0022-3565/02/3013-1151–1156$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 301:1151–1156, 2002
Vol. 301, No. 3
4755/985113
Printed in U.S.A.
Intimatan Prevents Arterial and Venous Thrombosis in a Canine
Model of Deep Vessel Wall Injury
JAMES K. HENNAN, TING-TING HONG, ARDAMANPAL K. SHERGILL, EDWARD M. DRISCOLL, ALAN D. CARDIN, and
BENEDICT R. LUCCHESI
University of Michigan Medical School, Department of Pharmacology, Ann Arbor, Michigan (J.K.H., T.-T.H., A.K.S., E.M.D., B.R.L.); and Celsus
Laboratories, Inc., Cincinnati, Ohio (A.D.C.)
Received November 13, 2001; accepted January 23, 2002
This article is available online at http://jpet.aspetjournals.org
Surface-bound thrombin is resistant to inhibition by the
heparin/antithrombin III complex, thereby limiting the efficacy of heparin in arterial vessel wall disease (Hogg and
Jackson, 1989; Weitz et al., 1990; Hogg et al., 1996; Becker et
al., 1999). Bound thrombin which remains resilient at the
site of injury after heparin treatment may then perpetuate
thrombin generation via a feedback loop to promote rethrombosis (Kumar et al., 1994). In contrast to the heparin/antithrombin III complex which inhibits various serine proteases
of the coagulation cascade, the activated form of heparin
cofactor II is specific for thrombin and is also an efficient
inhibitor of surface-bound thrombin (Liaw et al., 2001). As
such, improved agonists of heparin cofactor II may present
effective tools to elucidate more completely the role of thrombin in the promotion of vessel wall thrombosis and provide a
novel therapeutic approach for the treatment of the thromboocclusive disorders. Heparin cofactor II activity is accelerated
This study was supported by a grant from Celsus Laboratories, Inc. and by
the Cardiovascular Pharmacology Research Fund, University of Michigan.
J. K. H. is the recipient of a Research Fellowship from the Heart and Stroke
Foundation of Canada.
min) thrombosis, compared with control vessels (carotid artery,
87.1 ⫾ 7.9 min; jugular vein, 60.6 ⫾ 7.4 min). Vessel patency
was maintained in eight of eight jugular veins and seven of eight
carotid arteries during treatment with Intimatan. Dalteparin significantly increased time to carotid artery thrombosis (122.1 ⫾
17.5 min) compared with control (64.3 ⫾ 8.2 min), but did not
change the time to thrombosis in the jugular vein. Only one
carotid artery remained patent at the end of the dalteparin
protocol. The two drugs produced minimal increases in bleeding times, and Intimatan increased the activated partial thromboplastin time above that observed with dalteparin. The results
demonstrate that Intimatan is effective in preventing occlusive
arterial and venous thrombosis in an experimental model of
deep vascular wall injury.
by dermatan sulfate (Tollefsen et al., 1983). In contrast to the
heparin template-assisted assembly of the thrombin-antithrombin complex, the activation of heparin cofactor II occurs
via an allosteric mechanism that involves a change in conformation associated with enhanced anti-thrombin activity
(Liaw et al., 1999). Despite a favorable pharmacological profile, including less anticoagulant activity than heparin and
an increased venous antithrombotic action (Desnoyers et al.,
1989) the clinical use of dermatan sulfate has been limited by
its low potency and solubility (Boneu et al., 1992).
Intimatan is an improved heparin cofactor II agonist that
inhibits the bound conformation of thrombin and provides a
sustained inhibition of vessel wall thrombogenicity in rabbit
models of vascular injury (Buchanan and Brister, 2000;
Buchanan et al., 2001). The present study examines whether
Intimatan effectively prevents occlusive thrombus formation
in response to vascular wall injury in a canine model of
occlusive arterial/venous thrombosis. Experiments were designed to assess the efficacy of Intimatan to prevent or reduce
the incidence of carotid artery and jugular vein thrombus
formation in response to deep vessel wall injury. The results
of the study demonstrate that Intimatan prevents both oc-
ABBREVIATIONS: LCA, left carotid artery; RJV, right jugular vein; RCA, right carotid artery; LJV, left jugular vein; PRP, platelet-rich plasma; AA,
arachidonic acid; aPTT, activated partial thromboplastin time; ANOVA, analysis of variance; HR, heart rate; MABP, mean arterial blood pressure.
