©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Plasminogen Activator from Snake Venom
PURIFICATION, CHARACTERIZATION, AND MOLECULAR CLONING (*)

Yun Zhang (1) (2)(§), Anne Wisner (1), Yuliang Xiong (2), Cassian Bon (1)(¶)

From the (1) Unité des Venins, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris Cedex 15, France and the (2) Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native Glu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg-Val. Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl- p-guanidinobenzoate.

Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-glycosylation site at Asn. The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Agkistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.


INTRODUCTION

The native form of human plasminogen present in blood and in many tissue fluids is a single-chain glycoprotein with a molecular weight of 93,000, characterized by a N-terminal glutamic acid (Glu-plasminogen).() A modified form, Lys-plasminogen, is generated by plasmin by cleavage of the Lys-Lys or Lys-Val bond of Glu-plasminogen (Robbins et al., 1967). The main physiological function of plasmin is fibrinolysis. Conversion of plasminogen to plasmin, however, also plays a role in various other processes, such as ovulation, spermatogenesis, embryonic development, involution of mammary glands, prohormone processing, and tumor cell invasion (Dano et al., 1985; Saksela and Rifkin, 1988).

In all mammalian species so far investigated, two distinct plasminogen activators have been identified, urokinase-type plasminogen activator (u-PA, M 55,000) and tissue-type plasminogen activator (t-PA, M 65,000). They are both serine proteases with similar structural and catalytic properties. Both enzymes cleave plasminogen, either Glu-plasminogen or Lys-plasminogen, at the Arg-Val bond and generate plasmin, a serine protease with broad proteolytic activity. However, important functional differences exist between the two plasminogen activators. Whereas t-PA binds to fibrin, an interaction that dramatically increases its catalytic activity for clot-bound plasminogen (Hoylaerts et al., 1982), u-PA binds to specific membrane receptors of a variety of normal and transformed cell types (Blasi et al., 1987; Del Rosso et al., 1985; Dano et al., 1994). The activity of u-PA is thus localized at the cell surface, while that of t-PA is mainly directed against fibrin. Accordingly, the two plasminogen activators have distinct physiological roles.

Envenomations of mammals, including humans, by snakes, especially by those belonging to the Crotalidae and Viperidae families, are characterized by an inhibition of blood coagulation due to the action of fibrino(geno)lytic enzymes. This might be also caused by the activation of plasminogen into plasmin by venom components; however, none of the proteases from snake venoms that have been purified and studied until now are able to activate plasminogen in vitro (Stocker, 1990). In a previous study, it was observed that Trimeresurus stejnegeri venom significantly activates human plasminogen in vitro (Zhang et al., 1993). This report precisely describes the purification of a plasminogen activator (TSV-PA) which is responsible for this activity from T. stejnegeri venom. The major molecular and physiological properties of TSV-PA were characterized. The cDNA sequence encoding TSV-PA and the deduced amino acid sequence were also obtained and are reported here. This plasminogen activator is the first snake venom protease which specifically activates plasminogen.


EXPERIMENTAL PROCEDURES

Materials T. stejnegeri venom was from the stock of the Kunming Institute of Zoology, Chinese Academy of Sciences. The products for gel filtration and FPLC, Sephadex G-75 (Superfine), and Mono-Q 16/10 column were from LKB Pharmacia. p-Nitroanilide chromogenic substrates, H-DPhe-Pip-Arg- pNA (S-2238), H-DVal-Leu-Lys- pNA (S-2251), H-DPro-Phe-Arg- pNA (S-2302), Bz-Ile-Glu-Gly-Arg- pNA (S-2222), and H-DVal-Leu-Arg- pNA (S-2266), were obtained from Kabi Vitrum (Stockholm, Sweden); H-DLys(Cbo)-Pro-Arg- pNA (CBS-6525) was from Diagnostica Stago (Asnières, France). Sodium dodecyl sulfate (SDS), PMSF, p-nitrophenyl- p-guanidinobenzoate (NPGB), iodoacetamide, benzamidine, p-aminobenzamidine, aprotinin, soybean trypsin inhibitor (type I-S), and bovine serum albumin (fatty-acid free) were purchased from Sigma. PAGE gels and reagents for Phastsystem (fast PAGE system) were obtained from LKB Pharmacia. All other reagents were of the highest purity available from Merck or Prolabo (Paris, France).

Proteins

Proteins were from human origin except when indicated. Prothrombin, Lys-plasminogen, urokinase (two-chain form, M 33,000), plasmin, anti-thrombin III, factor X (bovine), and streptokinase ( Streptococcus) were obtained from Sigma. Purified Glu-plasminogen was a gift from Dr. Eduardo Angles-Cano (INSERM U.143, le Kremlin Bictre, France). Protein C, t-PA, and fibrin CNBr fragments (t-PA stimulator) were obtained from Diagnostica Stago. Fibrinogen (grade L) from Kabi Vitrum was pretreated with diisopropyl fluorophosphate according to the instructions of the manufacturer. The absence of plasminogen was confirmed by incubating the fibrinogen for 6 h at 37 °C with streptokinase (1000 unitsmg fibrinogen): no evidence of degradation was found by SDS-PAGE. The concentration of plasminogen was determined by measuring the absorbance at 280 nm, using E = 16.8 (Wallén and Wiman, 1972). Methods

Chromogenic Assays

The amidolytic activity of the enzymes was measured with a Kontron spectrophotometer in 1-cm path length plastic cuvettes. Assays were performed in 50 mM Tris-HCl, pH 7.8, 0.01% Tween 80 in a total volume of 500 µl. The reactions were initiated by the addition of the enzyme at a final concentration of 18 nM and the formation of p-nitroaniline was monitored continuously at 405 nm. The amount of substrate hydrolyzed was calculated from the absorbance at 405 nm by using a molar extinction coefficient of 10,000 Mcm for free p-nitroaniline. The kinetic parameters Kand k were determined by analysis of double-reciprocal plots of the initial velocity as a function of substrate concentration.

