Thrombosis Research 132 (2013) e135–e144

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Thrombosis Research

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Regular Article

Antithrombotic effects of a newly purified fibrinolytic protease fromUrechis unicinctus

Qingqing Bi, Baoqin Han ⁎, Yilin Feng, Zhongqing Jiang, Yan Yang, Wanshun LiuCollege of Marine Life Science, Ocean University of China, Qingdao, China

⁎ Corresponding author. Tel./fax: +86 532 8203 2105E-mail address: baoqinh@ouc.edu.cn (B. Han).

0049-3848/$ – see front matter © 2013 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.thromres.2013.07.001

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 4 March 2013Received in revised form 21 June 2013Accepted 4 July 2013Available online 24 July 2013

Keywords:Urechis unicinctusFibrinolytic proteaseThrombogenesisAntithrombotic

Introduction: The prevalence of thromboembolic disease, one of the top 3 leading causes of mortality world-wide, is being reported continually. More effective and safer antithrombotic drugs may overcome the under-lying problems in antithrombotic therapy. In the present work, antithrombotic effects of UFEIII, a newlypurified fibrinolytic protease from Urechis unicinctus were evaluated.Materials and methods: UFEIII was purified from the marine invertebrate, Urechis unicinctus, using anion ex-change and gel filtration chromatography. Molecular weight, fibrinolytic activity and fibrinogenolysis patternof UFEIII were determined. Furthermore, antithrombotic effects of UFEIII in vivo were investigated throughelectrical induced carotid arterial thrombosis in rats, FeCl3 induced carotid arterial thrombus model in rabbitsand stasis induced vena caval thrombus model in rats.Results: SDS-PAGE of the purified enzyme showed a single polypeptide chain with molecular weight of

20.8 kDa. In fibrin plate assays, UFEIII could not only directly degrade fibrin but also activate plasminogen.The fibrinogenolysis pattern of UFEIII was Aα-chains N Bβ-chains N γ-chain. Moreover, UFEIII could effec-tively prolong the time to occlusion in electrical induced carotid arterial thrombosis. Besides, both in rabbitsand rats, the administration of UFEIII not only prolonged the activated partial thromboplastin time (APTT)and thrombin time (TT) also decreased the fibrinogen (FIB) content. Further, the thrombus lysis wasobserved after administration of UFEIII both in rabbits and rats.Conclusion: UFEIII can possibly be a new potential source of fibrinolytic agent.

© 2013 Elsevier Ltd. All rights reserved.

Introduction

Thromboembolic diseases, involving cardiovascular disease, cere-brovascular disease and venous thromboembolism, are within thetop three leading causes of death as exposited in the global mortalityprojections to 2030 by WHO [1]. Thromboembolism often occurswhen part or all of a thrombus, the archcriminal of the whole com-plex disorders, breaks away from the vessel wall, travels to otherorgans with blood circulation, resulting in a myriad of pathologicalchanges wherever they arrive. Thrombus is generated when thephysiological equilibrium between coagulation system and fibrinolyt-ic system is disturbed. Regarding the treatment, administration ofthrombolytic drugs to dissolve the blood clot is the only alternativeof surgical interventions to remove or by pass the blockage [2]. How-ever, the thrombolytic drugs still exhibit many undesired side effectssuch as hemorrhage, low fibrin specificity and short half-lives afterthree generations developments. Consequently, searching for idealthrombolytic drugs has never been stopped.

Marine organisms, which constitute approximately one half of thetotal global biodiversity, are rich reservoirs of natural biofunctional

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rights reserved.

components [3], and numerous antithrombotic compounds have al-ready been discovered in the ocean [4–6]. Urechis unicinctus, whichbelongs to Echiuroidea, Xenopneusta, Urechidae, mostly inhabits inmarine intertidal and subtidal zones of North China, Korea andJapan. In our lab, a cluster of fibrinolytic proteases were discoveredfrom this marine worm and one 10.38 kDa protein [7] was purifiedand reported before.

In this present study, UFEIII, a newly purified fibrinolytc proteasefrom Urechis unicinctus was investigated from multiple perspectives in-cluding fibrinolytic activity, fibrinogenolysis pattern and antithromboticeffects in vivo.

Materials and Methods

Reagents and animals

Ehiuroid worms (U. unicinctus) were obtained from a local aquaticmarket in Qingdao, China. Sephacryl S-100 HR, Q-Sepharose FF andSephadex G-50 were purchased from GE Healthcare Bioscience AB(Uppsala, Sweden). Thrombin, fibrinogen and plasminogen, azocaseinand Folin-Ciocalteu’s phenol reagent were all purchased from Sigma-Aldrich (St.Louis, USA). Other reagents and chemicals used were ofanalytical grade and commercially available.

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Wistar rats (male and female half, weights 280 ± 20 g) and maleNew Zealand white rabbits (weights 3.5-4.0 kg) were purchased fromLukang Pharmaceutical Co. (Shandong, China). Kunming mice (maleand female half, weights 20 ± 1 g) were obtained from QingdaoExperimental Animal Center. All animals were accommodated to thenew environment for at least 1 week with conditions of moderatetemperature and good ventilation. Standard diet and tap water wereprovided freely. The animal experimental protocol was approved bythe Ethics Committee at Ocean University of China (Qingdao, China).

