Oral fibrinogen-depleting agent lumbrokinase for secondary ischemic stroke prevention: Results from a multicenter, randomized, parallelgroup and controlled clinical trial

Abstract

Elevated fibrinogen (Fg) level is a known risk factor for ischemic stroke. There are few clinical trials on oral fibrinogen-depleting therapies for secondary ischemic stroke prevention. We aimed to assess the effects of one-year therapy with oral lumbrokinase enteric-coated capsules on secondary ischemic stroke prevention.
This is a multicenter, randomized, parallel group and controlled study that began treatment in hospitalized patients with ischemic stroke and continued for 12 months. Patients were randomized to either the control group that received the standard stroke treatment or the fibrinogen-depleting group that received the standard stroke treatment plus enteric-coated lumbrokinase capsules. The NIH Stroke Scale scores (NIHSSs) and plasma Fg level were recorded. The carotid artery intima-media thickness (IMT) and status of plaques were examined through carotid ultrasound examination. Primary outcomes included all-cause mortality, any event of recurrent ischemic stroke/transient ischemic attack (TIA), hemorrhagic stroke, myocardial infarction and angina, and other noncerebral ischemia or hemorrhage. Kaplan-Meier survival analysis and the Long-rank test were used to compare total vascular end point incidence between the two groups. Comparison of median values between two groups was done by the Student t test, one-way analysis of variance (ANOVA), or non-parametric rank sum test.
A total of 310 patients were enrolled, 192 patients in the treatment group and 118 patients in the control group. Compared to the control group, the treatment group showed favorable outcomes in the Fg level, carotid IMT, the detection rate of vulnerable plaques, the volume of carotid plaques, NIHSS scores, and incidence of total vascular (6.78% and 2.08%, respectively) and cerebral vascular events (5.93% and 1.04%, respectively) (P < 0.05). In the treatment group, the volume of carotid plaques was significantly related to the carotid IMT, the plaque diameter, width and number (P = 0.000, 0.000, 0.000, 0.022; F = 13.51, 2.52, 11.33, -3.29, but there was a weak correlation with the Fg level (P = 0.056). After 1-year therapy, the incidence of overall vascular end points was reduced by 4.7%.
Long-term oral fibrinogen-depleting therapy may be beneficial for secondary ischemic stroke prevention.

Full text

Chin Med J 2013;126 (21)
4060
DOI: 10.3760/cma.j.issn.0366-6999.20131332
Department of Neurology, Second Affiliated Hospital of Soochow
University, Suzhou, Jiangsu 215004, China (Cao YJ, Zhang X and
Liu CF)
Department of Neurology, Kunshan No.1 People’s Hospital,
Kunshan, Jiangsu 215300, China (Wang WH)
Department of Neurology, Taicang No.1 People’s Hospital, Taicang,
Jiangsu 215400, China (Zhai WQ and Li RX)
Department of Neurology, Zhangjiagang Traditional Chinese
Medicine Hospital, Zhangjiagang, Jiangsu 215600, China (Qian JF)
Department of Neurology, Changshu No.1 People’s Hospital,
Changshu, Jiangsu 215500, China (Wang JS)
Department of Neurology, Wuxi Traditional Chinese Medicine
Hospital, Wuxi, Jiangsu 214000, China (Chen J)
Department of Neurology, Changshu No.2 People’s Hospital,
Changshu, Jiangsu 215500, China (You NX)
Department of Neurology, Suzhou Municipal Hospital, Suzhou,
Jiangsu 215000, China (Zhao Z)
Department of Neurology, Zhangjiagang No.1 People’s Hospital,
Zhangjiagang, Jiangsu 215600, China (Wu QY)
Department of Neurology, Wujiang No.1 People’s Hospital, Wujiang,
Jiangsu 215200, China (Xu Y)
Department of Neurology, Taicang Traditional Chinese Medicine
Hospital, Taicang, Jiangsu 215400, China (Yuan L)
Correspondence to: Pro. LIU Chun-feng, Department of Neurology,
Second Afliated Hospital of Soochow University, Suzhou, Jiangsu
215004, China (Tel: 86-512-67783307. Fax: 86-512-68284303.
Email: liucf20@hotmail.com)
Conict of interest: none.
