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The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis
Thrombosis Research (2008) 122, 683—690
www.elsevier.com/locate/thromres
REGULAR ARTICLE
The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis Jieying Fu a,b , Jianping Ren a , Libo Zou b , Guangxing Bian a , Ruifu Li a , Qiujun Lu a,⁎ a b
Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, China School of Life Science and Biological Pharmacy, Shenyang Pharmaceutical University, China
Received 10 September 2007; received in revised form 19 December 2007; accepted 5 January 2008 Available online 6 March 2008
KEYWORDS Miniplasmin; Recombinant tissue plasminogen activator; Femoral artery thrombosis; Thrombolytic therapy; Hemorrhage
Abstract Background and purpose: Miniplasmin was a des-kringle variant of plasminogen with potential pharmacological application. We investigated the thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis. Methods: In anesthetized dogs, a stable occlusive thrombus was formed by mechanical and electrolytic injury of the vessel wall, that the animals were later injected with miniplasmin (0.75 mg/kg, 1.5 mg/kg and 3.0 mg/kg, i.a.) and rt-PA (0.5 mg/kg, i.a.) intraarterially. Hemodynamic parameters and hemorrhage status were monitored for 2 h. Thrombin time, activated partial thromboplastin time, prothrombin time and fibrinogen concentration were tested at 2 h after administration. Fibrin degradation product and Ddimer concentration were tested by ELISA. Results: The incidence of reperfusion in the miniplasmin (3.0 and 1.5 mg/kg) groups was 100%, and time to reperfusion was (3.3 ± 1.0) and (7.0 ± 2.3) min, which was shorter than rt-PA. After reperfusion, none of the vessels in the miniplasmin (1.5 and 3.0 mg/kg) groups reoccluded, whereas 20% of vessels reoccluded in the rt-PA group. Rudimental thrombus mass in the miniplasmin (1.5 and 3.0 mg/kg) groups were smaller than rt-PA. The operative wounds in all miniplasmin groups had no hemorrhage within 2 h. There were no significant differences in thrombin time, activated partial thromboplastin time and prothrombin time. Fibrinogen concentration in the miniplasmin (3.0 mg/kg) group reduced significantly as compared with baseline and thrombosis values, whereas these values in the miniplasmin (1.5 and 0.75 mg/kg) groups were unchanged. Fibrin degradation product and D-dimer concentration increased significantly after thrombolysis. Conclusions: The results suggest that miniplasmin may be useful for the treatment of thrombosis and without complication of hemorrhage. This is in contrast to rt-PA, which intrinsically has a higher risk of occurring the hemorrhage risk. © 2008 Elsevier Ltd. All rights reserved.
⁎ Corresponding author. Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, No. 27 Taiping Road Beijing 100850, China. Tel.: +86 10 68180392; fax: +86 10 68214653. E-mail address: [email protected] (Q. Lu). 0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.01.007
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Introduction Therapeutic thrombolysis with plasminogen activators is an effective and potentially life-saving measure in the management of patients with acute myocardial infarction [1,2] and peripheral arterial or graft occlusion (PAO) [3]. However, every thrombolytic agent thus far utilized carries a tangible risk of life-threatening hemorrhage. While direct administration of the agent into a venous or arterial thrombus induces more efficient thrombolysis, bleeding at remote sites cannot be entirely avoided, probably because the activator is distributed systemically during and after vascular reperfusion. Thus, the incidence of intra-cranial hemorrhage (ICH) is 0.4–2.0% of patients with acute myocardial infarction, deep venous thrombosis or pulmonary embolism treated intravenously [4–6] and ICH occurs in 1–2% of patients with PAO who receive local infusions for up to 2 days [3,7], especially with concomitant heparin administration [8]. Agents that barely affect the plasma fibrinogen concentration can still cause bleeding [9], reinforcing the concept that fibrinolytic hemorrhage is the result of the haemostatic plug dissolution at vascular injury sites. Currently, no thrombolytic agent is demonstrated to have clinical efficacy without bleeding risk. Plasminogen is easily obtained from human plasma or plasma fractions. Its activation is affected by digestion of the peptide bond between arginine 561 and valine 562 (R561 V562) by tissue plasminogen activator (tPA) or urokinase (uPA) trapped in the blood clot. Plasminogen cannot readily be expressed in eukaryotic expression systems but has been obtained from the baculovirus cell system, which is however, not suitable for large-scale production. During the early investigations of thrombolytic approaches, plasmin was recognized as a direct fibrinolytic enzyme with potential clinical application. In the context of local administration, the theoretical limitations of plasmin, namely, inactivation by antiplasmin in the blood might actually provide a mechanism for safety from hemorrhage. Plasmin is unstable in neutral solutions, but can be stabilized by specific amino acids (such as lysine or tranexamic acid), acid pH (range 2–4) or glycerol (10–50%). Plasmin contains two chains. The chain A of the plasmin molecule consists of five triple-loop disulfide kringle (Kr) domains, while the chain B contains a “linker” region of 20 amino acids and a serine protease domain. Miniplasmin [10] in the study consists of only the kringle-5 domain, the linker, and the serine protease domain, which is different from recombinant microplasmin (μPlm) [11] that consists of only the linker and serine protease
J. Fu et al. domain. The inhibition of miniplasmin and μPlm by α2-antiplasmin (AP) occurs slowly when compared with plasmin, reflecting the absence of the noncovalent binding site of K1-3 [11,12]. The inhibition of miniplasmin is slower than μPlm due to the kringle-5 domain, which can slow down the action by increasing stereospecific blockade. MiniPlasminogen is refolded in Escherichia coli inclusion bodies and activated into miniplasmin by urokinase (uPA). Miniplasmin is stabilized in a dilute citrate buffer at pH 3–4. Miniplasmin in the study is prepared by PTI Company (USA), and spatial configuration modification is performed by Fuchun-zhongnan Company (China). The present study evaluates the thrombolytic effect of miniplasmin in a canine model of femoral artery thrombus and the hemorrhage risk of miniplasmin as compared with rt-PA. Materials and methods Drugs and reagents Miniplasmin was from Shanghai Fuchun-zhongnan Biotech Company (China). Recombinant tissue-type plasminogen activator (rt-PA; actilyse) was from Boehringer Ingelheim (Germany). All other reagents used in this study were obtained from commercial sources.
Animals Experiments were performed on adult male Beagle dogs weighing 9–12 kg, which were obtained from Academy of Military Medicine Sciences (Beijing, China). At all times, the animals received care in compliance with the criteria in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Animal procedures performed were in accordance to the guidelines of the Chinese Society of Laboratory Animal Sciences.
Surgical procedure Dogs were anesthetized with 30 mg/kg intravenous sodium pentobarbital. For replacement of fluid loss, 10 mL/kg/h saline was given continuously throughout the experiment. Oesophageal temperature was monitored and maintained at 37 ± 0.5 °C with an electric blanket and a heat lamp. The femoral thrombosis model used in this study was performed by injuring vascular wall [13,14]. The left femoral artery and femoral vein were dissected, a 3–4 cm section of each vessel and a side-branch of the femoral artery were carefully exposed. The side-branch was isolated for administration of drugs and contrast medium. The femoral artery was instrumented with a 3.0-mm ultrasonic flow probe (Transonic Systems, Ithaca, NY, USA) for continuous measurement of blood flow. The endothelium of the femoral artery was injured by gently squeezing with a hemostat clamp. After perfunctory injury, the Goldblatt clamp on the femoral artery was adjusted to reduce flow by approximately 80%, and a 150-μA anodal current electrode was placed around the femoral artery for 30 min due to thrombus formation. The sides of injured blood vessel were clipped by two bulldog clamps for 50–60 min, and then thrombus was performed if the flow was reduced to 0 mL/min.
