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Functional properties of a recombinant chimeric plasminogen activator with platelet-targeted fibrinolytic and anticoagulant potential
Molecular Genetics and Metabolism 82 (2004) 304–311 www.elsevier.com/locate/ymgme
Functional properties of a recombinant chimeric plasminogen activator with platelet-targeted Wbrinolytic and anticoagulant potential Leiliang Zhang, Jing Wang, Meimin Yu, and Binggen Ru¤ National Lab of Protein Engineering, College of Life Sciences, Peking University, Beijing 100871, PR China Received 22 February 2004; received in revised form 20 May 2004; accepted 20 May 2004 Available online 2 July 2004
Abstract The construction, puriWcation, and characterization of dscuPA33khC, a bifunctional protein designed for thrombosis treatment is described. The chimera was designed to consist of a decorsin (platelet aggregation inhibitor), a low molecular mass (33 kDa) singlechain urokinase (scuPA-33k), and a thrombin inhibitory domain. We have successfully produced this recombinant protein in the Escherichia coli expression system, in which the target protein exists in the form of inclusion bodies. After refolding by dilution in vitro, the chimeric protein was puriWed to homogeneity by immobilized metal aYnity chromatography, ion-exchange chromatography, and gel Wltration chromatography. The dscuPA33khC could directly activate plasminogen following Michaelis–Menten kinetics with Km D 1.52 M and K2 D 0.0024 s¡1. The speciWc activity of the chimera detected by Wbrin plate determination was 11,000 IU/mg, which suggested a high thrombolysis eVect. However, the chimeric dscuPA33khC bound the activated platelet and signiWcantly increased aYnity to platelet clots as compared to Wbrin clots. It was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner as well as it exhibits antithrombin activity. These results suggest that the chimeric protein not only has platelet-targeted thrombolytic activity but also obtains anti-thrombus function. 2004 Elsevier Inc. All rights reserved. Keywords: Decorsin; Hirudin; Platelet-targeted; Antithrombin activity
Introduction Urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) have been proved eVective in the treatment of myocardial infarction, but the agents have certain limitations such as bleeding or platelet-mediated reocclusion [1]. To solve these problems in thrombolytic therapy, the plasminogen activators should be combined with antiplatelet agents or strategies [2]. Based on these considerations, we want to construct a recombinant chimeric plasminogen activator with platelet inhibitory activity. The single chain urokinase (scu-PA) is composed of 411 amino acids with Mr 54,000, which can be converted
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Corresponding author. Fax: +86-10-62751842. E-mail address: [email protected] (B. Ru).
1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.05.008
to two-chain urokinase (tcuPA) with speciWc cleavage of Lys158-Ile159 by plasmin [3]. Scu-PA has a higher speciWc thrombolytic activity and a better selectivity for Wbrin than tcu-PA [4]. A low molecular weight form of scu-PA (scu-PA-33K), lacking the 135 NH2-terminal amino acids, has been obtained from cleavage between residues Lys135 and Lys136 [5]. ScuPA-33k has similar properties to scu-PA and can be used as a plasminogen activator. Decorsin is a 39-residue protein isolated from the leech Macrobdella decora, containing three intramolecular disulWde bonds and Arg-Gly-Asp (RGD) sequence [6,7]. Decorsin blocks Wbrinogen binding to the platelet glycoprotein IIb/IIIa (GpIIb/IIIa) and acts as a potent inhibitor of platelet aggregation [8]. Hirudin is the most potent thrombin-speciWc inhibitor, as it acts on both circulating and clot-bound thrombin [9,10]. Its N-terminal domain binds to the active site
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Fig. 1. Schematic representation of the composition of dscuPA33khC and the function of the individual components. The decorsin is linked to scuPA-33k and a thrombin inhibitory domain. The thrombin inhibitory domain comprised a 5-aa linker, a thrombin active site motif, part sequences of thrombin receptor, and C-terminal of hirudin.
of thrombin, whereas its C-terminal fragment covers the recognition site of Wbrinogen [11]. C-terminal fragments of hirudin can be used as thrombin inhibitors [2]. We designed a new polypeptide to combine Wbrinolytic activity, platelet aggregation inhibitory and thrombin inhibitory activity into one molecule. In this study (Fig. 1), a thrombin inhibitory domain was fused to the C-terminus of scuPA-33k; meanwhile decorsin was fused to the N-terminus of scuPA-33k. The thrombin inhibitory domain comprised a 5-aa linker (SPVVA), a thrombin active site motif (FPRP), partial sequences of thrombin receptor (FLLRNP), and the C-terminus (residues 54–65) of hirudin. It is expected that this chimeric plasminogen activator may have platelet-targeted thrombolytic activity with anti-thrombus function.
SP Sepharose, and Sephacryl S-200 were purchased from Amersham Biosciences (Piscataway, USA). Construction of E. coli expression vector of dscuPA33khC
Materials and methods
The DNA fragment encoding decorsin was obtained by PCR from the plasmid pMAL-decorsin with the primers A and B. The DNA fragment encoding scuPA-33k (Lys136–Leu411) was obtained by PCR from the plasmid pUC19-scuPA with primers C and D. The DNA fragment encoding the part of thrombin inhibitor was obtained from the synthesized three oligonucleotides: E, F, and G. Following, the chimeric cDNA (dscuPA33khC) was obtained by PCR (Table 1). Finally the chimeric cDNA was cloned into the pET29a vector from the NdeI and HindIII sites. The sequence of the desired recombinant vector pET29a-dscuPA33khC conWrmed by restriction analyses and dideoxynucleotide sequencing [12].