Original article
Oral brinogen-depleting agent lumbrokinase for secondary ischemic
stroke prevention: results from a multicenter, randomized, parallel-
group and controlled clinical trial
CAO Yong-jun, ZHANG Xia, WANG Wan-hua, ZHAI Wan-qing, QIAN Ju-fen, WANG Jian-sheng, CHEN Jun,
YOU Nian-xing, ZHAO Zhong, WU Qiu-yi, XU Yuan, YUAN Lei, LI Rui-xia and LIU Chun-feng
Keywords: ischemic stroke; lumbrokinase enteric-coated capsules; brinogen; carotid atherosclerosis
Background Elevated brinogen (Fg) level is a known risk factor for ischemic stroke. There are few clinical trials on oral
fibrinogen-depleting therapies for secondary ischemic stroke prevention. We aimed to assess the effects of one-year
therapy with oral lumbrokinase enteric-coated capsules on secondary ischemic stroke prevention.
Methods This is a multicenter, randomized, parallel group and controlled study that began treatment in hospitalized
patients with ischemic stroke and continued for 12 months. Patients were randomized to either the control group that
received the standard stroke treatment or the brinogen-depleting group that received the standard stroke treatment plus
enteric-coated lumbrokinase capsules. The NIH Stroke Scale scores (NIHSSs) and plasma Fg level were recorded. The
carotid artery intima-media thickness (IMT) and status of plaques were examined through carotid ultrasound examination.
Primary outcomes included all-cause mortality, any event of recurrent ischemic stroke/transient ischemic attack (TIA),
hemorrhagic stroke, myocardial infarction and angina, and other noncerebral ischemia or hemorrhage. Kaplan-Meier
survival analysis and the Long-rank test were used to compare total vascular end point incidence between the two groups.
Comparison of median values between two groups was done by the Student t test, one-way analysis of variance (ANOVA),
or non-parametric rank sum test.
Results A total of 310 patients were enrolled, 192 patients in the treatment group and 118 patients in the control group.
Compared to the control group, the treatment group showed favorable outcomes in the Fg level, carotid IMT, the detection
rate of vulnerable plaques, the volume of carotid plaques, NIHSS scores, and incidence of total vascular (6.78% and
2.08%, respectively) and cerebral vascular events (5.93% and 1.04%, respectively) (P <0.05). In the treatment group, the
volume of carotid plaques was signicantly related to the carotid IMT, the plaque diameter, width and number (P=0.000,
0.000, 0.000, 0.022; F=13.51, 2.52, 11.33, –3.29, but there was a weak correlation with the Fg level (P=0.056). After 1-year
therapy, the incidence of overall vascular end points was
reduced by 4.7%.
Conclusion L on g- te rm oral fib rinogen-dep le ti ng
therapy may be benecial for secondary ischemic stroke
prevention.
Chin Med J 2013;126 (21): 4060-4065
Elevated fibrinogen level is a known risk factor for
stroke.1 It was also suggested that higher fibrinogen
level measured in patients within 6 hours of stroke onset
was associated with poor functional outcome.2 However,
venous fibrinogen-depleting therapy failed to show any
benefit.3,4 There are few clinical trials on oral fibrinogen-
depleting therapy to treat ischemic stroke. Lumbrokinase
is an effective enzyme extracted by a method modified
from a Chinese traditional herb by Mihara in 1983. It has
been marketed in China for more than 10 years mainly
for the treatment of acute phase ischemic stroke. One
study showed good functional outcome in patients with
acute ischemic stroke when treated with lumbrokinase for
21days.5 Our study was to observe the efcacy of one-year
oral lumbrokinase enteric-coated capsules therapy for its
effectiveness on secondary ischemic stroke prevention.
METHODS
Lumbrokinase was provided by the Institute of Biophysics,

Chinese Medical Journal 2013;126 (21) 4061
Chinese Academy of Sciences, Beijing, China. From
May 2007 to June 2009, the second Afliated Hospital of
Soochow University and other 10 middle-sized hospitals in
the southern Jiangsu Province conducted this multicenter
clinical trial to explore the efficacy of long-term oral
lumbrokinase for secondary ischemic stroke prevention.
