1038
Kistrin, a Polypeptide Platelet GPIIb/IIIa
Receptor Antagonist, Enhances and Sustains
Coronary Arterial Thrombolysis With
Recombinant Tissue-Type Plasminogen
Activator in a Canine Preparation
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Tsunehiro Yasuda, MD; Herman K. Gold, MD; Robert C. Leinbach, MD;
Hiroyuki Yaoita, MD; John T. Fallon, MD, PhD; Luis Guerrero; Mary A. Napier, PhD;
Stuart Bunting, PhD; and Desire Collen, MD, PhD
Background. Kistrin is a 68-amino acid polypeptide from the venom of the Malayan pit viper
Agkistrodon rhodostoma, which inhibits the platelet GPIIb/IIIa receptor. Its effect on thrombolysis, reocclusion, and bleeding associated with administration of recombinant tissue-type
plasminogen activator (rt-PA) was studied in a canine model of coronary artery thrombosis.
Methods and Results. Coronary patency was monitored for 2 hours by ultrasonic flow probe
and repeated coronary angiography. The rt-PA was given as 0.45-mg/kg bolus injections at
15-minute intervals until recanalization or to a maximum of four boluses. Four groups of four
or five dogs were studied: a control group that received intravenous heparin (4,000-unit bolus
and 1,000 units each hour) and three groups that received heparin and 0.48, 0.24, or 0.12 mg/kg
kistrin, administered as a 10% bolus injection and an infusion during a 60-minute period. In
the control group, reflow occurred in four of five dogs within 37±+47 minutes but was followed
by cyclic reflow and reocclusion. Kistrin at a dose of 0.48 and 0.24 mg/kg reduced the time to
reflow to 6+5 and 10+3 minutes, respectively, and abolished reocclusion. With 0.12 mg/kg
kistrin, reflow occurred in all four animals, within 27+±23 minutes, and reocclusion occurred in
two animals. Kistrin induced a dose-related prolongation of the template bleeding time: with
0.48 mg/kg kistrin, the bleeding time was prolonged from 3.8±+1.3 minutes before infusion to
29+2 minutes during infusion, but it was shortened to 8.3 ±2.6 minutes at 90 minutes after the
end of infusion. Kistrin also caused a dose-related inhibition of platelet aggregation with ADP
and collagen: with 0.48 mg/kg kistrin, platelet aggregation was abolished during the infusion
but had partially recovered toward the end of the observation period. Pathological examination
of recanalized coronary arterial segments of dogs given 0.48 or 0.24 mg/kg kistrin revealed
widely patent arteries with some platelets layered on the damaged intimal surface.
Conclusions. Kistrin increases the rate and extent of thrombolysis with a reduced dose of
rt-PA, and it prevents reocclusion. At an effective dose, it is associated with a transient
prolongation of the bleeding time and inhibition of platelet aggregation. Kistrin may offer
promise as adjunctive treatment to thrombolytic agents in patients with acute myocardial
infarction. (Circulation 1991;83:1038-1047)
Tnhrombolytic therapy of acute myocardial infarction has become an established procedure for reperfusion of occluded coronary
arteries, resulting in reduction of infarct size and
preservation of left ventricular function.' Despite
their widespread use, the currently available throm-
From the Cardiac Unit and the Department of Pathology,
Massachusetts General Hospital and Harvard Medical School,
Boston; Genentech, Inc., South San Francisco, Calif.; and the
Departments of Biochemistry and Medicine, University of Vermont College of Medicine, Burlington.
Supported in part by Ischemia Specialized Center of Research
(SCOR) grant HL-26205 and Thrombosis SCOR grant HL-35058
from the National Heart, Lung, and Blood Institute, Bethesda,
Md.; and by a sponsored research agreement between Massachusetts General Hospital and Genentech, Inc.
Address for correspondence: Herman K. Gold, MD, Massachusetts General Hospital, ACC 4, Room 480, 15 Parkman Street,
Boston, MA 02114.
Received July 23, 1990; revision accepted October 30, 1990.
Yasuda et al Kistrin and Thrombolysis
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bolytic agents and strategies suffer from a number of
significant limitations, including resistance to reperfusion in approximately 25% of patients,23 occurrence of acute coronary reocclusion in 5-25%,4,5 a
See p 1115
prolonged time to restoration of antegrade coronary
flow of an average of 45 minutes,6-8 and a bleeding
tendency with a frequency of intracerebral bleeding
of approximately 0.5%.9-11 Various approaches to
overcome the present limitations of thrombolytic
therapy are currently under investigation in laboratory animal models.
Platelet-mediated thrombosis is a major pathogenic mechanism underlying the limited efficacy of
thrombolytic therapy.12-16 Several approaches have
been investigated to prevent platelet-mediated coronary occlusion including the use of aspirin,17 prostaglandin E1,'8 selective thromboxane A2 synthase
inhibitors,19 selective thromboxane A2 receptor antagonists in combination with serotonin receptor
antagonists,20,21 combined thromboxane A2 synthase
inhibitors/prostaglandin endoperoxide receptor antagonists,22 or selective thrombin inhibitors.23-26
The platelet receptor GPIIb/111a mediates platelet
aggregation by most physiological agonists27 and,
therefore, may constitute a preferred target for therapeutic intervention in thromboembolic disease. The
GPI1b/IIIa receptor may be inhibited by several
compounds, including monoclonal antibodies28 that
exhibit potent antithrombotic effects in vivo29-32 but
also by peptides derived from the gamma chain of
fibrinogen33 or Arg-Gly-Asp-containing (RGD) peptides34'35 that prevent thrombosis in vivo.36-40 Kistrin
is a polypeptide (Mr, 7,334) composed of 68 amino
acids, isolated from the venom of the Malayan pit
viper Agkistrodon rhodostoma, which contains the
typical RGD recognition sequence.41 It belongs to a
family of proteins from pit viper venoms that are
specific human platelet GPIIb/IIIa receptor antagonists that inhibit platelet aggregation.42-45 Kistrin is
100-1,000 times more potent than the pentapeptide
GRGDS for the inhibition of fibrinogen binding to
platelets and for platelet aggregation, and its binding
to platelets is both rapid and fully reversible.41
In the present study, we evaluated the effects of
adjunctive use of kistrin with recombinant tissue-type
plasminogen activator (rt-PA), in a canine model of
coronary artery thrombosis with superimposed high
grade stenosis, on: 1) the rate and extent of thrombolysis, 2) the frequency of reocclusion, 3) prolongation of the bleeding time, and 4) inhibition of ex vivo
platelet aggregation.46
Methods
Reagents
The single-chain rt-PA used was Activase, supplied
by Genentech, Inc., South San Francisco, Calif. Kistrin was purified from the venom of Agkistrodon
rhodostoma and characterized as described elsewhere,41 and was also supplied by Genentech. Heparin was purchased from Sinn, Cherry Hill, N.J.