Electrophoretic Studies

SDS-PAGE, native-PAGE, and isoelectric focusing were performed in a Phastsystem apparatus (LKB Pharmacia) following the manufacturer's directions and recommended separation methods. For SDS-PAGE, samples were pretreated in 2.5% SDS alone (nonreducing conditions) or in 2.5% SDS and 5% -mercaptoethanol (reducing conditions) at 100 °C for 10 min. Gels were stained with 0.1% Coomassie Brilliant Blue R in methanol/acetic acid/water (3/1/6) and destained in the same solution. The molecular weights were estimated by interpolation from a linear semilogarithmic plot of relative molecular mass versus distance of migration, using the following proteins as standards: phosphorylase b (94, 0) , bovine serum albumin (67, 0) , ovalbumin (43, 0) , carbonic anhydrase (30, 0) , soybean trypsin inhibitor (20, 100) , and -lactalbumin (14, 400) . For isoelectric focusing, protein markers were used for calibration of the gel (isoelectric focusing calibration kit, Pharmacia). Gels were fixed with 20% trichloroacetic acid, stained in methanol/acetic acid/water (3/1/6) containing 0.02% Coomassie Brilliant Blue R and 0.1% CuSO, and destained as described above.

Plasminogen Activation Assays

The initial rate of plasminogen activation was measured with a Kontron spectrophotometer. Glu-plasminogen or Lys-plasminogen was incubated at a final concentration of 1.3 µM in 50 mM Tris-HCl, pH 7.8, 0.01% Tween 80. The reaction was initiated by the addition of TSV-PA at a final concentration of 15-120 nM, in a total reaction volume of 250 µl. Aliquots (50 µl) were taken at various time intervals and assayed for plasmin activity. They were introduced into plastic cuvettes which contained 450 µl of S-2251 (0.3 mM) in 50 mM Tris-HCl, pH 7.8, 0.01% Tween 80 and the formation of p-nitroaniline was monitored at 405 nm. One unit of activity was defined as the amount of TSV-PA which, activating plasminogen during 1 min under the conditions above, produced a plasmin amidolytic activity of 1 milliabsorbance unit per min.

Fibrinogenolytic Assays with or without Plasminogen

Fibrinogen was incubated in 50 mM Tris-HCl, pH 7.8, at a final concentration of 5 mgml with TSV-PA (final concentration 6-120 nM), in the presence or absence of Lys-plasminogen (final concentration 1.3 µM), at 37 °C for different time intervals. Thrombin (final concentration 2.5 NIH unitsml) was then added and incubation was continued at room temperature for 15 min to allow the formation of the fibrin clot. The clot was centrifuged at 3,200 g for 20 min and the protein content of the supernatant was determined by the method of Folin (Lowry et al., 1951), using bovine serum albumin as a standard.

Plasminogen Cleavage by TSV-PA and u-PA

Glu-plasminogen (11 µM) was incubated with TSV-PA (1.2 µM) or urokinase (2.4 µM) in 50 mM Tris-HCI, pH 7.8, in the presence of soybean trypsin inhibitor (7.1 µM, in the case of TSV-PA) or aprotinin (10 TIUml, in the case of u-PA) at 37 °C for various times. Aliquots (5 µl) were taken to determine the amidolytic activity on S-2251 in 500 µl (final volume) of 50 mM Tris-HCI, pH 7.8, containing 0.01% Tween 80. Samples were then analyzed by SDS-PAGE.

Inhibitor Studies

The effects of various protease inhibitors specific for different classes of proteases were examined. Stock solutions of inhibitors were freshly prepared as follows: NPGB and PMSF were dissolved in dimethyl sulfoxide, hirudin, antithrombin III, soybean trypsin inhibitor, aprotinin, p-aminobenzamidine, iodoacetamide, benzamidine, and EDTA were dissolved in 50 mM Tris-HCl, pH 7.8, 0.1 M NaCl. TSV-PA (18 nM) was incubated 30 min at room temperature in 50 mM Tris-HCl, pH 7.8, in the absence or presence of the inhibitors at the indicated concentrations. The initial rate of amidolysis was measured after addition of 50 µl of 2 mM S-2238. In the case of NPGB, PMSF, L-cysteine, and iodoacetamide, mixtures containing 0.6 µM TSV-PA in the absence or presence of the indicated concentrations of the inhibitors were incubated for 30 min at 37 °C, then 15-µl aliquots were taken to determine amidolytic activity on S-2238.

Effect of TSV-PA on Blood Coagulation Factors

Assays for activation of prothrombin and factor X were performed according to the methods described by Hofmann and Bon (1987a, 1987b). Purified prothrombin (0.7 µM) was incubated at 37 °C in 50 mM Tris-HCI, pH 7.8, 0.1 M NaCl with TSV-PA (60 nM). Aliquots (50 µl) were taken at various times and their amidolytic activity was assayed in 500 µl (final volume) of the same buffer containing 0.2 mM S-2238. Purified factor X (bovine) (0.43 µM) was incubated at 37 °C in 50 mM Tris-HCl, pH 7.8, 0.1 M NaCl, containing 5 mM CaCl with TSV-PA (60 nM). Aliquots (50 µl) were taken at various times and their amidolytic activity was immediately assayed in 500 µl of 50 mM Tris-HCl, pH 7.8, 0.1 M NaCl, containing 0.2 mM S-2222. After incubating prothrombin or factor X (bovine) with TSV-PA for 3 h at 37 °C and at a molar ratio of 50/1, samples were analyzed by SDS-PAGE both under reducing and nonreducing conditions. In these experiments, Bothrops atrox venom was used as a positive control for activation and cleavage (Hofmann and Bon 1987a, 1987b). Protein C activation was assayed by the method described by Orthner et al. (1988): protein C (80 nM) was incubated with TSV-PA (60 nM) in 50 mM Tris-HCl, pH 7.8, containing 1 mgml polyethylene glycol and 5 mM EDTA at 37 °C. At various times, aliquots (50 µl) were taken to determine the amidolytic activity on CBS65-25 (final concentration 0.3 mM). In parallel, A. contortrix contortrix venom was used as a positive activation control (Kisiel et al., 1987).