Study characteristics and patient samples
Patients diagnosed with anterior circulation ischemic
stroke or transient ischemic attack (TIA) within 6 months
from the 11 participating hospitals were recruited into the
study. Noncomatose subjects were eligible if they were
between 40 and 80 years of age and had carotid circulation
ischemic stroke conrmed by brain CT or MRI within 72
hours of symptom onset and the level of brinogen (Fg) is
higher than 2.0 g/L. Carotid TIA patients were diagnosed
when they complained with transient hemiparalysis,
unilateral sensory disturbance, aphasia or monocular visual
impairment lasting less than 24 hours. The research protocol
was approved by the local Ethics Committee of Soochow
University and all patients or family members signed
informed consents. Patients with the following conditions
were excluded: cardiogenic embolism and hemorrhagic
transformation, on or need to be on anticoagulant therapy,
severe hepatic, renal, hematopoietic and endocrine diseases,
allergy to lumbrokinase, planning to have a major operation
or carotid angioplasty, and pregnancy.
This was a multicenter, parallel group, open label,
controlled trial with 2:1 simple randomization by random
number table. Patients assigned with odd numbers were
included in the treatment group and even numbers in the
control group. Patients in the treatment group were given
oral enteric-coated lumbrokinase capsules (600 000 units
a time, three times a day, 30 minutes before meals) for
one year. The control group was given capsules without
lumbrokinase for one year. Both groups received standard
stroke treatment otherwise. All patients had monthly
followed-up by telephone and visits at the hospitals 6
and 12 months post discharge. At the two follow-up
visits, data on patient’s current medication, any disease
history, the NIH Stroke Scale scores (NIHSSs), carotid
ultrasonography, blood biochemical and coagulation tests,
blood and plasma viscosity, the incidence of vascular
endpoint events were collected.
End points and denitions
The primary endpoints included all-cause mortality, any
event of recurrent ischemic stroke/TIA, hemorrhagic
stroke, myocardial infarction and angina, and other
noncerebral ischemia or hemorrhage. Other noncerebral
ischemia or hemorrhage means hemorrhagic and ischemic
events of other organs excluding brain, which was dened
as a primary endpoint. In case of a suspected recurrent
cerebrovascular or cardiovascular event, conrmation was
sought from the treating doctor or hospital. If a patient had
a noncerebral ischemia or hemorrhagic event, the treating
doctor or hospital would need to confirm the diagnosis.
When a patient died during the following-up period, the
cause of death was recorded according to the medical
records and death certificate. Safety end points included
mortality, intracranial hemorrhage, major bleeding, and
review of other physical and laboratory measurements. The
safety committee received reports of all deaths and serious
adverse events as planned for review.
Carotid ultrasound examination
Ultrasonography was performed by using a GEVivid 7D
(GE Company, USA) system with a 7.5-MHz transducer,
including carotid intima-media thickness (IMT), diameter,
the number and distribution of plaques of both carotid
arteries. All examinations were performed by one trained
radiologist in each hospital who had no knowledge of the
clinical history and prole of the subjects. The IMT of the
distal wall of the carotid artery was measured at 2.0, 2.5
and 3.0 cm proximal to the carotid bifurcation in each of
the right and left common carotid arteries. Measurements
were made on longitudinal scans obtained in the anterior
oblique, lateral and posterior oblique views. The IMT
was defined as the distance between two echogenic lines
separated by a hypoechoic or anechoic space, with the
outer line corresponding to the media-adventitia border
and the inner line representing the lumen-intima border.
The mean IMT was calculated as the average value of the
IMT measurements for the six sites in the carotid arteries.
Plaque demonstrates a thickness of IMT >1.2 mm.6 The
vulnerable plaques were defined to show hypoechoic, or
both hypoechoic and hyperechoic signals. Those showing
hyperechoic with a smooth surface were dened as stable
plaques.7-9 The plaque volume was calculated as “the
average plaque diameter × the average plaque width ×
IMT”.
Statistical analysis
SPSS 13.0 software (IBM Company, USA) was used for
statistical analysis. Sample size was calculated based on
the incidence of vascular events of 3% in the trial group
and 12% in the placebo group, with a probability of type
I error of 0.05 and type II error of 0.10. Results were
expressed as a percentage or mean ± standard deviation
(SD). Differences in frequencies were compared using the
chi-square test. Comparison of median values between two
groups was done by the Student’s t test, one-way analysis
of variance (ANOVA), or non-parametric rank sum test.
Forward stepwise multiple linear regression analysis was
performed to detect factors that influenced the plaque
volume. Comparison of total vascular end point incidence
between the two groups was evaluated by Kaplan-Meier
survival analysis and the Long-rank test. All statistical
analyses were 2-tailed. A P value <0.05 was considered
statistically signicant in all analyses.