1039
Canine Left Anterior Descending Coronary
Arterial Thrombosis
The experimental model was used essentially as
previously described.46 Adult mongrel dogs (20-25
kg) were anesthetized with pentobarbital (30 mg/kg
body wt i.v. and with additional doses as required).
The dogs were intubated and were placed on a
respirator with a tidal volume of 10-15 ml/kg. Procainamide, 1.5 g i.m., and lidocaine, 0.1 mg/kg/min
i.v., were given for prophylaxis of arrhythmias. The
left carotid artery was exposed through an incision in
the neck and was cannulated with a 7-1 modified
Amplatz Judkins coronary angiographic catheter.
Thoracotomy was performed through the left fifth
intercostal space, with cannulation of the left internal
mammary artery for continuous blood pressure recording. The pericardium was opened and was suspended to create a pericardial cradle. The left anterior descending coronary artery was dissected, and a
2.5 -cm segment was isolated distal to the first diagonal branch. A 0.7-mm internal diameter catheter was
inserted into a side branch of the isolated left anterior descending coronary artery segment, and an
ultrasonic flow probe (T101 Transsonic System Inc.,
Ithaca, N.Y.) was placed on the proximal portion of
the artery for continuous blood flow monitoring.
Selective angiography of the left anterior descending
coronary artery was obtained with 1-2 ml meglumine
diatrizoate with videotape recording. One milliliter
of blood was drawn for thrombus formation.
The dog was then given heparin intravenously
(bolus of 4,000 units and 1,000 units at hourly intervals). A 2-mm wide plastic wire tie (Mass Gas and
Electric Supply, Watertown, Mass.) was progressively
constricted around the left anterior descending coronary artery, just distal to the proposed site of
thrombus formation, to limit the blood flow to
40+10% of baseline. A control angiogram was then
obtained.
The isolated left anterior descending coronary
artery segment was traumatized by four consecutive
external compressions with blunt forceps during 3-5
seconds. This procedure was performed to damage
the endothelium and, thereby, to promote thrombus
adherence. Snare occlusions were made distal to the
flow probe and proximal to the constriction site.
Thrombin, 0.1 ml of 100 units/ml (Thrombinar, Armour Pharmaceutical, Kankakee, Ill.) mixed with 0.3
ml blood was injected through the side branch catheter into the emptied coronary artery segment. After
5 minutes, the proximal snare was released; 2 minutes later, the distal tourniquet was released. An
angiogram was obtained 30 minutes after thrombus
formation to confirm total occlusion of the artery as
demonstrated by the ultrasonic flow probe.
Infusion Protocols and Evaluation of Coronaty
Artery Patency
Four treatment groups were studied: 1) a control
group given heparin alone; 2) three groups given
1040
Circulation Vol 83, No 3 March 1991
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heparin and an intravenous infusion of kistrin at a
dose of 0.48, 0.24, or 0.12 mg/kg, which was given as
a bolus of 10% of the total dose followed by infusion
of the remainder during a 60-minute period (rate of
8, 4, or 2 ,ug/kg/min, respectively). Intravenous infusion was performed with a constant-rate infusion
pump (Harvard Apparatus, South Natick, Mass.),
starting after stable complete occlusion for 30 minutes had been confirmed by coronary angiography.
Fifteen minutes later, rt-PA was given as an intravenous bolus injection of 0.45 mg/kg at 15-minute
intervals until recanalization of the thrombosed coronary artery was achieved, or to a maximum of four
boluses. Angiograms were obtained every 15 minutes
to monitor occlusion and additionally when the flow
probe showed evidence of reflow. Reflow was monitored by angiography and flow probe for at least 2
hours after the initial angiographic confirmation of
stable coronary artery occlusion. The animals were
killed, on average, 150 minutes after the start of the
kistrin infusion.
The reflow time was recorded as the time from the
first rt-PA bolus injection until recanalization was
documented by the return of blood flow in the artery
to at least 25% of that before thrombus formation
and was confirmed by complete angiographic filling
of the apex with rapid clearance of the dye in four
heart beats or less. After recanalization was achieved,
blood flow was monitored for evidence of reocclusion,
defined as less than 25% of baseline flow; the final
confirmation was obtained by angiography that
showed dye clearance in more than five cycles. The
reocclusion time was defined as the interval between
documented reflow and reocclusion. Frequently, cyclic
reflow occurred, which was interspersed with periods
of reocclusion.
The coronary artery patency status was categorized
as follows: 1) persistent occlusion: no reflow; 2) cyclic
reflow and reocclusion: alternating reocclusion and
recanalization after initial reflow; 3) persistent patency:
persistent flow without reocclusion after initial reflow.
Blood Analyses
Venous blood samples for platelet and plasma assays
were collected in 0.01 M citrate and 200 kallikrein
inhibitor units (KIU) aprotinin/ml (Sigma Chemical
Co., St. Louis, Mo.). Platelet-rich plasma was prepared
immediately and was tested for platelet aggregation
induced with 11 ,uM ADP or 0.1 mg/ml collagen
(Sigma). Plasma was prepared from blood kept on ice
until the end of the experiment; then, it was centrifuged
at room temperature and was stored at -20°C. Fibrinogen was measured by a coagulation rate assay47,48 that
is insensitive to therapeutic concentrations of heparin,49 and t-PA antigen was measured with a monoclonal antibody-based enzyme-linked immunosorbent
assay (ELISA)50 that does not cross-react with canine
t-PA. Venous blood samples collected in EDTA were
also obtained for measurement of the platelet count.