685 Compared to thrombosis value, miniplasmin had enhanced significantly femoral artery blood flow for 2 h after the infusions. Values are expressed as mean ± SEM. n = 6. ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs thrombosis values.
0.2 ± 0.4 42.3 ± 40.8⁎ 59.8 ± 21.0⁎⁎⁎ 56.8 ± 13.0⁎⁎⁎ 26.0 ± 21.4⁎ 0.2 ± 0.4 43.3 ± 36.6⁎ 65.8 ± 26.7⁎⁎⁎ 54.2 ± 13.1⁎⁎⁎ 27.0 ± 21.1⁎ 0.3 ± 0.8 38.5 ± 36.9⁎ 61.7 ± 26.2⁎⁎⁎ 59.3 ± 14.4⁎⁎⁎ 29.3 ± 23.6⁎ 0.3 ± 0.8 29.3 ± 33.5 59.7 ± 22.1⁎⁎⁎ 64.0 ± 19.1⁎⁎⁎ 34.7 ± 21.0⁎⁎ 0.2 ± 0.4 33.5 ± 31.4⁎ 60.3 ± 21.0⁎⁎⁎ 62.8 ± 19.4⁎⁎⁎ 22.7 ± 18.3⁎
15 min Thrombosis Baseline
Blood flow (mL/min)
Dose (mg/kg) Group
Blood was obtained from femoral vein at 2 h after administration. Serum was separated by centrifugation at 3000 rpm for 15 min at 4 °C. FDP (FDP ELISA Kit, SHAIHAI SAN BIOTECH CO. LTD, China) was measured using ELISA method. FDP concentration was calculated with the calibrated regression line set between the optical density and the known concentration of FDP. Blood was obtained from femoral vein at 2 h after administration. The blood samples were anticoagulated with 3.8% trisodium citrate solution (9:1, v/v). Plasma was separated by centrifugation at 3000 rpm for 15 min at 4 °C. D-dimer (D-dimer ELISA Kit, SHAIHAI SAN BIOTECH CO. LTD, China) was measured using ELISA method. D-dimer concentration was calculated with the calibrated regression line set between the optical density and the known concentration of D-dimer.
Femoral artery blood flow during the thrombolysis process
Measurement of fibrin degradation product (FDP) concentration and D-dimer by ELISA
Table 1
Blood was obtained from femoral vein at 2 h after administration. The blood samples were anticoagulated with 3.8% trisodium citrate solution (9:1, v/v). Plasma was separated by centrifugation at 3000 rpm for 15 min at 4 °C. TT, APTT, PT and FIB concentration were measured by optical method on a coagulometer (PABER-I, Steellex Scientific Instrument Co., China). Briefly, TT was measured by incubating 200 μL plasma with 200 μL thrombin agent. APTT was measured by incubating 100 μL plasma with 100 μL αPTT-SA agent for 3 min at 37 °C, and followed by addition of 100 μL CaCl2. PT was measured by incubating 100 μL plasma with 200 μL prewarmed prothrombin agent. FIB clotting time was measured by incubating 200 μL plasma which was diluted by 10-fold with 100 μL thrombin agent. At last, FIB concentration was calculated with the calibrated regression line set between the clotting time and the known concentration of FIB.
30 min
Measurement of coagulation parameters
0.3 ± 0.8 0.5 ± 0.8 0.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
The observation of hemorrhage was performed in operative wounds after administration for 2 h. The hemorrhage status was photographed using a digital camera (SONY, Japan).
66.7 ± 15.6 73.5 ± 14.6 66.2 ± 25.4 58.8 ± 16.3 52.5 ± 18.5
45 min
Hemorrhage transformation
0.75 1.5 3.0 0.5
60 min
90 min
Thirty animals were randomly allocated into model control, miniplasmin (0.75 mg/kg), miniplasmin (1.5 mg/kg), miniplasmin (3.0 mg/kg) and rt-PA (0.5 mg/kg) treated group, six dogs in each group. Thirty minutes after thrombus formation, a catheter was gently advanced from the side-branch of the femoral artery into the thrombus, miniplasmin and rt-PA were infused 15 min through the catheter using an injection pump (TCI-II, Slgo high-tech development Co., China), whereas control rats were given infusion of saline. The blood flow was recorded at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min intervals after administration. Reperfusion was defined as 50% of baseline blood flow for 5 min, and time to reperfusion was recorded. The time to reperfusion was reported as 120 min if flow remained at 0 mL/min during the 2 h period after initiation of current. Reocclusion was defined as any occurrence of zero flow after termination of drugs or saline. At the end of the experiment the vessel segments between the proximal and the distal ends of the point of injury were excised. The vessels were opened longitudinally, in which the thrombus was removed and weighed.
Control Mini-Plm Mini-Plm Mini-Plm Rt-PA
120 min
Experimental protocol
0.2 ± 0.4 47.7 ± 44.8⁎ 61.7 ± 21.9⁎⁎⁎ 56.3 ± 15.0⁎⁎⁎ 24.3 ± 21.0⁎
The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis
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Table 2 Effect of miniplasmin on occlusion parameters at 2 h after administration in a canine model of femoral artery thrombus Group Control Miniplasmin Miniplasmin Miniplasmin Rt-PA
Dose (mg/kg)
Incidence of reperfusion n (%)
Time of reperfusion (min)
Incidence of reocclusion n (%)
Thrombus mass (mg)
0.75 1.5 3.0 0.5
0/6 (0) 4/6 (67) 6/6 (100) 6/6 (100) 5/6 (83)
14.4 ± 9.0 7.0 ± 2.3 3.3 ± 1.0 11.3 ± 6.8
0/0 1/4 0/6 0/6 1/5
83.7 ± 25.7 16.2 ± 7.3⁎⁎⁎ 4.8 ± 4.1⁎⁎⁎ 1.3 ± 1.6⁎⁎⁎ 14.0 ± 6.4⁎⁎⁎
(0) (25) (0) (0) (20)
Values are expressed as mean ± SEM. ⁎⁎⁎P b 0.001 vs Control group.
Statistical analysis
Results
All data were expressed as mean ± SEM. Data among the groups were assessed by one-way ANOVA. Among control group and treatment groups, thrombus mass was compared with Dunnett's test. To compare baseline and post-treatment values, TT, APTT, PT and FIB concentration, Dunnett's test was used. To compare thrombosis and post-treatment values, blood flow, FIB, FDP and D-dimer concentration, Dunnett's test was used. We assumed statistical significance at P b 0.05.
Femoral artery blood flow Before the induction of electrolytic injury, blood flow in all the group was similar and ranged from 52.5±18.5 to 73.5 ± 14.6 mL/min (P N 0.05). Electrolytic injury produced a progressive decline in flow and subsequent thrombotic occlusion of the femoral artery. Infusion of saline did not increase blood flow during the process (Table 1). Infusion of rt-PA restored the blood flow to only 43.2–
Figure 1 Photographs showed hemorrhage status in operative wounds after administration of drugs. (a) 0.75 mg/kg miniplasmin, (b) 1.5 mg/kg miniplasmin, (c) 3.0 mg/kg miniplasmin and (d) 0.5 mg/kg rt-PA. From these pictures, none of miniplasmin groups exhibited any visible hemorrhage. However, four of six animals in the rt-PA group showed fierce hemorrhage in the operative wound within 2 h after administration. The other two dogs were bleeding for about 1 h after infusion of rt-PA.
