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Introduction
Staphylokinase (SAK) is an extracellular protein of 136 amino acids and is excreted by staphylococcus aureus. Its ability
to dissolve thrombus was first discovered in 1948, which intrigued most scholars in the field. However, its application has not
been adapted in the clinical setting due to poor purity of production and lack of natural resources. Recombinant staphylokinase
(r-SAK) is a third generation thrombolytic agent produced by genetic
engineering[1,2], and it has not yet been introduced to
the market both in China and abroad. The r-SAK of interest in this research was created by the Shanghai Institute of Botany
and Biophysiology (Shanghai, China) and was certified as the type I national new drug. This study aims to investigate the
thrombolytic efficacy, ideal dosage and administration route of native
r-SAK in animal experiments, which may offer basic evidence for clinical application.
Materials and methods
Study subjects Forty New Zealand male rabbits, 2.0 to
2.5 kg, were randomly assigned into 8 groups of 5 rabbits each: control, r-SAK low-dose group, medial-dose group, high-dose
group, r-SAK single bolus group, allied therapy group (r-SAK+heparin), r-SK group, and the UK group.
Thrombolytic agents r-SAK freeze-dry powder was produced by the Shanghai Institute of Botany and Biophysio-logy
(China) and Chengdu Jingpeng Bioengineering Co Ltd (Chengdu, Sichuan, China; batch
No 980601); 2.6 mg/ramus (6×104
U/mg); r-SK asepsis freeze-dry powder was produced by the Shanghai Shiyeyida Biotechnology Co Ltd (Shanghai, China;
batch No 990702; 150×104 U/ramus); UK freeze-dry powder was produced by the Medical Company Ltd (Nanjing, Jiangsu,
China) affiliated to Nanjing University (batch
No 20000704-2; 10×104 U/ramus).
Establishment of rabbit right femoral artery thrombosis
model[3] The rabbit was first anesthetized with 3% pentobarbital
sodium (40 mg/kg) and then fixed onto the operation table. The left femoral vein and bilateral femoral arteries were isolated
and a catheter was placed into the left femoral vein for collection of blood sample. Two other catheters were placed in the
femoral arteries at the distal end and then connected to a pressure monitor to record the baseline pressures of the femoral
arteries. Afterwards, the catheter in the right femoral artery was removed and a balloon of (2.0_2.5 mm)×20.0 mm was inserted
into the proximal part of the femoral artery. The balloon was then dilated with
8-12 atm and pushed and pulled 3 times to injure the endothelium of the femoral artery. Finally, the balloon was removed and a catheter was inserted once again for
dynamically monitoring the pressure.
Thrombolytic therapy The drugs were infused through the parallel-ear vein via a pump 2 h post balloon injury as follows:
the control group was given normal sodium (NS) 10 mL iv for 30 min; the r-SAK low-dose group was given
r-SAK 0.25 mg/kg iv for 30 min; the r-SAK medial-dose group was given r-SAK at 0.5 mg/kg iv for 30 min; the r-SAK high-dose
group was given r-SAK 1.0 mg/kg iv for 30 min; the r-SAK single bolus group was given r-SAK 0.5
mg/kg iv for 2 min; the allied therapy group was given 200 U/kg heparin, followed by an intravenous infusion of r-SAK 0.5 mg/kg for 30 min, and a
subsequent infusion of heparin 50
U·kg_1·h_1 for 4.5 h; the r-SK group were given r-SK
3.0×104 U/kg iv for 30 min; and the UK
group were given UK 3.0×104 U/kg iv for 30 min. The blood pressures in the rabbits' femoral arteries were monitored
consistently during the experiment for 5 h in order to evaluate the patency status of the femoral arteries described by
Rebello[4].
Femoral artery angiogram Some of the rabbits received femoral artery angiogram. The selected rabbits underwent skin
preparation, sterilization and local anesthesia. The right common carotid artery of the rabbit was then isolated, and a catheter
was carefully introduced into the lower part of the abdominal aorta through the right common carotid artery and aortic arch.
