KR20000018933A - Kringle proteins derived from human prothrombin having inhibitory activity of growth of endothelial cell - Google Patents

Abstract

The present invention relates to a human prothrombin Kringle-1, -2 protein derived from human prothrombin and having endothelial cell growth inhibitory activity and a gene encoding the same. We isolated and purified prothrombin protein from human plasma, hydrolyzed it with factor Xa, a prothrombin activating enzyme, and separated and purified human prothrombin Kringle-1 and -2 from electrolysates using ion exchange chromatography. It was. Subsequently, cell growth experiments using bovine capillary endothelial cells and chick chorioallantoic membrane (CAM) analysis showed that human prothrombin kringle-1 and -2 had excellent endothelial growth inhibitory activity and angiogenesis inhibitory function. Furthermore, cDNAs of human prothrombin kringle-1 and -2 were obtained by polymerase chain reaction using human prothrombin cDNA as a template, and the cDNAs were cloned into E. coli expression vectors, respectively. Human prothrombin Kringle-1 and -2 of the present invention have excellent endothelial growth inhibitory activity and directly inhibit angiogenesis, and thus can be effectively used for the treatment of rheumatism or cancer, and also prothrombinase during blood coagulation. Inhibiting the activity of the complex may be useful as a therapeutic agent for diseases caused by blood clots.

Description

Human prothrombin kringle protein and endogenous cell growth inhibitory activity

The present invention relates to a human prothrombin kringle protein having endothelial cell growth inhibitory activity and a gene encoding the same. More specifically, the present invention comprises two Kringle structures, derived from human prothrombin and having endothelial growth inhibitory activity, named 'human prothrombin kringle-1' and 'human prothrombin kringle-2', respectively. Eggplant proteins and genes encoding them.

Prothrombin, a precursor of thrombin, a major coagulant in the coagulation process, is a glycoprotein in plasma with a molecular weight of 72 kDa, synthesized in hepatocytes, and glycosylation and gamma-carboxylation (γ). It is secreted into the blood vessel through transformation after transcription such as -carboxylation. At the onset of coagulation, the inactive form of prothrombin is hydrolyzed by Factor Xa into an N-terminus containing two kringle structures and an active form of thrombin, whereby the generated thrombin changes blood by converting fibrinogen into fibrin. Induce coagulation In this process, the prothrombin fragments 1 and 2, which form the N-terminus of prothrombin, are cut out. The fragment 1 portion is glycosylated through post-translational modification, while the fragment-2 portion retains the unmodified Kringle structure. (Esmon, CT, Owen, WG and Jackson, CM, J. Biol. Chem., 249: 8045 (1974)).

Kringle refers to a domain commonly found in serine protease-based proteins involved in blood coagulation and thrombosis, and contains three disulfide bonds that form a unique tertiary structure. It consists of about 80 amino acid residues, including Furie, B. and Furie, CB, Cell, 53: 505-518 (1988). This structure was first discovered in prothrombin, which plays an important role in the coagulation process. In addition, it is involved in blood coagulation and hemolysis of plasminogen, urokinase, tissue-type plasminogen activator (tPA), and Factor XII. Are found in a wide variety of proteins in the blood, from proteolytic enzymes to apolipoprotein A and human hepatocyte growth factor, which play an important role in the fat transfer process. have. In addition, the Kringle structure has high similarity not only between proteins but also between species, and is considered to have been preserved for at least 500 million years. On the other hand, since Kringle structure is found in blood proteins intensively, it is assumed that it is very likely to be a tool for the regulation of blood coagulation and intercellular recognition. The report was insignificant.

Recently, however, researchers such as Folkman (J) and others have found that kringle-structured proteins, such as angiostatin and endostatin, inhibit the growth of endothelial cells. (Cao, Y., Ji, RW, Davidson, D., Schaller, J., Marti, D., Sohndel, S., McCance, SG, O'Reilly, MS, Llinas, M. and Folkman, J., J. Biol. Chem., 271: 29461-29467 (1996)). According to them, angiostatin with a molecular weight of 38 kDa including four kringle structures, as well as each kringle region constituting the protein, also depends on bFGF (basic fibroblast growth factor) for growth of endothelial cells. It has been shown to inhibit by 50%, and the growth of bFGF-dependent endothelial cells is directly related to abnormal angiogenesis, which is drawing more attention.