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ABSTRACT
Resistance of fibrin-bound thrombin to inactivation by the heparin/antithrombin III complex is considered a limitation in the
use of heparin as an antithrombotic agent. Intimatan (dermatan
4,6-di-O-sulfate) is a heparin cofactor II agonist that inhibits
both free and bound forms of thrombin. The present study
examines the hypothesis that Intimatan prevents thrombotic
occlusion in response to vascular wall injury in a canine model
of carotid artery/jugular vein thrombosis. The left carotid artery
and right jugular vein served as vehicle-treated control vessels,
whereas the right carotid artery and left jugular vein were subjected to electrolytic injury after administration of Intimatan (9
mg/kg bolus ⫹ 300 ␮g/kg/min infusion, i.v.) or dalteparin (Fragmin) (400 IU/kg, s.c.). Intimatan significantly increased time to
carotid artery (226.0 ⫾ 14.0 min) and jugular vein (240.0 ⫾ 0.0
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Hennan et al.
clusive arterial and venous thrombosis in the dog at doses
that minimally affect the bleeding time.
Model of Arterial and Venous Occlusion
Experimental Protocol
Fifteen purpose-bred beagle dogs, weighing 9 to 13 kg, were anesthetized with sodium pentobarbital (30 mg/kg, i.v.), intubated, and
ventilated with room air using a Harvard respirator (Harvard Apparatus, Holliston, MA), adjusted to deliver a tidal volume of 30
ml/kg at a frequency of 12 breaths/min. A catheter was inserted into
the right femoral vein for drug administration. Blood pressure was
recorded from the right femoral artery using a Strathum Transducer
(Gould Inc., Cardiovascular Products, Oxnard, CA). A standard limb
lead II electrocardiogram was recorded continuously to monitor
heart rate. A carotid artery and jugular vein were isolated, and each
was fitted with Transonic ultrasonic flow probes (Transonic Systems,
Inc., Ithaca, NY) for continuous recording of blood flow. Recordings of
blood pressure, lead II electrocardiogram, mean, and phasic blood
flow from the carotid artery and jugular vein were obtained on a
Grass model 7 polygraph recorder (Grass Instrument Division, Astro-Med Inc., West Warwick, RI).
A C-shaped mechanical constrictor was placed around each vessel
and served to produce a narrowing of the vessel by adjusting a Teflon
screw to produce a regional stenosis. The flow was adjusted until the
For each experiment, the left carotid artery (LCA) and right jugular vein (RJV) served as control vessels, while the right carotid
artery (RCA) and left jugular vein (LJV) served as drug-treated
vessels. Animals were allowed 30 min to stabilize while the RCA and
LJV were isolated and prepared for induction of electrolytic injury.
Figure 1 illustrates the experimental protocol in detail. Before
initiation of electrolytic injury in control vessels, animals were
treated with either an intravenous “loading dose” and intravenous
infusion of saline (Intimatan control) or a subcutaneous injection of
saline (dalteparin control). Electrolytic injury was initiated in the
LCA and RJV 10 min after the start of the infusion in the Intimatan
group (9 mg/kg bolus ⫹ 300 ␮g/kg/min infusion, i.v.) and 2 h after the
subcutaneous injection in the dalteparin group (400 IU/kg, s.c.). The
optimal dose of Intimatan required to increase the time to thrombosis was determined in preliminary studies. The only parameter used
to determine an optimal bolus and i.v. infusion for Intimatan was the
inhibition of arterial and venous thrombosis. We began with an
initial dose of 3 mg/kg followed by an infusion of 30 ␮g/kg/min. This
dosing regimen was based upon preliminary data in the pig (unpub-
Materials and Methods
Guidelines for the Use and Care of Experimental Animals
The procedures used in this study are in accordance with the
guidelines of the University of Michigan University Committee on
the Use and Care of Animals and conform to the standards in The
Guide for Care and Use of Laboratory Animals (National Institutes
of Health publication no. 86-23). Veterinary care was provided by the
University of Michigan Unit for Laboratory Animal Medicine.