The angiogram of the bilateral femoral arteries was produced by digital subtraction angiography with a contrast medium of
iopromide.
Pathological examination The balloon-injured segment of the femoral arteries was dissected 5 h subsequent to
throm-bolysis. The segments were retained in 10% formaldehyde solution, imbedded in paraffin, sliced and stained by
hemagglutinin-esterase. The pathological results were acquired under microscope.
Statistical analysis Data were expressed as mean±SD. Student's
t-test was used to evaluate the changes of pressures
pre and post thrombolysis and the quantified variables of patency status in the different groups. Four fold table exact
probability ratio was used to compare the recanalization rates in the different groups. Kruskall-Wallis test was used to
analyze the nonparametric variables of the patency status post thrombolysis in each group.
Results
Changes of the pressure of left femoral
artery There was no significant change of the pressure of the left femoral artery
in each group after balloon injury or in the control group after NS infusion. The diastolic and mean pressures significantly
decreased 75 min and 60 min post thrombolysis respectively in the r-SAK low-dose group
(P<0.05). However, the systolic and pulse pressures did not change. In the
r-SAK medial-dose group, the diastolic and mean pressures significantly decreased 45 min post thrombolysis
(P<0.05) and the systolic pressure significantly decreased 150 min post thrombolysis
(P<0.05), while the mean pressure did not change. In
the r-SAK high-dose group, the diastolic and mean pressures significantly decreased 30 min and 45 min post thrombolysis
respectively, and the systolic pressure significantly decreased 75 min post thrombolysis, while the pulse pressure did not
change. In the r-SAK single bolus group, the systolic, diastolic and mean pressures significantly decreased 45, 70, and 30 min
post thrombolysis respectively (P<0.05), and the pulse pressure did not change. In the r-SAK allied therapy group, the
systolic pressure significantly decreased 120 min post thrombolysis
(P<0.05), while the diastolic, mean and pulse pressures
did not change. There was no significant change of the pressure of the left femoral artery in the r-SK group post thrombolysis.
The diastolic and mean pressures significantly decreased 150 and 210 min respectively post thrombolysis in the UK group
(P<0.05), while the systolic and pulse pressures did not change.
Changes of the pressure of right femoral artery
The pulse, systolic, and mean pressures significantly decreased after
balloon injury in the control group, while the diastolic pressure first increased and then decreased. The pulse pressure
decreased to 1.0±0.7 mmHg 120 min post balloon injury in the control group, which was lower than 10% of the pulse pressure
of the left femoral artery. There was no evidence of
recovery after NS infusion. The pressures of the right
femoral arteries in the study groups followed the same tendency of changes as in the control after balloon injury. However, some study groups
displayed a peak summit in the pressure-time curve post thrombolysis with the pulse pressures reaching more than 50% of
the left femoral arteries in some rabbits. The numbers are: 2 in the r-SAK low-dose group, 5 in the medial-dose group, 5 in the
high-dose group, 4 in the single bolus group, 5 in the allied therapy group, and 0 in both the UK and r-SK groups. The pulse
pressures of these arteries all decreased to less than 50% of the left femoral arteries 5 h post thrombolysis (Figure 1).
The recanalized femoral arteries after thrombolytic
therapy The following is the artery reopening rate in each group: 0
in the control group, 2 in the r-SAK low-dose group, 5 in the medial-dose group, 5 in the high-dose, 4 in the single bolus, 5 in
the allied therapy group, and 0 in both the UK and r-SK groups. The reopening rates in the r-SAK medial and high-dose
groups (P<0.01) were significantly higher than that of the control, and there was a tendency of increasing of the reopening
rate as the dosage of r-SAK given increased. The reopening rate reached 100% when the dosage exceeded 0.5 mg/kg. There
was no statistical difference concerning the reopening rate, reopening time and reperfusion time among the different r-SAK
dosage groups and the same dosage groups with different administration manners. The reperfusion time in the r-SAK single
bolus group and the reopening time in the allied therapy group had a tendency to be shorter. The reopening rate was
significantly higher in the r-SAK group than in both the r-SK and UK groups
administered with the corresponding dosage. All the
recanalized arteries reoccluded at the end of the study (Table
1; Figure 2).