In general, endogenous cells rarely divide or grow during normal adult development (see Engerman, RL, Pfaffenbacvh, D. and Davis, MD, Lab Invest., 17: 738-743 (1967). )). However, when new angiogenesis is required in connection with the treatment of a wound or female reproduction, or when growth and metastasis of malignant cells occurs, certain growth factors are secreted and these growth factors are almost stopped growing. By stimulating endothelial cells to induce division and growth, new angiogenesis occurs (see Auerbach, W. and Auerbach, R., Pharmacol. Ther., 63: 265-311 (1994); Cockerill, GW, Gamble, JR and Vadas, MA, Int. Rev. Cytol., 159: 113-160 (1995); Adams, JM and Cory, S., Science, 254: 1161-1167 (1991)). In addition, it is known that abnormal angiogenesis in differentiated adults is closely related to relatively high incidence and difficult to treat diseases such as rheumatoid arthritis and hemangioma. It is an increasing trend in the field.

Therefore, by artificially controlling the angiogenesis process, it is believed that various diseases associated with abnormal angiogenesis can be treated and prevented. In particular, in the case of malignant tumors, the angiogenesis process is essential for the growth and metastasis of tumor cells, the development of angiogenesis inhibitors is expected to be an effective response to malignancies. Indeed, Isaiah, J, et al. Are developing methods to prevent cancer metastasis by inhibiting angiogenesis (see Isaiah, J. and Ellis, LM, Cell, 79: 185-188 (1994); Wu, Z., O'Reilly, MS, Folkman, J. and Shing, Y., Biochem.Biophys.Res.Commun., 236: 651-654 (1997); Abraham, JA, Mergia, A., Whang, JL, Tumolo, A., Friedman, J., Hjerrild, KA, Gospodarowicz, D. and Fiddes, JC Science, 233: 545-548 (1986)).

On the other hand, the inventors found prothrombin Kringle-2 protein that inhibits the growth of endothelial cells from the serum of New Zealand white rabbit sensitized with Bacillus calmet-gerin and treated with endotoxin, and purely isolated the electric protein, The amino acid sequence was determined, and the cDNA of rabbit prothrombin Kringle-2 was cloned (see Korean Patent Application No. 97-82347; Korean Patent Application No. 97-82346).

Therefore, the present inventors expect that the N-terminal fragment ("Kringle protein") containing the Kringle structure, derived from human prothrombin, also has endothelial cell growth inhibitory activity, and as a result, the human prothrombin Kringle- After confirming that the 1 and -2 proteins have potent endothelial growth inhibitory activity, the cDNAs of human prothrombin kringle-1 and -2 were cloned and the present invention was completed.

Finally, it is an object of the present invention to provide two proteins derived from human prothrombin and having excellent endothelial growth inhibitory activity, human prothrombin kringle-1 and human prothrombin kringle-2.

Another object of the present invention is to provide cDNAs of electric human prothrombin Kringle-1 and -2.

It is another object of the present invention to provide a recombinant expression vector comprising cDNAs of human prothrombin Kringle-1 and -2, respectively.

1 is a gel photograph of SDS-PAGE analysis of purified human prothrombin from human plasma.

FIG. 2 is a gel photograph of electrophoresis of peptides generated when purified human prothrombin is cleaved with Factor Xa. FIG.

FIG. 3 is a chromatogram showing the distribution of each protein eluted when the peptides produced when cleaving human purified prothrombin by Factor Xa are separated by ion exchange chromatography.

4 is a graph showing endothelial cell growth inhibitory activity of human prothrombin-derived peptides.

5a, 5b and 5c are photographs showing the angiogenesis inhibitory activity of human prothrombin Kringle-1 and -2.

6a and 6b show the nucleotide sequence of human prothrombin cDNA.

7 is a photograph of cDNA of human prothrombin Kringle-1 and -2 amplified by DNA polymerase chain reaction by 1.5% agarose gel electrophoresis.

8 is a photograph of the recombinant expression vectors pEF-hK1 and pEF-hK2 containing human prothrombin Kringle-1 and -2 cDNA, respectively, treated with restriction enzymes and analyzed by 1.5% agarose gel electrophoresis.