Reagents
Fig. 1. Diagrammatic representation of the experimental protocol used to investigate the effects of Intimatan and dalteparin on carotid artery and jugular
vein thrombosis. Hemodynamic parameters including
heart rate, blood pressure, carotid artery, and jugular
vein blood flow were monitored throughout the protocol.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017
Intimatan was supplied by Celsus Laboratories, Inc. (Cincinnati,
OH) and dissolved in 0.9% sodium chloride solution for injection
(saline). Dalteparin (Fragmin) was purchased from the University of
Michigan Hospital Pharmacy as formulated for clinical use. All other
reagents were purchased from Sigma-Aldrich (St. Louis, MO).
pulsatile flow pattern was reduced by 50% without altering the mean
carotid or jugular blood flow. An intraluminal electrode consisting of
a 30-gauge, Teflon-insulated, silver-coated, copper wire attached to
the tip of a 25-gauge hypodermic needle. The needle-electrode assembly was inserted through the vessel wall and positioned in a
manner that allowed the noninsulated segment of the electrode to
remain in direct contact with the intimal surface of the vessel. Proper
positioning of the electrode in the respective vessels was confirmed
by visual inspection at the end of each experiment.
The intravascular electrode was connected to the positive pole
(anode) of a dual channel stimulator (Grass S88 stimulator and
Grass Constant Current Unit, model CCU1A; Grass Instrument
Division, Astro-Med Inc.). The cathode was placed in a distant subcutaneous site. Application of an anodal d.c. current to the intimal
surface of the carotid artery or jugular vein resulted in a deep
vascular wall electrolytic lesion with exposure of subendothelial
components. The current delivered to the vessel was monitored continuously with an ammeter and was maintained at 300 ␮A for a
period of 3 h or was discontinued 30 min after a stable occlusive
thrombus had formed.
1153
In Vivo Antithrombotic Effects of Intimatan
Hematologic Determinations
Ex Vivo Platelet Aggregation Studies. Blood was taken for
platelet aggregation studies at baseline and at specific time points,
as indicated in Fig. 1. Venous blood (10 ml) was withdrawn from the
right femoral vein into a plastic syringe containing 3.7% sodium
citrate as the anticoagulant [1:10 citrate to blood (v/v)]. Platelet-rich
plasma (PRP) was obtained by collecting the supernatant from whole
blood centrifuged at 140g for 5 min. Platelet-poor plasma was prepared from the same blood sample by further centrifugation at 2000g
for 10 min. Ex vivo platelet aggregation was assessed at 37°C with a
four-channel platelet aggregometer (Bio-Data-PAP-4; Bio-Data, Hatboro, PA) by recording the increase in light transmission through a
stirred suspension of PRP adjusted to 200,000 platelets/␮l. Aggregation was induced with arachidonic acid (AA, 0.65 mM), ADP (20 ␮M),
and ␥-thrombin (25 nM). A subaggregatory concentration of epinephrine (550 nM) was used to prime the platelets before the agonists
were added. Values are expressed as percentage of aggregation,
representing the percentage of light transmission standardized to
PRP and platelet-poor plasma samples yielding 0% and 100% light
transmission, respectively.
Tongue Bleeding Times . Bleeding times were determined with
the use of a SurgiCut device, which makes a uniform incision 5 mm
long and 1 mm deep on the upper surface of the tongue. The tongue
lesion was blotted with a filter paper every 20 s until the transfer of
blood to the filter paper was no longer apparent. The interval, from
the time of the tongue incision until the time that blood is no longer
transferred to the filter paper, was recorded as the “tongue bleeding
time”.