Patency status of right femoral arteries post
thrombolysis The patency status of the right femoral arteries were
classified quantitatively according to pulse pressures in reference to studies published by
Rebello[4] and Collen[5]. Statistical
analysis showed that the patency status in the different r-SAK dosage groups had no differences during each time period,
but the ranks in both the r-SAK medial and high-dose groups were higher than that in the low dose group (all
P<0.01), and there was no significant difference between the medial and high-dose groups
(P=0.053). There were also no differences in the
patency status of the right femoral arteries among the groups which received the same dosage of r-SAK, but administered in
different manners, while the ranks of the patency status during each period were significantly higher in the r-SAK infusion
and allied therapy groups than that in the single bolus group
(P<0.01 and P<0.05, respectively), and there was no difference
between the
r-SAK infusion group and the allied therapy group
(P=
0.60; Table 2).
Angiogram of the femoral artery
An angiogram showed that the rabbits' femoral arteries were undamaged with fluent
blood flow originally. The right femoral arteries were totally occluded within 120 min after balloon injury which indicated that
occlusive thrombus formed at the site. Some arteries were reopened after r-SAK thrombolysis, but all the reopened arteries
were reoccluded at the end of the experiment (Figure 3).
Pathology of the artery A pathological test showed that the femoral arteries were totally occluded 120 min after balloon
injury (Figure 4A), and some of the arteries were reopened after r-SAK thrombolysis (Figure 4B). A pathological slice from the
unopened artery in the single bolus group showed that the undissolved thrombus was enriched with degenerated platelets
and neutrophils (Figure 4C). The slices from the UK and r-SK groups after thrombolysis showed that the lumen was filled
with degenerated platelets, neutrophils and erythrocytes, which formed the mixed thrombus (Figure 4D). All the recanalized
arteries reoccluded 5 h post thrombolysis and the re-formed thrombus contained mainly degenerated platelets and
neutrophils (Figure 4E, 4F).
Discussion
r-SAK is a new type, third generation thrombolytic agent, which was first produced by genetic engineering in
1985[1,2].
Some studies showed that r-SAK induced early, complete, and sustained coronary artery patency at least as frequently as the
accelerated recombinant tissue type plasminogen activator without associated fibrinogen
degradation[6,7]. Native r-SAK was created by the Shanghai Institute of Botany and Biophysiology (China) in 1990, which is a molecular cloning of the SAK gene
in Escherichia coli, and is highly purified and crystallized in white color freeze-dry powder with biological characteristics
very similar to natural SAK[8]. It has recently been certified as the type I national new drug. Yet its efficacy has not been fully
evaluated both experimentally and clinically. Therefore, this study aims to investigate the thrombolytic efficacy, ideal dosage
and administration of native r-SAK through animal experimentation, which may offer basic evidence for clinical use.
The purposes of observing the pressure of the left femoral artery are: (1) to act as a control for the pressure of the right
femoral artery; and (2) to observe the effect of native
r-SAK on systemic blood pressure. As a result, the pressure of the left femoral artery in each r-SAK dosage group decreased
significantly. This may be due to the following factors: (1) capillary hemorrhage occurred in the field surrounding the
procedure post thrombolysis, which may lead to hypovolemia and decreased blood pressure; (2) many blood samples were
collected for tests and there was a lack of transfusion and nourishment given to the subjects; and (3) native r-SAK caused the
decrease of the blood pressure directly. The decrease of blood pressure did not threaten the lives of the animals and all the
rabbits were kept alive until the end of each experiment.