RESULTS
A total of 310 patients were enrolled: 192 patients in the
treatment group and 118 patients in the control group. All
were included in the analysis. About 88.0% patients in the
treatment group and 89.0% in the placebo group completed

Chin Med J 2013;126 (21)
4062
the therapy (Figure 1). Their average age was (67.10±9.27)
years. There was no statistical difference in age, sex, blood
pressure, combined medications, and past history between
two groups (P >0.05). The usage rate of antihypertensive,
antiplatelet and statin drugs of both groups had no signicant
difference (Table 1).
Fg Changes (Table 2)
The baseline Fg levels of the treatment and control group
were (3.47±1.07) g/L, (3.53±1.19) g/L, respectively.
After 1 year, in the treatment group, Fg level decreased
to (2.68±0.95) g/L but unchanged in control group
((3.56±0.86) g/L, P <0.05); CRP level decreased
(P=0.013); D-dimer level signicantly deceased (P=0.013);
t-PA activity was elevated (P=0.018); blood and plasma
viscosity apparently improved (both P <0.05). In both
groups, the content of PT and APTT were unchanged (P
>0.05).
Plaque number and echo intensity changes
Baseline plaque number of both carotid arteries in
treatment group was 1.12±0.66 and 1.08±0.89 per patient
and in control group 0.93±0.45 and 1.06±0.70, with no
significant difference. After 1 year of therapy, there was
a significant reduction of the plaque number in treatment
group (P=0.043) but a significant elevation in the control
group (P=0.023).
There were mainly hyperechoic plaques in both groups.
In the treatment group, the baseline vulnerable plaque
rates consisting of hypoechoic and mixed echo plaques
were 28% in the left side and 17.50% in the right side.
After 1 year, the rates were reduced to 7.90% and 9.50%,
respectively (left side, χ2=16.49, P <0.001; right side,
χ2=3.05, P=0.080). Detection rate of vulnerable plaques
(%) = (number of low echo plaques + number of mix-echo
plaques) × 100% / total number of echo plaques.
IMT and plaque volume changes
In treatment group, bilateral carotid IMT was reduced
significantly after 1 year (left side, (1.26±0.49) mm vs.
(1.09±0.40) mm, right side (1.25±0.51) mm vs. (1.06±0.35)
mm, both sides P <0.001). In control group, the IMT
thickness increased after 1 year (right side, (1.40±0.79) mm
vs. (1.59±0.69) mm, P=0.055), especially on the left side
((1.38±0.52) mm vs. (1.62±0.56) mm, P=0.001).
There was no signicant difference in the baseline plaque
volume between two groups. After 1 year of therapy, the
carotid plaque volume reduced, falling by 41.84% in the
left side and 51.30% in the right side (both sides P=0.02) in
the treatment group. However, the carotid plaque volume
increased by 103.38% in the left side and 80.04% in the
right side in the control group (left side P=0.008, right side
P=0.038).
Multiple linear regression analysis of the carotid plaque
volume
Forward stepwise multiple linear regression analysis was
performed to assess the factors influencing the carotid
Table 1. Baseline characteristics of the study groups
Variables Treatment (n=192) Control (n=118)
Gender (n (%))
Male 143 (74) 74 (63)
Female 49 (26) 44 (37)
Age (years) 66.61±8.87 67.89±9.88
Blood pressure (mmHg)
Systolic blood pressure 148.97±23.73 148.14±22.71
Diastolic blood pressure 85.68±12.91 89.88±16.06
Concomitant medications (n (%))
Baseline usage of antiplatelet drugs 154 (80.21) 98 (83.05)
Baseline usage of statin drugs 56 (29.17) 38 (33.20)
Baseline usage of antihypertensive drugs 56 (29.17) 37 (31.36)
1-year usage of antiplatelet drugs 104 (54.17) 62 (52.54)
1-year usage of statin drugs 41 (21.35) 24 (20.34)
1-year usage of antihypertensive drugs 53 (27.60) 36 (30.51)
Past history (n (%))
None 58 (30.21) 29 (24.58)
Hypertension 86 (44.79) 62 (52.54)
Diabetes mellitus 8 (4.17) 4 (3.39)
Hyperlipidemia 1 (0.52) 2 (1.70)
Hypertension+diabetes 26 (13.54) 20 (16.95)
Hypertension+hyperlipidemia 4 (2.08) 1 (0.85)
Hypertension+CHD 2 (1.04) 4 (3.39)
Hypertension+diabetes+CHD 2 (1.04) 1 (0.85)
There was no statistically significant difference in age, sex, blood pressure,
combined medications and past history between two groups (P >0.05). CHD:
coronary heart disease.