Activated Partial Thromboplastin Time (aPTT). aPTT is a
measure of the intrinsic coagulation pathway that involves all the
coagulation factors, except factors VIII and VII. The aPTT determinations were performed using 2 ml of citrated whole blood [1:10
citrate/blood (v/v)] injected into OneStep aPTT tubes placed in a
Hemochron whole blood coagulation instrument (International Technidyne Corp., Edison, NJ), and time to coagulation was determined
automatically.
Statistical Analysis
Data are expressed as mean ⫾ S.E. for all experiments. Comparisons between heart rate and mean arterial blood pressure were
performed using a one-way ANOVA followed by Student-NewmanKeuls multiple comparison test. Comparisons between the incidence
of occlusion in saline and drug-treated vessels were performed using
a ␹2 test. Changes in time to thrombosis between saline and drugtreated vessels were carried out using paired t tests. Differences in
time to thrombosis between Intimatan- and dalteparin-treated animals were made using Student’s t test. Platelet aggregation values,
bleeding times, and aPTT values were compared with respective
baseline using a one-way ANOVA followed by Dunnett’s post hoc
test. All results were considered significant when p ⬍ 0.05.
Results
Hemodynamic Data. Table 1 summarizes the systemic
hemodynamic data for animals treated with Intimatan or
dalteparin. Heart rate (HR) and mean arterial blood pressure
(MABP) were unchanged from their respective baseline in
each group studied.
Carotid Artery and Jugular Vein Thrombosis. Electrolytic injury to the carotid artery and jugular vein resulted
in typical cyclic flow reductions that progressed to form an
occlusive thrombus and cessation of blood flow in all control
vessels. As shown in Table 2, left carotid artery and right
jugular vein time to thrombosis averaged 87.1 ⫾ 7.9 min and
60.6 ⫾ 7.4 min, respectively, in the Intimatan control group
(animals treated with saline, i.v.). In the dalteparin control
group (animals treated s.c. with saline), time to thrombosis
averaged 64.3 ⫾ 8.2 min in the left carotid artery and 70.3 ⫾
9.8 min in the right jugular vein.
Intravenous administration of Intimatan (9 mg/kg bolus ⫹
300 ␮g/kg/min infusion) prevented the development of occlusive thrombi in seven of eight right carotid arteries and eight
of eight left jugular veins. After 3 h of electrolytic injury,
Intimatan infusion and anodal current delivery to the right
carotid artery and left jugular vein were terminated. Vessel
patency was monitored for an additional hour. Vessels that
were patent at the time of drug and current cessation (180
min) continued to maintain blood flow at 240 min, at which
time the protocol was concluded. Despite continued arterial
or venous blood flow in the patent injured (seven of eight
arterial and eight of eight venous) vessels of the Intimatantreated group, time to thrombosis was recorded as 240 min at
the termination of the experiment for the purpose of statistical analysis (see Table 2).
In dalteparin-treated animals (400 IU/kg, s.c.), occlusive
TABLE 1
Effect of Intimatan (9 mg/kg bolus ⫹ 300 ␮g/kg/min infusion, i.v.) and
dalteparin (400 IU/kg, s.c.) on HR and MABP in anesthetized dogs
Data represent mean ⫾ SE for n ⫽ 8 with Intimatan and n ⫽ 7 with dalteparin.
Intimatan
Baseline
70 min saline
70 min Intimatan
130 min Intimatan
Dalteparin
Baseline
70 min saline
70 min dalteparin
130 min dalteparin
HR
MABP
bpm
mm Hg
156.8 ⫾ 8.5
152.8 ⫾ 10.0
142.5 ⫾ 12.5
140.5 ⫾ 11.7
134.4 ⫾ 14.2
138.8 ⫾ 12.4
129.8 ⫾ 14.9
126.9 ⫾ 14.6
135.3 ⫾ 5.7
145.9 ⫾ 5.3
144.9 ⫾ 6.8
136.0 ⫾ 8.1
132.1 ⫾ 9.5
134.3 ⫾ 9.7
142.9 ⫾ 11.5
140.7 ⫾ 11.8
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lished data, Celsus Laboratories). Unfortunately, we did not observe
inhibition of thrombosis with this dose. The dose was increased
progressively in three separate experiments, and a final dosing regimen of 9 mg/kg bolus followed by an infusion of 300 ␮g/kg/min was
established. The predetermined optimal dose of Intimatan was used
throughout the study.