Purification of UFEIII

All fractionation steps were carried out at 4 °C. Washed ehiuroidworms (U. unicinctus 2000 g) were laparotomized to remove bodywalls and viscera, the rest coelomic fluid was centrifuged at8000 × g for 40 min. Then, the supernatant was pooled and filteredto remove insoluble lipid. After the soluble extracts were dialyzedby membranes with a MWCO of 3500-5000 Da (Spectrum ChemicalMfg. Corp., USA), the semifinished product was obtained by a freezedry system (Labconco corp., USA). The crude enzyme solution (7.5%)was applied to a Sephacryl S-100 HR column (3.5 cm × 80 cm) equili-brated with triple-distilled water at a flow rate of 1 ml/min. Themajor active fractions were pooled and introduced into a Q-sepharoseFF column (2.5 cm × 20 cm) previously equilibrated with 20 mMTris–HCl buffer (pH 8.0) and the bound proteins were eluted with alinear gradient of 0-0.5 M NaCl in the same buffer at a flow rate of5 ml/min. Fractions containing the highest fibrinolytic activity wereconcentrated by lyophilization after desalting. The concentrated samplewas dissolved in a small volume of 20 mMTris–HCl buffer (pH 8.0) andloaded onto a Sephadex G-50 column (1.6 cm × 80 cm) equilibratedwith the same sample dissolving buffer at aflow rate of 1 ml/min. Finally,the active fraction was desalted, lyophilized and used as the purifiedenzyme preparation.

Protease activity was measured by azocasein assay [8] and chro-mogenic assay [9] using S-2444 (pyro-Glu-Gly-Arg-p-nitroanilide)as a substrate at 405 nm. Protein concentration was estimated bythe method of Bradford [10]. After column chromatography, the pro-tein concentration was estimated by measuring the absorbance at280 nm.

Determination of purity and molecular weight

High-performance liquid chromatography (Shimadzu LC system,Japan) was employed to analyze the purity of UFEIII using a ShodexOHPak SB-806 HQ (8 mm × 300 mm) column (Shodex, Japan)at 30 °C, with an elution of 0.1 M Na2SO4, a flow rate of 1 ml/minand 20 μl sample loaded.

The molecular weight of the purified enzyme was determined bySDS-PAGE using 5% (w/v) stacking and 12% (w/v) resolving poly-acrylamide gels. Following electrophoresis, the gel was stained withCoomassie Brilliant Blue R250 for 3 h and destained with a solutioncontaining methanol: glacial acetic acid: distilled water (1:1:8 byvolume). A LMW standard protein marker (Takara, Japan) was usedfor calibration.

Determination of amidolytic activity

Amidolytic activity of UFEIII was measured using chromogenicsubstrates according to previous report [11] with slight modifications.The reaction mixture (1 ml) contained 20 μl of 0.5 mg/ml enzymesolution, 1 mM substrate solution and 0.1 M Tris-HCl buffer(pH7.4). After incubation for 15 min at 37 °C, absorbance of releasedp-nitroaniline at 405 nm was determined with a TU-1810 spectro-photometer (Purkinje General, China). Then, the amount of liberatedp-nitroaniline was calculated from the change in absorbance. One

unit of amidolytic activity (AU) was defined as micromoles ofsubstrate hydrolyzed per min/ml by UFEIII.

Fibrinolytic and fibrinogenolytic activities

Fibrinolytic activitywas determined using the fibrin platemethod ofAstrup and Mullertz [12] with minor modifications. The plasminogen-rich fibrin plate was prepared by pouring the solution composed of0.68 mg/ml human fibrinogen in 50 mM Tris–HCl buffer (pH 7.4) con-taining 0.15 M NaCl, 1% agarose, 1.25 U/ml thrombin and 0.25 U/mlhuman plasminogen into a sterile petri dish with a diameter of120 mm. The solution in the plate was left for 1 h to form fibrin clotand then 3.5 mm diameter wells were made in the plate for sampleapplication. The plasminogen-freefibrin plate contained noplaminogenand was heated at 85 °C for 30 min in addition. To observe the fibrino-lytic activity, 100 μl of sample solution was carefully dropped into eachwell and incubated for 18 h at 37 °C.

Analysis of fibrinogenolytic activity was performed following thereported method [13] with slight modifications. 200 μl fibrinogen(1 μg/μl) in 10 mM Tris–HCl buffer (pH 7.4) containing 0.15 M NaClwas incubated at 37 °C with 10 μg UFEIII for various time durations.A portion of the incubated sample (20 μl) was withdrawn at eachconsidered time interval, boiled for 3 min to terminate the reaction,and then analyzed by SDS-PAGE.

Hemorrhagic test of UFEIII

Hemorrhagic evaluation was carried out according to previousmethod [14] with slight modifications. Different doses of the purifiedUFEIII were subcutaneously injected into the back and belly region ofmice. As a positive control, urokinase was also injected into mice.24 h later, the mice were sacrificed and inspected for the emergenceof hemorrhagic halos in the back and belly skins.

Thrombolysis activity of UFEIII in vitro

Blood was collected from the central ear artery of New Zealandwhite rabbit to a sterilized petri plate. After spontaneous coagulation,the blood clot was blotted up and cut into small pieces. 0.9% saline,lumbrokinase solution (500 U/ml) and UFEIII solution (500 U/ml)were used as the negative control, the positive control and the testinggroup, respectively. Every group included three parallel tubes, each ofwhich contained 2 g blood clot with 1 ml 0.9% saline, lumbrokinasesolution and UFEIII solution. Then, the tubes were incubated in a37 °C thermostat shaker (60 r/min) for 1 h, 2 h and 3 h. At eachconsidered time interval, a little supernatant fluid was withdrawnto determine the amount of the released red cells microscopically.At last, the residual clot was weighed to calculate the dissolve rate ofblood clot. Dissolve rate (%) = (Massbefore dissolve - Massafter dissolve)/Massbefore dissolve × 100%.