Bleeding times were measured before the first injection, 20 minutes after the first injection, and 60 minutes
after the last injection of rt-PA; measurements were
made with a spring-loaded blade device (Surgicutt
International, Technidyne Corp., Edison, N.J.) applied
to a shaved foreleg. Kistrin levels in plasma were
assayed by specific ELISA.
Pathological Examination
At the end of the experiment, the dogs were killed
with an overdose of pentobarbital. The heart was
removed; persistently occluded and reoccluded arteries were perfusion fixed with 5% buffered formalin in
situ,51 and the whole heart was fixed in 5% formaldehyde. The thrombosed, stenotic, and poststenotic
segments of the left anterior descending coronary
artery with contiguous 0.5 cm of proximal artery and
the distal stenosis were then removed intact, were
embedded in paraffin blocks, and were sectioned
longitudinally. Sections were stained with hematoxylin and eosin and were examined microscopically for
the presence of intraluminal or mural thrombi. Persistently patent arteries were subjected to perfusion
fixation with 0.1 M cacodylate-buffered 2.5% glutaraldehyde for scanning electron microscopy as previously described.2551 The extent of thrombosis was
semiquantitatively graded on a scale of 1 to 4: 1, no or
minimal mural thrombus; 2, mural thrombus occupying less than 50% of the luminal diameter over a
section greater than the diameter of the arterial
segment; 3, thrombus occupying more than 50% but
less than 95% of the luminal diameter; and 4, completely occlusive thrombus or occupying more than
95% of the luminal diameter. The composition of the
thrombus was characterized as erythrocyte-rich,
platelet-rich, or mixed with interlaced platelet-rich
and erythrocyte-rich zones.
Statistical Analysis
The values are reported as mean+±SD. The significance of differences between groups was determined
with Student's t test for paired or unpaired values.
Fisher's exact test was used to compare the occurrence of reflow and reocclusion in the various groups.
A Kruskal-Wallis nonparametric analysis of variance
was performed52 on ranks of the ordered variable of
arterial patency, which ranges from 0 (persistent
occlusion) to 1 (cyclic reflow and reocclusion), and to
2 (persistent patency, as determined with the blood
flow meter). A similar analysis was performed on
arterial patency, graded on the pathological analysis
as described above. This form of analysis of variance
was selected because of the non-Gaussian distribution of the patency-state variables.
Results
Coronary Artery Reflow and Reocclusion
The results of coronary blood flow and coronary
artery patency status, categorized as persistent occlusion, cyclic reflow and reocclusion, and persistent
patency, as defined in "Methods," are summarized in
Table 1. The patency status in the individual animals
Yasuda et al Kistrin and Thrombolysis
1041
TABLE 1. Results of Coronary Blood Flow and Coronary Artery Patency Status
Coronary patency status
Blood flow (ml/min)
Poststenotic
Postreflow
3.6+1.0
5.1+0.9
4.9±1.4
3.2+1.2
6.6±1.0
2.5±0.4
5.0±0.8
2.3±0.5
Dose
(mg/kg)
Agent
Control
Kistrin
Baseline
n
5
5
5
4
-
0.48
0.24
0.12
13+2.4
11±0.9
16±1.4
11±1.1
(frequency)
CR
PO
1
0
0
0
4
1
2
2
PP
0
4
3
2
Data are mean±SD.
PO, persistent occlusion; CR, cyclic reflow; PP, persistent patency.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
is also schematically represented in Figure 1. The
external constrictor reduced the blood flow in the
different groups on average to 39-46% of baseline,
from a mean value of 11-16 ml/min to 5-6.6 ml/min
before thrombus formation (Table 1).
Bolus injection of rt-PA in control animals without
kistrin induced recanalization in four of five animals
(Figure 1). Reflow was achieved within 37+±47 minutes, with a median time of 25 minutes and a range of
3-120 or more minutes (Table 2). Cyclic reflow and
reocclusion occurred in all four dogs. The median
number of rt-PA boluses administered was two, with
a range of one to four. Bolus injection of rt-PA
induced recanalization in all five animals given 0.48
mg/kg kistrin (Figure 1). Reflow was achieved in all
animals with a single bolus of rt-PA, with a median
time of 6 minutes and a range of 3-15 minutes (Table
2). Reocclusion did not occur, except for one brief
cycle of reocclusion and reflow in one dog. A single
bolus of rt-PA, combined with 0.24 mg/kg kistrin,
induced reflow in all five dogs, with a median time of
10 minutes and a range of 7-15 minutes. Brief
periods of reocclusion occurred in two of these five
dogs. With 0.12 mg/kg kistrin, reperfusion required
more than one rt-PA bolus in two of four animals in
which reflow was followed by cyclic reflow and reocclusion. The median number of rt-PA boluses was
two, with a range of one to four.
Several significant differences in coronary arterial
patency were observed with the various infusion
TIME (min)
L
L
0
30
60
TIME (min)
90
120
KISTRIN 0.48 mg/kg
n
30
0
60
120
90
KISTRIN 0.12 mg/kg
v
v
_I
1
111
3_
1'
2_
HEM
2
3__
4v
4
5
n
KISTRIN 0.24 mg/kg
1
,
CONTROLS
v
v
v
v
,.
v
V
V
V
1
2
2__
D-v
-I
3
4
I__
_
_
2=
W= =
u. m
=
3 V5
E
M
v
v
IF
v
v
_
5
5
FIGURE 1. Schematic representation of the left anterior descending
coronary arterial patency status in
dogs receiving 0.45-mg/kg bolus injections of recombinant tissue-type
plasminogen activator (rt-PA) and
kistrin. v, rt-PA bolus injection;
1
continuous intravenous infusion of kistrin. Open bars, patency; solid bars, occlusion of the
_
~
m
M
_
1
1
coronary
artery.