Table 2. Blood indexes
Items Treatment group Control group
Baseline 1 year Baseline 1 year
Fg (g/L) 3.47±1.07 2.68±0.95 3.53±1.19 3.56±0.86
CRP (mg/L ) 5.91±1.33 3.22±1.67 5.13±1.62 3.84±1.59
t-PA (IU/ml) 116.21±10.68 121.37±13.34 118.76±14.10 105.85±4.36
PAI-1 (AU/ml) 83.26±9.48 81.67±6.74 75.55±8.24 72.48±10.11
D-Dimer (mg/L) 0.82±0.30 0.45±0.25 0.37±0.28 0.49±0.29
PT (seconds) 12.12±1.62 12.32±1.84 12.30±1.60 12.23±1.41
APTT (seconds) 30.04±5.76 30.30±5.12 30.45±4.66 30.73±4.32
INR 1.01±0.24 1.09±0.25 1.03±0.18 1.02±0.31
Blood viscosity
(low cut)
13.50±10.06 9.97±1.75 11.80±6.16 10.96±1.53
Blood viscosity
(medium cut)
9.48±8.36 5.97±0.97 8.70±5.89 6.73±0.90
Blood viscosity
(high cut)
7.49±6.22 4.49±0.80 6.78±4.84 5.23±0.67
Plasma viscosity
(mPa/s)
3.69±2.01 1.46±0.23 4.24±3.67 1.67±0.22
Figure 1. Trial profile. Missing doses indicates patients who
have missed more than 100 doses in total or 30 consecutive
doses. Medical problem indicates patients with other concurrent
diseases not suitable for continuation during the follow-up
period.

Chinese Medical Journal 2013;126 (21) 4063
plaque volume. NIHSS scores, Fg level, carotid IMT
and diameter, the plaque number, plaque diameter and
width were regarded as independent variables. Fg level,
carotid IMT, the plaque number, the plaque diameter and
width were entered into the regression equation (R2=0.85;
F=51.88; P=0.000). Carotid IMT, the plaque diameter, the
plaque width and the plaque number showed a signicant
association with average carotid plaque volume (t=4.25,
7.18, 10.63, –2.36; P=0.000, 0.000, 0.000, 0.022; F=13.51,
2.52, 11.33, –3.29). However, the Fg level was found to
be weakly associated with average carotid plaque volume
(t=1.95, P=0.056, F=3.53).
NIHSS scores (Table 3) and the incidence of vascular
end-point events (Table 4)
After 6 months of therapy, NIHSS scores were signicantly
improved in both groups (both P <0.001). After a year,
there was a further reduction of NIHSS scores in both
groups. Compared to control group, NIHSS scores in the
treatment group showed a significant reduction after one
year (both P <0.001).
At the 12-month follow-up, in the control group, 7
patients (5.93%) had cerebrovascular events: 2 TIA, 4
cerebral infarction, and 1 cerebral hemorrhage. There
was also 1 other organ ischemia event in the control
group. In the treatment group there were 2 (1.04%)
cerebrovascular events (1 TIA and 1 cerebral infaction),
1 angina pectoris, and 1 lower gastrointestinal bleeding.
There were signicant differences in total vascular events
and cerebrovascular events between two groups (χ2=3.97,
P=0.046; χ2=5.79, P=0.016). Kaplan-Meier survival
analysis showed a significant reduction of total vascular
events, falling by 4.7% in treatment group (Log-rank test,
χ2=4.33, P=0.038, Figure 2).
Safety assessment
There were 5 patients with adverse events in treatment
group (excluding vascular endpoints), 7 patients in the
control group. Three and two patients complained with
dizziness in the treatment and control group, respectively.
Three and four patients complained with nauseating and
vomiting in each group. All patients were not treated and
continued the treatment, with disappearance of symptoms
themselves. There was no significant difference in the
adverse event incidence between two groups (χ2=1.998,
P=0.157).