Determination of Partial Amino Acid Sequence of TSV-PA

Sequence determinations of proteins and peptides were performed by Edman degradation with a 470A gas-phase sequencer. Phenylthiohydantoin-derivatives were separated and identified by on-line reverse phase high pressure liquid chromatography in an RP column, with an Applied Biosystem 120-A analyzer. For internal amino acid sequence of TSV-PA, the purified protein was digested with trypsin, and the resultant digested fragments were separated by high pressure liquid chromatography in a C column. For determination of the cleavage site of plasminogen by TSV-PA, plasmin chains generated by TSV-PA were first submitted to SDS-PAGE and electrotransferred to an Immobilon membrane. The protein bands were analyzed with the protein sequencer.

PCR and RACE-RT-PCR

On the basis of the determined N-terminal and internal peptide sequences of TSV-PA, and the analysis of genetic code usage for the published venom protease cDNA sequences, corresponding to batroxobin (Itoh et al., 1987) and ancrod (Au et al., 1993), two oligonucleotide primers, P and P, were designed for the amplification of a cDNA internal fragment by PCR. Primer P (5`-CTTGTAGTCTTGTTCAA(CT)TCTAACGG-3`) is oriented in the sense direction and corresponds to the N-terminal residues 15-23 of TSV-PA. Primer P (5`-ACAGATGAGGGGTCCCCCAGAGTC-3`) is oriented in the antisense direction and corresponds to TSV-PA residues 179-186. Total RNA was prepared from T. stejnegeri venom glands according to the method of Chomczynski and Sacchi (1987). Single-stranded cDNAs were prepared from the mRNAs contained in total RNA (5 µg) by reverse transcriptase (Life Technologies, Inc.), using an oligo-d(T) primer. A first amplification by PCR with the primers P and P was carried out using Taq polymerase (Promega), under the following conditions: a first step of 5 min at 94 °C was followed by 30 cycles of 1 min at 94 °C, 1 min at 60 °C, and 1 min at 72 °C.

From the cDNA fragment sequence obtained from the first PCR amplification, two TSV-PA specific primers, P (5`-CAGTTGCTGTTTGGTGTG-3`) and P (5`-CACACCAAACAGCAACTG-3`), oriented in the sense (P) or the antisense (P) direction and corresponding to TSV-PA residues 48-53, were defined. They were used in 5` and 3` RACE-RT-PCR to obtain the full-length cDNA sequence encoding TSV-PA, as essentially described by Frohman (1990) with the following modifications. In the case of 5` RACE-RT-PCR amplification, the reverse transcription was performed by using primer P, and an oligonucleotide T (5`-GATCCCCTATAGTGAGTCG-3`) was ligated to the 3` end of the first cDNA strand. Then a first PCR amplification was carried out by using primers R (5`-AAGGATCCGTCGACATC-3`) and P in the presence of an adaptor primer E (5`-AAGGATCCGTCGACATCGATAATACGACTCACTATAGGG-3`) which contains the sequences of primers R, R (5`-GACATCGATAATACGAC-3`), and the reverse complementary sequence of oligonucleotide T. This PCR amplification was followed by a second one carried out using primers R and P. For amplification of the 3`-end, the reverse transcription was performed by using oligonucleotide primer E with an additional 3` poly(T) tail. Two consecutive PCR amplifications were then carried out using primers R and P, and primers R and P.

All oligonucleotide primers were synthetized by Genset Laboratory (Paris, France). PCR products were subcloned by the dideoxythymidine-tailed vector method into a pGEM vector (Promega). DNA sequencing was performed by the dideoxy termination method using a T sequencing kit (LKB Pharmacia).

Construction and Screening of a cDNA Library

mRNAs were prepared from the total RNA of T. stejnegeri venom glands by oligo(dT) cellulose chromatography. A directional cDNA library was constructed with a plasmid cloning kit (SuperScript plasmid system, Life Technologies, Inc.) with some modifications. Size-selected cDNAs were ligated to pTTD EcoRI/ NotI/BAP Phagemid vector arms (LKB Pharmacia) and used to transform MAX efficiency DH10B competent cells, producing a library of about 4 10 independent colonies. A PCR-based method for high stringency screening of DNA libraries (Israel, 1993) was used for screening and isolating TSV-PA clones. Two TSV-PA specific primers, P as described previously and P (5`-GCCACCTGCCGCCATGAATAAGC-3`) corresponding to TSV-PA residues 153-160 in the antisense orientation, and a vector T promoter primer located 5` of the cloned insert, were used in PCR reactions. The presence of an insert encoding TSV-PA and its size were examined by PCR analysis between P-P and T-P, under the following conditions: 5 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 55 °C, 1 min at 72 °C.