Materials
Expression and puriWcation of the chimeric protein
Plasmid pET-29a was purchased from Novagen (Madison, USA). Escherichia coli BL21-CodonPlus (DE3)-RIL was purchased from Stratagene (La Jolla, USA). IPTG, GSH, and GSSG were purchased from Promega (Madison, USA). Fibrinogen, plasminogen, plasmin, and thrombin were purchased from Sigma (St. Louis, USA). S-2444 (L-pyroGlu-Gly-L-Arg-p-nitroanilide) and S-2251 (D-Val-Leu-Lys-p-nitroanilide) were purchased from Kabi Vitrum (Stockholm, Sweden). Chromozym TH was purchased from Roche (Basel, Switzerland). Urokinase standard and scu-PA were obtained from NICPBP (Beijing, China). Zinc chelate–Sepharose,
The recombinant vector was transformed into E. coli BL21-CodonPlus (DE3)-RIL. The protein expression and refolding was performed as described by Jiao [13]. The refolding mixture was applied to a zinc chelate– Sepharose column equilibrated with 20 mM Tris–HCl, 500 mM NaCl, 0.01% Tween 80, pH 7.4, and eluted with the same buVer containing 50 mM imidazole. After ultraWltration the concentrated sample was subjected to SP Sepharose column in 50 mM NaAc (pH 4.5), with a linear gradient of 0–1.0 M NaCl. Then the sample was applied to Sephacryl S-200, which was developed with 20 mM Tris–HCl, 50 mM NaCl, pH 7.4. Protein concentration
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Table 1 The PCR steps of the chimeric cDNA of dscuPA33khC Step
Primersb
Template
Product
1 2 3a 4 5 6
A, B C, D A, D A, E A, F A, G
pMAL-decorsin pUC19-scuPA I, II III IV V
I II III IV V Final chimeric cDNA (dscuPA33khC)
A: 5⬘-GGGAATTCCATATGGCTCCACGTCTG-3⬘ NdeI " B: 5⬘-TTCTTCCGGCGGGCTGCTCGGTTTTTCACAGTATGGATC-3⬘ C: 5⬘-AGCAGCCCGCCGGAAGAATTAAAATTTCAGTGT-3⬘ D: 5⬘-TGATCAAGCTTCTGATGGA-3⬘ E: 5⬘-CAGGAACGGTCTCGGGAAAGCAACAACCGGGCTGAGGGCCAGGCCATT-3⬘ F: 5⬘-CGGGATTTCTTCGAAGTCACCCGGATTTCTCAGCAGGAACGGTCTCG-3⬘ G: 5⬘-TGACTAAGCTTTTACTGCAGGTATTCTTCCGGGATTTCTTCGAA-3⬘ HindIII " Step 3 is a recombinant PCR. b Oligonucleotides sequences used in PCR. a
was measured as described by Bradford [14], using bovine serum albumin as standard. Activity determination Fibrinolytic activity was determined on bovine Wbrin plates with an overnight incubation at 37 °C by comparison with International reference preparation for urokinase [15]. Amidolytic activity was measured before and after plasmin treatment by using chromogenic substrate S-2444 at 37 °C [16]. SDS–PAGE and Western blot analysis SDS–PAGE was performed according to Laemmii [17] with 5% stacking gels and 12% separating gels. Western blot analysis, utilizing a mouse monoclonal antibody against the B-chain of urokinase as the primary antibody and goat-anti-mouse IgG-alkaline phosphatase as the secondary antibody, was carried out as described by Towbin et al. [18]. Activation of plasminogen Activation of Glu-plasminogen (0.1–2 M) was measured with scu-PA or dscuPA33khC (10 nM) in 50 mM Tris–HCl, pH 7.4, 38 mM NaCl, and 0.01% Tween 80 at 37 °C. Generation of plasmin was monitored at 405 nm with S-2251 (1.5 mM). The reaction rate was measured by monitoring the OD increase over time squared as previously described [19]. The kinetic constants were determined from Lineweaver–Burk plots.
Inhibition of platelet aggregation Human blood was drawn on 3.8% sodium citrate from healthy volunteer who had not taken aspirin or related medication for at least two weeks. Blood was centrifuged at 1000g for 10 min at room temperature and platelet-rich plasma (PRP) was decanted. Platelet-poor plasma (PPP) was prepared from the remaining blood by centrifugation at 3000g for 20 min. The reaction mixture with scuPA-32k or dscuPA plus PRP (2 £ 108/ml) was incubated for 3 min at 37 °C. Light transmittance was recorded and ADP (10 M) was added to initiate platelet aggregation [6]. The inhibition of platelet aggregation was measured at the maximum aggregation response. Determination of platelet binding activity by ELISA Microtiter plates were Wrst coated overnight with the activated human platelets induced by thrombin solution (0.2 IU/ml), then blocked with 2 mg/ml BSA for 2 h. Add 100 l dscuPA33khC in the buVer A (50 mM Tris–HCl, 100 mM NaCl, 2 mM CaCl2, pH 7.4) and incubated it for 3 h at 37 °C. After washing the plate with PBST for three times, bound it with RAH urokinase multiclonal antibodies and HRP-SAR IgG orderly. Substrate solution of TMB was added to develop the color and was stopped with 1 M H2SO4. Finally, the optical density was read at 450 nm. We chose the scu-PA as the negative control. The protein concentration in the tests were 10, 1, and 0.1 g, respectively.
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Thrombin inhibition by dscuPA33khC Ki value for dscuPA33khC was determined as follows [20]. A 10¡9M solution of thrombin was prepared in the Tris buVer containing 50 mM Tris–HCl (pH 8.3), 100 mM NaCl, and 0.1% PEG6000 at 25 °C. Varying concentrations of dscuPA33khC were taken, and the assays were started by adding Chromozym TH. The recorded initial rate velocities were used to construct Dixon plots. The relationship of the inverse of the initial velocities versus inhibitor concentration was analyzed using linear regression, with the Ki value being determined by standard procedures using the equation that describes competitive inhibition [21]. Thrombin time test Platelet-poor plasma (PPP, 240 l) was mixed with 30 l physiological salt solution at 37 °C, then 30 l thrombin solution was added and clotting time was recorded. Adjustment of the thrombin concentration was made to restrict the thrombin time to 15 s. The thrombin time was determined by varying the concentration of dscuPA33khC and scu-PA. Measurement of aYnities with thrombus Fibrin clot was generated in the presence of 2 IU thrombin, 1.5 mg Wbrinogen, and 60 l PBS (pH 7.0). Platelet-rich clot was generated in the presence of 2 IU thrombin, 1.5 mg Wbrinogen, and 60 l human platelet. Scu-PA or dscuPA33khC were mixed with the clots, respectively, for 15 min at room temperature. The clots were washed subsequently with 0.1 M PBS containing 0.05 M NaCl for 10 min. Then, the clot and 0.9 U plasminogen were incubated in 0.3 ml of the same buVer at 37 °C for 1 h. The absorbance at 280 nm was measured and the mass of the absorbed proteins can be determined. Fibrinolytic eYciency Clot plus (1.5 mg) 1 U plasminogen and 200 IU urokinase or samples were incubated in 3 ml PBS (pH 7.0) at 37 °C for 1.5 h. The Wbrinolytic eYciencies of scu-PA and dscuPA33khC were compared with each other according to the absorbance of Wbrin at 280 nm. It was deWned to be at 100% when clot was lysed entirely [22].
Results Construction of the expression vector The plasmid pET29a was chosen to construct the expression vector pET29a-dscuPA33khC, mainly because
307
of the high expression level in E. coli that can be achieved using a T7 promoter. The chimeric cDNA was inserted into the appropriate sites of pET29a (the NdeI and HindIII sites). The DNA sequence, encoding the target protein of 342 amino acids, was veriWed by dideoxynucleotide sequencing. Expression and puriWcation The E. coli BL21-CodonPlus (DE3)-RIL strain containing the expression vector was cultivated in LB medium and induced with IPTG. The supernatant of the cell lysate was examined by SDS–PAGE and densitometric scanning (not shown) to check the expression level. The bacteria were grown at 37 °C until the OD600 reached 0.6. Protein expression was induced by addition of with 0.3 mM IPTG and cultured at 37 °C for 5 h. The cells were harvested and managed to increase the expression amount of dscuPA33khC to about 22% of total cellular proteins (Fig. 2A). Then expression was performed in 2YT medium and induced by IPTG. The cell lysate had nearly no u-PA activity, indicating that the chimeric protein was packaged in inclusion bodies. To obtain the active form of protein, the inclusion bodies were isolated, denatured, and refolded. The refolded product was puriWed by a combination of zinc chelate–Sepharose, SP Sepharose, and Sephacryl S-200 chromatography. After puriWcation, approximately 30.7 mg of dscuPA33khC protein was obtained from 1 L culture medium. Physicochemical characterization of puriWed decorsin/ scuPA-32k Analysis of puriWed dscuPA33khC by SDS–polyacrylamide gel electrophoresis (Fig. 2B) in the presence of dithiothreitol revealed a single protein band at 38 kDa. Its migration behavior suggests that dscuPA33khC was obtained as single-chain forms. Western blot (Fig. 2C) after SDS–PAGE under reducing condition revealed that the chimeric protein had a similar antigen binding capacity to urokinase. The Mr of dscuPA33khC obtained by MALDI-TOF analysis was 38,628 Da, which was consistent with the values calculated from amino acid sequence (38,616 Da). The speciWc activity of the puriWed dscuPA33khC was 11,000 IU/mg (means § SEM, n D 4) according to Wbrin plate determination (standard urokinase as the control). It showed that chimeric protein kept the enzymatic activity of scuPA-33k. The chimeric protein had low amidolytic activity (380 IU/mg) as determined with the substrate S-2444, however it increased dramatically when treated by plasmin. These data also demonstrated that the puriWed product was in the single-chain form.