The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis
687
66.1% of the baseline value (Table 1). The effect of miniplasmin, however, was better than that of rt-PA. After the treatment of miniplasmin, blood flow in the 3.0 mg/kg group was similar to baseline value (Table 1).
hemorrhage (Fig. 1a–c). However, four of six animals in the rtPA group had fierce hemorrhage in the operative wound within 2 h after administration (Fig. 1b), and the other two dogs had been bleeding for about 1 h after infusion of rt-PA.
Occlusion parameters
Effect of miniplasmin on coagulation parameters
Infusion of saline in the control group did not dissolve thrombus in six arteries (Table 2). Infusion of rt-PA in the rt-PA treated group, 30 min after persistent occlusion, resulted in lysis of the thrombus in five of six arteries (83%) (Table 2). The incidence of reperfusion during miniplasmin (0.75 mg/kg) infusion was 67%. Miniplasmin (1.5 and 3.0 mg/kg) further increased the incidence of reperfusion to 100%. In the rt-PA group, one of five arteries (20%) that reperfused initially, reoccluded after the termination of rt-PA infusion. Similarly, one artery (25%) in the 0.75 mg/kg miniplasmin group reoccluded after the end of infusion. All arteries in the 1.5 and 3.0 mg/kg groups did not reocclude after reperfusion. The thrombus mass in the control group was (83.7 ± 25.7) mg. The thrombus mass in the miniplasmin groups decreased significantly compared to the saline control group, and the reduction was dose-dependent (Table 2).
Hemorrhage transformation Hemorrhage status in operative wounds was showed in Fig. 1. The wounds of miniplasmin groups did not visibly exhibit any
The effect of miniplasmin on TT, APTT and PT was presented in Table 3. Administration of miniplasmin had no significant effect on TT, APTT and PT as compared with baseline values, however, rt-PA (0.5 mg/kg) prolonged TT, APTT (P b 0.001), whereas PTwas unchanged. The effect of miniplasmin on FIB concentration was presented in Table 3. FIB concentration between 0.75 mg/kg miniplasmin and 1.5 mg/kg miniplasmin was unchanged. Compared with baseline and thrombosis values, administration of miniplasmin (3.0 mg/kg) reduced significantly FIB concentration by about 20%, while rt-PA (0.5 mg/kg) decreased FIB concentration by 1-fold (P b 0.001).
Effect of miniplasmin on FDP and D-dimer concentration The effect of miniplasmin on FDP and D-dimer concentration was presented in Table 4. FDP concentration increased significantly after administration of miniplasmin and rt-PA, which was compared with thrombosis values. D-dimer concentration increased significantly after administration of
Table 3 Effect of miniplasmin on TT, APTT, PT and FIB concentration at 2 h after administration in a canine model of femoral artery thrombus Group
Dose (mg/kg)
Control Baseline Thrombosis 2h Miniplasmin Baseline Thrombosis 2h
0.75
Miniplasmin Baseline Thrombosis 2h
1.5
Miniplasmin Baseline Thrombosis 2h
3.0
rt-PA Baseline Thrombosis 2h
0.5
TT (s)
APTT (s)
PT (s)
FIB (mg/dL)
12.4 ± 3.3 11.0 ± 2.5 9.5 ± 2.3
22.2 ± 4.4 24.6 ± 5.4 25.0 ± 5.0
8.2 ± 2.9 8.0 ± 3.0 7.9 ± 2.4
22.1 ± 3.0 23.4 ± 2.2 23.6 ± 2.3
15.7 ± 6.1 16.1 ± 1.9 18.3 ± 5.7
26.3 ± 6.9 23.6 ± 6.1 29.1 ± 8.2
7.2 ± 1.4 6.5 ± 0.4 8.8 ± 3.5
22.6 ± 1.0 23.6 ± 0.8 21.6 ± 3.9
12.1 ± 4.8 13.8 ± 5.4 13.4 ± 4.1
27.9 ± 3.8 23.9 ± 5.1 25.7 ± 3.7
6.7 ± 0.5 7.2 ± 1.4 7.5 ± 2.0
23.5 ± 1.7 24.2 ± 0.9 23.7 ± 1.8
10.3 ± 1.4 10.9 ± 1.3 14.8 ± 5.2
21.9 ± 8.3 22.9 ± 9.1 26.3 ± 11.8
8.2 ± 4.2 7.0 ± 2.3 7.9 ± 1.8
24.7 ± 1.9 24.9 ± 1.8 EE, ⁎⁎ 19.6 ± 2.6
10.5 ± 2.7 10.7 ± 2.3 74.2 ± 5.9⁎⁎⁎
23.7 ± 4.2 21.2 ± 4.8 87.9 ± 6.4⁎⁎⁎
6.3 ± 0.6 6.0 ± 0.3 6.8 ± 1.0
24.1 ± 1.4 24.6 ± 0.5 EEE 11.4 ± 4.2 , ⁎⁎⁎
Values are expressed as mean ± SEM. EEP b 0.01, EEEP b 0.001 vs Baseline values. ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs Thrombosis values.
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Table 4 Effect of miniplasmin on FDP and D-dimer concentration at 2 h after administration in a canine model of femoral artery thrombus Group
Dose (mg/kg)
Control Baseline Thrombosis 2h
FDP (μg/mL) 5.3 ± 0.8 6.5 ± 0.7 6.7 ± 0.6
Miniplasmin Baseline Thrombosis 2h
0.75
Miniplasmin Baseline Thrombosis 2h
1.5
Miniplasmin Baseline Thrombosis 2h
3.0
rt-PA Baseline Thrombosis 2h
0.5
D-dimer (μg/mL) 0.5 ± 0.1 0.5 ± 0.1 0.6 ± 0.1
5.1 ± 1.1 6.6 ± 0.8 10.6 ± 1.3⁎⁎⁎
0.5 ± 0.1 0.6 ± 0.1 1.0 ± 0.1⁎⁎⁎
5.4 ± 0.5 6.8 ± 0.6 13.1 ± 0.9⁎⁎⁎
0.5 ± 0.1 0.6 ± 0.1 1.6 ± 0.1⁎⁎⁎
5.3 ± 0.8 6.9 ± 0.6 16.1 ± 0.6⁎⁎⁎
0.5 ± 0.1 0.6 ± 0.1 2.2 ± 0.2⁎⁎⁎
5.2 ± 0.5 6.8 ± 0.5 12.1 ± 1.7⁎⁎⁎
0.5 ± 0.1 0.7 ± 0.1 1.2 ± 0.1⁎⁎⁎
Values are expressed as mean ± SEM. ⁎⁎⁎P b 0.001 vs Thrombosis values.
miniplasmin and rt-PA, which was compared with thrombosis values.