The bacterium library was titered. The culture was diluted with LB broth/100 µgml ampicilline, plated in an 8 8 matrix (64 wells total, 100 µl/well) in a 96-well multiwell plate (Costar), and amplified at 37 °C overnight. Titers of approximately 500 and 30 bacteria/well were used for the first round screening and the second round screening, respectively. Aliquots of amplified bacteria from 8 wells across a row or 8 wells down a column were pooled. The matrix of 64 wells was thus reduced to 16 pools which were used as templates for PCR analysis, as described above. Bacteria from positive wells in the second round screening were plated out. Single colonies were picked out and analyzed by PCR to obtain individual positive clones.

Computer analysis of protein sequences and nucleotide acid sequences were performed with the Clustal V sequence software package.


RESULTS

Purification of TSV-PA

The progress of the purification was followed by determining the following for each fraction: activation of Lys-plasminogen, amidolytic activities on S-2238 and S-2251, fibrinogen clotting activity, fibrinogenolytic activity, procoagulant and anti-coagulant activities, activation and inhibition of platelet aggregation, and finally phospholipase A activity. The methods used to assay platelet aggregation and phospholipase A were those as described by Born (1962) and Lôbo de Araujo and Radvanyi (1987), respectively.

Gel filtration of T. stejnegeri venom in Sephadex G-75 resulted in the separation of seven protein peaks (Fig. 1 A). The plasminogen activation activity was predominantly associated with fraction IV and was separated from procoagulant and phospholipase A activities. Furthermore, no plasminogen activation activity was found in other fractions. Phospholipase A activity and inhibition of the platelet aggregation induced by thrombin were concentrated in fractions V to VII; fibrinogen clotting activity was found in fraction II, and fraction III showed a strong fibrinogenolytic activity (not shown in Fig. 1A for clarity).


Figure 1: Purification of TSV-PA. A, gel filtration of T. stejnegeri venom in a Sephadex G-75 column. Lyophilized T. stejnegeri venom (2 g) was dissolved in 20 ml of 50 mM Tris-HCl buffer, pH 7.8, containing 0.1 M NaCl. The solution was centrifuged, and the small pellet was discarded. The supernatant was applied to a Sephadex G-75 column (5 100 cm), and equilibrated at 4 °C with the same buffer. The elution was achieved with the same buffer at a flow rate of about 24 ml h, collecting fractions of 8 ml. The protein concentration was estimated from the absorbance at 280 nm (). Plasminogen activating activity (), and amidolytic activity on S-2238 () and S-2251 () were determined as described under ``Experimental Procedures.'' The fractions were pooled as indicated. B, purification of TSV-PA by FPLC on a Mono-Q preparative column (HR16/10). Fraction IV of Sephadex G-75 (158 mg, 1.5 mgml) was dialyzed against 20 mM Tris-HCl, pH 7.8, for 24 h and applied to a Mono-Q (16/10) preparative column (40 mg of proteins were loaded in each run). The elution was performed at a flow rate of 5.5 mlmin with the indicated NaCl gradient. The absorbance of the eluant was monitored at 280 nm. The plasminogen activating activity was mainly associated with protein peak 7, indicated by an arrow in the figure. C, peak 7 of the first Mono-Q column, pH 7.8, was lyophilized, dissolved in 10 ml of distilled water, and dialyzed against 20 mM Tris-HCI, pH 8.8, for 24 h. A total of 11 mg of proteins in 12 ml was obtained and loaded again on the Mono-Q HR16/10 preparative column previously equilibrated with the same buffer. Elution was performed at a flow rate of 5.5 mlmin with the NaCl gradient, as shown in the figure. The plasminogen activation activity was found in peak 4.



Fraction IV, containing the activity for plasminogen activation, was dialyzed against 20 mM Tris-HCl, pH 7.8, for 24 h and applied on a FPLC Mono-Q preparative column (HR 16/10) equilibrated with the same buffer. Elution was achieved with a linear NaCl gradient, yielding 12 protein peaks (Fig. 1 B). The plasminogen activation activity was concentrated in peak 7, associated with amidolytic activities on S-2238 and S-2251. SDS-PAGE revealed that fraction 7 contained a major polypeptide of M 30,000 ± 3000, in both reducing and nonreducing conditions. PAGE carried out in native conditions revealed, however, the presence of other small protein components.

Peak 7, obtained from the Mono-Q column run at pH 7.8, was pooled, lyophilized, dissolved in water, and dialyzed for 24 h against 20 mM Tris-HCl, pH 8.8. The sample was again applied to a Mono-Q preparative column (HR 16/10), eluted with a shallow linear NaCl gradient. Four peaks were thus obtained. The plasminogen activation activity was only found in the largest one, peak 4 (Fig. 1 C), associated again with amidolytic activities on S-2238 and S-2251. This protein fraction is referred to as TSV-PA. On the other hand, peak 1 had no proteolytic activity, while peaks 2 and 3 possessed amidolytic activity on S-2238, but not on S-2251.

The purification procedure of TSV-PA is summarized in . TSV-PA was purified 200-fold from crude venom, with a yield higher than 40%. It represents a minor component of the venom, about 0.5% of total venom proteins.

Characterization of TSV-PA

Fig. 2A shows the SDS-PAGE analysis of the samples from the various steps of the purification. The purified TSV-PA appeared as a single band and therefore consisted of a single polypeptide chain of 33,000. Purified TSV-PA was also analyzed by PAGE in native conditions (Fig. 2 B) and by isoelectric focusing (Fig. 2 C). It appeared as a homogeneous protein in both conditions, with an apparent isoelectric point of 5.2. PAGE, combined with Schiff's reagent staining (Van-Seuningen and Davril, 1992), showed that TSV-PA is a glycoprotein.