DISCUSSION
Plasma fibrinogen level is strongly associated with risks
of developing CHD, stroke, vascular mortality, and
nonvascular mortality in healthy middle-aged adults. The
age- and sex- adjusted hazard ratio per 1-g/L increase in
usual fibrinogen level for all the events is around 2.0.1
Our study has provided new information on the use of
fibrinogen-depleting therapy for ischemic stroke. The
present trial is a relatively early study to demonstrate the
benefit of long-term oral fibrinogen-depleting therapy on
the secondary prevention of ischemic stroke.
Lumbrokinase is extracted from Lumbricus rubellus,
which is heat-stable and displays a very broad optimal
pH range. It has six fractions and acts as plasminogen
activator. It dissolves brin clot by converting plasminogen
to plasmin.10 It has selective affinity for Fg and directly
hydrolyzes Fg to soluble brinogen degradation products.
Lumbrokinase can also reduce platelet aggregation rate
and ameliorate blood and plasma viscosity.11,12 t-PA and
PAI are both key enzymes of Fibrinolytic system. T-PA is
an activator, however, PAI is an inhibitor for fibrinolysis
through integration with t-PA. Decrease of ratio of t-PA/
PAI increases the risk of ischemic stroke.13,14 Our study
showed that after treatment, the activity of t-PA was
significantly increased, which supported the findings
in the animal study.15 Therefore, lumbrokinase has
indirectly increased t-PA activity and directly stimulated
vascular endothelial cells to secrete t-PA. However, PAI-1
(Plasminogen activator inhibitor-1) did not change. A high
serum Fg concentration produces a high hypercoagulable
and hyperviscosity state. Our study showed that Fg
concentration was decreased but not too low after 1 year
treatment (Fg level >0.7 g/L after 1 year). Meanwhile,
Table 4. Vascular endpoints (n (%))
Vascular events Treatment group Control group χ2 values P values
Cerebrovascular events 2 (1.04) 7 (5.93) 5.79 0.016
TIA 1 (0.52) 2 (1.69) 1.03 0.311
Cerebral infarction 1 (0.52) 4 (3.39) 3.45 0.056
Cerebral hemorrhage 0 1 (0.85) 1.62 0.203
Cardiovascular events
Angina pectoris 1 (0.52) 0 1.62 0.203
Myocardial infarct 0 0 - -
Other organ vascular events
Ischemia 0 1 (0.85) 1.62 0.203
Hemorrhage 1 (0.52) 0 1.62 0.203
Total vascular events 4 (2.08) 8 (6.78) 3.97 0.046
Table 3. The changes of NIHSS scores between two groups
NIHSS scores Treatment group Control group P values
NIHSS (baseline) 5.71±3.78*,‡ 6.18±5.49§,|| 0.369
NIHSS (a half year) 3.72±2.76*,† 3.78±3.63§0.178
NIHSS (one year) 2.35±2.16†,‡ 3.62±3.53 || <0.001
*,‡,§,||P <0.001, †P=0.001.
Figure 2. Kaplan-Meier survival analysis. Solid line = control
group; broken line = lumbrokinase group. There was a signicant
difference between the control group and lumbrokinase group (*P
<0.05 by the log-rank test).

Chin Med J 2013;126 (21)
4064
blood and plasma viscosity improved. Such changes may
have the benefit of stopping the progression of arterial
atherosclerosis. However, the content of PT and APTT did
not change, which means lumbrokinase does not affect
coagulation system. It may reduce the risk of hemorrhage.
There were no obvious adverse events in the treatment
group with any difference in adverse events incidence
compared to the control group. So it is a safe drug.
As a principal degradation fragments of fibrin, D-dimer
level is an important predictive factor for the diagnosis and
prognosis of arterial atherosclerosis. Our study showed that
the D-dimer level was decreased, which was different from
previous reports.5 This may be explained by the possible
ceiling effect of lumbrokinase activity. After reaching a
certain point, Fg level would not drop further. Consequently
Fg level was not too low after 12 months of treatment. The
other explanation is that Lumbrokinase may degrade both
brinogen and brin deposits, which leads to the decrease
of D-dimer level. CRP, a major inflammatory index, is
notably inhibited by lumbrokinase.
Fg is an independent risk factor for atherosclerosis and
related to the severity of disease.16 Our study showed that
debrinogenation therapy improved carotid atherosclerosis.
After 1 year of treatment, we found that in the treatment
group, IMT notably was thinned, average plaque number
and vulnerable plaque rate were decreased, and plaque
volume was significantly reduced. These benefits may
be related to the anti-platelet effect of Lumbrokinase
through the elevation of cAMP level and Ca2 + release.