Antithrombotic effects of UFEIII in vivo

Electrical induced carotid arterial thrombosis in ratsThis model was a clot prevention model and UFEIII was intro-

duced before thrombogenesis. Wistar rats were anesthetized withintraperitoneal injection of 3% pentobarbital sodium (1 ml/kg)after overnight fasting. Through a midline cervical incision, approxi-mately 15 mm of the right carotid artery was surgically exposed viablunt dissection, and a silkiness membrane was placed under thevessel to isolate it from the vagus nerve and connective tissue.Thrombogenesis was achieved using a BT87-3 Animal ThrombosisGenerator (Baotou Medical College, China) following the methodpreviously described with minor modification [15]. Primarily, 0.9%saline, heparin (300 U/kg), high (14 mg/kg), medium (7 mg/kg)and low dosage (3.5 mg/kg) of UFEIII were administrated intravascularly

Table 1Purification summary of the UFEIII.

Purificationstep

Totalprotein(mg)

Specific activity(U/mg)

Total fibrinolyticactivity (U)

Purificationfold

Yield(%)

Crude extract 1500 126.9 190410 1.00 100.0SephacrylS-100HR

316.7 410.7 130076 3.27 68.3

Q-Sepharose FF 63.8 974.8 62196 7.68 32.7Sephadex G-50 42.7 1382.0 59011 10.90 30.9

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in the right femoral vein. 20 min after the administration, the tempera-ture sensor and electrode of the thrombosis generator were laid on thedistal and proximal end of carotid artery, respectively. Then, a 1.5 mAconstant current was delivered by the electrode continuously. Alongwith thrombogenesis, the temperature of carotid blood was abruptlydecreased. Meanwhile, the time to occlusion (OT) was recorded ontimer of the generator, which accompanied an alarm from the tempera-ture sensor and the termination of electric stimulation on carotid artery.The rate of thrombosis inhibition was calculated as follows: thrombosisinhibition (%) = (OTtesting groups – OTnegative group)/OTnegative group × 100%.

FeCl3 induced carotid arterial thrombus model in rabbitsThis model is a treatment model and the UFEIII was administered

after thrombogenesis. The model was established on the basis ofmethod reported before [16]. Overnight fasted before surgery, maleNew Zealand white rabbits were anesthetized with intravascularinjection of sodium pentobarbital (30 mg/kg). After a midline incisionextending from mandible to suprasternal notch, the exposed anteriorcervical triangle was blunt dissected to free a 2 cm long segment ofthe right carotid artery. Thrombogenesis was induced by applying apiece of filter paper (2 cm × 2.5 cm) saturated with 35% FeCl3 salinesolution. The paper was placed around the external surface of thevessel for 40 min. Afterwards, the cervical incision was sutured andcleansed with iodophors carefully. Then, after thrombus occurred 3,24, 48 and 96 h, the rabbits were administrated with 0.9% saline,urokinase (5000 U/kg), high (7.5 mg/kg) and low dosage (2.5 mg/kg)of UFEIII through the marginal ear vein at every considered time inter-val. Moreover, since the primary administration, 24, 48 and 96 h later,3 ml blood was collected from central ear artery of each rabbit into3.8% sodium citrate, respectively, and then APTT, TT, PT and fibrinogenconcentration were determined by clinical laboratory of The AffiliatedHospital of Medical College, Qingdao University. At last, some rabbitswere sacrificed and several histology sections stained with HE of thevessels containing thrombus were made to observe the thrombolysiseffect of UFEIII.

Stasis induced vena caval thrombus model in ratsThis model is a treatment model and the UFEIII was administered

after thrombogenesis. The stasis caused thrombogenesis was actualizedin accordance with manner described before [17,18]. Wistar rats wereanesthetized by the same way as 2.8.1 described. The rats were fixedon a temp-controlled heating pad (38 °C) tomaintain the body temper-ature. The abdomen was opened by making an incision along the lineaalba towards the sternum. Afterwards, the intestines were gentlymoved to one side and covered with saline moistened gauze. Then asegment of the abdominal vena cava beneath the left renal vein andabove the iliac veins was cleared of adherent tissue by blunt dissection.Subsequently, 1 mm distance above the bifurcation of vena iliaca andvena cava, the free vein was ligated for 1 h using 4-0 sutural line(Shinva Medical, China) to induce blood stasis. Warm saline wassprayed over tissues. Besides, muscle layer and skin were provisionallyclosed with clamps. After accomplished thrombogenesis, 4-0 suturallinewas loosed and the ratswere administrated intravascularly throughthe right femoral vein with 0.9% saline, urokinase (7000 U/kg), high(14 mg/kg), medium (7 mg/kg) and low dosage (3.5 mg/kg) of UFEIII.40 min after the administration, the abdomen was unwrapped againand 3 ml blood was collected from abdominal aorta of each rat into3.8% sodium citrate, detecting APTT, TT, PT and fibrinogen concentra-tion in the mentioned clinical laboratory. Finally, the ligated venoussegments were excised and the clots were removed, blotted of excessblood, dried in a 37 °C oven, and immediately weighed.