Discussion The present study was designed to evaluate the thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis. In this study, administered miniplasmin dissolved thrombus of the femoral artery and did not affect coagulation parameters afterward. The thrombolytic effect of miniplasmin was demonstrated in this study as evidenced by the high incidence of reperfusion, with high-grade arterial blood flow, for the duration of the study after thrombolysis. Thrombolytic agents have been clinically employed since 1955 [15]. However, the risk of serious bleeding complications was very high so that the procedure of systemic infusion of thrombolytic agents in the treatment of PAO was largely neglected in favor of catheter-direct thrombolytic therapy. Recent advances in thrombolytic therapy have, in part, been driven by significant technical advances in catheter design and delivery, permitting local drug administration directly into a clot. Intra-
arterial thrombolysis with catheter means that the drug is administered into a blood vessel nearby thrombus or thrombus through a catheter. This therapy can reduce dose of drug and hemorrhage risk, extend the “window of treatment” beyond the 3 h for intravenous (IV) use. Ouriel [16] thinks that intraarterial thrombolysis with catheter therapy is the only way for the recanalization blood vessel. However, negative results were also reported in IA treatment with rt-PA [17,18]. In recent years, many investigations indicate that cerebral hemorrhage caused by rt-PA is related to matrix metalloproteinase (MMP), which damaged blood brain barrier (BBB) [19–21]. However, rt-PA can enhance expression of the MMP [22]. So it is possible that rt-PA causes cerebral hemorrhage through activation of the MMP. Further investigation of IA thrombolytic treatment with rt-PA is therefore warranted. In this study, the drugs including saline, miniplasmin and rt-PA were administered into blood vessel nearby thrombus through a catheter. The results indicated incidence of reperfusion in the miniplasmin (1.5 and 3.0 mg/kg) groups was 100%, which was higher than that in rt-PA group, and time of reperfusion was faster than rt-PA. Some previous reports indicated that clot lysis in the absence of an adjunctive agent will often be followed by rethrombosis. But the data in this study indicated none of the vessels in the miniplasmin (1.5 and 3.0 mg/kg) groups reoccluded after reperfusion, whereas 20% of vessels reoccluded in the rt-PA group. The result suggested that miniplasmin (1.5 and 3.0 mg/kg) might dissolve thrombus soundly, and the hypothesis was also demonstrated by the following result. The clots in 3.0 mg/kg miniplasmin group were nearly dissolved, the rudimental thrombus mass was only (1.3 ± 1.6) mg, while the thrombus mass in rt-PA group was (14.0 ± 6.4) mg. The most feared complication of intra-arterial thrombolysis with plasminogen activators is, however, intra-cranial hemorrhage that occurs in 1–2% of treated patients. The major reason is fibrinolytic system accentuation caused by thrombolytic drugs. Local infusion of plasmin obtained from plasma plasminogen has been shown in a rabbit model to dissolve arterial clots with less bleeding time prolongation than rt-PA [23]. The present study confirms and extends these observations to miniplasmin. The results showed that miniplasmin doesn't prolong TT, APTT and PT after administration. However, rt-PA could prolong TT by 5- and 10fold and APTT by 3- and 5-fold, respectively, whereas PT was unchanged. FIB concentration in miniplasmin (0.75 and 1.5 mg/kg) had no significant difference. But compared with baseline and thrombosis values, administration of miniplasmin
The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis (3.0 mg/kg) reduced significantly FIB concentration by about 20%, whereas, rt-PA (5 mg/kg) decreased FIB concentration to about 50% of baseline value. The reduction of FIB concentration was profitable for thrombolysis [24], while the reduction was excessive, which would increase permeability of vascular and hemorrhage risk. The results above indicate that intra-arterial administration of miniplasmin has no significant influence on fibrinolytic and coagulation parameters, and thus miniplasmin is unlikely to cause hemorrhage. The inhibitory action of AP and α2-macroglobulin (α2-M) is the mechanism that explains why miniplasmin doesn't lead to hemorrhage. In the blood, miniplasmin is inhibited by AP and α2-M after thrombolysis, which is identical to plasmin. For miniplasmin, local administration escapes inhibition by AP and α2-M, presumably by competitive fibrin binding which affords protection and a free hand at fibrin lysis. The explanation for absent bleeding with systemic miniplasmin is explained by AP and α2-M neutralization in the circulation [25]. So long as AP and α2-M are present in the blood, miniplasmin would not circulate freely, would not reach fibrin in vulnerable haemostatic plugs, and would not induce bleeding from the injured vessel. It is reasonable to anticipate that larger dosages of miniplasmin than that were tested here could achieve plasma concentrations that would consume AP and α2-M, and result in bleeding from vascular injury sites. The study in dogs indicates that the direct fibrinolytic enzyme, miniplasmin, may cure PAO effectively. Moreover, miniplasmin did not increase hemorrhage risk, it may be a safer alternative to rtPA for stroke therapy. This advantage indicates that miniplasmin is a promising candidate for the development as a treatment for thrombosis.
Acknowledgments The study was supported by funding and a supply of miniplasmin from Fuchun-zhongnan Biotech Company (Shanghai, China). We acknowledge the technical assistance of Beijing Institute of Radiation Medicine. The authors thank Bing Han for assistance with the operative procedure and Ruiting Liu for her insightful comment.
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[2] Wilcox RG, von der Lippe G, Olsson CG, Jensen G, Skene AM, Hampto JR. Effects of alteplase in acute myocardial infarction: 6-month results from the ASSET study. Anglo-Scandinavian Study of Early Thrombolysis. Lancet 1990;335:1175—8. [3] Ouriel K, Shortell CK, DeWeese JA, Green RM, Francis CW, Azodo MVU, et al. A comparison of thrombolytic therapy with operative revascularization in the initial treatment of acute peripheral arterial ischemia. J Vasc Surg 1994;19:1021—30. [4] An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. The GUSTO Investigators. N Engl J Med 1993;329:673—82. [5] Gore JM, Stoan M, Price TR, Randall AM, Bovill E, Collen D, et al. Intracerebral hemorrhage, cerebral infarction, and subdural hematoma after acute myocardial infarction and thrombolytic therapy in the thrombolysis in myocardial infarction study. Thrombolysis in Myocardial Infarction, Phase II, pilot and clinical trial. Circulation 1991;83:448—59. [6] Kanter DS, Mikkola KM, Patel SR, Parker JA, Goldhaber SZ. Thrombolytic therapy for pulmonary embolism. Frequency of intracranial hemorrhage and associated risk factors. Chest 1997;111:1241—5. [7] Ouriel K, Gray B, Clair DG, Olin J. Complications associated with the use of urokinase and recombinant tissue plasminogen activator for catheter-directed peripheral arterial and venous thrombolysis. J Vasc Interv Radiol 2000;11:295—8. [8] Ouriel K, Veith FJ, Sasahara AA. A comparison of recombinant urokinase with vascular surgery as initial treatment for acute arterial occlusion of the legs. Thrombolysis or Peripheral Arterial Surgery Investigators. N Engl J Med 1998;338:1105—11. [9] Single-bolus tenecteplase compared with front-loaded alteplase in acute myocardial infarction: the ASSENT-2 doubleblind randomized trial. Assessment of the Safety and Efficacy of a New Thrombolytic Investigators. Lancet 1999;354:716—22. [10] Medynski D, Tuan M, Liu W, Wu S, Lin X. Refolding, purification, and activation of miniplasminogen and microplasminogen isolated from E. coli inclusion bodies. Protein Expr Purif 2007;52:395—402. [11] Nagai N, Demarsin E, Vanhoef B, Wouters S, Cingolani D, Laroche Y, et al. Recombinant human microplasmin: production and potential therapeutic properties. J Thromb Haemost 2003;1:307—13. [12] Steven L, Nancy L. α2-Macroglobulin is the primary inhibitor of miniplasmin in vitro and in vivo in the mouse. Biochem J 1988;255:725—30. [13] Kageyama S, Yamamoto H, Nakazawa H, Yoshimoto R. Antihuman vWF monoclonal antibody, AjvW-2 Fab, inhibitsrepetitive coronary artery thrombosis without bleeding time prolongation in dogs. Thromb Res 2001;101:395—404. [14] Eobello SS, Blank HS, Lucchesi BR. Antithrombotic efficacy of single subcutaneous administration of a recombinant nematode anticoagulant peptide (rNAP5) in a canine model of coronary artery thrombolysis. Thromb Res 2000;98:531—40. [15] Tillet WS, Johnson AJ, McCarthy WR. The intravenous infusion of the streptococcal fibrinolytic principle (streptokinase) into patients. J Clin Invest 1955;34:169—85. [16] Ouriel K. Current status of thrombolysis for peripheral arterial occlusive disease. Ann Vasc Surg 2002;16(6):797—804. [17] Hilger T, Niessen F, Diedenhofen M, Hossmann KA, Hoehn M. Magnetic resonance angiography of thromboembolic stroke in rats: indicator of recanalization probability and tissue survival after recombinant tissue plasminogen activator treatment. J Cereb Blood Flow Metab 2002;22:652—62. [18] Niessen F, Hilger T, Hoehn M, Hossmann KA. Thrombolytic treatment of clot embolism in rat: comparison of intraarterial and intravenous application of recombinant tissue plasminogen activator. Stroke 2002;33:2999—3005.
