Background Art Up to now, various enzymes, inhibitory factors and their complex which participate in blood clotting and platelet aggregation have been separated from snake venom (Reference: Stoker, K. F., Medical Use of Snake Venom Protein, Boston, CRC Press, 97-160,1990). For example, thrombin-like enzyme coagulating fibrinogen, fibrino (geno) lytic enzyme degrading fibrin (fibrinogen), platelet aggregation activator, platelet aggregation inhibitory factor and so on have been disclosed (Reference: Holleman, W. H. and Weiss, L. J., J. Biol. Chem., 251 : 1663-1669, 1976; Itoh, N. et al., J. Biol. Chem., 262: 3132-3135,1987; Markland, F. S. Jr., Thromb. Haemost., 65: 438-443,1991). It is also reported that these enzymes consist in 2 subunits which contain C-type lectin motif and are linked with a disulfide bond. However, the accurate mechanism that the enzymes work on platelet aggregation has not been clarified yet (Reference: Drickamer, K., J. Biol. Chem., 263: 9557-9560,1998).

Meantime, thrombin is a multi-functional protease belonging to the serine family

and catalyzes the fibrinogen coagulation. Concretely it participates in blood clotting, activation of anti-aggregation factor and other physiological regulations (Reference: Esmon, C. T., Annu. Rev. Cell. Biol., 9: 1-26,1993). In these functions, external binding site of thrombin as well as active site of thrombin plays an important role to combine blood clotting factors with thrombin (Reference: Guillin, M. C. et al., Thromb. Haemost., 74: 129-133, 1995). Practically, fibrinogen has been known to attach onto the external binding site of thrombin, exosite 1 (Reference: Bouton, M. C. et al., Thromb. Haemost., 80 : 310-315,1998).

For the activation of thrombin, prothrombin should be changed into thrombin, which is a leading process in the hemostasis. Especially, prothrombinase is a catalyst complex which works on the above conversion process and is composed of factor Xa as a serine family protease, co-factor Va, phospholipid and calcium.

On the other hand, the blood clotting process described above is closely related with thrombus and cardiovascular diseases caused by thrombus such as coronary disease, cerebrovascular disease, vascular thrombosis and the like. Therefore, it is necessary to inhibit the activation of fibrinogen and thrombin for preventing and treating thrombotic diseases.

Disclosure of Invention To satisfy the foregoing and other purposes, we, the inventors of the present invention have separated a new protein which is related with blood clotting and can prevent and treat thrombus and have examined its use for an pharmaceutically effective component of anti-thrombus agents. Concretely, the venom of Korean adder, Agkistrodon halys brevicaudus was collected and then processed for purifying an effective protein by performing anion exchange resin chromatography and gel filtration and again anion exchange resin chromatography. As a result, the present invention has confirmed that the protein

prepared above can inhibit fibrinogen clotting potently.

Precisely, it is an object of the present invention to provide a cDNA molecule of new protein from Korean adder venom which has the inhibitory activity against fibrinogen clotting.

It is another object of the present invention to provide a new protein with the inhibitory activity against fibrinogen clotting which is separated from Korean adder venom and contains the amino acid sequence deduced from said cDNA.

It is a third object of the present invention to provide a process for preparing said protein with the inhibitory activity against fibrinogen clotting by using said venom.

Further objects and advantages of the present invention will appear clearly hereinafter.

Precisely, the present invention provides new protein with the inhibitory activity against fibrinogen clotting and its cDNA molecule as described below.

Above all, the inventors of the present invention have adopted the venom of Korean adder, Agkistrodon halys brevicaudus and have prepared a novel protein with the inhibitory activity against fibrinogen clotting by the method comprising the steps: (1) collecting the snake venom; and (2) processing with anion exchange chromatography and gel filtration and again with anion exchange chromatography for the purification.

Preferably, Q-Sepharose FPLC column and Mono Q FPLC column are chosen for the anion exchange chromatography and Superdex 75 FPLC column is adopted for the gel filtration preferably. This will not limit the scope of the present invention.

