in each experiment.19 20 21
Reagent
APAP (MW, 571.1) was a gift from Daiichi
Pharmaceutical Co, Ltd
(JP 5-208946-A). The structural formula of APAP is shown in Fig
1
. For
parenteral administration, APAP was dissolved in 60 mL saline and
infused for 60 minutes proximal to the thrombogenic devices. For oral
administration, APAP (50 mg/kg) was dissolved in 20 mL of water and
administered directly into the stomach by gavage under light ketamine
anesthesia.
Laboratory Procedures
ELISAs for ß-thromboglobulin
(ß-TG),
platelet factor 4 (PF 4), and thrombin–antithrombin III complex
(TAT) and radioimmunoassays for fibrinopeptide A (FPA)
were performed as described
previously19 20 21 on blood
samples drawn immediately before and at the end of each 60-minute
study.
Whole blood for measuring the activated partial thromboplastin time (aPTT) and anti-FXa activity was collected in 3.8% sodium citrate (9 volumes blood into 1 volume citrate before, 30 minutes after, and 60 minutes after infusion was initiated). At 30 and 60 minutes, blood was drawn from sites both proximal and distal to the site of drug infusion to evaluate both local and systemic effects of the infused APAP. aPTT (Ortho-Diagnostic Systems) was performed with a fibrometer (Fibrosystem; Becton Dickinson).
Catalytic activity of FXa bound to thrombus formed in vivo was measured by FXa chromogenic substrate determinations (Spectrozyme FXa, American Diagnostica). In this assay, segments of Dacron vascular graft were incorporated into chronic exteriorized femoral AV shunts for 15 minutes, as described.23 24 The thrombus-bearing segments of vascular graft were then removed, perfused with cold buffer (calcium- and magnesium-free PBS) at 20 mL/min for 5 minutes, and incubated with 200 µL 0.25 mmol/L FXa chromogenic substrate in 0.05 mol/L Tris buffer and 0.15 mol/L NaCl, pH 8.2, for 30 minutes at room temperature. The Spectrozyme Xa solution was filtered through a 30 000-Mr exclusion filter (Centricon 30, Amicon Corp) by centrifugation at 3000g for 15 minutes to remove potentially confounding hemoglobin. The filtrate (200 µL) was assayed for amidolytic product and related to a purified bovine FXa standard (a gift from Dr Sriram Krishnaswamy, Emory University, Atlanta, Ga). To correct for the non-FXa amidolytic activity in this determination, a parallel segment was carried out incubating the thrombus-bearing segments with 2 mg/mL tick anticoagulant peptide (TAP), a potent specific inhibitor of FXa. The difference in amidolytic activity between TAP-untreated and TAP-treated thrombus-bearing segments, ie, TAP-inhibitable activity, was assumed to represent the FXa specific activity bound to the graft thrombus. To evaluate the ability of heparin to inactivate bound FXa, washed thrombus-bearing segments were incubated with citrated plasma containing 1 U/mL bovine lung heparin (Upjohn) for 30 minutes before chromogenic substrate was added.
The effects of incubating 1 and 100 mg/L APAP with thrombus-bearing segments for 15 minutes before the addition of chromogenic substrate were determined in relation to the TAP-inhibitable bound FXa enzyme. To assess nonspecific effects of APAP for bound thrombin, similar measurements were performed, except that thrombin chromogenic substrate, Spectrozyme TH, was substituted for the FXa substrate in the assay, and thrombin-specific activity was defined as the hirudin-inhibitable amidolytic activity.
The plasma
concentration of APAP was determined for each individual
animal by use of the anti-FXa activity and appropriate calibration
curves with dilutions prepared from autologous citrated
platelet-poor plasma. Plasma clearance rates (T50
and T50ß) after intravenous infusion,
Cmax, and area under the curve after oral
administration of APAP were estimated by established
methodologies.25
Data Analysis
For comparative analyses, the two-tailed
Student's
t test for paired or unpaired groups was used when the data
were normally distributed. Otherwise, the Wilcoxon sign-rank test was
used. Multivariate data were compared by ANOVA for
repeated measures (SYSTAT, Systat, Inc). All data are
given as the mean±SEM.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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To establish the inherent antithrombotic potency of APAP in baboons, thrombus formation was evaluated while APAP was administered locally via continuous infusion at rates of 15 and 120 µg/min for 60 minutes into exteriorized AV shunts proximal to interposed thrombogenic devices. These doses were selected on the basis of preliminary dose-ranging infusion studies that documented intermediate and maximal antithrombotic effects for venous-type thrombosis with these doses. The corresponding peak plasma levels of APAP achieved in the thrombogenic devices by these infusions were 0.5±0.1 and 4.3±0.4 mg/L, respectively (Table 1
; Fig 2A); plasma
concentrations were greater at 60 minutes versus 30 minutes (Table
1).