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Fig. 2. SDS–PAGE and Western blot analyses of puriWed dscuPA33khC. (A) Coomassie blue-stained SDS–polyacrylamide gel. Lane 1 is the molecular weight marker. Lane 2 is bacterial sample after IPTG induction. Lane 3 is bacterial sample before IPTG induction. (B) Coomassie blue-stained SDS–polyacrylamide gel. Lane 1 is the puriWed dscuPA33khC. Lane 2 is the molecular weight marker. (C) Western blot probed with a mouse monoclonal antibody against the B-chain of urokinase. Table 2 Kinetics of plasminogen activation by scu-PA and dscuPA33khC Sample
Km (mol £ L¡1)
k2 (s¡1)
k2/Km (mol¡1 £ s¡1 £ L)
scu-PA dscuPA33khC
1.36 1.52
0.0028 0.0024
0.0021 0.0016
Data obtained from three experiments.
Activation of Glu-plasminogen Kinetic constants analysis revealed that the activation of plasminogen to plasmin by scu-PA or dscuPA33khC also obeyed Michaelis–Menten kinetics. Determined by linear regression analysis from Lineweaver–Burk plots, the kinetic constants (Table 2) were Km D 1.52 M and K2 D 0.0024 s¡1 for dscuPA33khC (n D 2, r D 0.99), and Km D 1.36 M and K2 D 0.0021 s¡1 for scu-PA (n D 2, r D 0.99). Thus the catalytic eYciency (K2/Km) of dscuPA33khC (0.016 M¡1 £ s¡1) was comparable to that of scu-PA (0.021 M ¡1 £ s¡1). Inhibition of platelet aggregation
Fig. 3. Inhibition of ADP-induced platelet aggregation. 䉫, dscuPA33khC; 䊐, scu-PA; and 䉭, decorsin. Platelets were stimulated with 10 M ADP. Data represent means § SEM obtained from four experiments.
As shown in Fig. 3, the dose-dependent inhibition of human platelet aggregation by dscuPA33khC was measured by inhibition of ADP-induced platelet aggregation in PRP. Inhibition of approximately 50% aggregation (IC50) was achieved at a concentration of approximate 0.35 M, with similar inhibition potency to that of decorsin (approximate 0.5 M). However, under the same condition, scu-PA had little potent inhibition on ADP-induced platelet aggregation. Platelet binding OD450 rose linearly with the increase of the concentration of dscuPA33khC (Fig. 4), which conWrmed the
Fig. 4. ELISA for the platelet binding. 䉭, dscuPA33khC; 䊐, scu-PA. Data represent means § SEM obtained from three experiments.
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platelet-binding ability of dscuPA33khC. As shown in Fig. 5, scu-PA had no platelet-binding activity. Thrombin inhibition by dscuPA33khC The dscuPA33khC was a competitive inhibitor to thrombin. Its Ki was 0.16 M, determined from the plot of 1/v against concentration of inhibitor (Dixon plot) as shown in Fig. 5. Thrombin time test The thrombin time was prolonged from 15 to 19 s by adding dscuPA33khC (0.12 M). However, scu-PA alone at the same concentration could not prolong the thrombin time. It reveals that after fusion into one protein, the thrombin inhibitory domain and the anti-platelet aggregation domain (decorsin) of the dscuPA33khC can function well. AYnities with thrombus The quantity of dscuPA33khC absorbed by platelet clot was higher than that of scu-PA when the amount of added proteins was similar (Fig. 6). However, the absorbed quantity of scu-PA or dscuPA33khC was comparable in Wbrin clot assays. It means that dscuPA33khC could bind to platelet clots more strongly and resolve a thrombus more eYciently. Fibrinolytic eYciency Whether the enhanced platelet binding ability by dscuPA33khC could be translated to a better clot lysis was investigated through a clot lysis assay. Data in Table 3 showed that the aYnities of the chimera towards platelet colts were higher than toward Wbrin clots, while the
Fig. 5. Dixon plot of thrombin inhibition by dscuPA33khC at three chromagenic substrate concentrations: (䉭) 20 M, (䊐) 40 M, and (䉫) 80 M. Points are experimental, and the lines represent the best Wt by linear regression. In all case, the linear regression coeYcients were over 0.99.
Fig. 6. Platelet or Wbrin clot binding of scu-PA or dscuPA33khC. 䉫, scuPA with Wbrin clot; 䊐, dscuPA33khC with Wbrin clot; 䉭, scu-PA with platelet clot; and *, dscuPA33khC with platelet clot. Data represent means § SEM obtained from three experiments. The quantity of dscuPA33khC absorbed by platelet clot was higher than that of scu-PA. Table 3 Lysis eYciency of scu-PA and dscuPA33khC Lysis eYciency scu-PA dscuPA33khC
Platelet-clot (%) 54 § 1
Fibrin-clot (%) 48 § 1.2
68 § 1.2
45 § 0.8
The entire lysis is set as 100%. Similar moles of samples were added to equal clots. Data are obtained from three experiments. It showed that dscuPA33khC was more speciWc to platelet-clot than to Wbrin-clot in comparison with scu-PA.
Wbrinolytic eYciencies of scu-PA on the two kinds of clot were similar. Furthermore, the dscuPA33khC kept a majority of speciWc aYnity with Wbrin in comparison with scu-PA.