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J. Fu et al. teinase by tissue plasminogen activator. Nat Med 2003;9:1313—7. [23] Marder VJ, Landskroner K, Novokhatny V, Zimmerman T, Koig M, Kanouse JJ. Plasmin induces local thrombolysis without causing hemorrhage: a comparison with tissue plasminogen activator in the rabbit. Thromb Haemost 2001;86:739—45. [24] Ajjan R, Grant PJ. Coagulation and atherothrombotic disease. Atherosclerosis 2006;186:240—59. [25] Senior K. Thrombolysis without bleeding. DDT 2001;6: 1246—8.
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REGULAR ARTICLE
The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis Jieying Fu a,b , Jianping Ren a , Libo Zou b , Guangxing Bian a , Ruifu Li a , Qiujun Lu a,⁎ a b
Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, China School of Life Science and Biological Pharmacy, Shenyang Pharmaceutical University, China
Received 10 September 2007; received in revised form 19 December 2007; accepted 5 January 2008 Available online 6 March 2008
KEYWORDS Miniplasmin; Recombinant tissue plasminogen activator; Femoral artery thrombosis; Thrombolytic therapy; Hemorrhage
Abstract Background and purpose: Miniplasmin was a des-kringle variant of plasminogen with potential pharmacological application. We investigated the thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis. Methods: In anesthetized dogs, a stable occlusive thrombus was formed by mechanical and electrolytic injury of the vessel wall, that the animals were later injected with miniplasmin (0.75 mg/kg, 1.5 mg/kg and 3.0 mg/kg, i.a.) and rt-PA (0.5 mg/kg, i.a.) intraarterially. Hemodynamic parameters and hemorrhage status were monitored for 2 h. Thrombin time, activated partial thromboplastin time, prothrombin time and fibrinogen concentration were tested at 2 h after administration. Fibrin degradation product and Ddimer concentration were tested by ELISA. Results: The incidence of reperfusion in the miniplasmin (3.0 and 1.5 mg/kg) groups was 100%, and time to reperfusion was (3.3 ± 1.0) and (7.0 ± 2.3) min, which was shorter than rt-PA. After reperfusion, none of the vessels in the miniplasmin (1.5 and 3.0 mg/kg) groups reoccluded, whereas 20% of vessels reoccluded in the rt-PA group. Rudimental thrombus mass in the miniplasmin (1.5 and 3.0 mg/kg) groups were smaller than rt-PA. The operative wounds in all miniplasmin groups had no hemorrhage within 2 h. There were no significant differences in thrombin time, activated partial thromboplastin time and prothrombin time. Fibrinogen concentration in the miniplasmin (3.0 mg/kg) group reduced significantly as compared with baseline and thrombosis values, whereas these values in the miniplasmin (1.5 and 0.75 mg/kg) groups were unchanged. Fibrin degradation product and D-dimer concentration increased significantly after thrombolysis. Conclusions: The results suggest that miniplasmin may be useful for the treatment of thrombosis and without complication of hemorrhage. This is in contrast to rt-PA, which intrinsically has a higher risk of occurring the hemorrhage risk. © 2008 Elsevier Ltd. All rights reserved.
⁎ Corresponding author. Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, No. 27 Taiping Road Beijing 100850, China. Tel.: +86 10 68180392; fax: +86 10 68214653. E-mail address: [email protected] (Q. Lu). 0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2008.01.007
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Introduction Therapeutic thrombolysis with plasminogen activators is an effective and potentially life-saving measure in the management of patients with acute myocardial infarction [1,2] and peripheral arterial or graft occlusion (PAO) [3]. However, every thrombolytic agent thus far utilized carries a tangible risk of life-threatening hemorrhage. While direct administration of the agent into a venous or arterial thrombus induces more efficient thrombolysis, bleeding at remote sites cannot be entirely avoided, probably because the activator is distributed systemically during and after vascular reperfusion. Thus, the incidence of intra-cranial hemorrhage (ICH) is 0.4–2.0% of patients with acute myocardial infarction, deep venous thrombosis or pulmonary embolism treated intravenously [4–6] and ICH occurs in 1–2% of patients with PAO who receive local infusions for up to 2 days [3,7], especially with concomitant heparin administration [8]. Agents that barely affect the plasma fibrinogen concentration can still cause bleeding [9], reinforcing the concept that fibrinolytic hemorrhage is the result of the haemostatic plug dissolution at vascular injury sites. Currently, no thrombolytic agent is demonstrated to have clinical efficacy without bleeding risk. Plasminogen is easily obtained from human plasma or plasma fractions. Its activation is affected by digestion of the peptide bond between arginine 561 and valine 562 (R561 V562) by tissue plasminogen activator (tPA) or urokinase (uPA) trapped in the blood clot. Plasminogen cannot readily be expressed in eukaryotic expression systems but has been obtained from the baculovirus cell system, which is however, not suitable for large-scale production. During the early investigations of thrombolytic approaches, plasmin was recognized as a direct fibrinolytic enzyme with potential clinical application. In the context of local administration, the theoretical limitations of plasmin, namely, inactivation by antiplasmin in the blood might actually provide a mechanism for safety from hemorrhage. Plasmin is unstable in neutral solutions, but can be stabilized by specific amino acids (such as lysine or tranexamic acid), acid pH (range 2–4) or glycerol (10–50%). Plasmin contains two chains. The chain A of the plasmin molecule consists of five triple-loop disulfide kringle (Kr) domains, while the chain B contains a “linker” region of 20 amino acids and a serine protease domain. Miniplasmin [10] in the study consists of only the kringle-5 domain, the linker, and the serine protease domain, which is different from recombinant microplasmin (μPlm) [11] that consists of only the linker and serine protease
J. Fu et al. domain. The inhibition of miniplasmin and μPlm by α2-antiplasmin (AP) occurs slowly when compared with plasmin, reflecting the absence of the noncovalent binding site of K1-3 [11,12]. The inhibition of miniplasmin is slower than μPlm due to the kringle-5 domain, which can slow down the action by increasing stereospecific blockade. MiniPlasminogen is refolded in Escherichia coli inclusion bodies and activated into miniplasmin by urokinase (uPA). Miniplasmin is stabilized in a dilute citrate buffer at pH 3–4. Miniplasmin in the study is prepared by PTI Company (USA), and spatial configuration modification is performed by Fuchun-zhongnan Company (China). The present study evaluates the thrombolytic effect of miniplasmin in a canine model of femoral artery thrombus and the hemorrhage risk of miniplasmin as compared with rt-PA. Materials and methods Drugs and reagents Miniplasmin was from Shanghai Fuchun-zhongnan Biotech Company (China). Recombinant tissue-type plasminogen activator (rt-PA; actilyse) was from Boehringer Ingelheim (Germany). All other reagents used in this study were obtained from commercial sources.