Statistical Analysis

All parametric data were expressed as means ± standard devia-tion (SD). Statistical comparisons between means were performed

by one-way analysis of variance (ANOVA), and a value of p b 0.05 wasconsidered significant (computed by SPSS version 13.0 for Windows).

Results

Purification of UFEIII

Purification of UFEIII was accomplished by a combination of threecolumn chromatography steps which were summarized in Table 1.Gel filtration of the crude extract on Sephacryl S-100 HR yielded twofractions with fibrinolytic enzymes (Fig. 1a). The major peak withfibrinolytic activity was pooled, concentrated and loaded onto theQ-Sepharose FF column, which yielded three active peaks in the elutedfractions (Fig. 1b). The first peakwas further purified by SephadexG-50,and then a clear, unique peak with high fibrinolytic activity wasappeared (Fig. 1c). In addition, the crude extract contained too muchhemoglobin, which was hard to elute from the ion exchange stuffingeven using 2.0 M NaCl solution. Therefore, we attempted to add a gelfiltration step before the ion exchange, which efficiently removed theobstruction of hemoglobin on the purification. Overall, 10.9-foldpurification and 30.9% activity recovery (yield) were achieved aftercompletion of all purification steps.

Purity, molecular weight and amidolytic activity

The result of gel filtration HPLC (Fig. 2a) indicated that UFEIII waspurified to homogeneity higher than 97%. Native-PAGE of the purifiedenzyme showed one single band in the gel (Fig. 2b). SDS-PAGE of theenzyme was performed in order to verify the subunit composition andcalculate the molecular weight. The protein migrated as a single band(Fig. 2c), which revealed that the molecule of UFEIII consisted of onesingle polypeptide chain, and the molecular weight was determinedto be 20.8 kDa (Fig. 2d).

The amidolytic activity of UFEIII was investigated with severalsynthetic substrates (summarized in Table 2). UFEIII exhibitedspecificity for the substrates of Pyro-Glu-Gly-Arg-p-nitroanilide(for urokinase) and H-D-Val-Leu-Lys-p-nitroanilide (for plasmin),and less effects for the substrate H-D-Ile-Pro-Arg-p-nitroanilide(for t-PA). Besides, UFEIII showed low specificity for the substrateN-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (for subtilisin).

Fibrinolytic and fibrinogenolytic activities

As shown in Fig. 3a, a visible clear circle emerged around the wellcontaining UFEIII solution, which indicated UFEIII could degrade fibrinin plasminogen-rich plate. Besides, in plasminogen-free plate (Fig. 3b),UFEIII also generated clear circle, which suggested that UFEIII possesseda direct fibrin-degrading activity.

Fibrinogenolytic activity of UFEIII was investigated by SDS-PAGE. Asshown in Fig. 3d, the fibrinogenolysis pattern of UFEIII was Aα N Bβ N γ.UFEIII completely hydrolyzed the Aα-chains and Bβ-chains within20 min and 30 min, respectively. γ-chain was the last subunit to bedegraded.

Fig. 1. Purification of UFEIII by Sephacryl S-100 HR (a), Q Sepharose FF (b) and Sephadex G-50 (c). Target fractions were pooled (↔).

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KDa

97.2kD

66.4kD

44.3kD

29.0kD

20.1kD

14.3kD

1 M

0 2 4 6 8 10 12 14Minutes

16 18 20 22 24 26 28 30

0.00

000

0.00

005

0.00

010

Vol

ts

0.00

015

0.00

020

0.00

025

Time: 30.0207 Minutes - Amplitude: --Volts

0.00000

0.00005

0.00010

Vol

ts

0.00015

0.00020

0.00025

b c d

a

Fig. 2. (a) The purity of UFEIII was tested by HPLC using a refractive index detector produced by Shimadzu. (b) Native-PAGE of UFEIII (1 mg/ml), using 5% (w/v) stacking and 12%(w/v) resolving polyacrylamide gels. (c) SDS-PAGE of UFEIII. Lane 1, the purified UFEIII at 1 mg/ml; Lane M, LMW standard protein marker. (d) Determination of the molecularweight on logarithmic plot.

Table 2Amidolytic activity of UFEIII with different substrates.

Synthetic substrate Characteristics Concentration(mM)

Amidolyticactivity (AU)

H-D-Val-Leu-Lys-p-nitroanilide plasmin 1 49.6pyro-Glu-Gly-Arg-p-nitroanilide urokinase 1 58.7H-D-Ile-Pro-Arg-p-nitroanilide t-PA 1 42.2N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide

subtilisin 1 6.1

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In vitro thrombolysis activity

As displayed in Fig. 4, the liberated red cells of the UFEIII groupsignificantly exceeded the lumbrokinase and saline tubes at eachidentical time interval. Furthermore, the dissolve rate of blood clotby UFEIII was 60.5% (Table 3), which was significantly higher thanthat of lumbrokinase and negative control. All mentioned above indi-cated that the thrombolysis activity in vitro of UFEIII was superior tolumbrokinase under the same dosage and conditions. In addition, thepictures of microscopical examination showed the red cells allremained intact, which implied that UFEIII was harmless to red cell.