1
1
Ion MINEETRILE
5 a a Wirir-in
TABLE 2. Effects of Kistrin on Time to Left Anterior Descending Coronary Artery Recanalization and Number of rt-PA
Boluses Required to Achieve Recanalization
Reperfusion
Dose
Time
(min)
rt-PA boluses (n)
Median
Median
Mean
n/total n
Range
Range
1-4
2
3-120
25
37±47
4/5
1-1
1
6
3-15
6+5
5/5
0.48
1
1-1
10
7-15
10+3
0.24
5/5
1-4
2
7-55
21
27±23
0.12
4/4
for
the
total
are
values
and
in
and
the
median
Data are mean±SD of values responsive animals,
range
group.
rt-PA, recombinant tissue-type plasminogen activator.
Agent
Control
Kistrin
(mg/kg)
1042
Circulation Vol 83, No 3 March 1991
TABLE 3. Effect of Kistrin on Template Bleeding Time Induced With Recombinant Tissue-Type Plasminogen Activator
60 min after
20 min after
last rt-PA boluis
End
Before
first rt-PA bolus
3.6+1.3
5
3.8+0.8
3.8+0.9
4.9+1.1
26+4.7
5
0.48
3.8+1.3
29+2.2
8.3+2.6
5
3.9+1.3
0.24
18±+1
17+11
6.5±1.9
4
4.1+1.5
3.0±0.4
6.0+1.1
0.12
Data, expressed in minutes, represent mean±SD. Results >30 minutes are given a 3-minute value for calculation of
mean±SD.
rt-PA, recombinant tissue-type plasminogen activator.
Dose
(mg/kg)
Agent
Control
Kistrin
n
protocols. A Kruskal-Wallis analysis of all experiments with patency status ordered in the sequence of
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
persistent occlusion, cyclic reflow and reocclusion,
and persistent patency yielded a p value of 0.01.
Comparison of the patency status in the group with
heparin and 0.48 mg/kg kistrin with that of the
control group given heparin alone gave a p value of
0.006. Persistent patency occurred more frequently
with the combination of kistrin and heparin than with
heparin alone (five of five versus zero of five dogs,
respectively, p=0.008 by Fisher's exact tests).
The time to reflow was significantly shorter in the
groups receiving 0.48 mg/kg kistrin than in the group
receiving heparin alone (p=0.05). Likewise, these
groups receiving kistrin (0.48 or 0.24 mg/kg)
achieved reperfusion with fewer rt-PA boluses than
did the group given heparin alone (p=0.02).
Hemostasis Analysis
Serial template bleeding times were measured in
all animals with results summarized in Table 3. In
control animals given heparin and rt-PA, the template bleeding time did not change significantly
throughout the experiment. Kistrin in combination
with rt-PA prolonged the bleeding time: with 0.48mg/kg kistrin, bleeding time was prolonged from
3.8±1.3 minutes before injection to 29+2.2 minutes
within 20 minutes after the first rt-PA bolus injection
(30 minutes into the kistrin infusion); with 0.24
mg/kg kistrin, the bleeding time was prolonged from
3.9+1.3 minutes before infusion to 18±11 minutes
during infusion; and with 0.12 mg/kg of kistrin,
bleeding time was prolonged from 3.0±0.4 minutes
before infusion to 6.0±1.1 minutes during infusion.
The bleeding time prolongation was, however, rapidly reversible, for near normalization occurred
within 90 minutes after the end of the kistrin infusion
(at the end of the experiment) (Table 3).
Table 4 summarizes the results of platelet function
tests. Platelet counts did not markedly decrease
except moderately at the highest dose of kistrin.
ADP-induced and collagen-induced platelet aggregation were impaired to an extent proportional to the
kistrin dose, with partial recovery toward the end of
the experiment. In the control group, platelet aggregation remained essentially unaltered throughout the
experiment.
Results of plasma kistrin levels, rt-PA antigen and
fibrinogen levels, and of hematocrit determination
are summarized in Table 5. Infusion of 0.48 mg/kg
kistrin resulted in a steady-state plasma level of
0.51 +±0.19 ,ug/ml. This was followed by rapid clearance of kistrin from plasma to reach near-background levels toward the end of the experiment.
Plasma rt-PA levels increased to 3-4.5 gg/ml, measured 1 minute after bolus injection, and decreased
to 0.3-0.5 ,ug/ml within 10 minutes. The fibrinogen
level in the different groups of animals decreased
moderately but remained greater than 50% of baseline in all groups. The hematocrit value decreased
moderately (<30%) in all groups.
Pathology
The results of pathological analysis are summarized in Table 6. The extent of arterial thrombosis
was graded, and the thrombus composition was characterized as described in "Methods" and in the
legend of Table 6. Segments not recanalized primarily contained occlusive red cell clots (Figure 2);
segments with reocclusion after initial reflow contained occlusive thrombi composed of red cell and
platelet-rich clots; and segments with persistent pa-
TABLE 4. Effect of Kistrin on Platelet Function Parameters
Platelet aggregation (7%)
Platelet count
Dose
Agent
Control
Kistrin
(mg/kg)
-
0.48
0.24
0.12
n
5
5
5
2
Data represent mean+SD.
Before
390+140
350+120
380+83
330+110
(x 1O3mm3)
60 min
340+130
210-+-160
340+79
340±90
End
270+150
250+150
340+75
360+90
ADP
Before
77+5
73+9
77+16
73+6
30 min
65+23
0
35+30
52+21
Collagen
End
66+17
21+23
67+14
68+13
Before
30 min
70±4
42+38
63+35
0+0
-
-
-
40+46
21+30
53±48
End
80+34
16+32
Yasuda
et
al Kistrin and Thrombolysis
1043
TABLE 5. Results of Blood Analyses
rt-PA antigen level
(rg/ml)
Dose
Agent (mg/kg)
Control
Kistrin
0.48
0.24
0.12
Plasma kistrin level
10 min
40 min
0.25+0.15 0.51±0.19
-
(,ug/kg)
End
0.07±0.03
-
1
min after
rt-PA
3.0± 1.2
3.3±1.0
4.6±1.2
3.1±1.2
10
min after
rt-PA
0.31±1.9
0.30±6.7
0.54+0.20
0.54±0.15
Data are mean±SD.
rt-PA, recombinant tissue-type plasminogen activator.