The dose of dalteparin used in this study was adjusted from the
recommended human clinical dose used for treatment of venous
thrombosis (70 IU/kg). The pharmacokinetics of dalteparin in humans and dogs are similar (Grebe at al., 2000). The bioavailability of
dalteparin after subcutaneous injection is 100% in beagles and 90%
in humans (Grebe et al., 2000). Preliminary studies in the dog did not
produce inhibition of either arterial or venous thrombosis with 70
IU/kg; thus, additional pilot studies were done in an effort to achieve
an antithrombotic effect with dalteparin. At 400 IU/kg, a dose well
above that used clinically, we observed an increase in time to thrombosis; however, complete inhibition of thrombosis was not observed.
All remaining experiments were performed with 400 IU/kg dalteparin. The subcutaneous route of administration was selected since
this is how the drug is administered clinically for prevention of
venous thrombosis. In preliminary studies with dalteparin, electrolytic injury was initiated 30 min after subcutaneous injection, and no
drug effect was observed. When the time from injection was extended
to 2 h, time to thrombosis was extended, indicating an effect with
dalteparin.
If thrombotic occlusion developed in less than 3 h, the current and
infusion (Intimatan only) were discontinued after 30 min of zero
blood flow. If occlusive thrombosis did not occur and blood flow
persisted after 3 h of electrolytic stimulation, the current and infusion (Intimatan only) were discontinued and vessel patency was
monitored for an additional 2 h.
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Hennan et al.
TABLE 2
Incidence of carotid artery and jugular vein occlusion and time to thrombosis after treatment with Intimatan (9 mg/kg bolus ⫹ 300 ␮g/kg/min
infusion, i.v.) and dalteparin (400 IU/kg, s.c.)
Data represent mean ⫾ S.E. for n ⫽ 8 Intimatan and n ⫽ 7 Dalteparin.
Incidence of CA
Occlusion
CA Time to
Thrombosis
Incidence of JV
Occlusion
min
Saline i.v.
Intimatan i.v.
Saline s.c.
Dalteparin s.c.
8
1
7
6
/8 (100%)
/8 (12.5%)*,**
/7 (100%)
/7 (86%)
JV Time to
Thrombosis
min
87.1 ⫾ 7.9
226.0 ⫾ 14.0*,**
64.3 ⫾ 8.2
122.1 ⫾ 17.5*
8
0
7
7
/8 (100%)
/8 (0%)*,**
/7 (100%)
/7 (100%)
60.6 ⫾ 7.4
240.0 ⫾ 0.0*,**
70.3 ⫾ 9.8
91.9 ⫾ 12.2
CA, carotid artery; JV, jugular vein.
* Significant difference from respective saline control.
** Significant difference between Intimatan and dalteparin (p ⬍ 0.05).
Fig. 3. Effect of saline (vehicle) on LCA and RJV blood flow, and effect of
dalteparin (400 IU/kg, s.c.) on RCA and LJV blood flow during electrolytic
injury. Each time point represents the mean ⫾ S.E.M. for n ⫽ 7 experiments.
bin (25 nM) was determined before and after administration
of Intimatan or dalteparin in all animals studied. As shown
in Fig. 4, Intimatan decreased significantly platelet responses to ␥-thrombin at 70 and 130 min after infusion and to
ADP 70 min after initiating the loading dose and infusion.
Figure 5 presents a summary of the platelet aggregation
responses obtained after treatment with dalteparin. Dalteparin reduced significantly platelet responses to ␥-thrombin
at 70 and 130 min after subcutaneous injection. Ex vivo
aggregation responses to ADP were also reduced significantly at 130 min after drug administration. Both Intimatan
and dalteparin significantly increased aPTT above the respective baseline values at 1 and 2 h after drug treatment
(see Fig. 6).
The effects of Intimatan and dalteparin on tongue bleeding
time are summarized in Fig. 7. Both drugs produced only
limited increases in bleeding time that were not statistically
significant from baseline.