Animals Experiments were performed on adult male Beagle dogs weighing 9–12 kg, which were obtained from Academy of Military Medicine Sciences (Beijing, China). At all times, the animals received care in compliance with the criteria in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Animal procedures performed were in accordance to the guidelines of the Chinese Society of Laboratory Animal Sciences.
Surgical procedure Dogs were anesthetized with 30 mg/kg intravenous sodium pentobarbital. For replacement of fluid loss, 10 mL/kg/h saline was given continuously throughout the experiment. Oesophageal temperature was monitored and maintained at 37 ± 0.5 °C with an electric blanket and a heat lamp. The femoral thrombosis model used in this study was performed by injuring vascular wall [13,14]. The left femoral artery and femoral vein were dissected, a 3–4 cm section of each vessel and a side-branch of the femoral artery were carefully exposed. The side-branch was isolated for administration of drugs and contrast medium. The femoral artery was instrumented with a 3.0-mm ultrasonic flow probe (Transonic Systems, Ithaca, NY, USA) for continuous measurement of blood flow. The endothelium of the femoral artery was injured by gently squeezing with a hemostat clamp. After perfunctory injury, the Goldblatt clamp on the femoral artery was adjusted to reduce flow by approximately 80%, and a 150-μA anodal current electrode was placed around the femoral artery for 30 min due to thrombus formation. The sides of injured blood vessel were clipped by two bulldog clamps for 50–60 min, and then thrombus was performed if the flow was reduced to 0 mL/min.
685 Compared to thrombosis value, miniplasmin had enhanced significantly femoral artery blood flow for 2 h after the infusions. Values are expressed as mean ± SEM. n = 6. ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs thrombosis values.
0.2 ± 0.4 42.3 ± 40.8⁎ 59.8 ± 21.0⁎⁎⁎ 56.8 ± 13.0⁎⁎⁎ 26.0 ± 21.4⁎ 0.2 ± 0.4 43.3 ± 36.6⁎ 65.8 ± 26.7⁎⁎⁎ 54.2 ± 13.1⁎⁎⁎ 27.0 ± 21.1⁎ 0.3 ± 0.8 38.5 ± 36.9⁎ 61.7 ± 26.2⁎⁎⁎ 59.3 ± 14.4⁎⁎⁎ 29.3 ± 23.6⁎ 0.3 ± 0.8 29.3 ± 33.5 59.7 ± 22.1⁎⁎⁎ 64.0 ± 19.1⁎⁎⁎ 34.7 ± 21.0⁎⁎ 0.2 ± 0.4 33.5 ± 31.4⁎ 60.3 ± 21.0⁎⁎⁎ 62.8 ± 19.4⁎⁎⁎ 22.7 ± 18.3⁎
15 min Thrombosis Baseline
Blood flow (mL/min)
Dose (mg/kg) Group
Blood was obtained from femoral vein at 2 h after administration. Serum was separated by centrifugation at 3000 rpm for 15 min at 4 °C. FDP (FDP ELISA Kit, SHAIHAI SAN BIOTECH CO. LTD, China) was measured using ELISA method. FDP concentration was calculated with the calibrated regression line set between the optical density and the known concentration of FDP. Blood was obtained from femoral vein at 2 h after administration. The blood samples were anticoagulated with 3.8% trisodium citrate solution (9:1, v/v). Plasma was separated by centrifugation at 3000 rpm for 15 min at 4 °C. D-dimer (D-dimer ELISA Kit, SHAIHAI SAN BIOTECH CO. LTD, China) was measured using ELISA method. D-dimer concentration was calculated with the calibrated regression line set between the optical density and the known concentration of D-dimer.
Femoral artery blood flow during the thrombolysis process
Measurement of fibrin degradation product (FDP) concentration and D-dimer by ELISA
Table 1
Blood was obtained from femoral vein at 2 h after administration. The blood samples were anticoagulated with 3.8% trisodium citrate solution (9:1, v/v). Plasma was separated by centrifugation at 3000 rpm for 15 min at 4 °C. TT, APTT, PT and FIB concentration were measured by optical method on a coagulometer (PABER-I, Steellex Scientific Instrument Co., China). Briefly, TT was measured by incubating 200 μL plasma with 200 μL thrombin agent. APTT was measured by incubating 100 μL plasma with 100 μL αPTT-SA agent for 3 min at 37 °C, and followed by addition of 100 μL CaCl2. PT was measured by incubating 100 μL plasma with 200 μL prewarmed prothrombin agent. FIB clotting time was measured by incubating 200 μL plasma which was diluted by 10-fold with 100 μL thrombin agent. At last, FIB concentration was calculated with the calibrated regression line set between the clotting time and the known concentration of FIB.
30 min
Measurement of coagulation parameters
0.3 ± 0.8 0.5 ± 0.8 0.2 ± 0.4 0.0 ± 0.0 0.0 ± 0.0
The observation of hemorrhage was performed in operative wounds after administration for 2 h. The hemorrhage status was photographed using a digital camera (SONY, Japan).
66.7 ± 15.6 73.5 ± 14.6 66.2 ± 25.4 58.8 ± 16.3 52.5 ± 18.5
45 min
Hemorrhage transformation
0.75 1.5 3.0 0.5
60 min
90 min
Thirty animals were randomly allocated into model control, miniplasmin (0.75 mg/kg), miniplasmin (1.5 mg/kg), miniplasmin (3.0 mg/kg) and rt-PA (0.5 mg/kg) treated group, six dogs in each group. Thirty minutes after thrombus formation, a catheter was gently advanced from the side-branch of the femoral artery into the thrombus, miniplasmin and rt-PA were infused 15 min through the catheter using an injection pump (TCI-II, Slgo high-tech development Co., China), whereas control rats were given infusion of saline. The blood flow was recorded at 15 min, 30 min, 45 min, 60 min, 90 min and 120 min intervals after administration. Reperfusion was defined as 50% of baseline blood flow for 5 min, and time to reperfusion was recorded. The time to reperfusion was reported as 120 min if flow remained at 0 mL/min during the 2 h period after initiation of current. Reocclusion was defined as any occurrence of zero flow after termination of drugs or saline. At the end of the experiment the vessel segments between the proximal and the distal ends of the point of injury were excised. The vessels were opened longitudinally, in which the thrombus was removed and weighed.