Discussion Despite the wide use of thrombolytic agents, limitations have been observed in term of system haemorrhage and platelet-mediated reocclusion therapy of thromboembolic disorders can be improved by the application of proteins that simultaneously dissolve the thrombus and inhibit the formation of blood clots [23–25]. These goals can be achieved by the combination of a plasminogen activator, platelet aggregation inhibitor, and thrombin inhibitor to one molecular [22]. In this report, scuPA-33k is the part of thrombolytic agent that can activate plasminogen to plasmin. Decorsin is an RGD-containing protein, which is characterized as a potent antagonist of platelet protein GpIIb/IIIa. The C-terminus of hirudin, thrombin active site motif, and part of thrombin receptor provided the function of a potent and speciWc thrombin inhibitor. After denaturing the inclusion body, refolding, and puriWcation, the speciWc activity of the chimeric protein
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was 11,000 IU/mg, revealing that the chimera maintained the speciWc Wbrinolytic activity. The activation of plasminogen by dscuPA33khC conWrmed that it had an appreciable intrinsic plasminogen activity. The lower K2/Km suggested that the dscuPA33khC was somehow impaired in its enzymatic activity. Perhaps the reason of lower K2/Km was that the decorsin and thrombin inhibitory domain interacted the scuPA-33k domain. The freshly formed blood clots are rich in active platelet. ELISA for the platelet binding indicted that dscuPA33khC could bind the active platelet in a dosedependent manner. Platelet or Wbrin clot binding assays suggested that dscuPA33khC could bind the platelet clot with a higher aYnity. Platelet clot lysis eYciency of dscuPA33khC was higher than that of Wbrin clot lysis, while there were no signiWcant diVerences between the two clots for scu-PA. Based on these results, we can conclude that the fusion protein accumulates on the platelet surface and may enrich the thrombin-inhibitory activity at the site of the thrombus, thus preventing more general systemic anticoagulant activity. Furthermore, if less dscuPA33khC is required to achieve the same degree of clot lysis, the risk for side eVects could be minimized. The thrombin time was prolonged by dscuPA33khC. The fusion protein was enriched on the platelet surface and showed more favorable kinetics of plasmin formation, which was the partial reason why thrombin time prolonged. There were two reasons for anti-thrombus function of the fusion protein. One was that dscuPA33khC showed high inhibition eVect on the ADPinduced platelet aggregation. The other is that it had signiWcant inhibitory eVect on thrombin. As a result, it would allow the use of less frequent administration of the agent. It is known that simple RGD containing peptides are able to inhibit platelet aggregation via their interaction with GPIIb–IIIa; however these peptides are considerably less potent than decorsin. Not only the RGD motif sequence, but also its conformation (maintained by the appropriate cysteine paring or located at the top of a mobile loop) was important for the disintegrin activity. The higher aYnity observed for decorsin peptide is likely due to a speciWc conformation of it. DscuPA33khC has the higher platelet aggregation inhibitory activity than that of previous chimeras [22]. Our study provides the Wrst piece of evidence to show that the scuPA can be further improved by introducing the full decorsin, in which the RGD motif has appropriate conformation. Each functional domain in dscuPA33khC was preserved. The present study demonstrates that dscuPA33khC not only exhibits higher platelet-targeted thrombolytic activity compared with scu-PA but also obtains anti-thrombus function. Further testing in experimental animal models will assess the potential of dscuPA33khC as an improved thrombolytic agent.
Acknowledgment This work is supported by grant from National Natural Science Foundation of China (No.30200057). References [1] D. Collen, Thrombolytic therapy, Throm. Haemost. 78 (1997) 742–746. [2] H.R. Lijnen, S. Wnendt, J. Schneider, E. Janocha, B. Van Hoef, D. Collen, G.J. SteVens, Function properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogenactivating potential, Eur. J. Biochem. 234 (1995) 350–357. [3] W.E. Holmes, D. Pennica, M. Blaber, M.W. Rey, W.W. Guenzler, G.J. SteVens, H.L. Heyneker, Cloning and expression of the gene for pro-urokinase in Escherichia coli, Biotechnology 3 (1985) 923–929. [4] G.A.W. De Munk, D.C. Rijken, Fibrinolytic properties of single chain urokinase-type plasminogen activator (pro-urokinase), Fibrinolysis 4 (1990) 1–9. [5] G.J. SteVens, W.A. Gunzler, F. Otting, E. Frankus, L. Flohe, The complete amino acidsequence of low molecular mass urokinase from human urine, H-S. Z. Physiol. Chem. 363 (1982) 1043–1058. [6] J.L. Seymour, W.J. Henzel, B. Nevins, J.T. Stults, R.A. Lazarus, Decorsin—a potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech Macrobdella decora, J. Biol. Chem. 265 (1990) 10143–10147. [7] A.M. Krezel, G. Wagner, J. Seymour-Ulmer, R.A. Lazarus, Structure of the RGD protein decorsin: conserved motif and distinct function in leech proteins that aVect blood clotting, Science 264 (1994) 1944–1947. [8] P. Polverino de Laureto, E. Scaramella, V. De Filippis, O. Marin, M.G. Doni, A. Fontana, Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation, Protein Sci. 7 (1998) 433–444. [9] J. Schneider, R. Hauser, H.-H. Hennies, J. Korioth, G. SteVens, S. Wnendt, A novel chimaric derivative of saruplase, rscu-PA-40kD/ Hir, binds to thrombin and exerts thrombus-speciWc Wbrinolysis in arterial and venous thrombosis in dogs, Thromb. Haemast. 77 (1997) 535–539. [10] H.D. White, C.J. Ellis, J.K. French, P. Aylward, Hirudin (desirudin) and hirulog (bivalirudin) in acute ischaemic syndromes and the rationale for the hirulog/early reperfusion occlusion (HER02) study, NZ. J. Med. 28 (1998) 551–554. [11] J.Y. Chang, Production, properties, and thrombin inhibitory mechanism of hirudin amino-terminal core fragments, J. Biol. Chem. 265 (1990) 22159–22166. [12] M.A. Innis, K.B. Myambo, D.H. Gelfand, M.A. Brow, DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-ampliWed DNA, Proc. Natl. Acad. Sci. 85 (1988) 9436–9440. [13] Jiao Jianwei, Yu Meimin, Ru Binggen, Construction and characterization of a recombinant chimeric plasminogen activator consisting of a Wbrin peptide and a low molecular mass single-chain urokinase, Biochimie 83 (2001) 1049–1055. [14] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [15] T. Astrup, S. Müllertz, The Wbrin plate method for estimating the Wbrinolytic activity, Arch. Biochem. Biophys. 40 (1952) 346– 351. [16] T.C. Wun, W.D. Schleuning, E. Reich, Isolation and characterization of urokinase from human plasma, J. Biol. Chem. 257 (1982) 3276–3283. [17] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685.
L. Zhang et al. / Molecular Genetics and Metabolism 82 (2004) 304–311 [18] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350–4354. [19] Dang Xin, Ji Jianguo, Ru Qiang, Yang Jingxin, Yu Meimin, Ru Binggen, A new method of kinetic study on the react ion of plasminogen activation, Chin. J. Biochem. Mol. Biol. 17 (2001) 75–79. [20] J.L. Richardson, P. Fuentes-Prior, J. Evan Sadler, R. Huber, W. Bode, Characterization of the residues involved in the human Thrombin–haemadin complex: an exosite II-binding inhibitor, Biochemistry 41 (2002) 2535–2542. [21] I.H. Segel, Enzyme Kinetics, Wiley, New York, 1975. [22] Q. Bi, X.D. Cen, Y.X. Huang, S.G. Zhu, Construction and characterization of trifunctional single-chain urokinase-type plasmino-
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gen activators-trifunctional plasminogen activators, Eur. J. Biochem. 269 (2002) 1708–1713. [23] Q. Lian, S.J. Szarka, K.K.S. Ng, S.L. Wong, Engineering of a staphylokinase-based Wbrinolytic agent with antithrombotic activity and targeting capability toward thrombin-rich Wbrin and plasma clots, J. Biol. Chem. 278 (2003) 26677–26686. [24] C. Icke, B. Schlott, O. Ohlenschlager, M. Hartmann, K.H. Guhrs, E. Glusa, Fusion proteins with anticoagulant and Wbrinolytic properties: functional studies and structural considerations, Mol. Pharmacol. 62 (2002) 203–209. [25] F. Wirsching, C. Luge, A. Schwienhorst, Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential, Mol. Genet. Metab. 75 (2002) 250–259.
