Vol. 39, No. 4, July 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 797-803 A LOW MOLECULAR WEIGHT UROKINASE DERIVATIVE WITH ENHANCED FIBRIN AFFINITY Xiao-Chun Chen, Zi-Chun Hua ~', De-Xu Zhu Pharmaceutic Biotechnology Key Laboratory, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China Received April 26, 1996 Summary: A Gly-Pro-Arg-Pro tetrapepfide, homologous to amino-terminal segment of the human fibrin a chain after the release of the fibrinopeptide A, was covalently coupled to peptide A of low molecular weight urokinase. The resulting derivative gained increased affinity for fibrin. In caseinolytic assay, fibrin can stimulate the derivative to activate plasminogen. The derivative had twofold greater fibrinolytic potency than native low molecular weight urokinase and its affinity for fibrin clot was 3.9-fold higher than that of low molecular weight urokinase. Human fibrinogen is transformed into fibrin by the thrombin-catalyzed release of small polar peptides (fibrinopeptides A and B) from the amino terminal of the a and 13 chains. Upon release of these peptides, polymerization of the fibrin monomer units occurs spontaneously to form a noncovalently bonded gel. The polymer can be covalently cross linked by another enzyme, factor XIII, which activated by thrombin (i). Short peptides beginning with the sequence Gly-Pro-Arg---, which corresponds to the amino-terminal segment of the fibrin a chain after the release of the fibrinopeptide A, can prevent the polymerization of fibrin monomers. These peptides also bind to fibrinogen and the plasrnin-generated fragment D (2). Furthermore Gly-Pro-Arg was found to be an effective inhibitor of polymerization. When addition ofa proline as a fourth residue, the peptide Gly- Pro-Arg-Pro (GPRP) can significantly increase both the binding and the inhibitory activity (3). Urokinase catalyzes the conversion of the proenzyme plasminogen to the proteolytic enzyme of fibrin, plasmin. The single-chain form pro-urokinase (pro-uk) can be converted into an active twochain form urokinase by a single cleavage between Lys158 and I1e159 by plasmin (4,5). A low molecular weight form ofurokinase (LUK), lacking the epidermal growth factor domain and the *To whom correspondence should be addressed. Present correspondence address: 17 Bradford Street, Albany, NY 12206, USA. After May 25, 1996, please contact Dr. Zi-chun Hua at: Room 301, 2 Baijishancun, Tianshan Road, Nanjing 210008, People's Republic of China. 797 1039-9712/96/040797~)7505.00/0 Copyright 1996 'by Academic Press Australia. All rights of reproduction in any form reserved.
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL kringle domain, is proteolytically derived from high molecular weight urokinase (HUK) by plasmin which cleaves between Lys135 and Lys136 (6). The growth factor domain is responsible for the interaction of pro-uk with its specific high-affinity cell receptor (7). This interaction can induce tyrosine phosphorylation and signal transduction (8), possibly affecting cell growth and differentiation (9,10,11). This interaction could possibly produce unwanted side-effects during thrombolytic therapy. So LUK might have some advantage over pro-uk and HUK as a drug for thrombolytic therapy. But the lack of fibrin specificity prevents LUK from applying. In the present study we describe generation of a LUK derivative, Gly-Pro-Arg-Pro-LUK (GPRP-LUK), and assess its fibrin affinity and fibrinolytic properties. MATERL4~LS AND METHODS Materials: Low molecular weight urokinase with specific enzyme activity 100,000I.U./mg was obtained from Biochemical Product Factory of Nanjing University. EDC was purchased from Shanghai Dongfong Biochemical Reagent Factory. Plasminogen was from Sigma. Thrombin was from Tianjing Biochemical Product Factory. Benzamidine-Sepharose 6B was purchased from Pharmacia. Synthesis of GPRP tetrapeptide: Gly-Pro-Arg-Pro tetrapeptide was synthesized on Applied Biosystems 430A peptide synthesizer using solid phase methodology and Boo-protected amino acids. The compositions of tetrapeptide were determined by amino acid analysis and were similar to theoretical. Preparation of peptide A and B chain of LUK: Preparation ofpeptide A and B chain from L- UK was performed according to Robbins (12). 