Control Mini-Plm Mini-Plm Mini-Plm Rt-PA
120 min
Experimental protocol
0.2 ± 0.4 47.7 ± 44.8⁎ 61.7 ± 21.9⁎⁎⁎ 56.3 ± 15.0⁎⁎⁎ 24.3 ± 21.0⁎
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Table 2 Effect of miniplasmin on occlusion parameters at 2 h after administration in a canine model of femoral artery thrombus Group Control Miniplasmin Miniplasmin Miniplasmin Rt-PA
Dose (mg/kg)
Incidence of reperfusion n (%)
Time of reperfusion (min)
Incidence of reocclusion n (%)
Thrombus mass (mg)
0.75 1.5 3.0 0.5
0/6 (0) 4/6 (67) 6/6 (100) 6/6 (100) 5/6 (83)
14.4 ± 9.0 7.0 ± 2.3 3.3 ± 1.0 11.3 ± 6.8
0/0 1/4 0/6 0/6 1/5
83.7 ± 25.7 16.2 ± 7.3⁎⁎⁎ 4.8 ± 4.1⁎⁎⁎ 1.3 ± 1.6⁎⁎⁎ 14.0 ± 6.4⁎⁎⁎
(0) (25) (0) (0) (20)
Values are expressed as mean ± SEM. ⁎⁎⁎P b 0.001 vs Control group.
Statistical analysis
Results
All data were expressed as mean ± SEM. Data among the groups were assessed by one-way ANOVA. Among control group and treatment groups, thrombus mass was compared with Dunnett's test. To compare baseline and post-treatment values, TT, APTT, PT and FIB concentration, Dunnett's test was used. To compare thrombosis and post-treatment values, blood flow, FIB, FDP and D-dimer concentration, Dunnett's test was used. We assumed statistical significance at P b 0.05.
Femoral artery blood flow Before the induction of electrolytic injury, blood flow in all the group was similar and ranged from 52.5±18.5 to 73.5 ± 14.6 mL/min (P N 0.05). Electrolytic injury produced a progressive decline in flow and subsequent thrombotic occlusion of the femoral artery. Infusion of saline did not increase blood flow during the process (Table 1). Infusion of rt-PA restored the blood flow to only 43.2–
Figure 1 Photographs showed hemorrhage status in operative wounds after administration of drugs. (a) 0.75 mg/kg miniplasmin, (b) 1.5 mg/kg miniplasmin, (c) 3.0 mg/kg miniplasmin and (d) 0.5 mg/kg rt-PA. From these pictures, none of miniplasmin groups exhibited any visible hemorrhage. However, four of six animals in the rt-PA group showed fierce hemorrhage in the operative wound within 2 h after administration. The other two dogs were bleeding for about 1 h after infusion of rt-PA.
The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis
687
66.1% of the baseline value (Table 1). The effect of miniplasmin, however, was better than that of rt-PA. After the treatment of miniplasmin, blood flow in the 3.0 mg/kg group was similar to baseline value (Table 1).
hemorrhage (Fig. 1a–c). However, four of six animals in the rtPA group had fierce hemorrhage in the operative wound within 2 h after administration (Fig. 1b), and the other two dogs had been bleeding for about 1 h after infusion of rt-PA.
Occlusion parameters
Effect of miniplasmin on coagulation parameters
Infusion of saline in the control group did not dissolve thrombus in six arteries (Table 2). Infusion of rt-PA in the rt-PA treated group, 30 min after persistent occlusion, resulted in lysis of the thrombus in five of six arteries (83%) (Table 2). The incidence of reperfusion during miniplasmin (0.75 mg/kg) infusion was 67%. Miniplasmin (1.5 and 3.0 mg/kg) further increased the incidence of reperfusion to 100%. In the rt-PA group, one of five arteries (20%) that reperfused initially, reoccluded after the termination of rt-PA infusion. Similarly, one artery (25%) in the 0.75 mg/kg miniplasmin group reoccluded after the end of infusion. All arteries in the 1.5 and 3.0 mg/kg groups did not reocclude after reperfusion. The thrombus mass in the control group was (83.7 ± 25.7) mg. The thrombus mass in the miniplasmin groups decreased significantly compared to the saline control group, and the reduction was dose-dependent (Table 2).
Hemorrhage transformation Hemorrhage status in operative wounds was showed in Fig. 1. The wounds of miniplasmin groups did not visibly exhibit any
The effect of miniplasmin on TT, APTT and PT was presented in Table 3. Administration of miniplasmin had no significant effect on TT, APTT and PT as compared with baseline values, however, rt-PA (0.5 mg/kg) prolonged TT, APTT (P b 0.001), whereas PTwas unchanged. The effect of miniplasmin on FIB concentration was presented in Table 3. FIB concentration between 0.75 mg/kg miniplasmin and 1.5 mg/kg miniplasmin was unchanged. Compared with baseline and thrombosis values, administration of miniplasmin (3.0 mg/kg) reduced significantly FIB concentration by about 20%, while rt-PA (0.5 mg/kg) decreased FIB concentration by 1-fold (P b 0.001).
Effect of miniplasmin on FDP and D-dimer concentration The effect of miniplasmin on FDP and D-dimer concentration was presented in Table 4. FDP concentration increased significantly after administration of miniplasmin and rt-PA, which was compared with thrombosis values. D-dimer concentration increased significantly after administration of
Table 3 Effect of miniplasmin on TT, APTT, PT and FIB concentration at 2 h after administration in a canine model of femoral artery thrombus Group
Dose (mg/kg)
Control Baseline Thrombosis 2h Miniplasmin Baseline Thrombosis 2h
0.75
Miniplasmin Baseline Thrombosis 2h
1.5
Miniplasmin Baseline Thrombosis 2h
3.0
rt-PA Baseline Thrombosis 2h
0.5
TT (s)
APTT (s)
PT (s)
FIB (mg/dL)
12.4 ± 3.3 11.0 ± 2.5 9.5 ± 2.3
22.2 ± 4.4 24.6 ± 5.4 25.0 ± 5.0
8.2 ± 2.9 8.0 ± 3.0 7.9 ± 2.4
22.1 ± 3.0 23.4 ± 2.2 23.6 ± 2.3
15.7 ± 6.1 16.1 ± 1.9 18.3 ± 5.7
26.3 ± 6.9 23.6 ± 6.1 29.1 ± 8.2
7.2 ± 1.4 6.5 ± 0.4 8.8 ± 3.5
22.6 ± 1.0 23.6 ± 0.8 21.6 ± 3.9
12.1 ± 4.8 13.8 ± 5.4 13.4 ± 4.1
27.9 ± 3.8 23.9 ± 5.1 25.7 ± 3.7
6.7 ± 0.5 7.2 ± 1.4 7.5 ± 2.0
23.5 ± 1.7 24.2 ± 0.9 23.7 ± 1.8
10.3 ± 1.4 10.9 ± 1.3 14.8 ± 5.2
21.9 ± 8.3 22.9 ± 9.1 26.3 ± 11.8
8.2 ± 4.2 7.0 ± 2.3 7.9 ± 1.8
24.7 ± 1.9 24.9 ± 1.8 EE, ⁎⁎ 19.6 ± 2.6
10.5 ± 2.7 10.7 ± 2.3 74.2 ± 5.9⁎⁎⁎
23.7 ± 4.2 21.2 ± 4.8 87.9 ± 6.4⁎⁎⁎
6.3 ± 0.6 6.0 ± 0.3 6.8 ± 1.0
24.1 ± 1.4 24.6 ± 0.5 EEE 11.4 ± 4.2 , ⁎⁎⁎
Values are expressed as mean ± SEM. EEP b 0.01, EEEP b 0.001 vs Baseline values. ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 vs Thrombosis values.