The background value measured in plasma before kistrin infusion is 0.05±0.04
tency (grade 1) showed a platelet layer adherent to
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
the vessel wall (Figure 3). All vessels showed significant mural disruption with intramural hemorrhage.
Scanning electron microscopy of persistently patent
left anterior descending coronary arterial segments
revealed an irregular intimal surface, denuded of
endothelium, and areas covered with platelets with
few neutrophils and red cells.
A Kruskal-Wallis analysis of the graded categories
of the extent of thrombosis yieldedp=0.22 for significant overall differences between the groups and
p=0.15 for the difference between the 0.48 mg/kg
kistrin group and the control group.
Discussion
Platelets play a central role in the current limitations of thrombolytic therapy.12-16 Inhibition of plate-
Fibrinogen level (g/l)
Baseline
1.4±0.54
1.2±0.39
1.8±0.14
2.6±0.63
End
0.84±0.49
0.79±0.32
1.4+0.23
2.4±0.84
Hematocrit
End
Baseline
40+7
36±10
42±3
32±8
43±4
35±5
41±9
43±10
gg/ml.
let aggregation with the potent monoclonal antiplatelet GPI1b/IIIa antibody 7E3 overcomes resistance of
platelet-rich thrombus to thrombolysis54 and accelerates and sustains coronary artery reperfusion with
reduced doses of rt-PA.31,32 However, in the setting
of high-grade stenosis and endothelial damage, these
effects are only obtained at extensive saturation of
platelet GPI1b/111a receptors, resulting in protracted
inhibition of platelet aggregation and prolongation of
the bleeding time.32
Many peptides and proteins, derived from the
RGD sequence or the gamma-chain recognition sequences of fibrinogen, have binding affinity for the
GPI1b/IIIa receptor.27,55 These compounds compete
with fibrinogen binding to the GPIIb/IIIa receptor,
which constitutes a common final pathway in platelet
TABLE 6. Pathological Analysis of Left Anterior Descending Coronary Arterial Segments
Agent
Control
Dose
(mg/kg)
Dog
number
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
Analysis
SEM
HE
SEM
SEM
HE
SEM
SEM
SEM
SEM
SEM
SEM
SEM
SEM
SEM/HE
SEM
SEM
Patency
status
2
1
4
1
4
1
1
1
1
2
3
2
1
3
2
2
2
1
1
Description
ER
Clean vessel
ER
Platelet layer
PR
Platelet monolayer
0.48
Kistrin
Platelet layer, some fibrin
Platelet layer
Platelet layer
ER
ER+fibrin
0.24
PR at stenosis
Some platelets
ER
ER, adherent platelet
MPE, ER
0.12
PR
2
SEM
MPE
SEM
3
Platelet monolayer
4
SEM
SEM, analysis by scanning electron microscopy; HE, analysis by histologic analysis after hematoxylin and eosin
staining; ER, erythrocyte-rich thrombus; PR, platelet-rich thrombus; MPE, mixed thrombus with interlaced plateletrich and erythrocyte-rich zones.
Patency status: 1, no or minimal mural thrombus; 2, mural thrombus occupying less than 50% of the luminal diameter
over a section greater than the diameter of the arterial segment; 3, thrombus occupying more than 50% but less than
95% of the luminal diameter; and 4, completely occlusive thrombus or occupying more than 95% of the luminal
diameter.
1044
Circulation Vol 83, No 3 March 1991
FIGURE 2. Scanning electron micrograph of a left anterior descending coronary arterial segment from a dog
treated with heparin and recombinant
tissue-type plasminogen activator (rtPA), which resulted in cyclic reflow.
Thrombus is erythrocyte-rich clot. Orig-
inal magnification, x 1, 900.
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
aggregation. Several RGD-containing snake venom
polypeptides inhibit fibrinogen binding and platelet
aggregation 100-1,000 times more efficiently than
does the GRGDS pentapeptide.39Al One of these
polypeptides, kistrin, obtained from the venom of
Agkistrodon rhodostroma was recently characterized.41
It binds with a high affinity (Kd in the nanomolar
range) to both resting and ADP-activated human
platelets, and it inhibits ex vivo platelet aggregation
reversibly after in vivo injection in rabbits.
We have used a canine model of coronary artery
thrombosis with superimposed high-grade stenosis,
which has anatomical similarities to the coronary
artery occlusion in patients with acute myocardial
infarction,46 to study the effects of kistrin on the
efficacy and speed of thrombolysis with rt-PA, on
reocclusion, on the bleeding tendency as revealed by
the template bleeding time, and on ex vivo platelet
aggregation. Simultaneous systemic therapeutic heparin anticoagulation was used because it is mandatory
in achieving the full effect of the monoclonal GPIIb/
Illa antibody56 (and unpublished observations).
Intravenous infusion of kistrin at a dose of 0.48 or
0.24 mg/kg infused during 1 hour, which prolonged
the template bleeding time from approximately 4 to
20 minutes or more and which abolished ADP- and
collagen-induced ex vivo platelet aggregation, markedly accelerated coronary arterial thrombolysis with
negligible residual thrombus demonstrated on pathological examination. These observations are in agree-
FIGURE 3. Scanning electron micrograph of a persistently patent left anteNior descending coronary artery segment in a dog given one bolus of 0.45
mg/kg of recombinant tissue-type plasminogen activator with 0.48 mg/kg kistrin. Intimal surface reveals numerous
platelets and a few red cells but no
fibrin strand.s. Original magnification,
x2,200.