The protein purified by the above process is verified to have 27kDa molecular weight under non-reduced state and to be a heterodimeric protein which consists of two chains of 15 kDa and 14kDa molecular weights respectively under reduced state. Then, the chains of the protein are named'A chain'and'B chain'independently and identified to have

7 residues of cystein.

In order to examine the characteristics of the purified protein with the inhibitory activity against blood coagulation, SDS-Polyacrylamide gel electrophoresis (SDS-PAGE), amino acid sequence analysis and so on are performed. Concretely, cDNA library of venom gland from the above adder is manufactured and cDNA of the above protein is cloned. Then the N-terminal amino acid sequence of A peptide and B peptide are determined and on the basis of these sequences, complementary primers are synthesized so as to be utilized for the selection of cDNA clones. Also, on the basis of this cDNA, antisense primers are prepared so as to choose an accurate cDNA clone. As a result, the nucleotide sequence of the cDNA clone is determined and its amino acid sequence is deduced from the nucleotide sequence.

Precisely, both A peptide chain and B peptide chain contain a hydrophobic signal peptide with 23 amino acid residues and are composed of 131 and 122 amino acid residues respectively, which shows 45% of sequence homology between two chains. In addition, the protein purified above is identified to have a sequence homology in the primary amino acid sequence compared with snake venom C-type lectin protein and to be new. Especially, it has 45-65% of sequence homology in the coding region and thus is named'Salmorin'.

In detail, the present invention provides the cDNA molecule of Salmorin protein from Korean adder venom which consists of Salmorin A chain and Salmorin B chain. The nucleotide sequence encoding Salmorin A chain is shown in SEQ ID NO: 5 and the nucleotide sequence encoding Salmorin B chain is shown in SEQ ID NO: 7.

In addition, the present invention provides the Salmorin protein which is composed of Salmorin A chain with the amino acid sequence deduced from the nucleotide sequence of SEQ ID NO: 5 and Salmorin B chain with the amino acid sequence deduced from the nucleotide sequence of SEQ ID NO: 7.

Besides, the Salmorin protein is investigated to make clear the mechanism that it

affects thrombin, a catalyst of blood coagulation. Consequently, the Salmorin is thought to prevent the association of thrombin and fibrinogen by binding onto an exosite, not active site and makes two molecular complex with prothrombin so as to inhibit the activation of prothrombin to thrombin and to delay the blood coagulation.

Therefore, the Salmorin protein of the present invention can be utilized to treat thrombosis efficiently since it represses the fibrinogen clotting potently by binding onto prothrombin and thrombin so as to delay the blood coagulation.

Brief Description of the Drawings The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which; FIG. la depicts cDNA nucleotide sequences of Salmorin A chain and amino acid sequences deduced from the cDNA sequence.

FIG. 1b depicts cDNA nucleotide sequences of Salmorin B chain and amino acid sequences deduced from the cDNA sequence.

FIG. 2 depicts the inhibitory activity of Salmorin protein against fibrinogen clotting.

FIG. 3 depicts the graph that Salmorin protein represses the inhibitory activity of hirudin against thrombin.

FIG. 4a depicts Salmorin protein and prothrombin which are analyzed by performing polyacrylamide gel electrophoresis (PAGE).

FIG. 4b depicts the effect of Salmorin protein in the activation of prothrombin mediated by factor Xa.

FIG. 5a depicts the prothrombin activity mediated by factor Xa in the presence of

Salmorin protein.

FIG. 5b depicts the time delay of fibrinogen clotting caused by Salmorin protein of the present invention.

Best mode for carrying out the invention EXAMPLES Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples.