30 20 15 10 5 FIB Marker

Incubation time (min)

b

c

a

Fig. 3. (a) Fibrinolytic activity of UFEIII on plasminogen-rich fibrin plate. Numericallymarked lytic circles were: 1-5, urokinase standard solutions (0 U, 40 U, 80 U, 120 U,160 U, respectively); 6, purified UFEIII (100 μg). (b) Fibrinolytic activity onplasminogen-free fibrin plate. Lytic circle 1, purified UFEIII (100 μg); circle 2, urokinase(160 U). (c) Fibrinogenolysis pattern of UFEIII detected by SDS-PAGE.

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Effect on electrical induced carotid arterial thrombosis in vivo

In this test, administration of heparin and UFEIII could effectivelyprolong the time to occlusion as shown in Fig. 5. The thrombosis

inhibition of low, medium and high dosage of UFEIII were 36.40%,54.04% and 70.22%, respectively, presented a dose-related tendency.Besides, the administration doses of heparin and UFEIII in this testwere translated from clinical dose according to relative standard[19] and adopted after several preliminary experiments.

Effect of FeCl3 induced carotid arterial thrombus model in rabbits

As shown in Fig. 6b, the carotid artery of rabbit administrated withsaline was totally blocked due to the clot induced by FeCl3 exposed,which verified the reliability of this thrombosis model. Fortunately,the administration of UFEIII (both high and low dosage) could breakup parts of the clots and some tiny spaces emerged in the clot asdisplayed in Fig. 6c and d. This phenomenon suggested that UFEIIIcould attenuate arterial thrombosis. Moreover, to evaluate effects onanimal coagulation system by UFEIII, coagulation parameters weredetected. On the first day after thrombus occurred, administation oflow dosage UFEIII prolonged APTT from 18.05 ± 0.21 s to 26.4 ±3.48 s and TT from 14.70 ± 0.28 s to 16.33 ± 0.95 s. Still on thefirst day, low dosage UFEIII decreased fibrinogen concentration from5.57 ± 0.61 g/l to 4.23 ± 0.62 g/l. More detailed coagulation systemparameters were all appeared in Table 4.

Effect on stasis induced rat vena caval thrombus model in rats

As shown in Fig. 7, dry weights of the residual thrombus removedfrom the occlusive vessels of rats were all measured. Compared withthe negative control, the positive control urokinase decreased thethrombus weight from 36.42 ± 4.36 mg to 26.52 ± 4.97 mg, andUFEIII decreased the thrombus weight to 28.54 ± 5.72 mg, 24.96 ±3.95 mg and 20.84 ± 2.63 mg at doses of 3.5 mg/kg, 7 mg/kg and14 mg/kg, respectively, presented a dose-dependent fashion. Further,as presented in Table 5, intravenous administration of UFEIII at dosageof 7 mg/kg prolonged APTT and TT to a higher extent than dosage of14 mg/kg and 3.5 mg/kg. Besides, the 7 mg/kg dosage of UFEIII wasthe most effective one on decreasing fibrinogen concentration of ratblood. PT kept on undergoing a slight change in this detection.

Discussion

In this presentwork, a new fibrinolytic protease, UFEIII, was purifiedto electrophoretic homogeneity using column chromatography fromthe marine invertebrate, Urechis unicinctus. Biochemical characteristicsof UFEIII were investigated from multiple perspectives. Molecularweight of UFEIII was determined to be 20.8 kDa, which was higherthan that (10.38 kDa) of the one in our previous work [7], but lowerthan the six fibrinolytic enzymes (23.0 kDa-29.6 kDa) from Lumbricusrubellus [20], the seven fibrinolytic enzymes (23.0 kDa-29.7 kDa) fromEisenia fetida [21]. Amidolytic assay indicated that UFEIII selectivelyhydrolyzed the protein lysine and arginine residues, which was similarto the hydrolysis pattern of trypsin-like serine protease. Many otherfibrinolytic enzymes such as PPFE-I from Paenibacillus polymyxa EJS-3[11] and EfP from Eisenia fetida [22] were determined to be trypsin-like serine protease. According to fibrin plate assays, UFEIII had a directfibrin-degrading activity. NJF [23] and NJP [24] from Neanthes japonicawere both reported to be enzymes only with direct fibrin-degradingactivity. Nevertheless, fibrinolytic enzymes from Eisenia fetida [21] andLumbricus rubellus [25] were considered to be enzymes with bothplasminogen activator activity and direct fibrin degradation action.Moreover, the fibrinogenolysis pattern of UFEIII was determined to beAα- N Bβ- N γ-, which was similar to N-V protease [9], NJP [24] andCSP [26], but absolutely different from that of FP84 [27] and subtilisinFS33 [28], where Bβ-chains got hydrolyzed first.

Furthermore, a hemorrhagic test of UFEIII was carried out and therewas nohemorrhagic halo on the backnor the belly skins in every testingmouse after subcutaneous injection of purified UFEIII (up to 100 μg)

Fig. 4. Microscopical examination of each experimental tube for several time durations. (200×, Nikon Eclipse E200).

e141Q. Bi et al. / Thrombosis Research 132 (2013) e135–e144

into mice. However, there were hemorrhagic halos on the skins ofpositive control urokinase group (data not shown). The results prelim-inarily indicated that UFEIII was devoid of acute hemorrhagic effect andsome more assessments are still underway.

Subsequently, antithrombotic effects of UFEIII were evaluatedthrough considerable experiments. Although the studies referred rarelyto antithrombotic mechanisms of UFEIII, the abundant data of throm-bolysis tests in vitro and thrombosis models in vivo might providesome meaningful references for further research and development.