Subsequently, oral dose–bracketing studies revealed that 50 mg/kg of
oral APAP achieved blood levels midway within this range, ie, 2.4±0.8
mg/L after completion of the 60-minute graft study (Table 1).
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, APAP 10
mg · kg-1 · h-1 for 1.5 hours
(T
, APAP 2.5
mg · kg-1 · h-1 for 1.5 hours
(T
The disappearance rates of APAP from blood after discontinuation
of the infusions of 2.5 and 10
mg · kg-1 · h-1 for 1.5 hours
were
T50, 14.1 and 6.3 minutes, and T50ß, 94
and 99 minutes, respectively (Fig 2A). The pattern of APAP
plasma
levels after oral administration of 50 mg/kg is shown in Fig
2B.
Assuming blood volumes of 70 mL/kg and plasma volumes of 40 mL/kg,
clearance rates of T50 6.3 minutes and
T50ß 99 minutes, and a Cmax value of 6.9 mg/L
at 10 minutes, the bioavailability of APAP in four animals was
estimated from the Cmax to be approximately 5.5%; the
estimation of bioavailability from area-under-the-curve calculations
was 12.3%.
The effects of APAP on tests of coagulation during the infusion of APAP
were determined from blood collected both systemically and from AV
shunts distal to the thrombogenic devices (Table 1).
Prolongations in
the aPTT corresponding to the two doses of APAP were directly related
to the drug concentrations in plasma (Table 1;
P<.05). For
example, when APAP was infused locally into the shunt for 1 hour at 120
µg/min, the aPTT was double baseline values, corresponding to a
plasma APAP level of 4.3±0.4 mg/L, with lesser effects observed at the
smaller dose and in samples obtained systemically (Table 1). As
expected, the systemic plasma concentration of APAP was about one
fourth of that achieved locally in the device. Bleeding time
measurements were not prolonged by 3.8 mg/L APAP (Table 1). In
addition, no detectable cardiovascular or other adverse
side effects were observed during APAP infusion or after oral
administration.
In control studies (saline infusion alone), segments of Dacron vascular
graft and the expanded chambers accumulated comparable numbers of
deposited platelets and fibrin (Table 2). The
infusion of APAP directly into AV shunts proximal to thrombogenic
devices (15 and 120 µg/min for 1 hour) reduced platelet
deposition in a dose-dependent manner for venous-type thrombus in
the chambers (Fig 3), ie, the control value of
2.47±0.66x109 fell to
0.90±0.24x109 and
0.06±0.03x109 platelets (P=.073 and
P<.01, respectively, by t-test analysis
and P=.150 and P<.05, respectively, by ANOVA).
125I-Fibrin accumulation was also decreased in a
dose-dependent fashion (control value of 4.48±0.67 decreased to
1.92±0.39 and 0.56±0.20 mg fibrin; P<.05 and
P<.01, respectively). However, neither platelet
deposition nor fibrin accumulation was reduced significantly for
platelet-rich arterial-type thrombus forming on the
segments of vascular graft (Fig 4), ie, a control value
of 3.23±0.45x109 versus
2.96±0.36x109 and
2.15±0.58x109 platelets (P>0.1 in both
cases) and a control value of 2.18±0.27 versus 2.40±0.76 and
1.42±0.29 mg fibrin (P>0.1 in both cases).
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, APAP 120 µg/min for
60
minutes (n=4). B, The deposition of fibrin is shown in the chambers
after 60 minutes of blood exposure; infused APAP also suppressed fibrin
accumulation. The effects of oral APAP on thrombus formation within the
device chamber are shown in C and D. APAP was given 1 hour before the
thrombogenic device was interposed in the AV shunt. C, The accumulation
of platelets is depicted in their chambers during 60 minutes of
blood exposure; oral APAP reduced platelet accumulation.