Figure 2: PAGE and isoelectric focusing of fractions during purification of TSV-PA. A, SDS-PAGE (20% acrylamide): lane 1, crude venom; lane 2, fraction IV of Sephadex G-75 column; lane 3, fraction 7 of the first Mono-Q column, pH 7.8; lanes 4 and 5, TSV-PA fraction of the second Mono-Q column. Minus () and plus (+) indicate that SDS-PAGE was performed under nonreducing conditions and reducing conditions (5% -mercaptoethanol), respectively. Proteins were stained with Coomassie Brilliant Blue. The polarity of the electrodes is indicated by + and . B, PAGE in native conditions at pH 8.5 (20% acrylamide): lane 1, crude venom; lane 2, fraction IV of Sephadex G-75 column; lane 3, fraction 7 of the first Mono-Q column; lane 4, purified TSV-PA. C, isoelectric focusing of TSV-PA in the pH 3-9 range. Lane 1, TSV-PA; lane 2, LKB Pharmacia isoelectric focusing calibration kit pH 3-10.



Amidolytic Activity of TSV-PA

Purified TSV-PA possesses amidolytic activity toward several synthetic substrates. A detailed investigation was carried out by measuring the reaction velocities at different substrate concentrations and determining the Kand k parameters of TSV-PA by Lineweaver-Burk plots for the following synthetic substrates: S-2238, S-2251, and S-2302. Among the compounds tested, S-2238 was the best substrate for TSV-PA (). It can be seen from these studies that TSV-PA has a preference for arginine over lysine at the position that is cleaved. But TSV-PA also differentiates among substrates possessing an arginine at the cleavage site, since its activity was 30-fold lower with S-2302 than with S-2238, whereas its amidolytic activity on S-2222, which is very sensitive to trypsin, was 250-fold lower than on S-2238 (data not shown).

In general, the amidolytic activities of u-PA and t-PA are much lower than that of TSV-PA on different chromogenic substrates (data not shown). For example, whereas TSV-PA hydrolyzed S-2251 significantly, u-PA had undetectable activity on this substrate in our assay system.

Activation of Plasminogen by TSV-PA

The activation of human Glu- and Lys-plasminogen by TSV-PA was determined by measuring the amidolytic activity of activated plasminogen on S-2251. Fig. 3A shows that the initial rate of activation of human Glu- and Lys-plasminogen by TSV-PA was proportional to the concentration of the venom activator. In addition, the activation of Lys-plasminogen was 2-3-fold higher than that of Glu-plasminogen at the same concentration of TSV-PA. This difference was also observed in the case of plasminogen activation by t-PA (Fig. 3 B). The addition of CNBr fibrin fragments to the assay medium increased the rate of plasminogen activation of t-PA about 5-fold, as reported by Hoylaerts et al. (1982), but did not significantly modify the activation catalyzed by TSV-PA (Fig. 3 A). While the amidolytic activity of TSV-PA toward small peptide substrates is higher than that of t-PA, activation of plasminogen by TSV-PA was about 50-fold lower than that observed with t-PA stimulated by fibrin fragments.


Figure 3: Plasminogen activation by TSV-PA. Activation of purified human Glu-plasminogen and Lys-plasminogen (1.3 µM) by TSV-PA (120 nM) ( A) and t-PA (50 IUml, 2.8 nM) ( B) was determined in the absence ( open symbols) or in the presence ( closed symbols) of fibrin CNBr fragments (250 µgml). , : Glu-plasminogen; , : Lys-plasminogen. Inset, initial rate of plasminogen activation at different concentrations of the activator. , Glu-plasminogen; , Lys-plasminogen. C, purified human Glu-plasminogen (11 µM) was incubated at 37 °C in 50 mM Tris-HCl, pH 7.8, with TSV-PA (1.2 µM) and soybean trypsin inhibitor (7.1 µM) or with urokinase (2.4 µM) and aprotinin (10 TIUml). Samples removed at the indicated incubation times were analyzed by SDS-PAGE (20% acrylamide) under nonreducing ( left part) or reducing ( right part) conditions. C, lanes 7, plasminogen control: Glu-plasminogen incubated in the buffer without activators at 37 °C for 3 h.



The catalytic cleavage of the human Glu-plasminogen polypeptide by TSV-PA was further investigated by SDS-PAGE. In these experiments, soybean trypsin inhibitor was added to the activation medium in order to prevent the proteolytic action of the plasmin generated, since the inhibitor blocks plasmin but not TSV-PA activity (I). Electrophoretic analysis of the samples removed during incubation of Glu-plasminogen with TSV-PA in the presence of soybean trypsin inhibitor showed no modification in the molecular weight of Glu-plasminogen under nonreducing conditions (Fig. 3 C, left). Under reducing conditions, the polypeptide band of apparent molecular weight of 94,000 corresponding to Glu-plasminogen progressively disappeared, with the concomitant appearance of two bands of apparent molecular weight of 68,000 and 27,000 corresponding to Glu-plasmin (Fig. 3 C, right). This pattern of activation is identical to that observed with u-PA (Fig. 3 C, right). Analysis of the N-terminal sequences of the plasmin chains generated by TSV-PA demonstrated that TSV-PA cleaved plasminogen at the Arg-Val bond, located in a loop formed by a disulfide bridge. The position of cleavage is identical to that occurring upon activation by u-PA and t-PA (Robbins et al., 1967), therefore forming two chain Glu-plasmin.