Lumbrokinase can also inhibit ICAM-1 expression
that may have an antithrombotic effect. Furthermore,
lumbrokinase reduces cellular apoptosis activity through
activation of JAK1/STAT1 pathways.17 The key point
is that lumbrokinase can significantly reduce the Fg
level, which plays an important role in the progression
of atherosclerosis. There are several ways in which Fg
participates in atherosclerosis progression. Firstly, in vivo
Fg can be converted to fibrin and insoluble degradation
products, which deposit in the vascular wall and promote
the aggregation and adhesion of white blood cells to
accelerate inflammatory process of atherosclerosis.18
Secondly, increasing Fg level affects the endothelial
cell function to stimulate its synthesis and secretion of
plasminogen inactivators, which leads to delayed remove
of local microembolism. Endothelium can be injured
and cholesterol is easy to inltrate.19,20 Thirdly, Fg and its
degradation products stimulate smooth muscle cells and
can link to platelets to activate them.21 Fourthly, high Fg
level can alter the permeability of microphages cap to make
plaques vulnerable and thrombus formation. Fifthly, high
Fg level increases blood viscosity and substrates, which can
be risk factors of small vessel obstruction. Additionally, we
found that Fb, Fg, and FDPs are involved in the progression
of the instability of atherosclerotic plaques via increasing
the expression of MMPs and VEGF. This effect might be
mediated by the NF-κB pathway.22
Our study is a multicenter, randomized, parallel-group,
open label and controlled clinical trial, but not a double-
blinded study. For the practical reason, serum Fg, d-dimer
and t-PA levels were not checked on a schedule time
interval and therefore it was difficult to detect how and
when exactly these change of indexes took place.
Our trial has found that after 1 year of lumbrokinase
therapy, the incidence of overall vascular end points was
reduced by 4.7%. There is clearly a close relationship
between the plasma Fg level and atherosclerosis or stroke.23
And the trial may provide new evidences for clinical use of
brinogen-depleting therapy for the prevention of ischemic
stroke. However, more prospective randomized large
clinical trials are needed to provide better evidence of the
role of lumbrokinase in reducing ischemic strokes.
Acknowledgement: The authors are grateful to FENG Ping, the chief
of Department of Clinical Laboratories and PENG Chun-lei, the chief
of Blood Laboratory in the Second Afliated Hospital of Soochow
University for the help in detection of blood biochemical parameters.
We also thank Prof. David Wang, the Professor of Neurology
Department in the College of Medicine, at the University of Illinois
in the USA for the revision of the manuscript.
REFERENCES
1. Danesh J, Lewington S, Thompson SG, Lowe GD, Collins
R, Kostis JB, et al. Plasma brinogen level and the risk of major
cardiovascular diseases and nonvascular mortality: an individual
participant meta-analysis. JAMA 2005; 294: 1799-1809.
2. del Zoppo GJ, Levy DE, Wasiewski WW, Demchuk
AM, Trammel J, Demaerschalk BM, et al. Hyperbrinogenemia
and functional outcome from acute ischemic stroke. Stroke
2009; 40: 1687-1691.
3. Sherman DG, Atkinson RP, Chippendale T, Levin KA, Ng
K, Futrell N, et al. Intravenous ancrod for treatment of acute
ischemic stroke: the STAT study: a randomized controlled trial.
JAMA 2000; 283: 2395-2403.
4. Hennerici MG, Kay R, Bogousslavsky J, Lenzi GL, Verstraete
M, Orgogozo JM. Intravenous ancrod for acute ischaemic
stroke in the European Stroke Treatment with Ancrod Trial: a
randomised controlled trial. Lancet 2006; 368: 1871-1878.
5. Jin LR, Jin HM, Zhang GP, Xu G. Changes in coagulation and
tissue plasminogen activator after the treatment of cerebral
infarction with lumbrokinase. Clin Hemorheol Microcirc 2000;
23: 213-218.
6. Zhi G. Ultrasonic diagnostics of coronary atherosclerotic heart
disease, 1st ed. Beijing: The People’s Military Medical Press;
2001: 131-132.
7. Sawayama Y, Shimizu C, Maeda N, Tatsukawa M, Kinukawa
N, Koyanagi S, et al. Effects of probucol and pravastatin on
common carotid atherosclerosis in patients with asymptomatic
hypercholesterolemia: Fukuoka atherosclerosis trial (FAST). J
Am Coll Cardiol 2002; 39: 610-616.