The inactive form of prothrombin is hydrolyzed by factor Xa into an N-terminus comprising two kringle structures and an active thrombin, wherein the electrical N-terminus each comprises one Kringle structure. It is cut into fragments 1 and 2. Hereinafter, for convenience, the electric fragments 1 and 2 will be referred to as 'prothrombin kringle-1' and 'prothrombin kringle-2', respectively.

We isolated and purified prothrombin protein from human plasma, hydrolyzed it with factor Xa, a prothrombin activating enzyme, and separated and purified human prothrombin Kringle-1 and -2 from electrolysates using ion exchange chromatography. It was. Subsequently, cell proliferation experiments using bovine capillary endothelial cells and chick chorioallantoic membrane (CAM) analysis showed that human prothrombin kringle-1 and -2, which were electropurified, had excellent endothelial growth inhibitory activity and angiogenesis inhibitory function. It was. Furthermore, cDNAs of human prothrombin kringle-1 and -2 were obtained by polymerase chain reaction using human prothrombin cDNA as a template, and the cDNAs were cloned into E. coli expression vectors, respectively.

Hereinafter, the present invention will be described in more detail.

To separate and purify human prothrombin Kringle-1 and -2, the present inventors first separate and purify human prothrombin from human plasma, and hydrolyze the purified prothrombin with factor Xa, a prothrombin activating enzyme in vivo, -1 and Kringle-2 were generated. The protein hydrolyzate was then subjected to mono-Q ion exchange resin chromatography, and pure thrombin, thrombin, human prothrombin kringle-1 and human prothrombin kringle-2 were purified purely using a sodium chloride straightener. . In order to investigate the endothelial growth inhibitory activity of the purified human prothrombin-derived proteins, each protein and purified prothrombin were added to bovine capillary endothelial cells (BCE cells) at different concentrations and treated with basic fibroblast growth factor (bFGF). After inducing cell growth, the cell number was measured after a certain time, and it was confirmed that only prothrombin Kringle-1 and -2 effectively inhibited endothelial cell growth. Based on these results, we anticipate that human prothrombin kringle proteins may inhibit angiogenesis, and chick chorioallantoic membrane (CAM) analysis revealed that only kringle-1 and -2 blood vessels, as in electric cell growth experiments, It was confirmed to directly inhibit the formation. Thus, the inventors have used human prothrombin cDNA templates using oligonucleotide primers synthesized based on the N-terminal and C-terminal amino acid sequences of human prothrombin kringle-1 and -2 to secure human prothrombin kringle gene. The polymerase chain reaction was performed to amplify cDNA fragments encoding human prothrombin Kringle-1 and -2, respectively, and cloned the above cDNA into E. coli expression vectors, respectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples in accordance with the gist of the present invention. .

Example 1 Purification of Human Prothrombin

Human prothrombin was isolated and purified according to the method of Walker (Falker, F.J.) et al. (Weinstein, R.E., Rickles, F.R. and Walker, F.J., Trombosis Res., 59: 759 (1990)). First, sodium citrate was added to human plasma purchased from a blood source (Severance Hospital, 134 Sinchon-dong, Seodaemun-gu, Seoul) to a final concentration of 0.85% (w / v) to prevent blood coagulation. Centrifugation was performed for minutes to remove residual blood cells and insoluble matters in the plasma solution. Then, benzamidine-HCl and P-PACK (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone) were added to a final concentration of 5 mM and 1 M, respectively, and a kind of ion exchange resin per 350 ml of sample. 10 g of phosphorus QMA Accell (Pharmacia, Sweden) was added. The solution was stirred at room temperature for 1 hour to allow proteins in the sample to adhere to the ion exchange resin, precipitate the ion exchange resin and then remove the supernatant. Wash the ion-exchange resin with 4 ~ 5 times of electric protein with washing solution (20mM Tris-HCl, 100mM NaCl, 5mM benzamidine-HCl, 1μM P-PACK, pH 7.5), and eluate (0.6M NaCl, 20mM Tris). Protein was eluted with -HCl, 5 mM benzamidine-HCl, 1 μM P-PACK, pH 7.5). Sodium citrate was added to the eluted protein to a final concentration of 0.6% (w / v /), and 10M barium chloride solution was added stepwise. The electric protein solution was stirred at 4 ° C. for 1 hour and then centrifuged at 4,500 × g for 20 minutes to obtain a protein precipitate, which was dissolved in a 5 mM benzamidine-HCl solution containing 0.2 M EDTA and tris at 4 ° C. Dialysis against the solution. The dialysed protein solution was loaded onto an FPLC column and the protein was eluted by forming a sodium chloride linear thickener ranging from 0.2M to 0.4M. 1 is a gel photograph showing the results of analyzing the human prothrombin purified by the above-described process by SDS-PAGE.