Functional properties of a recombinant chimeric plasminogen activator with platelet-targeted Wbrinolytic and anticoagulant potential Leiliang Zhang, Jing Wang, Meimin Yu, and Binggen Ru¤ National Lab of Protein Engineering, College of Life Sciences, Peking University, Beijing 100871, PR China Received 22 February 2004; received in revised form 20 May 2004; accepted 20 May 2004 Available online 2 July 2004
Abstract The construction, puriWcation, and characterization of dscuPA33khC, a bifunctional protein designed for thrombosis treatment is described. The chimera was designed to consist of a decorsin (platelet aggregation inhibitor), a low molecular mass (33 kDa) singlechain urokinase (scuPA-33k), and a thrombin inhibitory domain. We have successfully produced this recombinant protein in the Escherichia coli expression system, in which the target protein exists in the form of inclusion bodies. After refolding by dilution in vitro, the chimeric protein was puriWed to homogeneity by immobilized metal aYnity chromatography, ion-exchange chromatography, and gel Wltration chromatography. The dscuPA33khC could directly activate plasminogen following Michaelis–Menten kinetics with Km D 1.52 M and K2 D 0.0024 s¡1. The speciWc activity of the chimera detected by Wbrin plate determination was 11,000 IU/mg, which suggested a high thrombolysis eVect. However, the chimeric dscuPA33khC bound the activated platelet and signiWcantly increased aYnity to platelet clots as compared to Wbrin clots. It was found to inhibit ADP-induced platelet aggregation in a concentration-dependent manner as well as it exhibits antithrombin activity. These results suggest that the chimeric protein not only has platelet-targeted thrombolytic activity but also obtains anti-thrombus function. 2004 Elsevier Inc. All rights reserved. Keywords: Decorsin; Hirudin; Platelet-targeted; Antithrombin activity
Introduction Urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) have been proved eVective in the treatment of myocardial infarction, but the agents have certain limitations such as bleeding or platelet-mediated reocclusion [1]. To solve these problems in thrombolytic therapy, the plasminogen activators should be combined with antiplatelet agents or strategies [2]. Based on these considerations, we want to construct a recombinant chimeric plasminogen activator with platelet inhibitory activity. The single chain urokinase (scu-PA) is composed of 411 amino acids with Mr 54,000, which can be converted
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Corresponding author. Fax: +86-10-62751842. E-mail address: [email protected] (B. Ru).
1096-7192/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.05.008
to two-chain urokinase (tcuPA) with speciWc cleavage of Lys158-Ile159 by plasmin [3]. Scu-PA has a higher speciWc thrombolytic activity and a better selectivity for Wbrin than tcu-PA [4]. A low molecular weight form of scu-PA (scu-PA-33K), lacking the 135 NH2-terminal amino acids, has been obtained from cleavage between residues Lys135 and Lys136 [5]. ScuPA-33k has similar properties to scu-PA and can be used as a plasminogen activator. Decorsin is a 39-residue protein isolated from the leech Macrobdella decora, containing three intramolecular disulWde bonds and Arg-Gly-Asp (RGD) sequence [6,7]. Decorsin blocks Wbrinogen binding to the platelet glycoprotein IIb/IIIa (GpIIb/IIIa) and acts as a potent inhibitor of platelet aggregation [8]. Hirudin is the most potent thrombin-speciWc inhibitor, as it acts on both circulating and clot-bound thrombin [9,10]. Its N-terminal domain binds to the active site
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Fig. 1. Schematic representation of the composition of dscuPA33khC and the function of the individual components. The decorsin is linked to scuPA-33k and a thrombin inhibitory domain. The thrombin inhibitory domain comprised a 5-aa linker, a thrombin active site motif, part sequences of thrombin receptor, and C-terminal of hirudin.
of thrombin, whereas its C-terminal fragment covers the recognition site of Wbrinogen [11]. C-terminal fragments of hirudin can be used as thrombin inhibitors [2]. We designed a new polypeptide to combine Wbrinolytic activity, platelet aggregation inhibitory and thrombin inhibitory activity into one molecule. In this study (Fig. 1), a thrombin inhibitory domain was fused to the C-terminus of scuPA-33k; meanwhile decorsin was fused to the N-terminus of scuPA-33k. The thrombin inhibitory domain comprised a 5-aa linker (SPVVA), a thrombin active site motif (FPRP), partial sequences of thrombin receptor (FLLRNP), and the C-terminus (residues 54–65) of hirudin. It is expected that this chimeric plasminogen activator may have platelet-targeted thrombolytic activity with anti-thrombus function.
SP Sepharose, and Sephacryl S-200 were purchased from Amersham Biosciences (Piscataway, USA). Construction of E. coli expression vector of dscuPA33khC
Materials and methods
The DNA fragment encoding decorsin was obtained by PCR from the plasmid pMAL-decorsin with the primers A and B. The DNA fragment encoding scuPA-33k (Lys136–Leu411) was obtained by PCR from the plasmid pUC19-scuPA with primers C and D. The DNA fragment encoding the part of thrombin inhibitor was obtained from the synthesized three oligonucleotides: E, F, and G. Following, the chimeric cDNA (dscuPA33khC) was obtained by PCR (Table 1). Finally the chimeric cDNA was cloned into the pET29a vector from the NdeI and HindIII sites. The sequence of the desired recombinant vector pET29a-dscuPA33khC conWrmed by restriction analyses and dideoxynucleotide sequencing [12].