In addition to S-2251, fibrinogen was used as a substrate for activated plasminogen. First, it was observed that unlike most other venom proteases, TSV-PA has no direct fibrinogenolytic activity in the absence of plasminogen (Fig. 4 A). SDS-PAGE analysis revealed that in the absence of plasminogen and after long incubation times (150-180 min) at 37 °C, there was no significant degradation of fibrinogen, TSV-PA producing only traces of fragments derived from the A chain of fibrinogen (Fig. 4 B). Recent studies showed that physiological plasminogen activators (t-PA and u-PA) not only hydrolyze a specific arginine-valine bond in plasminogen, but also cleave other proteins such as fibronectin and directly degrade fibrinogen (Weitz et al., 1988; Weitz and Leslie 1990). When plasminogen was incubated together with TSV-PA and fibrinogen (Fig. 4, A and C), the content of clottable protein greatly decreased after a short incubation time. PAGE analysis showed that the A, B, and also chains of human fibrinogen disappeared within 30 min, and were replaced by degradation fragments which were similar to those obtained with plasmin in control experiments. This result was consistent with the assay of clottable protein content (Fig. 4 A), which showed that the clottable protein completely disappeared in the presence of plasminogen within 30 min. The results indicate that TSV-PA converted plasminogen to its activated form and that activated plasminogen rapidly degraded fibrinogen.


Figure 4: Analysis of direct and indirect fibrinogenolytic activity of TSV-PA. A, human fibrinogen (15 µM) was incubated at 37 °C with TSV-PA 6 nM (, ), 24 nM (, ), or 120 nM (, ) in the presence ( closed symbols) or absence ( open symbols) of human plasminogen (final concentration 1.3 µM) in 50 mM Tris-HCl, pH 7.8, 0.1 M NaCl. The clottable protein remaining at the indicated time was assayed on aliquots as described under ``Experimental Procedures.'' B and C, human fibrinogen samples incubated with TSV-PA (120 nM) in the absence of plasminogen ( B) or in the presence of plasminogen ( C) were also analyzed by SDS-PAGE in reducing conditions. B, lane 7, control in the absence of TSV-PA. C, lanes 6 and 7, positive control: fibrinogen incubated with human plasmin (1 unitml). C, lane 8, negative control: fibrinogen incubated with plasminogen at 37 °C for 120 min.



Effect of Protease Inhibitors on TSV-PA

I shows the effects of reversible and irreversible protease inhibitors on the activity of TSV-PA. The activity of TSV-PA was not affected by a metal chelator (EDTA), nor by thiol group reagents. In contrast, NPGB, a specific alkylating agent of the active-site serine residue of serine proteases, completely inhibited the amidolytic activity of TSV-PA (I) and its plasminogen activation activity (data not shown). The amidolytic activity of TSV-PA was also inhibited by the irreversible serine protease inhibitor PMSF, while the reversible inhibitor aprotinin partially inhibited its activity, and soybean trypsin inhibitor had little or no effect (I). Antithrombin III/heparin or hirudin were also ineffective. The amidolytic activity of TSV-PA was inhibited by -aminobenzamidine and benzamidine. These observations show that TSV-PA is not a thiol protease nor a metalloprotease and indicate that it is a serine protease.

Effect of TSV-PA on Various Coagulation Factors

The possible actions of TSV-PA on prothrombin, factor X, and protein C were examined. Blood coagulation factors were incubated with TSV-PA (60 nM) at 37 °C for different time intervals with bovine factor X (0.43 µM, 5 mM CaCl), human prothrombin (0.7 µM), or human protein C (80 nM). In contrast with positive controls carried out with A. contortrix contortrix venom for activation of protein C (Kisiel et al., 1987) and with B. atrox venom for activation of prothrombin and factor X (Hofmann and Bon, 1987a, 1987b), no activation was observed with any of these proteins. Furthermore, after incubation with TSV-PA, prothrombin, and factor X could still be activated by B. atrox venom and achieved full activation (results not shown). As expected, SDS-PAGE analysis, both under reducing and nonreducing conditions, showed that prothrombin or factor X were not cleaved after a 3-h incubation with TSV-PA, at a substrate/enzyme ratio of 50/1 (results not shown).

Molecular Cloning and Sequence Analysis of TSV-PA

The purified protein was subjected to amino acid sequencing. The N-terminal amino acid sequence and internal peptide sequences of tryptic fragments of TSV-PA, obtained by Edman degradation, showed high similarity with the sequence of snake venom serine proteases such as batroxobin, a thrombin-like enzyme from B. atrox venom (Itoh et al., 1987).

The specific oligonucleotide primers P (in the sense direction and corresponding to TSV-PA N-terminal residues 15-23) and P (in the antisense direction and corresponding to residues 179-186, in a region conserved among serine proteases) were designed to amplify by PCR a partial cDNA sequence of 510 bases. In a second step, this cDNA sequence was used to design TSV-PA specific primers for 5` and 3` RACE-RT-PCR to obtain the complete cDNA sequence encoding TSV-PA. The sequences obtained by PCR greatly facilitated the subsequent molecular cloning of TSV-PA. A cDNA library constructed from T. stejnegeri venom glands was screened at high stringency, as described under ``Methods,'' by an efficient and rapid PCR-based procedure. A positive clone (clone D16), which contained a 1.6-kilobase insert, was thus identified and isolated. Both strands of clone D16 were sequenced. The complete nucleotide sequence of TSV-PA cDNA and the deduced amino acid sequence are shown in Fig. 5 .


Figure 5: Nucleotide sequence of TSV-PA cDNA and predicted amino acid sequence of the TSV-PA precursor protein. The nucleotide residues are numbered in the 5` to 3` direction. The predicted amino acid sequence is shown above; the numbering starts at the amino-terminal amino acid of mature TSV-PA (valine). Peptides corresponding to determined amino acid sequences are underlined. A potential glycosylation site was boxed.