Discussion
Fig. 2. Effect of saline (vehicle) on LCA and RJV blood flow and Intimatan (9 mg/kg bolus ⫹ 300 ␮g/kg/min infusion, i.v.) on RCA and LJV blood
flow during electrolytic injury. Each time point represents the mean ⫾
S.E.M. for n ⫽ 8 experiments.
This study demonstrates that Intimatan is an effective
antithrombotic agent in preventing formation of arterial and
venous thrombi in response to deep vessel wall injury in the
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thrombus formation was prevented in only one right carotid
artery. All remaining right carotid arteries (six of seven) and
left jugular veins (seven of seven) progressed to form an
occlusive thrombus in the presence of dalteparin. Although
dalteparin did not inhibit the development of an occlusive
thrombus, it did prolong the time to thrombosis significantly
in the right carotid artery (122.1 ⫾ 17.5 min), compared with
saline-treated controls (64.3 ⫾ 8.2 min). No significant
change in time to thrombosis was observed in the left jugular
vein after treatment with dalteparin.
The effects of Intimatan and dalteparin on blood flow are
summarized in Table 2 and shown graphically in Figs. 2 and
3. Although Intimatan prevented the development of occlusive thrombosis, there was a decrease in right carotid artery
and left jugular vein blood flow during the 3 h of electrolytic
injury (see Fig. 2). Dalteparin did not prevent occlusive
thrombosis; thus, the decrease in blood flow during electrolytic injury approached that of the vehicle-treated controls
(see Fig. 3).
Hematologic Measurements. Ex vivo platelet aggregation in response to AA (0.65 mM), ADP (20 ␮M), and ␥-throm-
In Vivo Antithrombotic Effects of Intimatan
Fig. 5. Effect of saline (vehicle) and dalteparin (400 IU/kg, s.c.) on ex vivo
platelet aggregation induced by arachidonic acid (650 ␮M), ADP (20 ␮M),
and ␥-thrombin (25 nM). Venous blood samples were collected in sodium
citrate. Values are expressed as mean ⫾ S.E.M. for n ⫽ 7 experiments for
AA and ADP and n ⫽ 2 experiments for ␥-thrombin. Asterisks indicate a
significant change from respective baseline (p ⬍ 0.05). Groups were
compared using a one-way ANOVA followed by Dunnett’s post hoc test.
dog. Furthermore, the antithrombotic effects of Intimatan
were achieved with only a minimal increase in bleeding time.
Dalteparin, a clinically approved glycosaminoglycan with inhibitory effects on factor Xa and thrombin, had little or no
effect on arterial and venous thrombosis in our model.
Thrombin is produced predominately on the surface of
circulating platelets by the proteolytic activation of prothrombin (Fenton, 1986). When tissue damage occurs, factor
VII is activated, which activates factor X, which in turn binds
Fig. 6. Effect of Intimatan (9 mg/kg bolus ⫹ 300 ␮g/kg/min infusion, i.v.)
and dalteparin (400 IU/kg, s.c.) on whole blood aPTT. Venous blood
samples were collected in sodium citrate. Values are expressed as mean ⫾
S.E.M. for n ⫽ 8 experiments (Intimatan) and n ⫽ 7 experiments (dalteparin). Asterisks indicate a significant change from respective baseline
(p ⬍ 0.05). Groups were compared using a one-way ANOVA followed by
Dunnett’s post hoc test.
Fig. 7. Effect of Intimatan (9 mg/kg bolus ⫹ 300 ␮g/kg/min infusion, i.v.)
and dalteparin (400 IU/kg, s.c.) on tongue-template bleeding time. Values
are expressed as mean ⫾ S.E.M. for n ⫽ 8 experiments (Intimatan) and
n ⫽ 7 experiments (dalteparin). Groups were compared using a one-way
ANOVA followed by Dunnett’s post hoc test. No statistically significant
differences in bleeding times were observed.