8. Pignoli P, Tremoli E, Poli A, Oreste P, Paoletti R. Intimal plus
medial thickness of the arterial wall: a direct measurement with
ultrasound imaging. Circulation 1986; 74: 1399-1406.
9. Touboul PJ, Hennerici MG, Meairs S, Adams H, Amarenco P,

Chinese Medical Journal 2013;126 (21) 4065
Bornstein N. Mannheim carotid intima-media thickness and
plaque consensus (2004-2006-2011). Cerebrovasc Dis 2012; 34:
290-296.
10. Mihara H, Sumi H, Yoneta T, Mizumoto H, Ikeda R, Seiki M, et
al. A novel brinolytic enzyme extracted from the earthworm,
Lumbricus rubellus. Jpn J Physiol 1991; 41: 461-472.
11. Ryu GH, Park S, Kim M, Han DK, Kim YH, Min B.
Antithrombogenicity of lumbrokinase-immobilized
polyurethane. J Biomed Mater Res 1994; 28: 1069-1077.
12. Peng XF, Yang JY, Tao JN, Peng YT, Bao JK. Purication of ve
brinolytic enzymes from the earthworm (Eisenia Foelide) and
studies on brinogen enzymdysis. West China J Pharm Sci (Chin)
1991; 14: 16-18.
13. Jaulmes A, Sansilvestri-Morel P, Rolland-Valognes G, Bernhardt
F, Gaertner R, Lockhart BP, et al. Nox4 mediates the expression
of plasminogen activator inhibitor-1 via p38 MAPK pathway in
cultured human endothelial cells. Thromb Res 2009; 124: 439-
446.
14. Tkachuk VA, Plekhanova OS, Parfyonova YV. Regulation of arte
rial remodeling and angiogenesis by urokinase-type plasminogen
activator. Can J Physiol Pharmacol 2009; 87: 231-251.
15. Jin HJ, Zhang G, Zhang M, Cao X, Qian R. Anticoagulation and
brinolysis mechanism of lumbrokinase and its relation to t-PA.
Chin J Geriatr (Chin) 1998; 6: 366-369.
16. Tataru MC, Schulte H, von Eckardstein A, Heinrich J, Assmann
G, Koehler E. Plasma fibrinogen in relation to the severity of
arteriosclerosis in patients with stable angina pectoris after
myocardial infarction. Coron Artery Dis 2001; 12: 157-165.
17. Ji H, Wang L, Bi H, Sun L, Cai B, Wang Y, et al. Mechanisms
of lumbrokinase in protection of cerebral ischemia. Eur J
Pharmacol 2008; 590: 281-289.
18. Bots ML, Elwood PC, Salonen JT, Sivenius J, Di Carlo A, Nikitin
Y. Level of fibrinogen and risk of fatal and non-fatal stroke.
EUROSTROKE: a collaborative study among research centers
in Europe. J Epidemiol Communith Health 2002; 56: i14-i18.
19. Moons KG, Bots ML, Salonen JT, Elwo od PC, F reire de
Concalves A, Nikitin Y, et al. Prediction of stroke in the general
population in Europe(EUROSTROKE): is there a role for
fibrinogen and electrocardiography? J Epidemiol Community
Health 2002; 56: i30-i36.
20. Cao YJ, Wu YH, Liu CF. Effect of brinogen,brin and brin
(ogen) degradation products on the tissue plasminogen activator
and plasminogen activator inhibitor-1 expressions of vasculat
endothelial cells in coculture system. Chin J Cardiol (Chin)
2008; 36: 62-67.
21. Cao YJ, Wang YM, Zhang J, Zeng YJ, Liu CF. The effects of
antiplatelet agents on platelet-leukocyte aggregations in patients
with acute cerebral infarction. J Thromb Thrombolysis 2009; 27:
233-238.
22. Cao YJ, Zhou XM, Liu HH, Zhang YL, Yu XY, Liu CF. The
NF-κB pathway: regulation of the instability of atherosclerotic
plaques activated by Fg, Fb, and FDPs. Mol Cell Biochem 2013;
Epub ahead of print.
23. Liu M, Counsell C, Zhao XL, Wardlaw J. Fibrinogen depleting
agents for acute ischemic stroke. Cochrane Database Syst Rev
2003; (3): CD000091.
(Received May 20, 2013)
Edited by JI Yuan-yuan