Yasuda et al Kistrin and Thrombolysis
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
ment with the much higher affinity of kistrin for the
platelet GPI1b/IIIa receptor41 compared with tetrapeptides such as RGDY.s7 Indeed, the effective
dose of 4 ,ug/kg/min to prevent reocclusion is 1,000
times lower than that of the tetrapeptide.57
Reocclusion was abolished during the 1-hour follow-up period after the infusion of kistrin, during
which the prolongation of the bleeding time and the
inhibition of platelet aggregation returned toward
normal. The persistence of coronary patency, despite
the near normalization of the bleeding time, suggests
either that the time required for passivation of the
damaged endothelial surface is relatively short or
that the extensive dissolution of the occluding thrombus by the combination of rt-PA, heparin, and kistrin
resulted in a nonthrombogenic vessel segment. Indeed, residual thrombus has been shown to be a
more potent thrombogenic stimulus than a damaged
vessel wall.58
In the present study, we administered kistrin as a
loading dose of 10% followed by a continuous intravenous infusion at a rate of 2, 4, or 8 ,ug/kg/min for 60
minutes. In preliminary experiments (not shown), this
dose range was determined to result in partial-tocomplete inhibition of platelet aggregation and in
marginal-to-extensive prolongation of the bleeding
time. The infusion of kistrin was started 15 minutes
before the bolus injections of rt-PA to allow evaluation of its effect on time to recanalization under
steady-state plasma concentrations. The dose of heparin was selected to maintain the "activated" partial
thromboplastin time more than twofold throughout
the experimental observation period. Insufficient heparin anticoagulation, indeed, reduces the ability of
rt-PA to recanalize occluded arteries, and adequate
heparinization is required for sustained patency after
the administration of GPIIb/IIIa antagonist (unpublished observations).
Thus, acceleration of lysis and prevention of reocclusion with the combination of a reduced dose of
rt-PA, heparin, and a short-lived reversible platelet
GPIIb/I1Ia antagonist can be accomplished with
transient prolongation of the bleeding time, possibly
resulting in a less-increased risk of bleeding than
would have occurred with protracted platelet inhibition. However, potent combined antiplatelet, anticoagulant, and thrombolytic therapy, as evaluated in
this experimental animal study, is expected to be
inherently associated with some increased bleeding
risk, which will have to be carefully documented in
humans. Provided that the observations of the present study can be extrapolated to humans, this combination therapy may result in improved efficacy to
toxicity ratios for pharmacological recanalization of
occluded coronary arteries in patients with acute
myocardial infarction.
Acknowledgment
We thank Ms. Missy Stanton for excellent secretarial assistance.
1045
References
1. Collen D, Stump DL, Gold HK: Thrombolytic therapy. Annu
Rev Med 1988;39:405-423
2. Collen D: Coronary thrombolysis: Streptokinase or recombinant tissue-type plasminogen activator? Ann Intern Med 1990;
112:529-538
3. Rentrop KP: Thrombolytic therapy in patients with acute
myocardial infarction. Circulation 1985;71:627-631
4. Gold HK, Leinbach RC, Palacios IF, Yasuda T, Block PC,
Buckley MJ, Akins CW, Daggett WM, Austen WG: Coronary
reocclusion after selective administration of streptokinase.
Circulation 1983;68(suppl I):I-50-I-54
5. Johns JA, Gold HK, Leinbach RC, Yasuda T, Gimple LW,
Werner W, Finkelstein D, Newell JB, Ziskind AA, Collen D:
Prevention of coronary artery reocclusion and reduction in
late coronary artery stenosis after thrombolytic therapy in
patients with acute myocardial infarction: A randomized study
of maintenance infusion of recombinant human tissue-type
plasminogen activator. Circulation 1988;78:546-556
6. Anderson JL, Rothbard RL, Hackworthy RA, Sorensen SG,
Fitzpatrick PG, Dahl CF, Hagan AD, Brown KF, Symkoviak
GP, Menlove RL, Barry WH, Eckerson HW, Marder VJ:
Multicenter reperfusion trial of intravenous anisoylated plasminogen streptokinase activator complex (APSAC) in acute
myocardial infarction: Controlled comparison with intracoronary streptokinase. JAm Coll Cardiol 1988;11:1153-1163
7. Rogers WJ, Mantle JA, Hood WP, Baxley WA, Whitlow PL,
Reeves RC, Soto B: Prospective randomized trial of intravenous and intracoronary streptokinase in acute myocardial
infarction. Circulation 1983;68:1051-1061
8. Simoons ML, Arnold AER, Betriu A, De Bono DP, Col J,
Dougherty FC, von Essen R, Lambertz H, Lubsen J, Meier B,
Michel PL, Raynaud P, Rutsch W, Sanz GA, Schmidt W,
Serruys PW, Thery C, Uebis R, Vahanian A, Van de Werf F,
Willems GM, Wood D, Verstraete M: Thrombolysis with
tissue plasminogen activator in acute myocardial infarction:
No additional benefit from immediate percutaneous coronary
angioplasty. Lancet 1988;1:197-203
9. Rovelli F, De Vita C, Feruglio GA, Lotto A, Selvini A,
Tognoni G: GISSI trial: Early results and late follow up. JAm
Coll Cardiol 1987;10:33B-39B
10. Wilcox RG, von der Lippe 0, Olsson CG, Jenssen G, Skene
AM, Hampton JR: Trial of tissue plasminogen activator for
mortality reduction in acute myocardial infarction: AngloScandinavian study of early thrombolysis (ASSET). Lancet
1988;2:525-530
11. The International Study Group: In-hospital mortality and
clinical course of 20,891 patients with suspected acute myocardial infarction randomised between alteplase and streptokinase with or without heparin. Lancet 1990;336:71-75
12. Friedman M, Van den Bovenkamp GJ: The pathogenesis of a
coronary thrombus. Am J Pathol 1966;48:19-44
13. Davies MJ, Thomas AC: Plaque fissuring-The cause of acute
myocardial infarction, sudden ischemic death, and crescendo
angina. Br Heart J 1985;53:363-373
14. Fuster V, Chesebro JH: Mechanisms of unstable angina. N
Engl J Med 1986;315:1023-1025
15. Willerson JT, Hillis LD, Winniford M, Buja LM: Speculation
regarding mechanisms responsible for acute ischemic heart
disease syndromes. J Am Coll Cardiol 1986;8:245-250
16. Collen D, Gold HK: Fibrin-specific thrombolytic agents and
new approaches to coronary arterial thrombolysis, in Julian D,
Kubler W, Norris RM, Swan HJC, Collen D, Verstraete M
(eds): Thrombolysis in Cardiovascular Disease. New York,
Marcel Dekker Inc, 1989, pp 45-67
17. Folts JD, Crowell EB, Rowe GG: Platelet aggregation in
partially obstructed vessels and its elimination with aspirin.