activated Factor V on the platelet membrane surface to form
the prothrombinase complex (see Rosenburg and Bauer, 1994
for review). Membrane-bound prothrombinase catalytically
activates prothrombin. Thrombin modulates thrombus formation by activating platelets, converting fibrinogen to fibrin, and by activating blood coagulant factors V, VII, and
VIII, thereby promoting systemic hypercoagulation and increased vessel wall thrombogenicity (see Schafer, 1994 for
review). Thrombin activated at distant sites in the systemic
circulation also participates in thrombus formation. Two
plasma protease inhibitors, antithrombin III and heparin
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Fig. 4. Effect of saline (vehicle) and Intimatan (9 mg/kg bolus ⫹ 300
␮g/kg/min infusion, i.v.) on ex vivo platelet aggregation induced by arachidonic acid (650 ␮M), ADP (20 ␮M) and ␥-thrombin (25 nM). Venous
blood samples were collected in sodium citrate. Values are expressed as
mean ⫾ S.E.M. for n ⫽ 8 experiments with AA and ADP and n ⫽ 3
experiments with ␥-thrombin. Asterisks indicate a significant change
from respective baseline (p ⬍ 0.05). Groups were compared using a
one-way ANOVA followed by Dunnett’s post hoc test.
1155
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Hennan et al.
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Address correspondence to: Dr. Benedict R. Lucchesi, Department of Pharmacology, University of Michigan Medical School, 1301 Medical Science Research Building III, Ann Arbor, MI 48109-0632. E-mail: [email protected]
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cofactor II, regulate thrombin activity. Heparin cofactor II
combines with thrombin at exosite I on the enzyme surface to
form a complex in which the catalytic site is blocked (Becker
et al., 1999). Intimatan targets the vessel wall and fibrin
clots, then catalyzes the inhibition of surface-bound thrombin
by specific activation of heparin cofactor II (Buchanan and
Brister, 1998, 1999, 2000). The prevention of arterial and
venous thrombosis by Intimatan supports the hypothesis
that inhibition of surface-bound thrombin represents an important pharmacological target for achieving an effective antithrombotic effect. Intimatan increased aPTT, along with an
anticipated, albeit modest, increase in tongue bleeding time.
The latter observations may be significant in that Intimatan
was effective in preventing fibrin-dependent venous thrombosis, while at the same time having a beneficial effect in
modulating platelet-dependent arterial thrombus formation.
Preclinical studies suggest that Intimatan is more effective
than heparin as an inhibitor of thrombin generation in a pig
model of cardiopulmonary bypass, of neointimal hyperplasia
in balloon-injured rabbit aortae, and following injury to the
carotid artery (Brister et al., 1996; Schwartz et al., 1998;
Yang et al., 1999). It was also determined that Intimatan can
influence activated protein C activity (Fernandez et al.,
1999).
Subcutaneous low molecular weight heparins such as
dalteparin are effective alternatives to intravenous unfractionated heparin for treatment of venous thrombosis (Koopman et al., 1996; Levine et al., 1996). The low molecular
weight heparins have the added advantage of use outside of
the hospital setting without the need for laboratory monitoring. Results of clinical studies, however, demonstrate that
dalteparin is only as effective as heparin (Lopaciuk et al.,
1992; Koopman et al., 1996; Levine et al., 1996). In this study
the dose of dalteparin (400 IU/kg, s.c.) was approximately 5
times greater than the recommended human clinical dose (70
IU/kg). Dalteparin altered ex vivo platelet reactivity and
produced a modest increase in tongue bleeding time, but
failed to prevent occlusive thrombosis, despite having been
administered in what may be viewed as an excessive dose.
The apparent lack of efficacy may be related to the experimental model in which the extent of electrolytic injury results in deep vessel wall injury. In the same experimental
model, Intimatan, derived from chemical modification of dermatan sulfate, was observed to prevent both venous and
arterial thrombus formation. Dermatan sulfate has been suggested to promote fibrinolytic activity by inducing the release
of tissue plasminogen activator from endothelial cells (Abbadini et al., 1987). Whether Intimatan possesses a similar
action is not known. Thus, further investigation into the
profibrinolytic effects and antithrombotic effects of Intimatan is warranted.