4rag LUK was dissolved in 2ml 0.1M phosphate buffer, ph6.8, then was dispersed into Benzamidine-Sepharose 6B column and incubated in 0.1M [3- mercaptoethanol, 0.1M phosphate buffer, ph6.8 at 25 C overnight. Native peptide A was obtained by eluting with 5mM EDTA, 0.1M [3-mercaptoethanol, 0.1M phosphate buffer, ph6.8 and LUK B chain was eluted with 0.1M HAc, 5mM EDTA, 0.1M [3-mercaptoethanol, ph4.0. Synthesis of GPRP-peptide A derivative: 2ml GPRP tetrapeptide solution (91Jg/mi) in distilled water was adjusted to ph4.5 with HC1 and supplemented with a 100-fold molar excess of EDC which dissolved in lml 0.05M phosphate buffer, phs.0. LUK peptide A solution was added to the mixture and incubated at 4 C overnight. The resultant product was dialyzed against 0. IM phosphate buffer, phs.0 for 6 hours at 4 C. Generation of LUK derivative: The obtained LUK peptide A derivative was mixed with LUK B chain elution in the ratio of 1:1 and incubated at room temperature overnight., then dialyzed again 0.1M phosphate buffer, prig.0 to obtain LUK derivative, GPRP-LLrK. Fibrinolytic activity assay: Fibrinolytic activity was assayed on fibrin plate prepared as described by Ploug and Kieldgaard (13). Caseinolytic assay: 7ml casein solution (12mg/ml) in 0.1M phosphate buffer, ph7.4 with or without 7mg fibrinogen and 3IU thrombin was incubated at 37 C for 10 minutes and 12I,U. LUK or GPRP-LUK was added to the solution. The mixture were incubated at room temperature for 15 798
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL minutes and 0.15mg/ml plasminogen was added into the mixture, then incubated at 37 C. lmi Aliquots of reaction mixture were removed at intervals of 30 minutes and 0.5ml 10% perchloric acid was added to stop the reaction. After centrifugation, supernatant was collected and two-fold diluted for measurement of the absorbance at 280nm. Fibrin dot binding measurement: 1.5mg fibrin was incubated in solutions containing 300I.U./ml LUK or GPRP-LUK for 15 minutes and then washed with 0.1M phosphate buffer, ph7.4 for 10 minutes. The fibrin clots were placed in a 3ml light path cuvette, which containing 3ml 0.1M phosphate buffer, ph7.4, 0.05M NaCI and 0. lmg/rnl plasminogen. The absorbance changes at 280nm were then plotted versus time. Binding to Fibrin-Sepharose chromatography column: Fibrin-Sepharose was made by rinsing 5g Sepharose 4B with water and subsequently 0.05M phosphate buffer, ph7.4, then suspended in 20ml buffer I (0.05M phosphate buffer, ph7.4, 0.1M NaC1, lmm EDTA) after filtration. 50mg fibrinogen and 20IU thrombin were added to the mixture subsequently and then the mixture was stirred at 30 C for 15 minutes. The resulting Fibrin-Sepharose 4B beads were transferred into empty column and equilibrated with buffer II (0.01M phosphate buffer, ph7.4, lmm EDTA, 0.3M NaC1). Equal amount of LUK or GPRP-LUK was passed over the column, washed with buffer II and eluted with buffer II plus 0.2M arginine. The protein concentration of the effluent was determined as described by Bradford (14). Fibrin clot lysis: 2mg fibrin clots were placed in a 1.5ml light path cuvette containing 0.1M phosphate buffer, ph7.4, then 100I.U. LLrK or GPRP-I.UK and 0.1mg plasminogen were added. The increase in absorbance at 280nm was measured. RESULTS LUK peptide A and B chain were separated from LUK by 13-mercaptoethanol reduction on a Benzamidine-Sepharose column. GPRP tetrapeptide was synthesized and coupled to isolated peptide A of LUK. Alter oxidization, disulfide bond was re-formed between peptide A derivative and LUK B chain and LUK derivative, GPRP-LUK was obtained. In the caseinolytic assay, the presence or absence of fibrin had no influence on native LUK activating plasminogen to degrade casein. In contrast, fibrin could stimulate GPRP-LUK to activate