Due to its favorable reliability and reproducibility, the electrolyticmodel of arterial thrombosis is one of the most established and com-monly used preparations to determine the in vivo efficacy of novelantithrombotic drugs [29]. The electrolytic injury can be deliveredto the initial surface of artery and produce an occlusive thrombusmostly consisted of platelet. In this study, UFEIII could effectively pro-long the time to occlusion in a dosage-related way, which suggestedthat UFEIII had some abilities of preventing thrombogenesis.

Thrombolytic therapy is an acknowledged approach to the acutemanagement of ischemic stroke, pulmonary embolism and deep vein

Table 3Dissolve rate for blood clots in vitro.

Group Dissolve ratea (%)

Negative control 6.5 ± 0.12Lumbrokinase 39.0 ± 0.36UFEIII 60.5 ± 0.41

a Dissolve rate (%) = (Massbefore dissolve - Massafter dissolve)/Massbeforedissolve × 100%.

thrombosis, applied in an effort to achieve more rapid resolution ofthrombus burden [30]. Thrombolytic agents, categorized into threegenerations until now, have become the corner stone in thrombolytictherapy. During the procedure for discovering and validating noveldrug targets, selecting new agents for clinical evaluation, and providingdosing and safety information for clinical trials, animal models ofthrombosis have played a crucial role [31]. Arterial thrombosis, a fre-quent consequence of atherosclerosis, can occur in regions of moderateto high shear stress through adhesion and aggregation of platelets at theluminal surface of damaged vessels andmay lead to their occlusion [32].FeCl3 induced arterial thrombosis represents a widely used model ofthrombosis, in which the tissue damage initiated by iron-mediatedchemical oxidation predisposes the injured area to platelet adhesionand aggregation followed by coagulation activation and fibrin deposi-tion. Fig. 6 displayed the attenuation of artery thrombosis by UFEIII.Beyond that, due to the administration of UFEIII, color of the modelrabbits’ tongues and eyes gradually restored to the normal state ofbright red from the morbid state of dark red. Further, as presented inTable 4, administration of UFEIII effectively prolonged APTT, whichwas an indicator measuring the efficacy of both the intrinsic and thecommon coagulation pathways. Meanwhile, fibrinogen content inUFEIII treated rabbits were decreased, and the reduction of this sub-strate concentration led to a prolongation of TT. However, PT, whichwas usually used to measure the extrinsic pathway of coagulationcascade, underwent no significant change after administration of UFEIII.Venous thrombosis, which is associated with blood coagulation, is apathological condition appearing in post traumatic and post operativeperiods as deposits of fibrin and erythrocytes in regions of stasis orlow shear stress [32]. Consequently, thrombus that form in veins are

***

**

Fig. 5. Time to reach occlusion of the carotid artery in the electrical induced thrombosis in rats. Where 0.9% saline and heparin were used as negative and positive control, respec-tively. Values were presented as mean ± SD (n = 5-6), significance of the difference in comparison with negative control: *p b 0.05, **p b 0.01.

Fig. 6. Observation by histological sections of rabbits’ carotid arteries containing thrombus stained with HE. (a) The normal vessel wall. (b) Rabbit administrated with saline.(c) Rabbit administrated with UFEIII high dosage. (d) Rabbit administrated with UFEIII low dosage.

e142 Q. Bi et al. / Thrombosis Research 132 (2013) e135–e144

rich in fibrin and trapped red blood cells and are referred to as red clots.Deep vein thrombosis and pulmonary embolism, which are both lead-ing causes of mortality worldwide, are the most common appearancesof venous thrombosis. Deep vein thrombosis occurs most often in the

Table 4Coagulation parameters of rabbit blood in FeCl3 induced carotid arterial thrombus model in

Group PT(s)

Day1 Day2 Day3

0.9% saline 7.65 ± 0.31 7.20 ± 0.14 7.35 ± 0.27urokinase 8.30 ± 0.14⁎ 9.25 ± 0.35⁎⁎ 8.75 ± 0.78UFEIII low 8.47 ± 0.35⁎ 9.23 ± 0.23⁎⁎ 7.80 ± 0.40UFEIII high 8.30 ± 0.28⁎ 9.60 ± 0.56⁎⁎ 7.90 ± 0.44

Group APTT(s)

Day1 Day2 Day3

0.9% saline 18.05 ± 0.21 16.45 ± 0.35 15.70 ± 0.29urokinase 25.30 ± 0.99⁎ 21.40 ± 0.99⁎ 21.35 ± 2.47UFEIII low 26.4 ± 3.48⁎ 28.10 ± 1.78⁎⁎Δ 25.20 ± 3.24UFEIII high 23.50 ± 3.85⁎⁎ 21.93 ± 2.70⁎⁎ 22.35 ± 2.78

0.9% saline and urokinase were used as negative and positive control, respectively.Values were appeared as means ± SD (n = 3) *: p b 0.05 compared with control; **: p b 0

large veins of the legs. Pulmonary embolism is a complication of deepvein thrombosis that can occur if part of the thrombus breaks away,travels to the lungs and lodges in a pulmonary artery, resulting in thedisruption of blood flow [33]. To mimic the venous thrombosis, we

vivo.