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The oral administration of APAP (50 mg/kg) decreased platelet
deposition to 0.49±0.21x109 platelets and also
decreased fibrin deposition to 1.80±0.81 mg fibrin in venous-type
thrombus in the chambers (P<.01 and P<.05,
respectively, by t test analysis and
P=.069 and P<.05, respectively, by ANOVA) but
failed to decrease platelet deposition in arterial-type
thrombus on segments of vascular graft (2.08±0.74x109
platelets and 2.27±0.93 mg fibrin; P>.1 in both
cases). The effects of oral APAP on thrombus formation are shown in
Figs 3 and 4.
Blood markers of platelet activation (ß-TG and PF 4) and thrombin
production (FPA and TAT) were assayed on systemic blood and
blood-collected effluent from the thrombogenic devices (Table
2). In
saline control studies, the levels of these markers increased 3- to
10-fold after device insertion. Oral and infused APAP prolonged the
aPTT and prevented thrombus-dependent elevations in plasma FPA, TAT,
ß-TG, and PF 4 levels (Table 2). By contrast, bleeding time
measurements were not prolonged by APAP at any of the doses
administered (P>.5).
The effect of APAP on bound FXa activity was also evaluated. In
untreated control studies, bound FXa was maximal 15 minutes after
thrombus formation was initiated. By contrast, bound thrombin activity
achieved a maximal value at 30 minutes that was sustained for many
hours. While heparin failed to inhibit bound FXa activity, APAP
substantially reduced bound FXa activity in a dose-response manner
(Table 3). Moreover, APAP (100 mg/L) exhibited no
significant inhibition of bound thrombin activity (control value,
2.33±0.31 and APAP value, 2.07±0.10 ng/10 mm, n=8 and
n=4,
respectively; P>.1).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present study demonstrates in baboons that the parenteral and oral administration of APAP, a synthetic FXa antagonist, inhibits both soluble and bound FXa activity and produces dose-dependent anticoagulant and antithrombotic effects for venous-type fibrin-rich thrombus formation at drug levels ranging from 0.5 to 5 mg/L without impairing platelet hemostatic function. These findings support the premise that targeting FXa is an effective and safe antithrombotic strategy. Antithrombotic efficacy for oral synthetic inhibitors of FXa has not been previously reported in primates.
Thrombin is the critical mediator that recruits platelets and
produces fibrin in the formation of hemostatic plugs and thrombosis
after vascular
damage.21 26 27 28 29
Thrombin is produced by
FXa-mediated catalysis of prothrombin in the presence of FVa cofactor
activity and calcium on phospholipid surfaces. FXa is also formed by
assembly of macromolecular enzyme complexes via either the extrinsic or
intrinsic pathways.1 Thrombin amplifies its own
production through the activation of the cofactors FV and
FVIII.30 Thrombin mediates platelet recruitment at
sites of vascular injury by cleaving the extracellular amino terminus
of the platelet thrombin receptor to create a neo–amino terminal
activation sequence and subsequent autoactivation and initiation of
receptor signaling.31 Importantly, thrombin bound to
forming thrombus is also capable of proteolytically activating
platelets and cleaving
fibrinogen.32 33 34 Thrombus-bound
thrombin is the principal agonist mediating thrombus formation under
high-flow conditions.20 21 The resistance of
platelet-rich arterial thrombi to the antithrombotic
effects of heparin is largely attributable to the relative
inaccessibility of the heparin-binding domain of thrombus-bound
thrombin to inactivation by heparin–antithrombin III
complexes.35 36 Similarly, thrombus-bound FXa is
inaccessible to the inhibitory effects of
heparin–antithrombin III complexes (Table 3).