Circulation 1976;54:365-370
18. Sharma B, Wyeth RP, Heinemann FA, Kolath G: Adjunctive
use of intracoronary prostaglandin El with streptokinase
enhances coronary thrombolysis (abstract). Circulation 1987;
76(suppl IV):IV-181
1046
Circulation Vol 83, No 3 March 1991
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
19. Aiken JW, Shebuski RJ, Miller OV, Gorman RR: Endogenous prostacyclin contributes to the efficacy of a thromboxane
synthetase inhibitor for preventing coronary arterv thrombosis. JPharmacol Exp Ther 1981;219:299- 308
20. Golino P, Ashton JH, McNatt J, Glas-Greenwalt P, ShengKun Y, O'Brien RA, Buja LM, Willerson JA: Simultaneous
administration of thromboxane A2 and serotonin S2-receptor
antagonists markedly enhances thrombolysis and prevents or
delays reocclusion after tissue-type plasminogen activator in a
canine model of coronarv thrombosis. Circulation 1989;79:
911-919
21. DeClerck F, Xhonneux L, Van Gorp L, Beetens J, Janssen
PAJ: S2-serotonergic receptor inhibition (ketanserin), cornbined with thromboxane Ajendoperoxide receptor blockade
(BM 13.177): Enhanced anti-platelet effect (letter). Thromb
Haemost 1986;56:236
22. De Clerck F, Beetens J, Van de Water A, Vercammen E,
Janssen PAJ: R68070: Thromboxane A, synthetase inhibition
and thromboxane Ajprostaglandin endoperoxide receptor
blockade combined in one molecule: II. Pharmacological
effects in vivo and ex vivo. Thromb Haemost 1989:61:43-49
23. Hanson SR, Harker LA: Interruption of acute plateletdependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone. Proc Natd
Acad Sci USA 1988;85:3184-3188
24. Heras M, Chesebro JH, Penny WJ, Bailey KR, Badimon L,
Fuster V: Effects of thrombin inhibition on the development
of acute platelet-thrombus deposition during angioplasty in
pigs: Heparin versus recombinant hirudin, a specific thrombin
inhibitor. Circulation 1989;79:657-665
25. Jang IK, Gold HK, Ziskind AA, Leinbach RC, Fallon JT,
Collen D: Prevention of platelet-rich arterial thrombosis by
selective thrombin inhibition. Circulation 1990;81:219-225
26. FitzGerald DJ, FitzGerald GA: Role of thrombin and thromboxane A2 in reocclusion following coronary thrombolysis with
tissue-type plasminogen activator. Proc Natl Acad Sci USA
1989;86:7585-7589
27. Plow EF, Ginsberg MH: Cellular adhesion: GPIIb/IIIa as a
prototypic adhesion receptor. Prog Hemost Thromb 1989;9:
117-156
28. Coller BS, Scudder LE: Inhibition of dog platelet function by
in vivo infusion of F(ab')2 fragments of a monoclonal antibody
to the platelet glycoprotein IIb/lIla receptor. Blood 1985;66:
1456-1459
29. Coller BS, Folts JD, Scudder LE, Smith SR: Antithrombotic
effect of a monoclonal antibody to the platelet glycoprotein
IIb/lIla receptor in an experimental animal model. Blood
1986;68:783-786
30. Simpson PJ, Mickelson JK, Cronin M, Laywell ED, Hoff PT,
Lucchesi BR: Monoclonal F(ab)2 that binds to platelet
GPIIb/IIIa receptor prevents experimental coronary artery
thrombosis (abstract). Circulation 1988;78(suppl II):II-80
31. Yasuda T, Gold HK, Fallon JT, Leinbach RC, Guerrero JL,
Scudder LE. Kanke M, Shealy D, Ross MJ, Collen D, Coller
BS: Monoclonal antibody against the platelet glycoprotein
IIb/IIla receptor prevents coronary artery reocclusion following reperfusion with recombinant tissue-type plasminogen
activator in dogs. J Clin Invest 1988;81:1284-1291
32. Gold HK, Coller BS, Yasuda T, Saito T, Fallon JT, Guerrero
JL, Leinbach RC, Ziskind AA, Collen D: Rapid and sustained
coronary artery recanalization with combined bolus injection
of recombinant tissue-type plasminogen activator and monoclonal antiplatelet GPIIb/IIIa antibody in a canine preparation. Circulation 1988;77:670-677
33. Hawiger J, Timmons S, Kloczewiak M, Strong DD, Doolittle
RF: Gamma and alpha chains of human fibrinogen possess
sites reactive with human platelet receptors. Proc Natl Acad
Sci USA 1982;79:2068-2071
34. Ruoslahti E, Pierschbacher MD: Arg-Gly-Asp: A versatile cell
recognition signal. Cell 1986;44:517-518
35. Plow EF, Pierschbacher MD, Ruoslahti E, Marguerie GA,
Ginsberg MH: The effect of Arg-Gly-Asp-containing peptides
on fibrinogen and von Willebrand factor binding to platelets.
Proc NatlAcad Sci USA 1985;82:8057-8061
36. McGoff MA, Allen BT, Sicard GA, Anderson CB, Santoro
SA: Arg-Gly-Asp-containing peptides inhibit platelet deposition into small diameter vascular grafts in a canine model
(abstract). Circulation 1989;80(suppl II):II-33
37. Shebuski RJ, Ramjit DR, Bencen GH, Polokoff MA: Effect of
bitistatin, a potent RGD-containing peptide from the venom
of Bitis arietens, on platelet aggregation and bleeding time in
the dog (abstract). Circulation 1989;80(suppl II):I1-422
38. Shebuski RJ, Ramjit DR, Lumma PK, Garsky VM: Prevention
of canine coronary artery thrombosis with Echistatin, a potent
inhibitor of platelet aggregation from the venom of the viper,
Echis carinatus (abstract). Circulation 1989;80(suppl II):II-645
39. Cook JJ, Huang TF, Rucinski B, Tuma RF, Williams JA,
Niewiarowski S: Inhibition of platelet hemostatic plug formation by trigramin, a novel RGD containing peptide (abstract).