Materials
Expression and puriWcation of the chimeric protein
Plasmid pET-29a was purchased from Novagen (Madison, USA). Escherichia coli BL21-CodonPlus (DE3)-RIL was purchased from Stratagene (La Jolla, USA). IPTG, GSH, and GSSG were purchased from Promega (Madison, USA). Fibrinogen, plasminogen, plasmin, and thrombin were purchased from Sigma (St. Louis, USA). S-2444 (L-pyroGlu-Gly-L-Arg-p-nitroanilide) and S-2251 (D-Val-Leu-Lys-p-nitroanilide) were purchased from Kabi Vitrum (Stockholm, Sweden). Chromozym TH was purchased from Roche (Basel, Switzerland). Urokinase standard and scu-PA were obtained from NICPBP (Beijing, China). Zinc chelate–Sepharose,
The recombinant vector was transformed into E. coli BL21-CodonPlus (DE3)-RIL. The protein expression and refolding was performed as described by Jiao [13]. The refolding mixture was applied to a zinc chelate– Sepharose column equilibrated with 20 mM Tris–HCl, 500 mM NaCl, 0.01% Tween 80, pH 7.4, and eluted with the same buVer containing 50 mM imidazole. After ultraWltration the concentrated sample was subjected to SP Sepharose column in 50 mM NaAc (pH 4.5), with a linear gradient of 0–1.0 M NaCl. Then the sample was applied to Sephacryl S-200, which was developed with 20 mM Tris–HCl, 50 mM NaCl, pH 7.4. Protein concentration
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Table 1 The PCR steps of the chimeric cDNA of dscuPA33khC Step
Primersb
Template
Product
1 2 3a 4 5 6
A, B C, D A, D A, E A, F A, G
pMAL-decorsin pUC19-scuPA I, II III IV V
I II III IV V Final chimeric cDNA (dscuPA33khC)
A: 5⬘-GGGAATTCCATATGGCTCCACGTCTG-3⬘ NdeI " B: 5⬘-TTCTTCCGGCGGGCTGCTCGGTTTTTCACAGTATGGATC-3⬘ C: 5⬘-AGCAGCCCGCCGGAAGAATTAAAATTTCAGTGT-3⬘ D: 5⬘-TGATCAAGCTTCTGATGGA-3⬘ E: 5⬘-CAGGAACGGTCTCGGGAAAGCAACAACCGGGCTGAGGGCCAGGCCATT-3⬘ F: 5⬘-CGGGATTTCTTCGAAGTCACCCGGATTTCTCAGCAGGAACGGTCTCG-3⬘ G: 5⬘-TGACTAAGCTTTTACTGCAGGTATTCTTCCGGGATTTCTTCGAA-3⬘ HindIII " Step 3 is a recombinant PCR. b Oligonucleotides sequences used in PCR. a
was measured as described by Bradford [14], using bovine serum albumin as standard. Activity determination Fibrinolytic activity was determined on bovine Wbrin plates with an overnight incubation at 37 °C by comparison with International reference preparation for urokinase [15]. Amidolytic activity was measured before and after plasmin treatment by using chromogenic substrate S-2444 at 37 °C [16]. SDS–PAGE and Western blot analysis SDS–PAGE was performed according to Laemmii [17] with 5% stacking gels and 12% separating gels. Western blot analysis, utilizing a mouse monoclonal antibody against the B-chain of urokinase as the primary antibody and goat-anti-mouse IgG-alkaline phosphatase as the secondary antibody, was carried out as described by Towbin et al. [18]. Activation of plasminogen Activation of Glu-plasminogen (0.1–2 M) was measured with scu-PA or dscuPA33khC (10 nM) in 50 mM Tris–HCl, pH 7.4, 38 mM NaCl, and 0.01% Tween 80 at 37 °C. Generation of plasmin was monitored at 405 nm with S-2251 (1.5 mM). The reaction rate was measured by monitoring the OD increase over time squared as previously described [19]. The kinetic constants were determined from Lineweaver–Burk plots.
Inhibition of platelet aggregation Human blood was drawn on 3.8% sodium citrate from healthy volunteer who had not taken aspirin or related medication for at least two weeks. Blood was centrifuged at 1000g for 10 min at room temperature and platelet-rich plasma (PRP) was decanted. Platelet-poor plasma (PPP) was prepared from the remaining blood by centrifugation at 3000g for 20 min. The reaction mixture with scuPA-32k or dscuPA plus PRP (2 £ 108/ml) was incubated for 3 min at 37 °C. Light transmittance was recorded and ADP (10 M) was added to initiate platelet aggregation [6]. The inhibition of platelet aggregation was measured at the maximum aggregation response. Determination of platelet binding activity by ELISA Microtiter plates were Wrst coated overnight with the activated human platelets induced by thrombin solution (0.2 IU/ml), then blocked with 2 mg/ml BSA for 2 h. Add 100 l dscuPA33khC in the buVer A (50 mM Tris–HCl, 100 mM NaCl, 2 mM CaCl2, pH 7.4) and incubated it for 3 h at 37 °C. After washing the plate with PBST for three times, bound it with RAH urokinase multiclonal antibodies and HRP-SAR IgG orderly. Substrate solution of TMB was added to develop the color and was stopped with 1 M H2SO4. Finally, the optical density was read at 450 nm. We chose the scu-PA as the negative control. The protein concentration in the tests were 10, 1, and 0.1 g, respectively.
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Thrombin inhibition by dscuPA33khC Ki value for dscuPA33khC was determined as follows [20]. A 10¡9M solution of thrombin was prepared in the Tris buVer containing 50 mM Tris–HCl (pH 8.3), 100 mM NaCl, and 0.1% PEG6000 at 25 °C. Varying concentrations of dscuPA33khC were taken, and the assays were started by adding Chromozym TH. The recorded initial rate velocities were used to construct Dixon plots. The relationship of the inverse of the initial velocities versus inhibitor concentration was analyzed using linear regression, with the Ki value being determined by standard procedures using the equation that describes competitive inhibition [21]. Thrombin time test Platelet-poor plasma (PPP, 240 l) was mixed with 30 l physiological salt solution at 37 °C, then 30 l thrombin solution was added and clotting time was recorded. Adjustment of the thrombin concentration was made to restrict the thrombin time to 15 s. The thrombin time was determined by varying the concentration of dscuPA33khC and scu-PA. Measurement of aYnities with thrombus Fibrin clot was generated in the presence of 2 IU thrombin, 1.5 mg Wbrinogen, and 60 l PBS (pH 7.0). Platelet-rich clot was generated in the presence of 2 IU thrombin, 1.5 mg Wbrinogen, and 60 l human platelet. Scu-PA or dscuPA33khC were mixed with the clots, respectively, for 15 min at room temperature. The clots were washed subsequently with 0.1 M PBS containing 0.05 M NaCl for 10 min. Then, the clot and 0.9 U plasminogen were incubated in 0.3 ml of the same buVer at 37 °C for 1 h. The absorbance at 280 nm was measured and the mass of the absorbed proteins can be determined. Fibrinolytic eYciency Clot plus (1.5 mg) 1 U plasminogen and 200 IU urokinase or samples were incubated in 3 ml PBS (pH 7.0) at 37 °C for 1.5 h. The Wbrinolytic eYciencies of scu-PA and dscuPA33khC were compared with each other according to the absorbance of Wbrin at 280 nm. It was deWned to be at 100% when clot was lysed entirely [22].
Results Construction of the expression vector The plasmid pET29a was chosen to construct the expression vector pET29a-dscuPA33khC, mainly because
307
of the high expression level in E. coli that can be achieved using a T7 promoter. The chimeric cDNA was inserted into the appropriate sites of pET29a (the NdeI and HindIII sites). The DNA sequence, encoding the target protein of 342 amino acids, was veriWed by dideoxynucleotide sequencing. Expression and puriWcation The E. coli BL21-CodonPlus (DE3)-RIL strain containing the expression vector was cultivated in LB medium and induced with IPTG. The supernatant of the cell lysate was examined by SDS–PAGE and densitometric scanning (not shown) to check the expression level. The bacteria were grown at 37 °C until the OD600 reached 0.6. Protein expression was induced by addition of with 0.3 mM IPTG and cultured at 37 °C for 5 h. The cells were harvested and managed to increase the expression amount of dscuPA33khC to about 22% of total cellular proteins (Fig. 2A). Then expression was performed in 2YT medium and induced by IPTG. The cell lysate had nearly no u-PA activity, indicating that the chimeric protein was packaged in inclusion bodies. To obtain the active form of protein, the inclusion bodies were isolated, denatured, and refolded. The refolded product was puriWed by a combination of zinc chelate–Sepharose, SP Sepharose, and Sephacryl S-200 chromatography. After puriWcation, approximately 30.7 mg of dscuPA33khC protein was obtained from 1 L culture medium. Physicochemical characterization of puriWed decorsin/ scuPA-32k Analysis of puriWed dscuPA33khC by SDS–polyacrylamide gel electrophoresis (Fig. 2B) in the presence of dithiothreitol revealed a single protein band at 38 kDa. Its migration behavior suggests that dscuPA33khC was obtained as single-chain forms. Western blot (Fig. 2C) after SDS–PAGE under reducing condition revealed that the chimeric protein had a similar antigen binding capacity to urokinase. The Mr of dscuPA33khC obtained by MALDI-TOF analysis was 38,628 Da, which was consistent with the values calculated from amino acid sequence (38,616 Da). The speciWc activity of the puriWed dscuPA33khC was 11,000 IU/mg (means § SEM, n D 4) according to Wbrin plate determination (standard urokinase as the control). It showed that chimeric protein kept the enzymatic activity of scuPA-33k. The chimeric protein had low amidolytic activity (380 IU/mg) as determined with the substrate S-2444, however it increased dramatically when treated by plasmin. These data also demonstrated that the puriWed product was in the single-chain form.