However, it will be appreciated that those skilled in the art, in consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Preparation of the protein Crude snake venom was obtained from Korean adder, Agkistrodon halys brevicaudus. 256 mg of crude venom extract was diluted 10 times with 20 mM Tris-HCl buffer (pH 8.0) and poured into Q-Sepharose HP column (2.6 X 10 cm ; Pharmacia-LKB Biotechnology Inc., Sweden) equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 200 mM of NaCI so as to be eluted with 20 mM Tris-HCl buffer containing 200 mM of NaCl. Then, the eluted fractions exhibiting the inhibitory activity against fibrinogen clotting were collected and the fibrinogen clotting titers were measured by performing fibrinogen clotting assay (Reference: Hofmann, H. et al., Biochimie, 65: 201-210,1983). Concretely, the eluted fractions were pretreated with 0.1 ml of thrombin (Sigma Chemical Co., U. S. A.) at 37°C for 2 minutes to adjust 0.5 unit/ml of final thrombin concentration and then measured the time period required to coagulate fibrinogen by adding the eluted *action/thrombin into

0.5 ml of human fibrinogen (0.5 mg/ml, Green Cross company) dissolved in 20 mM Tris-HCl (pH 7. 5).

The fractions having the inhibitory activity against fibrinogen clotting were concentrated and the concentrated solution was added to Superdex 75 FPLC column (l X 30 cm, Pharmacia, Sweden) for a gel filtration chromatography. Again, the active fractions were collected and poured into Mono Q FPLC column (0.5 X 5 cm) and then eluted with linear concentration gradient by using an equilibrium buffer containing 200 mM-300 mM NaCl.

As a result, the proteins were obtained at about 265 mM of NaCl concentration with high purity. The final productivity of proteins was 0.5 mg and this corresponded to 0.2% of total venom proteins.

Example 2: Identification of purity in the protein The purified proteins were examined by performing 15% SDS-polyacrylamide gel electrophoresis in non-reduced state and in reduced state with 2-mercaptoethanol. As a result, the protein of 28kDa molecular weight was shown clearly as single band in non-reduced state and two polypeptide chains of 15kDa and 14kDa molecular weight were separated respectively in reduced state. Therefore, the purified protein of the present invention was confirmed to be a heterodimeric protein and to be composed of two different subunits.

Precisely, the polypeptide chain of 15kDa molecular weight was named'A chain'and the polypeptide chain of 14kDa molecular weight was called'B chain'.

Example 3: cDNA cloning and determination of nucleotide sequences The cDNA library of adder venom gland was constructed by using 7bZAPXR cDNA cloning kit (Stratagene, U. S. A.) and i71 vitro packaging kit (Amersham, U. S. A.) as described below. Above all, the N-terminal amino acid sequences of fibrinogen clotting

inhibitory peptide purified above were determined in order to make primers for selecting cDNA clones of the above protein (Applied Biosystems Precise Protein Sequencing System, U. S. A.) and on the basis of these, degenerate oligonucleotide primers were also synthesized.

In A chain, primer of SEQ ID NO: 1; 5'-GCTGATTT (T/C) TT (T/C) TG (T/C) CC-3' In B chain, primer of SEQ ID NO: 2; 5'-GATTGTCC (C/T) TC (A/C/G/T) GG (A/C/G/T) TGG-3' The cDNA library was chosen by performing polymerase chain reactivity with T7 primer and above synthetic primers. About 550 bp of PCR products corresponding to each polypeptide chain was obtained and inserted into the cloning vector pGEM-T (Promega, U. S. A). Then the nucleotide sequence of the PCR product cloned into the vector pGEM-T was determined by using Sequenase kit (USB) as follows. Concretely, antisense primers for A chain and B chain of the above protein were prepared on the basis of partial cDNA sequences in order to determine the complete nucleotide sequence to 5'-terminus of cDNA and the sequences of antisense primers were depicted below.

In A chain, primer of SEQ ID NO: 3; 5'-GACTAAGCCTCGCAGACGAA-3' In B chain, primer of SEQ ID NO: 4; 5'-CTAGGCCTGGAACTCGC-3'

The nucleotide sequence of cloned cDNA and the amino acid sequence deduced above were analyzed by using DNASIS, PROSIS and BLAST searching programs.