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Table 4 Effect of miniplasmin on FDP and D-dimer concentration at 2 h after administration in a canine model of femoral artery thrombus Group
Dose (mg/kg)
Control Baseline Thrombosis 2h
FDP (μg/mL) 5.3 ± 0.8 6.5 ± 0.7 6.7 ± 0.6
Miniplasmin Baseline Thrombosis 2h
0.75
Miniplasmin Baseline Thrombosis 2h
1.5
Miniplasmin Baseline Thrombosis 2h
3.0
rt-PA Baseline Thrombosis 2h
0.5
D-dimer (μg/mL) 0.5 ± 0.1 0.5 ± 0.1 0.6 ± 0.1
5.1 ± 1.1 6.6 ± 0.8 10.6 ± 1.3⁎⁎⁎
0.5 ± 0.1 0.6 ± 0.1 1.0 ± 0.1⁎⁎⁎
5.4 ± 0.5 6.8 ± 0.6 13.1 ± 0.9⁎⁎⁎
0.5 ± 0.1 0.6 ± 0.1 1.6 ± 0.1⁎⁎⁎
5.3 ± 0.8 6.9 ± 0.6 16.1 ± 0.6⁎⁎⁎
0.5 ± 0.1 0.6 ± 0.1 2.2 ± 0.2⁎⁎⁎
5.2 ± 0.5 6.8 ± 0.5 12.1 ± 1.7⁎⁎⁎
0.5 ± 0.1 0.7 ± 0.1 1.2 ± 0.1⁎⁎⁎
Values are expressed as mean ± SEM. ⁎⁎⁎P b 0.001 vs Thrombosis values.
miniplasmin and rt-PA, which was compared with thrombosis values.
Discussion The present study was designed to evaluate the thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis. In this study, administered miniplasmin dissolved thrombus of the femoral artery and did not affect coagulation parameters afterward. The thrombolytic effect of miniplasmin was demonstrated in this study as evidenced by the high incidence of reperfusion, with high-grade arterial blood flow, for the duration of the study after thrombolysis. Thrombolytic agents have been clinically employed since 1955 [15]. However, the risk of serious bleeding complications was very high so that the procedure of systemic infusion of thrombolytic agents in the treatment of PAO was largely neglected in favor of catheter-direct thrombolytic therapy. Recent advances in thrombolytic therapy have, in part, been driven by significant technical advances in catheter design and delivery, permitting local drug administration directly into a clot. Intra-
arterial thrombolysis with catheter means that the drug is administered into a blood vessel nearby thrombus or thrombus through a catheter. This therapy can reduce dose of drug and hemorrhage risk, extend the “window of treatment” beyond the 3 h for intravenous (IV) use. Ouriel [16] thinks that intraarterial thrombolysis with catheter therapy is the only way for the recanalization blood vessel. However, negative results were also reported in IA treatment with rt-PA [17,18]. In recent years, many investigations indicate that cerebral hemorrhage caused by rt-PA is related to matrix metalloproteinase (MMP), which damaged blood brain barrier (BBB) [19–21]. However, rt-PA can enhance expression of the MMP [22]. So it is possible that rt-PA causes cerebral hemorrhage through activation of the MMP. Further investigation of IA thrombolytic treatment with rt-PA is therefore warranted. In this study, the drugs including saline, miniplasmin and rt-PA were administered into blood vessel nearby thrombus through a catheter. The results indicated incidence of reperfusion in the miniplasmin (1.5 and 3.0 mg/kg) groups was 100%, which was higher than that in rt-PA group, and time of reperfusion was faster than rt-PA. Some previous reports indicated that clot lysis in the absence of an adjunctive agent will often be followed by rethrombosis. But the data in this study indicated none of the vessels in the miniplasmin (1.5 and 3.0 mg/kg) groups reoccluded after reperfusion, whereas 20% of vessels reoccluded in the rt-PA group. The result suggested that miniplasmin (1.5 and 3.0 mg/kg) might dissolve thrombus soundly, and the hypothesis was also demonstrated by the following result. The clots in 3.0 mg/kg miniplasmin group were nearly dissolved, the rudimental thrombus mass was only (1.3 ± 1.6) mg, while the thrombus mass in rt-PA group was (14.0 ± 6.4) mg. The most feared complication of intra-arterial thrombolysis with plasminogen activators is, however, intra-cranial hemorrhage that occurs in 1–2% of treated patients. The major reason is fibrinolytic system accentuation caused by thrombolytic drugs. Local infusion of plasmin obtained from plasma plasminogen has been shown in a rabbit model to dissolve arterial clots with less bleeding time prolongation than rt-PA [23]. The present study confirms and extends these observations to miniplasmin. The results showed that miniplasmin doesn't prolong TT, APTT and PT after administration. However, rt-PA could prolong TT by 5- and 10fold and APTT by 3- and 5-fold, respectively, whereas PT was unchanged. FIB concentration in miniplasmin (0.75 and 1.5 mg/kg) had no significant difference. But compared with baseline and thrombosis values, administration of miniplasmin
The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis (3.0 mg/kg) reduced significantly FIB concentration by about 20%, whereas, rt-PA (5 mg/kg) decreased FIB concentration to about 50% of baseline value. The reduction of FIB concentration was profitable for thrombolysis [24], while the reduction was excessive, which would increase permeability of vascular and hemorrhage risk. The results above indicate that intra-arterial administration of miniplasmin has no significant influence on fibrinolytic and coagulation parameters, and thus miniplasmin is unlikely to cause hemorrhage. The inhibitory action of AP and α2-macroglobulin (α2-M) is the mechanism that explains why miniplasmin doesn't lead to hemorrhage. In the blood, miniplasmin is inhibited by AP and α2-M after thrombolysis, which is identical to plasmin. For miniplasmin, local administration escapes inhibition by AP and α2-M, presumably by competitive fibrin binding which affords protection and a free hand at fibrin lysis. The explanation for absent bleeding with systemic miniplasmin is explained by AP and α2-M neutralization in the circulation [25]. So long as AP and α2-M are present in the blood, miniplasmin would not circulate freely, would not reach fibrin in vulnerable haemostatic plugs, and would not induce bleeding from the injured vessel. It is reasonable to anticipate that larger dosages of miniplasmin than that were tested here could achieve plasma concentrations that would consume AP and α2-M, and result in bleeding from vascular injury sites. The study in dogs indicates that the direct fibrinolytic enzyme, miniplasmin, may cure PAO effectively. Moreover, miniplasmin did not increase hemorrhage risk, it may be a safer alternative to rtPA for stroke therapy. This advantage indicates that miniplasmin is a promising candidate for the development as a treatment for thrombosis.
Acknowledgments The study was supported by funding and a supply of miniplasmin from Fuchun-zhongnan Biotech Company (Shanghai, China). We acknowledge the technical assistance of Beijing Institute of Radiation Medicine. The authors thank Bing Han for assistance with the operative procedure and Ruiting Liu for her insightful comment.
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