FIB(g/l)

Day1 Day2 Day3

5.57 ± 0.61 5.39 ± 0.42 5.83 ± 0.503.05 ± 0.27⁎⁎ 3.24 ± 1.03 3.28 ± 0.38⁎

4.73 ± 0.33Δ 4.94 ± 0.68 5.09 ± 0.41Δ⁎ 4.23 ± 0.62⁎ 4.45 ± 0.57 4.41 ± 0.33⁎Δ

TT(s)

Day1 Day2 Day3

14.70 ± 0.28 12.95 ± 0.21 13.80 ± 0.14⁎ 17.40 ± 0.74⁎⁎ 18.30 ± 0.85⁎⁎ 15.85 ± 1.06⁎⁎ 16.33 ± 0.95⁎⁎Δ 16.77 ± 0.90⁎⁎Δ 15.53 ± 1.07⁎⁎ 15.40 ± 0.99 18.03 ± 1.55⁎⁎ 15.75 ± 0.78

.01 compared with negative control; Δ: p b 0.01 compared with positive control.

Fig. 7. Thrombus weight in the stasis induced thrombus model in rats. Where 0.9% saline and urokinase were used as negative and positive control, respectively. Values werepresented as mean ± SD (n = 5-6).

e143Q. Bi et al. / Thrombosis Research 132 (2013) e135–e144

employed a stasis induced model, in which a section of inferior venacava was isolated and ligated so that the stasis in this region promotedthrombus generation. As shown in Fig. 7, the largest reduction ofresidual thrombus weights was achieved by high dosage UFEIII from36.42 ± 4.36 mg to 20.84 ± 2.63 mg. And the main component ofthese red thrombuses was fibrin [33], so these results verify thefibribolytic activity of UFEIII, too. Data of these animals experimentsshowed that UFEIII administration to both rabbits and rats could signif-icantly reduce the level of fibrinogen content in blood plasma, prolongthe APTT and TT. Further, UFEIII could attenuate arterial and venousthrombosis. All the results indicated that UFEIII may be a new potentialsource of fibrinolytic agent.

Taken as a whole, as a new fibrinolytic protease, UFEIII, which can notonly directly degrade fibrin and fibrinogen but also activate plasminogen,was purified from the marine invertebrate, Urechis unicinctus. UFEIIIexhibited antithrombotic effects in animal thrombosis model and alsohad no acute hemorrhagic effect in mice. In conclusion, UFEIII maybecome a new source of fibrinolytic agents and further safety and func-tional valuations are required and underway.

Conflict of Interest Statement

The authors of this manuscript have no conflict of interest todeclare.

Acknowledgements

The authors are very grateful to Bonnie Paxman (Utah, USA) forher help with language modification. We are also grateful to clinicallaboratory of The Affiliated Hospital of Medical College, QingdaoUniversity for their help with coagulation parameters detection.This study was financially supported by the National high-technologyresearch and development program (863 program) of China(No. 2009ZX09103-646).

Table 5Coagulation parameters of rat blood in stasis induced vena caval thrombus model invivo.

Group PT (sec) FIB (g/l) APTT (sec) TT (sec)

0.9% saline 8.40 ± 0.16 1.25 ± 0.04 16.18 ± 0.62 35.54 ± 0.74urokinase 8.96 ± 0.18⁎⁎ 1.14 ± 0.03⁎⁎ 18.28 ± 1.25⁎ 38.00 ± 2.20⁎

UFEIII low 8.64 ± 0.11⁎ 1.11 ± 0.04⁎⁎ 18.08 ± 0.74⁎⁎ 40.78 ± 1.79⁎⁎Δ

UFEIIImedium

9.04 ± 0.11⁎⁎ 1.07 ± 0.05⁎⁎Δ 19.38 ± 0.58⁎⁎ 47.90 ± 3.06⁎⁎Δ

UFEIII high 8.68 ± 0.20 1.16 ± 0.05⁎⁎ 18.42 ± 0.77⁎⁎ 37.40 ± 1.50⁎

Values were expressed as means ± SD (n = 5-6) *: p b 0.05 with respect to negativecontrol; **: p b 0.01 with respect to negative control; Δ: p b 0.01 with respect topositive control.

Appendix A. Supplementary Data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.thromres.2013.07.001.

References

[1] Mathers CD, Loncar D. Projections of Global Mortality and Burden of Disease from2002 to 2030. PLoS Med 2006;3:e442.

[2] Simkhada JR, Cho SS, Mander P, Choi YH, Yoo JC. Purification, biochemical proper-ties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model. Thromb Res 2012;129:176–82.

[3] Harnedy PA, FitzGerald RJ. Bioactive peptides from marine processing waste andshellfish: A review. J Funct Foods 2012;4:6–24.

[4] Drozd NN, Tolstenkov AS, Makarov VA, Kuznetsova TA, Besednova NN,Shevchenko NM, et al. Pharmacodynamic parameters of anticoagulants based onsulfated polysaccharides frommarine algae. Bull Exp Biol Med 2006;142:591–3.

[5] Rajapakse N, JungWK, Mendis E, Moon SH, Kim SK. A novel anticoagulant purifiedfrom fish protein hydrolysate inhibits factor XIIa and platelet aggregation. Life Sci2005;76:2607–19.

[6] Luppi E, Cesaretti M, Volpi N. Purification and characterization of heparin from theItalian clam Callista chione. Biomacromolecules 2005;6:1672–8.

[7] Wang DL, Han BQ, Liu WS, Xu RA. Biochemical and enzymatic properties of anovel marine fibrinolytic enzyme from Urechis unicinctus. Appl BiochemBiotechnol 2007;136:251–64.