FIG. la depicted cDNA nucleotide sequences of Salmorin A chain shown in SEQ ID NO: 5 and amino acid sequences deduced from the cDNA sequences shown in SEQ ID NO: 6. As illustrated in FIG. la, the cDNA encoding A chain of the purified protein was identified to consist in 462 bp of open reading frame, stop codon, 124 bp of 3'-untranslated region and poly A tail.

FIG. lb depicted cDNA nucleotide sequences of Salmorin B chain shown in of SEQ ID NO: 7 and amino acid sequences deduced from the cDNA sequences shown in of SEQ ID NO: 8. As illustrated in FIG. lb, the cDNA encoding B chain of the purified protein was identified to consist in 435 bp of open reading frame, stop codon, 126 bp of 3'- untranslated region and poly A tail. In addition, the matured N-termini of both A chain and B chain were verified to comprise a hydrophobic signal peptide with 23 amino acid residues.

As a result, it was confirmed that both A chain and B chain were composed of 131 and 122 amino acid residues respectively when the amino acid sequence is deduced from the nucleotide sequence. This showed 45% of sequence homology between two peptide chains and molecular weights of the two polypeptides were 14890 Da and 14341 Da in A chain and B chain respectively when calculated with the deduced amino acid sequence. The results corresponded to the molecular weights, which was examined by using SDS-polyacrylamide gel electrophoresis.

Example 4: Examination of fibrinogen clotting inhibition mechanism by the purified protein

Example 4-1: Effect of the protein upon thrombin activation In order to examine the effects of Salmorin protein upon thrombin catalyzing fibrinogen clotting directly, the fibrinogen clotting time of Salmorin was measured by the process described in Example 1. Concretely, Salmorin was reacted with thrombin at 37°C for 2 minutes and 0.5 ml of fibrinogen (0.5 mg/ml) dissolved in 20 mM Tris-HCl (pH 7.5) was added onto above Salmorin/thrombin so as to record the clotting time. FIG. 2 depicted the graph of Salmorin protein showing the inhibitory activity against fibrinogen clotting. As shown in FIG. 2, the inhibitory effect against fibrinogen clotting was examined to increase dose-dependently.

The inhibitory effect of Salmorin to thrombin activation was observed by using a coloring substrate, D-Phe-pipercolyl-Arg-p-nitroanilide (Referred to as'S-2238'hereinafter ; Sigma Chemical Co., U. S. A.) which enabled to calculate amidolytic activities. As a result, Salmorin was verified not to bind directly onto the active site of thrombin since it does not repress the degradation of S-2238.

Meanwhile, the function of hirudin which was known to inhibit the amidolytic activity of thrombin by binding onto thrombin active site and exosite 1 was studied for finding the effects of Salmorin (Reference: Rydel, T. J. et al., Science, 249: 277-280,1990).

FIG. 3 depicted the graph that Salmorin protein repressed the inhibitory activity of hirudin against thrombin. At that time, (A) indicated the result treating S-2238 and (A) indicated the result treating hirudin and S-2238 together. As shown in FIG. 3, the Salmorin of the present invention was identified to prevent the activity of hirudin and expected to compete with fibrinogen or hirudin by binding onto exosite 1 of thrombin.

Consequently, the Salmorin of the present invention was confirmed to repress fibrinogen clotting caused by thrombin but it does not affect the thrombin activity directly.

Example 4-2: Identification of prothrombin and Salmorin complex As illustrated in Example 4-1, the Salmorin of the present invention worked to repress the activation of prothrombin to thrombin by using factor Xa and thus examined whether it inhibited the above process by binding onto prothrombin directly as described below. Above all, prothrombin was pretreated with Salmorin in 50 mM Tris-HCl (pH 7.5) for 5 minutes and separated by performing 12% native polyacrylamide gel electrophoresis. Then the complex of Salmorin and prothrombin were analyzed by performing the native polyacrylamide gel electrophoresis in non-reduced state.