Example 2: Induction of Human Prothrombin Kringle-1 and -2

In order to cleave the purified human prothrombin obtained from Example 1 to Factor Xa, first, 10 mg of purified prothrombin was dialyzed against a Factor Xa reaction solution (10 mM Tris, 100 mM NaCl, 10 mM CaCl ₂ , pH 7.4). 0.5 units of Factor Xa (Sigma, USA) was added to the dialysis protein sample, and hydrolyzed prothrombin by reacting at 25 ° C. for 12 hours, followed by a 10,000-fold reaction stop solution (10 mM Tris, 100 mM NaCl, 1 mM PMSF). , pH7.4). Subsequently, cleavage of prothrombin was confirmed by electrophoresis (see FIG. 2).

Example 3: Purification of Human Prothrombin Kringle-1 and -2 by Ion Exchange Chromatography

As shown in FIG. 2, the hydrolyzate of prothrombin is mixed with non-hydrolyzed prothrombin and kringle-2 sites in addition to prothrombin kringle-1, -2 and thrombin. Therefore, in order to separate and purify only prothrombin Kringle-1 and -2 from the hydrolyzate of prothrombin, ion exchange chromatography was performed in the following manner: First, the hydrolyzate of prothrombin obtained from Example 2 was 10,000-fold. Dialysed against a volume of mono-Q buffer (50 mM sodium phosphate, 1 mM PMSF, pH 7.0), then loaded into a mono-Q column (Pharmacia, Sweden) equilibrated with mono-Q buffer and loaded at 50 mM to 700 mM. Proteins were eluted by forming a sodium chloride linear thickener. At this time, by measuring the absorbance at 280nm to recover the protein fractions expected to include prethrombin, thrombin, prothrombin Kringle-1 and -2 (see Fig. 3), SDS-PAGE for each fraction As a result, each fraction was represented as a single band, and it was confirmed that prethrombin, thrombin, prothrombin kringle-1 and -2 were successfully separated and purified, respectively. This result was also confirmed by the N-terminal amino acid sequencing of each peptide.

Example 4 Measurement of Endothelial Growth Inhibitory Activity of Human Prothrombin-Derived Peptides

Primary cultures of bovine capillary endothelial cells (BCE cells) were grown using a growth medium containing heat treated 10% bovine calf serum and 3 ng / ml recombinant human bFGF (R & D Inc., USA). And maintained in a plate coated with gelatin. Electrocultured cells were isolated by treatment with 0.05% trypsin solution and then diluted to about 12,500 cells per 0.5 ml with growth medium containing 10% calf serum. 0.5 ml of the diluted cell solution was added to each well of gelatin-coated 24-well-plate, incubated at 37 ° C. for 24 hours, and then the medium was replaced with 0.25 ml of fresh growth medium containing 5% calf serum. Purified prothrombin Kringle-1, -2, thrombin or prothrombin purified in Example 1 were then added to the electric cells at concentrations of 0.5, 1, 2, 4 and 10 μg / ml respectively. After 30 minutes, the volume of the medium was adjusted to 0.5 ml, and then bFGF was treated to a final concentration of 1 ng / ml. After 72 hours, the cells were separated by trypsin and the cell number was measured to investigate the endothelial growth inhibitory activity of various peptides derived from prothrombin. As a result, prothrombin and thrombin have little endothelial growth inhibitory activity, whereas prothrombin kringle-1 and -2 are similar to conventional endothelial growth inhibitory proteins such as rabbit prothrombin kringle-2 and angiostatin. Or it was confirmed that it has a somewhat high endothelial growth inhibitory activity (see Fig. 4). Table 1 below summarizes the endothelial cell growth inhibitory activity of human prothrombin Kringle-1, -2 and conventional endothelial cell growth inhibitory proteins.