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Fig. 2. SDS–PAGE and Western blot analyses of puriWed dscuPA33khC. (A) Coomassie blue-stained SDS–polyacrylamide gel. Lane 1 is the molecular weight marker. Lane 2 is bacterial sample after IPTG induction. Lane 3 is bacterial sample before IPTG induction. (B) Coomassie blue-stained SDS–polyacrylamide gel. Lane 1 is the puriWed dscuPA33khC. Lane 2 is the molecular weight marker. (C) Western blot probed with a mouse monoclonal antibody against the B-chain of urokinase. Table 2 Kinetics of plasminogen activation by scu-PA and dscuPA33khC Sample
Km (mol £ L¡1)
k2 (s¡1)
k2/Km (mol¡1 £ s¡1 £ L)
scu-PA dscuPA33khC
1.36 1.52
0.0028 0.0024
0.0021 0.0016
Data obtained from three experiments.
Activation of Glu-plasminogen Kinetic constants analysis revealed that the activation of plasminogen to plasmin by scu-PA or dscuPA33khC also obeyed Michaelis–Menten kinetics. Determined by linear regression analysis from Lineweaver–Burk plots, the kinetic constants (Table 2) were Km D 1.52 M and K2 D 0.0024 s¡1 for dscuPA33khC (n D 2, r D 0.99), and Km D 1.36 M and K2 D 0.0021 s¡1 for scu-PA (n D 2, r D 0.99). Thus the catalytic eYciency (K2/Km) of dscuPA33khC (0.016 M¡1 £ s¡1) was comparable to that of scu-PA (0.021 M ¡1 £ s¡1). Inhibition of platelet aggregation
Fig. 3. Inhibition of ADP-induced platelet aggregation. 䉫, dscuPA33khC; 䊐, scu-PA; and 䉭, decorsin. Platelets were stimulated with 10 M ADP. Data represent means § SEM obtained from four experiments.
As shown in Fig. 3, the dose-dependent inhibition of human platelet aggregation by dscuPA33khC was measured by inhibition of ADP-induced platelet aggregation in PRP. Inhibition of approximately 50% aggregation (IC50) was achieved at a concentration of approximate 0.35 M, with similar inhibition potency to that of decorsin (approximate 0.5 M). However, under the same condition, scu-PA had little potent inhibition on ADP-induced platelet aggregation. Platelet binding OD450 rose linearly with the increase of the concentration of dscuPA33khC (Fig. 4), which conWrmed the
Fig. 4. ELISA for the platelet binding. 䉭, dscuPA33khC; 䊐, scu-PA. Data represent means § SEM obtained from three experiments.
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platelet-binding ability of dscuPA33khC. As shown in Fig. 5, scu-PA had no platelet-binding activity. Thrombin inhibition by dscuPA33khC The dscuPA33khC was a competitive inhibitor to thrombin. Its Ki was 0.16 M, determined from the plot of 1/v against concentration of inhibitor (Dixon plot) as shown in Fig. 5. Thrombin time test The thrombin time was prolonged from 15 to 19 s by adding dscuPA33khC (0.12 M). However, scu-PA alone at the same concentration could not prolong the thrombin time. It reveals that after fusion into one protein, the thrombin inhibitory domain and the anti-platelet aggregation domain (decorsin) of the dscuPA33khC can function well. AYnities with thrombus The quantity of dscuPA33khC absorbed by platelet clot was higher than that of scu-PA when the amount of added proteins was similar (Fig. 6). However, the absorbed quantity of scu-PA or dscuPA33khC was comparable in Wbrin clot assays. It means that dscuPA33khC could bind to platelet clots more strongly and resolve a thrombus more eYciently. Fibrinolytic eYciency Whether the enhanced platelet binding ability by dscuPA33khC could be translated to a better clot lysis was investigated through a clot lysis assay. Data in Table 3 showed that the aYnities of the chimera towards platelet colts were higher than toward Wbrin clots, while the
Fig. 5. Dixon plot of thrombin inhibition by dscuPA33khC at three chromagenic substrate concentrations: (䉭) 20 M, (䊐) 40 M, and (䉫) 80 M. Points are experimental, and the lines represent the best Wt by linear regression. In all case, the linear regression coeYcients were over 0.99.
Fig. 6. Platelet or Wbrin clot binding of scu-PA or dscuPA33khC. 䉫, scuPA with Wbrin clot; 䊐, dscuPA33khC with Wbrin clot; 䉭, scu-PA with platelet clot; and *, dscuPA33khC with platelet clot. Data represent means § SEM obtained from three experiments. The quantity of dscuPA33khC absorbed by platelet clot was higher than that of scu-PA. Table 3 Lysis eYciency of scu-PA and dscuPA33khC Lysis eYciency scu-PA dscuPA33khC
Platelet-clot (%) 54 § 1
Fibrin-clot (%) 48 § 1.2
68 § 1.2
45 § 0.8
The entire lysis is set as 100%. Similar moles of samples were added to equal clots. Data are obtained from three experiments. It showed that dscuPA33khC was more speciWc to platelet-clot than to Wbrin-clot in comparison with scu-PA.
Wbrinolytic eYciencies of scu-PA on the two kinds of clot were similar. Furthermore, the dscuPA33khC kept a majority of speciWc aYnity with Wbrin in comparison with scu-PA.