The importance of FXa in thrombogenesis has been experimentally demonstrated in baboons by studies investigating the relative antithrombotic efficacy and hemostatic safety of FXa antagonists, as illustrated by the effects of activated protein C,37 and two specific natural polypeptide inhibitors of FXa, antistasin13 and TAP.12 Activated protein C downregulates the production of thrombin by inactivating the cofactor activities of factor VIIIa and factor Va.38 It produces dose-dependent interruption of thrombus formation while sparing hemostasis.37 Antistasin and TAP inhibit FXa directly.39 They were originally obtained from the leech Haementeria officinalis and the tick Ornithodoros moubata, respectively.12 13 Antistasin comprises 119 amino acid residues, inhibits FXa with a Ki value of 0.31 to 0.62 nmol/L, has Cys-Arg*Val-His at its reactive site,40 and appears to be unrelated to any other family of serine protease inhibitors. TAP, a 60-amino-acid protein, is specific for FXa, with a Ki of 0.59 nmol/L,39 and resembles other Kunitz-type inhibitors. Antistasin41 and TAP42 interrupt platelet deposition and fibrin accumulation in a dose-dependent manner with half-maximal inhibitor doses of 2 and 6 µg/kg per minute, respectively, and corresponding inhibitor concentrations of 1.2±0.04 and 4.3±0.39 mg/L, respectively. The present studies document the capacity of APAP to inactivate both soluble and thrombus-bound FXa catalytic activity, albeit with a Ki three orders of magnitude greater than for TAP.
Whereas direct inhibitors of thrombin, such as hirudin, impair platelet hemostatic function in parallel with their antithrombotic effects for platelet-dependent heparin-resistant thrombotic processes,19 20 21 27 neither antistasin nor TAP affects bleeding time determinations at fully antithrombotic doses.41 42 Importantly, surgical blood loss at sites of endarterectomy is markedly reduced by antithrombotic doses of TAP (18 µg/kg per minute) compared with hirudin.42 Thus, it appears that inhibitors of FXa, as a class of agents, produce dose-dependent interruption of thrombus formation with substantially less impairment of platelet hemostatic function than exhibited by the direct antithrombins.12 13 41 42
Because oral dosing of APAP achieves blood levels that interrupt the formation of venous-type fibrin-rich thrombus but does not attain levels capable of inhibiting the formation of arterial-type platelet-rich thrombus, it is appropriate to consider the potential clinical use of oral direct FXa antagonists in the prevention of venous-type thrombosis and thromboembolism. These agents could provide an alternative to conventional anticoagulation in the management of deep venous thrombosis, pulmonary embolism, and cardiogenic thromboemboli associated with atrial fibrillation, artificial heart valves, or myocardial infarction. On the basis of relative efficacy, safety, convenience, and cost, oral FXa antagonists may have particular advantages in several clinical settings of venous thrombotic or thromboembolic disease. First, because of their rapid onset of activity, oral FXa antagonists may permit patients with acute deep venous thrombosis to be effectively and safely managed as outpatients. Substitution of an oral FXa antagonist for heparin would obviate several disadvantages of heparin therapy, including the need for in-hospital parenteral administration, routine monitoring to verify efficacy and safety, failure to interrupt heparin-resistant thrombotic processes, and possible development of antiheparin antibodies. Similarly, oral FXa antagonists may permit patients with acute calf vein thrombosis to be managed out of hospital. In addition, oral FXa antagonists could be substituted for parenteral heparin anticoagulation during outpatient procedures involving cardiovascular devices, such as angiography, angioplasty, vascular surgery, and hemodialysis. Second, because of its inherent sparing of hemostasis, relatively higher doses of an oral FXa antagonist could be used for reducing thrombotic complications that resist usual warfarin anticoagulation, such as thrombosis complicating knee and hip surgery, some metastatic malignancies, and thrombo-occlusive failure of AV access grafts. Third, an oral FXa antagonist could be substituted for heparin during cardiopulmonary bypass and other procedures in patients with heparin-induced thrombocytopenia. Thus, oral inhibitors of FXa have a number of theoretical advantages over heparin and coumarin-type anticoagulants in the management of venous thrombosis and thromboembolism.
In summary, the oral FXa antagonist APAP inhibits both soluble and bound FXa activity and produces dose-dependent anticoagulant and antithrombotic effects for venous-type fibrin-rich thrombus formation without impairing platelet hemostatic function in baboons. Such agents may be beneficial in the safe and cost-effective outpatient management of venous thrombotic disease.
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Acknowledgments |
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This study was supported by grants from the National Institutes of Health (HL-41619, HL-31469, and HL-48667). We are indebted to Debbie White and Greg Annisette for expert technical assistance.
Received August 18, 1994; revision received January 11, 1995; accepted January 17, 1995.
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