[8] Mander P, Cho SS, Simkhada JR, Choi YH, Yoo JC. A low molecular weightchymotrypsin-like novel fibrinolytic enzyme from Streptomyces sp. CS624. ProcessBiochem 2011;46:1449–55.

[9] Zhang YL, Cui JY, Zhang R, Wang YP, Hong M. A novel fibrinolytic serine proteasefrom the polychaete Nereis (Neanthes) virens (Sars): Purification and characteri-zation. Biochimie 2007;89:93–103.

[10] Bradford MM. A rapid and sensitive method for the quantification of microgramquantities of protein utilizing the principle of protein–dye binding. Anal Biochem1996;72:248–54.

[11] Lu FX, Lu ZX, Bie XM, Yao ZY, Wang YF, Lu YP, et al. Purification and characteriza-tion of a novel anticoagulant and fibrinolytic enzyme produced by endophyticbacterium Paenibacillus polymyxa EJS-3. Thromb Res 2010;126:e349–55.

[12] Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity.Arch Biochem Biophys 1952;40:346–51.

[13] Matsubara K, Sumi H, Hori K, Miyazawa K. Purification and characterization oftwo fibrinolytic enzymes from a marine green alga, Codium intricatum. CompBiochem Physiol B 1998;119:177–81.

[14] Popov SG, Popova TG, Hopkins S, Weinstein RS, MacAfee R, Fryxell KJ, et al. Effectiveantiprotease-antibiotic treatment of experimental anthrax. BMC Infect Dis 2005;5:25.

[15] Tang ZY, Wang YY, Xiao YL, Zhao M, Peng SQ. Anti-thrombotic activity of PDR, anewly synthesized L-Arg derivative, on three thrombosis models in rats. ThrombRes 2003;110:127–33.

[16] Tseng MT, Dozier A, Haribabu B, Graham UM. Transendothelial migration of ferricion in FeCl3 injured murine common carotid artery. Thromb Res 2006;118:275–80.

[17] Peternel L, Drevensˇek G, Cˇerne M, Sˇtalc A. Evaluation of two experimental ve-nous thrombosis models in the rat. Thromb Res 2005;115:527–34.

[18] Chi LG, Saganek LJ, Rogers KL, Mertz TE, Metz AL, Uprichard A, et al. A novel modelof venous thrombosis in the vena cava of rabbits. J Pharmacol Toxicol Methods1998;39:193–202.

[19] Shaw SR, Nihal M, Ahmad M. Dose translation from animal to human studiesrevisited. FASEB J 2007;22:659–61.

[20] Nakajima N, Mihara H, Sumi H. Characterization of potent fibrinolytic enzymes inearthworm, Lumbricus rubellus. Biosci Biotechnol Biochem 1993;57:1726–30.

[21] Wang F, Wang C, Li M, Gui LL, Zhang JP, Chang WR. Purification, characterizationand crystallization of a group of earthworm fibrinolytic enzymes from Eiseniafetida. Biotechnol Lett 2003;25:1105–9.

[22] Wu JX, Zhao XY, Pan R, He RQ. Glycosylated trypsin-like proteases from earth-worm Eisenia fetida. Int J Biol Macromol 2007;40:399–406.

e144 Q. Bi et al. / Thrombosis Research 132 (2013) e135–e144

[23] Deng ZH,Wang SH, Li Q, Ji X, Zhang L, Hong M. Purification and characterization ofa novel fibrinolytic enzyme from the polychaete, Neanthes japonica (Iznka).Bioresour Technol 2010;101:1954–60.

[24] Wang SH, Deng ZH, Li Q, Ge X, Bo QQ, Hong M. A novel alkaline serine proteasewith fibrinolytic activity from the polychaete, Neanthes japonica. Comp BiochemPhysiol B 2011;159:18–25.

[25] Cho IH, Choi ES, Lim HG, Lee HH. Purification and characterization of six fibrino-lytic serine-proteases from earthworm Lumbricus rubellus. J Biochem Mol Biol2004;37:199–205.

[26] Li HP, Hu Z, Yuan JL, Fan HD, Zou GL. A novel extracellular protease with fibrino-lytic activity from the culture supernatant of Cordyceps sinensis: purification andcharacterization. Phytother Res 2007;21:1234–41.

[27] Simkhada JR, Mander P, Cho SS, Yoo JC. A novel fibrinolytic protease from Streptomycessp. CS684. Process Biochem 2010;45:88–93.

[28] Wang CT, Ji BP, Li B, Nout R, Li PL, Ji H. Purification and characterization of a fibrinolyticenzymeofBacillus subtilisDC33, isolated fromChinese traditional douch. J IndMicrobiolBiotechnol 2006;33:750–8.

[29] Bush LR, Shebuski RJ. In vivo models of arterial thrombosis and thrombolysis.FASEB J 1990;4:3087–98.

[30] Marder VJ. Thrombolytic therapy for deep vein thrombosis: potential applicationof plasmin. Thromb Res 2009;123(Suppl. 4):S56–61.

[31] Leadley RJ, Chi LG, Rebello SS, Gagnon A. Contribution of in vivomodels of thrombosisto thediscovery and development of novel antithrombotic agents. J Pharmacol ToxicolMethods 2000;43:101–16.

[32] Andriamampandry MD. Antithrombotic effects of (n-3) polyunsaturated fattyacids in rat models of arterial and venous thrombosis. Thromb Res 1999;93:9–16.

[33] Mackman N. Triggers, targets and treatments for thrombosis. Nature 2008;451:914–8.