Comparison of Endothelial Growth Inhibitory Activity of Human Prothrombin Kringle-1, -2 and Conventional Endothelial Growth Inhibitory Proteins protein ED ₅₀ (nM) * Human Prothrombin Kringle-1 100 Person Prothrombin Kringle-2 120 Rabbit Prothrombin Kringle-2 155 Angiostatin 140 Angiostatin Kringle-1 320 Angiostatin Kringle-2 460

*: 50% cell growth inhibition concentration

Example 5 Determination of Angiogenesis Inhibitory Activity of Human Prothrombin-Derived Peptides

Commercially available chickens were fertilized with eggs and washed with 70% ethanol, and then incubated for 48 hours at 37 ° C. and 3% CO ₂ with the blunt side up with an air pocket. A sterile syringe was used to puncture the pointed side of the fertilized egg, extract 2-3 ml of white matter, seal the hole with tape, and incubate for another 24 hours at 37 ° C. and 3% CO ₂ . Then, using a knife in the air bag portion of the fertilized egg using a knife to make a window about 2cm in diameter, and about 1cm in diameter fertilized egg formed with chick chorioallantoic membrane (CAM) was selected, 10μl 0.45% aqueous solution of methylcellulose and human prothrombin Samples prepared by mixing 25 μg of Kringle-1, -2, thrombin, prethrombin, or prothrombin, respectively, and drying in a clean bench were placed in an electric window and the window was sealed with tape, followed by 37 ° C., 3% CO ₂ conditions. Incubated for 48 hours more. Subsequently, Intralipose fat emulsion (Green Cross, Korea) was injected under the CAM, and the degree of angiogenesis under the electrical sample treatment site was observed. In this case, a fertilized egg treated with PBS was used instead of the electric sample as a control. As a result, it was confirmed that angiogenesis was not inhibited when prothrombin, prethrombin, and thrombin were treated, whereas angiogenesis was inhibited when prothrombin kringle-1 and -2 were treated (see FIG. 5A, 5b and 5c). Figure 5a is a control group treated with PBS, 5b is a 25μg prothrombin Kringle-1 treatment group, and Figure 5c shows a 25μg prothrombin Kringle-2 treatment group, respectively.

Example 6: Cloning of cDNAs of Human Prothrombin Kringle-1 and -2

To clone cDNAs of human prothrombin kringle-1 and -2, based on the N-terminal and C-terminal amino acid sequences of human prothrombin kringle-1 and -2, respectively, oligonucleotide primers having the following sequencing: Were synthesized:

Human Prothrombin Kringle-1:

N-terminal primer: 5'- GAATTCCTTCTGGGCCAAGTACACAGC -3 '

EcoRI

C-terminal primer: 5'- AAGCTTTTAGCGTGGAGTCATCGCTAC -3 '

HindIII

Human Prothrombin Kringle-2:

N-terminal primer: 5´- CGAATTCCGAAGGCTCCAGTGTG -3´

EcoRI

C-terminal primer: 5'- GAAGCTTCTAACGCCCTTCGATGGC -3 '

HindIII

Subsequently, using a human prothrombin cDNA having a nucleotide sequence shown in FIGS. 6A and 6B as a template and using electric primers, the polymerase chain reaction was performed 36 times for 1 minute at 94 ° C, 57 ° C, and 73 ° C, respectively. Repeatedly, cDNAs of human prothrombin Kringle-1 and -2 were amplified, respectively. The amplified cDNA fragments were electrophoresed with 1.5% agarose gel to confirm the size (see Figure 7, Lane 1: Kringle-1 cDNA; Lane 2: DNA size marker; Lane 3: Kringle-2 cDNA), These were cloned into the pET22b + vector (Invitrogen, USA), an E. coli protein expression vector, respectively, to prepare a pEF-hK1 vector containing human prothrombin Kringle-1 cDNA and a pEF-hK2 vector containing human prothrombin Kringle-2 cDNA. , Insertion of the target cDNA was confirmed by agarose gel electrophoresis followed by restriction enzyme treatment (see FIG. 8, lane 1: pEF-hK1; lane 2: pEF-hK2; lane 3: DNA size marker). Meanwhile, E. coli transformed with the pEF-hK2 vector was identified as' E. coli BL21 (DE3) / pLysS, phEF13 ', and the deposit number' KCCM-10139 'to the Korea Microbiological Conservation Center (134, Sinchon-dong, Seodaemun-gu, Seoul, Korea) as of 14 August 1998. Was deposited.