Discussion Despite the wide use of thrombolytic agents, limitations have been observed in term of system haemorrhage and platelet-mediated reocclusion therapy of thromboembolic disorders can be improved by the application of proteins that simultaneously dissolve the thrombus and inhibit the formation of blood clots [23–25]. These goals can be achieved by the combination of a plasminogen activator, platelet aggregation inhibitor, and thrombin inhibitor to one molecular [22]. In this report, scuPA-33k is the part of thrombolytic agent that can activate plasminogen to plasmin. Decorsin is an RGD-containing protein, which is characterized as a potent antagonist of platelet protein GpIIb/IIIa. The C-terminus of hirudin, thrombin active site motif, and part of thrombin receptor provided the function of a potent and speciWc thrombin inhibitor. After denaturing the inclusion body, refolding, and puriWcation, the speciWc activity of the chimeric protein
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was 11,000 IU/mg, revealing that the chimera maintained the speciWc Wbrinolytic activity. The activation of plasminogen by dscuPA33khC conWrmed that it had an appreciable intrinsic plasminogen activity. The lower K2/Km suggested that the dscuPA33khC was somehow impaired in its enzymatic activity. Perhaps the reason of lower K2/Km was that the decorsin and thrombin inhibitory domain interacted the scuPA-33k domain. The freshly formed blood clots are rich in active platelet. ELISA for the platelet binding indicted that dscuPA33khC could bind the active platelet in a dosedependent manner. Platelet or Wbrin clot binding assays suggested that dscuPA33khC could bind the platelet clot with a higher aYnity. Platelet clot lysis eYciency of dscuPA33khC was higher than that of Wbrin clot lysis, while there were no signiWcant diVerences between the two clots for scu-PA. Based on these results, we can conclude that the fusion protein accumulates on the platelet surface and may enrich the thrombin-inhibitory activity at the site of the thrombus, thus preventing more general systemic anticoagulant activity. Furthermore, if less dscuPA33khC is required to achieve the same degree of clot lysis, the risk for side eVects could be minimized. The thrombin time was prolonged by dscuPA33khC. The fusion protein was enriched on the platelet surface and showed more favorable kinetics of plasmin formation, which was the partial reason why thrombin time prolonged. There were two reasons for anti-thrombus function of the fusion protein. One was that dscuPA33khC showed high inhibition eVect on the ADPinduced platelet aggregation. The other is that it had signiWcant inhibitory eVect on thrombin. As a result, it would allow the use of less frequent administration of the agent. It is known that simple RGD containing peptides are able to inhibit platelet aggregation via their interaction with GPIIb–IIIa; however these peptides are considerably less potent than decorsin. Not only the RGD motif sequence, but also its conformation (maintained by the appropriate cysteine paring or located at the top of a mobile loop) was important for the disintegrin activity. The higher aYnity observed for decorsin peptide is likely due to a speciWc conformation of it. DscuPA33khC has the higher platelet aggregation inhibitory activity than that of previous chimeras [22]. Our study provides the Wrst piece of evidence to show that the scuPA can be further improved by introducing the full decorsin, in which the RGD motif has appropriate conformation. Each functional domain in dscuPA33khC was preserved. The present study demonstrates that dscuPA33khC not only exhibits higher platelet-targeted thrombolytic activity compared with scu-PA but also obtains anti-thrombus function. Further testing in experimental animal models will assess the potential of dscuPA33khC as an improved thrombolytic agent.
Acknowledgment This work is supported by grant from National Natural Science Foundation of China (No.30200057). References [1] D. Collen, Thrombolytic therapy, Throm. Haemost. 78 (1997) 742–746. [2] H.R. Lijnen, S. Wnendt, J. Schneider, E. Janocha, B. Van Hoef, D. Collen, G.J. SteVens, Function properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogenactivating potential, Eur. J. Biochem. 234 (1995) 350–357. [3] W.E. Holmes, D. Pennica, M. Blaber, M.W. Rey, W.W. Guenzler, G.J. SteVens, H.L. Heyneker, Cloning and expression of the gene for pro-urokinase in Escherichia coli, Biotechnology 3 (1985) 923–929. [4] G.A.W. De Munk, D.C. Rijken, Fibrinolytic properties of single chain urokinase-type plasminogen activator (pro-urokinase), Fibrinolysis 4 (1990) 1–9. [5] G.J. SteVens, W.A. Gunzler, F. Otting, E. Frankus, L. Flohe, The complete amino acidsequence of low molecular mass urokinase from human urine, H-S. Z. Physiol. Chem. 363 (1982) 1043–1058. [6] J.L. Seymour, W.J. Henzel, B. Nevins, J.T. Stults, R.A. Lazarus, Decorsin—a potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech Macrobdella decora, J. Biol. Chem. 265 (1990) 10143–10147. [7] A.M. Krezel, G. Wagner, J. Seymour-Ulmer, R.A. Lazarus, Structure of the RGD protein decorsin: conserved motif and distinct function in leech proteins that aVect blood clotting, Science 264 (1994) 1944–1947. [8] P. Polverino de Laureto, E. Scaramella, V. De Filippis, O. Marin, M.G. Doni, A. Fontana, Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation, Protein Sci. 7 (1998) 433–444. [9] J. Schneider, R. Hauser, H.-H. Hennies, J. Korioth, G. SteVens, S. Wnendt, A novel chimaric derivative of saruplase, rscu-PA-40kD/ Hir, binds to thrombin and exerts thrombus-speciWc Wbrinolysis in arterial and venous thrombosis in dogs, Thromb. Haemast. 77 (1997) 535–539. [10] H.D. White, C.J. Ellis, J.K. French, P. Aylward, Hirudin (desirudin) and hirulog (bivalirudin) in acute ischaemic syndromes and the rationale for the hirulog/early reperfusion occlusion (HER02) study, NZ. J. Med. 28 (1998) 551–554. [11] J.Y. Chang, Production, properties, and thrombin inhibitory mechanism of hirudin amino-terminal core fragments, J. Biol. Chem. 265 (1990) 22159–22166. [12] M.A. Innis, K.B. Myambo, D.H. Gelfand, M.A. Brow, DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-ampliWed DNA, Proc. Natl. Acad. Sci. 85 (1988) 9436–9440. [13] Jiao Jianwei, Yu Meimin, Ru Binggen, Construction and characterization of a recombinant chimeric plasminogen activator consisting of a Wbrin peptide and a low molecular mass single-chain urokinase, Biochimie 83 (2001) 1049–1055. [14] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [15] T. Astrup, S. Müllertz, The Wbrin plate method for estimating the Wbrinolytic activity, Arch. Biochem. Biophys. 40 (1952) 346– 351. [16] T.C. Wun, W.D. Schleuning, E. Reich, Isolation and characterization of urokinase from human plasma, J. Biol. Chem. 257 (1982) 3276–3283. [17] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685.
L. Zhang et al. / Molecular Genetics and Metabolism 82 (2004) 304–311 [18] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350–4354. [19] Dang Xin, Ji Jianguo, Ru Qiang, Yang Jingxin, Yu Meimin, Ru Binggen, A new method of kinetic study on the react ion of plasminogen activation, Chin. J. Biochem. Mol. Biol. 17 (2001) 75–79. [20] J.L. Richardson, P. Fuentes-Prior, J. Evan Sadler, R. Huber, W. Bode, Characterization of the residues involved in the human Thrombin–haemadin complex: an exosite II-binding inhibitor, Biochemistry 41 (2002) 2535–2542. [21] I.H. Segel, Enzyme Kinetics, Wiley, New York, 1975. [22] Q. Bi, X.D. Cen, Y.X. Huang, S.G. Zhu, Construction and characterization of trifunctional single-chain urokinase-type plasmino-
311
gen activators-trifunctional plasminogen activators, Eur. J. Biochem. 269 (2002) 1708–1713. [23] Q. Lian, S.J. Szarka, K.K.S. Ng, S.L. Wong, Engineering of a staphylokinase-based Wbrinolytic agent with antithrombotic activity and targeting capability toward thrombin-rich Wbrin and plasma clots, J. Biol. Chem. 278 (2003) 26677–26686. [24] C. Icke, B. Schlott, O. Ohlenschlager, M. Hartmann, K.H. Guhrs, E. Glusa, Fusion proteins with anticoagulant and Wbrinolytic properties: functional studies and structural considerations, Mol. Pharmacol. 62 (2002) 203–209. [25] F. Wirsching, C. Luge, A. Schwienhorst, Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential, Mol. Genet. Metab. 75 (2002) 250–259.