plasminogen to degrade casein (Fig. 1). To determine the affinity of GPRP-LUK for fibrin clot, fibrin clots were soaked in LUK or GPRP-LUK solutions and then after washing were placed in cuvette containing plasminogen to determine the degradation of fibrin by plasmin, which activated by LUK or GPRP-LUK absorbed on the surface of fibrin clots. The activation of plasminogen induced by LUK may be attributed to the non-specific absorbance of LUK to fibrin clot surface. Evidently, GPRP-LUK has better affinity for the fibrin clots, thus convert more plasminogen to plasmin to degrade fibrin more rapidly (Fig.2). 799
BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL I.I0 1.00 0.90 E o 0.80 0.70 0.60 0.50 J i I i i i i i i i 0 20 40 60 80 100 120140 1601802130 Time(rain) Fig. 1. +... +: o, o: Comparison of caseinolysis induced by native and GPRP-LUK. native LUK, with or without fibrin; GPRP-LUK, without fibrin; ~, GPRP-LUK, with fibrin. 0.23,c, 0.20 0.17 ~0.14 C,/ < 0.11./.// /. 0.08 0.05 i t t 600 2600 4-600 6600 Time(see) Fig. 2. Fibrin clot lysis induced by native LUK or GPRP-LUK. o o: native LUK; *,: GPRP-LUK. 800
Vol. 39, No. 4, "1996 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Comparison of affinity of GPRP-LUK for fibrin clots with that of native LUK revealed that GPRP- LUK has 3.9-fold greater affinity for fibrin clots. To compare the binding ability of LUK and its derivative to fibrin, LUK and GPRP-LUK were passed over Fibrin-Sepharose column. The bound protein was quantatively eluted by buffer containing arginine. Nearly all the applied LUK was collected in the flow-through fraction, while 8.5% of the applied GPRP-LUK was found bound to the Fibrin-Sepharose column as shown in Fig.3 and Table 1. GPRP-LUK has specific binding capacity for fibrin. For evaluation offibrinolytic potency ofgprp-luk, LUK or GPRP-LUK of equal amount of activity was used to activate plasminogen to degrade fibrin. The absorbance when full fibrinolysis 4.0 3.0 Buffer 1 ~i Bofir 2 0 2.0 1.0 0.0 I I 0 10 20 I 30 40 Volume (ml) I I I 50 60 70 Fig. 3. Buffer 1: Buffer 2: Binding of GPRP-LUK to Fibrin-Sepharose column. 0.01M phosphate buffer, ph7.4, lmm EDTA, 0.3M NaCI; 0.01M phosphate buffer, ph7.4, lmm EDTA, 0.3M NaCI, 0.2M arginine. Table 1. Comparison of binding of native LUK and GPRP-LUK to Fibrin-Sepharose column. Samples Protein in Protein in Protein bound (1200~g) flow-through elution to column (%) LUK ll80~tg 0 (1% GPRP-LUK 1060lag 90gtg 8.5% 801
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL CD O!.?.o r/l~- ol 1 2 Fig. 4. Comparison of fibrin clot lysis potency of LUK and GPRP-LUK. 1" GPRP-LUK; 2: native LUK. occurred was defined as 100% and the percentage of clot lysis calculated from the absorbance at 1.5 hours was used to represent the fibrinolytic potency. As shown in Fig.4, GPRP-LUK has two-fold greater fibrinolytic potency than native LUK. DISCUSSION Comparing with pro-uk and HUK, LUK is a smaller molecule consisting ofpeptide A (136-158aa) and B chain (159-41 laa) which linked by one disulfide bond between Cys148 and Cys279. Lacking the growth factor domain and kringle domain, LUK only contains a serine-protease domain and seven disulfide bonds while pro-uk and HUK contain three domains and twelve disulfide bonds. This advantage facilitates the large-scale production of LUK via recombinant DNA process, avoiding difficulties encountered in the high level expression ofpro-uk in various expression systems (15-21) and renaturation ofpro-uk from inclusion bodies ore coli (22-25). In this paper we describe when a Gly-Pro-Arg-Pro tetrapeptide was coupled to LUK, it could impart fibrin affinity to LUK and increase its fibrinolytic potency. The affinity of GPRP-LUK for fibrin can increase the selective fibrin lysis of LUK. These results seem to open a new alternative in thrombolytic therapy via recombinant DNA process_ Further study will be continued to produce and characterize the GPRP-LUK via recombinant DNA techniques. 802
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