The pressure of the right femoral artery in each group decreased significantly post balloon injury and the pulse pressure
decreased to 0 or less than 10% of the left femoral artery. This indicated that the thrombus had formed and the lumen of the
artery was occluded[4]. The pulse pressures of the right femoral arteries did not change in the control group after NS infusion,
which indicated that the arteries were still occluded. However, the pulse pressures returned to the baseline status or more
than 50% of the left femoral arteries in some of the animals in the study groups, and the systolic, diastolic and mean pressures
were all enhanced significantly, which indicated that the thrombus was dissolved and the arteries reopened. There was no
difference among the different r-SAK dosage groups concerning the reopening rate, the time to reopening and the reperfusion
time. The recanalization rate had a tendency to increase as the r-SAK dosage increased, and it reached 100% as the dosage
administered exceeded 0.5 mg/kg. The patency status of the recanalized arteries in the different dosage groups had no
difference during each period, but the ranks in both the r-SAK medial and high-dose groups were higher than that in the
low-dose group (all P<0.01), which indicated that the patency status may improve as the dosage given increased. The
recanalization rate reached 80% in the r-SAK single bolus group, which was significantly higher than that of the control. However,
concerning the time to reopening, the reopening rate, the reperfusion time and the patency status post thrombolysis, the
administration manner of single bolus did not show any superiority to infusion. The reopening rate reached 100% and the
time to reopening displayed a tendency to be shorter in the r-SAK allied therapy group, in addition, the reperfusion time had
no difference compared with the other groups, which may be due to a smaller sample size of subjects.
This study showed that the reopening rate of r-SAK was significantly higher than that of r-SK and UK when administered
at the same dosage. After thrombolysis by r-SK, the systolic, diastolic and mean pressures of the right femoral arteries
temporarily increased, but the pulse pressure did not change. This indicated that the thrombus might be partially dissolved,
but the arteries did not reopen in the end due to weaker potency of r-SK. The pressures of the right femoral arteries did not
change post thrombolysis in the UK group, which indicated that the thrombolytic potency of UK might be even less than that
of r-SK.
A specific phenomenon of the pressure waves was found in this experiment, which showed that the pulse pressure of the
right femoral arteries changed in a fluctuant manner (Figure 1). This phenomenon may be due to the creation of a local
hypercoagulable state produced by the release of various procoagulate factors of the thrombus dissolution. This may
contribute to the reformation of the thrombus and lead to the deterioration of blood flow. Thrombus formation and
dissolution is inversely related and one may increase as the other decreases. This can contribute to the constant alteration of the
lumen of the artery and account for the fluctuant manner of the pulse pressure. The pulse pressures of right femoral arteries
in all the reopened vessels decreased to 0 or less than 50% of the left femoral arteries 5 h post thrombolysis, which indicated
that the thrombus reformed in the lumens and the arteries became stenotic or reoccluded. This may be due to following
reasons: (1) the balloon created a deep injury of the femoral artery, which increased the likelihood of thrombus
formation[9]; (2) the catheter blocked the distal part of the artery, and this might have promoted thrombus formation; and (3) anti-platelet
drugs were not used in the experiment.
Pathological testing of the reformed thrombus showed that it was enriched with degraded platelets and neutrophils,
which indicated that the platelet played a very important role in the mechanism of thrombus reformation. Similar to other fibrin
select thrombolytic agents, r-SAK allied therapy with anti-platelet and anticoagulant drugs may be a necessary way for
avoiding thrombus reformation post thrombolysis.
In conclusion, native r-SAK has a definite thrombolytic effect on the femoral artery thrombus of rabbit. The recanalization
rate reached 100% as the dosage amount exceeding
0.5 mg/kg. Single bolus r-SAK is an effective administration manner, which may be used in emergent status such as
pre-hospital thrombolysis. r-SAK allied therapy with the addition of heparin may improve the thrombolytic results in terms of
shortening the time to recanalization. The thrombolytic efficacy of native r-SAK is superior to that of r-SK and UK when
administered at the same dosage.
Acknowledgements
We thank Mr Guo-ping YANG (Animal Experiment Center, The First Affiliated Hospital of Nanjing Medical University,
Nanjing, China) for his assistance in animal experiment and Prof Zheng-qing FENG (Department of Pathology, Nanjing
Medical University, Nanjing, China) for his assistance of pathological examination.
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