Circulation 1988;78(suppl I1):II-313
40. Shebuski RJ, Berry DE, Bennett DB, Romoff T, Storer BL,
Ali F, Samaner J: Demonstration of Ac-Arg-Gly-Asp-Ser-NH2
as an antiaggregatory agent in the dog by intracoronary
administration. Thromb Haemost 1989;61:183-188
41. Dennis MS, Henzel WJ, Pitti RM, Lipari MT, Napier MA,
Deisher TA, Bunting S, Lazarus RA: Platelet GPIIb/IIIa
protein antagonists from snake venoms: Evidence for a family
of platelet aggregation inhibitors. Proc Natl Acad Sci USA
1990;87:2471-2475
42. Ouyang C, Huang TF: Potent platelet aggregation inhibitor
from Trimeresurus gramineus snake venom. Biochim Biophys
Acta 1983;757:332-341
43. Huang TF, Holt JC, Lukasiewicz H, Niewiarowski S: Trigramin: A low molecular weight peptide inhibiting fibrinogen
interaction with platelet receptors expressed on glycoprotein
IIb/IIla complex. J Biol Chem 1987;262:16157-16163
44. Huang TF, Holt JC, Kirby EP, Niewiarowski S: Trigramin:
Primary structure and its inhibition of von Willebrand factor
binding to glycoprotein JIb/lIla complex on human platelets.
Biochemistry 1989;28:661-666
45. Gan ZR, Gould RJ, Jacobs JW, Friedman PA, Polokoff MA:
Echistatin: A potent platelet aggregation inhibitor from the
venom of the viper: Echis carinatus. J Biol Chem 1988;262:
1.9827-19832
46. Yasuda T, Gold HK, Fallon JT, Leinbach RC, Garabedian
HD, Guerrero JL, Collen D: A canine model of coronary
artery thrombosis with superimposed high grade stenosis for
the investigation of rethrombosis after thrombolysis. JAm Coll
Cardiol 1989;13:1409-1414
47. Clauss A: Gerinnungsphysiologische Schnellmethode zur
Bestimmung des Fibrinogens. Acta Haematol 1957;17:237-246
48. Vermylen C, De Vreker R, Verstraete M: A rapid enzymatic
method for assay of fibrinogen: The fibrin polymerization time
(FPT-test). Clin Chim Acta 1963;8:418-424
49. Stump DC, Topol EJ, Chen AB, Hopkins A, Collen D:
Monitoring of hemostasis parameters during coronary thrombolysis with recombinant tissue-type plasminogen activator.
Thromb Haemost 1988;59:133-137
50. Holvoet P, Cleemput H, Collen D: Assay of human tissue-type
plasminogen activator (t-PA) with an enzyme-linked immunosorbent assay (ELISA) based on three murine monoclonal
antibodies to t-PA. Thromb Haemost 1985;54:684-687
51. O'Gara PT, Guerrero JL, Feldman B, Fallon JT, Block PC:
Effect of dextran and aspirin on platelet adherence after
transluminal angioplasty of normal canine coronary arteries.
Am J Cardiol 1984;53:1695-1698
52. Hollander M, Wolfe DA: Nonparametric Statistical Methods.
New York, John Wiley & Sons Inc, 1973
53. Chesebro J, Knatterud G, Braunwald E: Correspondence
section. N Engl J Med 1988;319:1544
54. Yasuda T, Gold HK, Leinbach RC, Saito T, Guerrero JL, Jang
IK, Holt R, Fallon JT, Collen D: Lysis of plasminogen
activator resistant platelet-rich coronary arterial thrombus
with combined bolus injection of recombinant tissue-type
plasminogen activator (rt-PA) and anti-platelet GPIIb/IIIa
antibody. JAm Coll Cardiol (in press)
55. Ruggeri ZM, Houghten RA, Russell SR, Zimmerman TS:
Inhibition of platelet function with synthetic peptides designed
Yasuda et al Kistrin and Thrombolysis
to be high-affinity antagonists of fibrinogen binding to plate-
lets. Proc Natl Acad Sci USA 1986;83:5708-5712
56. Yasuda T, Gold HK, Yaoita H, Leinbach RC, Guerrero JL, Jang
IK, Holt R, Fallon JL, Collen D: Comparative effects of aspirin,
a synthetic thrombin inhibitor, and a monoclonal antiplatelet
GPIIb/IIIa antibody, on coronary artery reperfusion, reocclusion
and bleeding with recombinant tissue-type plasminogen activator
in a canine preparation. JAm Coll Cardiol 1990;16:714-722
57. Haskel EJ, Adams SP, Feigen LP, Saffitz JE, Gorczynski RJ,
Sobel BE, Abendschein DR: Prevention of reoccluding plate-
1047
let rich thrombi in canine femoral arteries with a novel peptide
antagonist of platelet glycoprotein JIb/lIla receptors. Circulation 1989;80:1775-1782
58. Badimon L, Lassila R, Badimon J, Vallabhajosula S, Chesebro
J, Fuster V: Residual thrombus is more thrombogenic than
severely damaged vessel wall (abstract). Circulation 1988;
78(suppl II):II-119
KEY WORDS * glycoprotein IIb/IIla * antiplatelet agent
thrombolytic therapy * reocclusion
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Kistrin, a polypeptide platelet GPIIb/IIIa receptor antagonist, enhances and sustains
coronary arterial thrombolysis with recombinant tissue-type plasminogen activator in a
canine preparation.
T Yasuda, H K Gold, R C Leinbach, H Yaoita, J T Fallon, L Guerrero, M A Napier, S Bunting
and D Collen
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Circulation. 1991;83:1038-1047
doi: 10.1161/01.CIR.83.3.1038
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1991 American Heart Association, Inc. All rights reserved.
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