The cDNA structure of TSV-PA is similar to that of batroxobin. It was found to contain a coding region of 774 nucleotides, a 5`-noncoding region of 179 nucleotides, and a 3`-noncoding region of 653 nucleotides. The encoded amino acid sequence corresponds to a polypeptide of 258 residues. The determined N-terminal and internal amino acid sequences were found exactly in the deduced sequence (Fig. 5, underlined), thereby unequivocally confirming the identity of the isolated cDNA clone. The N-terminal residue valine is preceded by 24 amino acid residues which are strictly identical to those reported for the signal peptide of batroxobin. This signal peptide consists of an amino-terminal hydrophobic pre-peptide (18 amino acids) and a hydrophilic pro-peptide (6 amino acids), as suggested in the case of batroxobin (Itoh et al., 1987). The predicted molecular weight of the mature TSV-PA (amino acids 1-234) is 25,609. The difference with the apparent molecular weight (33, 0) is consistent with the presence of sugars, since TSV-PA is a glycoprotein. Accordingly, a potential N-glycosylation site, Asn- X-Thr, is located at amino acid residues 161-163.

Sequence Homology

The amino acid sequence of mature TSV-PA was compared with those of venom serine proteases, rat trypsin I, and the protease domain of human t-PA and u-PA (Fig. 6). The amino acid sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom serine proteases: 66% with ACC-C, a protein C activator from A. contortrix contortrix venom; 63% with batroxobin; and 60% with RVV-V, a factor V activator from Vipera russelli venom. On the other hand, TSV-PA shows only 23 and 21% sequence identity with the serine protease domain of t-PA and u-PA. Unlike t-PA and u-PA, TSV-PA does not contain any additional domains, such as the kringle and growth factor domains. Furthermore, it is important to note that the N-terminal region of TSV-PA does not contain the positively charged amino acid sequences such as the motifs of t-PA (KHRR) and u-PA (RRHR) which have been demonstrated to be involved in their interaction with plasminogen activator inhibitor 1 (PAI-1) (Madison et al., 1989). The homology of TSV-PA with venom and mammalian serine proteases indicates that the amino acid residues which form the catalytic triad are His, Asp, and Ser (Fig. 6).


Figure 6: Amino acid sequence comparison of TSV-PA with snake venom serine proteases, t-PA, u-PA, and trypsin. The numbering starts at the amino-terminal amino acid of TSV-PA. Gaps have been introduced to optimize the sequence homology. Identical residues in all sequences are shown by asterisks. Sequences were from the following sources: batroxobin, Itoh et al. (1987); RVV-V, Tokunaga et al. (1988); ACC-C, McMullen et al. (1989); rat trypsin I, MacDonald et al. (1982), and the protease domain of human t-PA and u-PA, Pennica et al. (1983) and Steffens et al. (1982).




DISCUSSION

Snake venoms contain numerous proteases which act at almost all steps of the blood coagulation cascade, either by specific proteolytic activation or by nonspecific degradation of blood factors (for a review, see Stoker (1990)). But the existence of components that could activate plasminogen, a major zymogen responsible for fibrinolysis in vivo, had not been previously reported. Such a protease that catalyzes the direct activation of plasminogen in vitro has been purified from the venom of T. stejnegeri. This plasminogen activator, designated as TSV-PA, represents about 0.5% of the proteins in a venom pool collected from a large number of the snakes. In fact, TSV-PA appears to be a constant component of individual venoms: all the individual samples collected from 15 living snakes of T. stejnegeri (8 from Anhwei Province in the central part of China and 7 from Yunnan Province in South West China) had similar plasminogen activation activity in vitro.

Inhibition of TSV-PA by various inhibitors and sequence comparisons established that it is a serine protease, belonging to the family of venom serine proteases. The homology of TSV-PA with venom and mammalian serine proteases indicates that the amino acid residues which form the catalytic triad are His, Asp, and Ser (Fig. 6). The amino acid sequences surrounding these residues are well conserved among serine proteases, including TSV-PA (Fig. 6). Like batroxobin (Itoh et al., 1987), TSV-PA contains 12 cysteine residues and by homology with trypsin it could be predicted that they are paired as: Cys-Cys, Cys-Cys, Cys-Cys, Cys-Cys, Cys-Cys, and Cys-Cys.

Although trypsin could also activate plasminogen in vitro (Wohl et al., 1980), TSV-PA can be readily distinguished from trypsin by its highly specific substrate specificity and its insensitivity to soybean trypsin inhibitor.

The sequence of TSV-PA exhibits a high degree of identity (60-66%) with other snake venom proteases, such as RVV-V from V. russelli venom (Tokunaga et al., 1988), batroxobin from B. atrox venom (Itoh et al., 1987), and ACC-C from A. contortrix contortrix venom (McMullen et al., 1989). However, these venom serine proteases present considerable difference in their substrate specificity. In particular, and at variance with the other snake venom proteases, TSV-PA did not clot nor degrade fibrinogen and did not activate nor degrade factor X, prothrombin, protein C. Sequence alignment of venom serine proteases reveals several variable regions which may explain their different substrate specificity. Particularly, in the region 43-50, TSV-PA lacks the positively charged arginine residues which are conserved in other venom proteases. This might explain why, at variance with other venom proteases, fibrinogen is a poor substrate for TSV-PA. In addition, the presence of Lys, instead of Gln which was conserved in trypsin and other venom serine proteases, renders TSV-PA more similar to t-PA and u-PA, both of which have Arg residue at this position.

The comparison of 5`- and 3`-noncoding regions of cDNAs encoding the venom serine proteases, TSV-PA, batroxobin, and a serine protease from Calloselasma rhodostoma venom (Au et al., 1993), surprisingly indicates that their untranslated regions are more conserved than coding regions, with a degree of identity of about 85% compared with 60-65%, respectively. A similar observation has already been reported in the case of snake venom phospholipase A isozymes (Nakashima et al., 1993). The venom serine proteases which have been characterized so far are regarded as a family of proteins which specifically interact with very different proteins on which they exert their physiological action. A phenomenon of accelerated evolution in the protein-coding regions of venom serine proteases, allowing an adaptive evolution of proteases, could therefore be postulated, as has been done in the case of venom phospholipase A.