As described and demonstrated in detail above, the present invention provides human prothrombin Kringle-1 and human prothrombin Kringle-2 proteins derived from human prothrombin and having endothelial growth inhibitory activity and genes encoding the same. Both human prothrombin kringle-1 and human prothrombin kringle-2 of the present invention have excellent endothelial growth inhibitory activity and directly inhibit angiogenesis, and thus can be effectively used for the treatment of rheumatism or cancer. By inhibiting the activity of the prothrombinase complex of may be useful as a therapeutic agent for diseases caused by blood clots.

Claims (10)

Genes encoding human prothrombin kringle-1 protein having the following amino acid sequences: FWAKYTACET ARTPRDKLAA CLEGNCAEGL GTNYRGHVNI TRSGIECQLW 50 RSRYPHKPEI NSTTHPGADL QENFCRNPDS STTGPWCYTT DPTVRRQECS 100 IPVCGQDQVT VAMTPR 116 Gene encoding human prothrombin kringle-2 protein having the following amino acid sequence: SEGSSVNLSP PLEQCVPDRG QQYQGRLAVT THGLPCLAWA SAQAKALSKH 50 QDFNSAVQLV ENFCRNPDGD EEGVWCYVAG KPGDFGYCDL NYCEEAVEEE 100 TGDGLDEDSD RAIEGR 116 Human prothrombin kringle-1 cDNA having the following nucleotide sequence encoding human prothrombin kringle-1 protein of claim 1: TTCTGGGCCA AGTACACAGC TTGTGAGACA GCGAGGACGC CTCGAGATAA 50 GCTTGCTGCA TGTCTGGAAG GTAACTGTGC TGAGGGTCTG GGTACGAACT 100 ACCGAGGGCA TGTGAACATC ACCCGGTCAG GCATTGAGTG CCAGCTATGG 150 AGGAGTCGCT ACCCACATAA GCCTGAAATC AACTCCACTA CCCATCCTGG 200 GGCCGACCTA CAGGAGAATT TCTGCCGCAA CCCCGACAGC AGCAACACGG 250 GACCCTGGTG CTACACTACA GACCCCACCG TGAGGAGGCA GGAATGCAGC 300 ATCCCTGTCT GTGGCCAGGA TCAAGTCACT GTAGCGATGA CTCCACGC 348 Human prothrombin Kringle-2 cDNA having the following nucleotide sequence encoding human prothrombin Kringle-2 protein of claim 2: TCCGAAGGCT CCAGTGTGAA TCTGTCACCT CCATTGGAGC AGTGTGTCCC 50 TGATCGGGGG CAGCAGTACC AGGGGCGCCT GGCGGTGACC ACACATGGGC 100 TCCCCTGCCT GGCCTGGGCC AGCGCACAGG CCAAGGCCCT GAGCAAGCAC 150 CAGGACTTCA ACTCAGCTGT GCAGCTGGTG GAGAACTTCT GCCGCAACCC 200 AGACGGGGAT GAGGAGGGCG TGTGGTGCTA TGTGGCCGGG AAGCCTGGCG 250 ACTTTGGGTA CTGCGACCTC AACTATTGTG AGGAGGCCGT GGAGGAGGAG 300 ACAGGAGATG GGCTGGATGA GGACTCAGAC AGGGCCATCG AAGGGCGT 348 Recombinant expression vector pEF-hK1 comprising the human prothrombin Kringle-1 cDNA of claim 3. Recombinant expression vector pEF-hK2 comprising the human prothrombin Kringle-2 cDNA of claim 4. Recombinant Escherichia coli E. coli BL21 (DE3) / pLysS, phEF13 (KCCM-10139) transformed with the expression vector pEF-hK2 of claim 6. Isolating and purifying prothrombin protein from human plasma; Hydrolyzing the electropurified prothrombin with Factor Xa to produce prothrombin kringle-1 and prothrombin kringle-2; And, Applying the hydrolyzate to ion exchange resin chromatography, forming a sodium chloride linear thickener to elute the protein to obtain human prothrombin kringle-1 and human prothrombin kringle-2; Purification Method of Human Prothrombin Kringle-1 and Human Prothrombin Kringle-2. Method for inhibiting endothelial cell growth in mammals except humans using human prothrombin kringle-1. Method of inhibiting endothelial cell growth in mammals except humans using human prothrombin kringle-2.