FIG. 4a depicted the result of Salmorin protein and prothrombin analyzed with Polyacrylamide gel electrophoresis (PAGE). At that time, lane 1 indicated 30 pmole of prothrombin; lane 2,5 pmole of Salmorin; lane 3,30 pmole prothrombin pretreated with 15 pmole of Salmorin; and lane 4, prothrombin pretreated with 30 pmole of Salmorin. As shown in FIG. 4a, when Salmorin and prothrombin were reacted in the same molar ratio, two protein bands disappeared and single band appeared slowly. Thus the Salmorin of the present invention was identified to make two molecular complex with prothrombin.

Meanwhile, FIG. 4b depicted the effect of Salmorin protein in the prothrombin activation mediated by factor Xa. At that time, lane 1 indicated 10 pmole of factor Xa; lane 2, 5 pmole of Salmorin; and lane 3,10 pmole of factor Xa pretreated with 5 pmole of Salmorin. As shown in FIG. 4b, the Salmorin of the present invention was identified not to make complex with factor Xa.

In addition, the function of factor Xa toward Salmorin/prothrombin complex was examined as follows. Concretely, 10 pmole of prothrombin was reacted with Salmorin and 0.05 unit of factor Xa dissolved in 50 mM Tris-HCl (pH 7.5) for 37°C for 20 minutes and then the reactivity mixture was separated by performing 12% SDS-polyacrylamide gel electrophoresis.

FIG. 5a depicted the factor Xa mediated prothrombin activities in the presence of Salmorin protein. At that time, lane 1 indicated prothrombin; lane 2, prothrombin pretreated with factor Xa; lane 3, prothrombin pretreated with factor Xa in the presence of 1 pmol of Salmorin; and lane 4, prothrombin pretreated with factor Xa in the presence of 10 pmole of Salmorin. As shown in lane 2 of FIG. 5a, prothrombin was hydrolyzed by factor Xa and cleaved into meizothrombin fragment 1 and 2 and prethrombin 2. Besides, the hydrolysis was repressed completely when prothrombin and Salmorin existed in the same molar ratio as illustrated in lane 4.

Furthermore, the time period for fibrinogen clotting was measured by the procedure described in Example 1 in order to examine the inhibitory activity against prothrombin activation in the presence of Salmorin.

FIG. 5b depicted the time delay of fibrinogen clotting caused by the Salmorin of the present invention. At that time, (A) indicated the result treating the mixture of 10 nM prothrombin, Salmorin and factor Xa; (A) indicated the result treating the mixture of 30 nM prothrombin, Salmorin and factor Xa; (o) indicated the result treating the mixture of 50 nM prothrombin, Salmorin and factor Xa. As shown in FIG. 5b, the fibrinogen clotting was confirmed to be delayed according to molar concentration ratio of Salmorin/prothrombin.

Industrial Applicability As described clearly and confirmed above, the present invention provides novel venom protein derived from Korean adder, Agkist7-odoi7 halys brevicaudus which has the inhibitory activity against fibrinogen clotting, cDNA encoding said protein and process for preparing thereof.

The Salmorin protein of the present invention can be prepared by the method

comprising the steps: (1) collecting the Korean adder venom; and (2) processing with anion exchange resin chromatography and gel filtration and again with anion exchange chromatography for the purification. As a result, the Salmorin protein was separated with SDS-polyacrylamide gel electrophoresis (PAGE) under non-reduced state so as to be single band of about 27 kDa molecular weight and under reduced state, to be two bands of Salmorin A chain and B chain which contain 7 residues of cystein and make two molecular complex with prothrombin. In addition, the Salmorin protein of the present invention can be utilized widely as a pharmaceutically effective component for treating thrombosis since it represses the activation of prothrombin into thrombin and thus inhibits fibrinogen clotting dose-dependently and delays blood coagulation by competitively working with hirudin.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.