TSV-PA also shares a weak sequence identity with the serine protease domain of t-PA (23%) and u-PA (21%). In addition to the catalytic domain, t-PA and u-PA have been shown to contain one or two kringle domains, one epidermal growth factor domain, and a fibronectin finger-like domain (Pennica et al., 1983; Gunzler et al., 1982), whereas TSV-PA consists only of a serine protease domain. Unlike t-PA and u-PA, TSV-PA has a relatively strong activity on small synthetic peptide substrates, like S-2238 or S-2251, but its plasminogen activation activity is about 50-fold lower than that of t-PA in the presence of fibrin fragments in the experimental conditions of our in vitro assay system. Binding to and stimulation by fibrin are hallmarks of t-PA, but the molecular details of these two important and distinctive processes, however, remain unclear (Madison, 1994). The fibronectin finger-like and kringle 2 domains of t-PA have been proposed to be responsible for its binding to fibrin (Horrevoets et al., 1994). In contrast with t-PA, which possesses a selectivity for fibrin clots (Hoylaerts et al., 1982; Fischer and Will, 1990), the activating activity of TSV-PA is not increased by the presence of fibrin fragments. The molecular structure of TSV-PA, which does not contain any kringle or fibronectin finger-like domains, does in fact suggest the involvement of these regions in the interaction of t-PA with fibrin.

Another important feature of plasminogen activators is their circulating half-life in vivo. Site-specific mutagenesis studies suggested that critical determinants recognized by hepatic receptors may be present in the finger, epidermal growth factor, and kringle 1 domains. For example, the epidermal growth factor-like domain has been implicated in the uptake of t-PA by hepatic cells (Collen et al., 1988). Deletion of this domain in t-PA markedly prolonged its half-time in blood, from less than 5 min for natural t-PA to 15 min for the deleted mutant molecule. As other snake venom proteases, TSV-PA may be expected to have an even lower clearance rate: for example, a half-life of 6.2 h was observed for defibrinase, the thrombin-like enzyme from Agkistrodon acutus venom (Fukutake and Ukita, 1988).

The most important endogenous inhibitor of t-PA and u-PA is endothelial cell PAI-1, a member of the serpin family. The fast inactivation of t-PA in vivo is in fact mainly due to its interaction with PAI-1. The interaction site of PAI-1 with t-PA and u-PA has been demonstrated to be localized in the N-terminal part of the serine protease domain, which is rich in positively charged amino acids, including the sequences KHRR (t-PA) and RRHR (u-PA). Deletion or charge reversal of these basic residues in t-PA resulted in a more than 1000-fold reduction in the inhibition rate by PAI-1 (Madison et al., 1989; Bennett et al., 1991; Keijer et al., 1991). The sequence of TSV-PA indicates that it lacks these positively charged residues, the corresponding region being absent in this molecule. It is therefore expected that TSV-PA will be insensitive to PAI-1.

In conclusion, a serine protease plasminogen activator, TSV-PA, showing a high specificity for plasminogen has been characterized for the first time in snake venom. Such an activator should play an important role in the mechanism of snake envenomations through fibrinogenolysis and hemorrhage, which are common aspects of such envenomations (Stocker, 1990). On the other hand, TSV-PA appears to be an interesting pharmacological tool to investigate fibrinolysis in vitro as well as in vivo. Studies are in progress to precisely investigate its mechanism of action. TSV-PA might also be useful in the design of new generations of thrombolytic agents by protein engineering. Recently, it has been shown that the fibrin specificity of t-PA can be substantially increased by mutations at several sites in the protease domain (Bennett et al., 1991; Paoni et al., 1993). The sequence of TSV-PA, which possesses a high selectivity for plasminogen, suggests additional mutations which might prove to be interesting in this line. On the other hand, it might be possible to improve the efficiency of TSV-PA by adding a fibrin binding domain to this molecule, to direct it to fibrin clots.

  
Table: Purification of TSV-PA


  
Table: Kinetic parameters of TSV-PA for different amide substrates


  
Table: Effect of protease inhibitors on amidolytic activity of TSV-PA



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Fondation pour la Recherche Médicale.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U21903.

To whom correspondence should be addressed: Unité des Venins, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, Cedex 15, France. Tel.: 33-1-4568-8685; Fax: 33-1-4061-3057.

The abbreviations used are: Glu-plasminogen, native Glu-plasminogen; Lys-plasminogen, plasmin-derived Lys-plasminogen; u-PA, urokinase-type plasminogen activator; t-PA, tissue-type plasminogen activator; TSV-PA, Trimeresurus stejnegeri venom plasminogen activator; FPLC, fast performance liquid chromatography; PMSF, phenylmethanesulfonyl fluoride; NPGB, p-nitrophenyl- p-guanidinobenzoate; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RACE-RT-PCR, rapid amplification of cDNA ends-reverse transcription polymerase chain reaction; RVV-V, Vipera russelli venom factor V activator; ACC-C, Agkistrodon contortrix contortrix venom protein C activator; PAI-1, plasminogen activator inhibitor type 1; pNA, p-nitroanilide; Bz, benozyl.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Eduardo Angles-Cano for his helpful advice, Dr. David Ojcius (Biologie des Interactions Cellulaires, Institut Pasteur, Paris) for critical reading of the manuscript, and Dr. Jacques d'Alayer (Laboratoire de microséquence, Institut Pasteur, Paris) for performing the amino acid sequence analysis.


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