HUMAN PLASMINOGEN ACTIVATOR

Abstract: Proteins involved in plasminogen complexation or activation are provided. Portions include a six amino acid peptide which can be used as a plasminogen activator. The peptide is particularly useful when inserted into a plasminogen or plasmin sequence, especially when inserted between the amino acid residues corresponding to amino acid residues 644 and 645 of full length human plasminogen.

Description: HUMAN PLASMINOGEN ACTIVATOR Background of the Invention The present invention is generally in the field of thrombolytic agents and more particularly directed to a plasminogen activator. Plasminogen (SEQ ID NO:l) is the principal serine protease zymogen in the extracellular fluids of vertebrates, and its active form, plasmin, is implicated in pericellular proteolysis associated with a wide range of physiological and pathological processes, including the hydrolysis of fibrin into soluble degradation products and the suppression of tumors by angiogenesis inhibition (Gately. Proceedings of the National Academy of Sciences, USA 1997; 94: 10868-10872). In general, plasminogen expression is fairly stable and the regulation of the activity of the fibrinolytic system occurs mainly via up- and down- regulation of the expression of plasminogen activators and the inhibitors of these activators. Activation of plasminogen is a consequence of cleavage of the Arg 561 -Val 562 bond, to form a two-chain, disulfide linked plasmin. The two known physiological plasminogen activators are the serine proteases tissue-type plasminogen activator (t-PA) (SEQ ID NO:2), urokinase (u-PA or UK) (SEQ ID NO:3), and Desmodus rotundus salivary plasminogen activator (DSP A) (SEQ ID NO:4), which hydrolyze a peptide bond in human plasminogen to convert it to human plasmin. Plasminogen can also be activated by another completely different mechanism, which requires formation of an activator complex with a molecule such as streptokinase (SEQ ID NO: 5) or staphylokinase (SEQ ID NO: 6), which are not proteases. Streptokinase and staphylokinase both form one-to-one tight binding complexes with human plasminogen and the complexes become plasminogen activators, spontaneously converting plasminogen into plasmin. This complexed plasmin is then able to activate free plasminogen. Plasmin on its own cannot activate plasminogen. Streptokinase (SK) (SEQ ID NO: 5) is a single-peptide secretory protein of 414 amino acid residues produced by various strains of hemolytic Streptococcus (Jackson and Tang. Biochemistry 1982; 21: 6620-6625; Malke et al., Gene 1985; 34: 357-361). SK does not contain cysteine or carbohydrate. Proteolytic digestion, NMR and other biochemical- biophysical studies indicate that SK is a flexible multi-domain protein (Conejero-Lara et al., Protein Science 1996; 5: 2583-2591; Medved et al., European Journal of Biochemistry 1996; 239: 333-339; Parrado et al., Protein Science 1996; 5: 693-704; Rodriguez et al., European Journal of Biochemistry 1995; 229: 83-90). SK and human plasminogen can form an equimolar activator complex that catalyzes the conversion of plasminogen from different mammalian species to plasmin. This property renders SK a potent clinical agent for the treatment of blood clotting disorders, such as acute myocardial infarction and stroke (Coleman et al., Hemostasis and Thrombosis. Basic Principles and Clinical Practice J. B. Lippincott Co.: Philadelphia, 1994). The activation of human plasminogen by SK involves the formation of a streptokinase-plasminogen (SK-Plg) complex that alters the conformation of the catalytic domain of the zymogen to complete its enzyme-active center. The SK-Plg complex converts to a streptokinase- plasmin (SK-Plm) complex spontaneously. Both the SK-Plg and the SK-Plm complexes catalyze the hydrolysis of the specific activation bond, Arg 561 - Val 562 , of the substrate plasminogen, resulting in the formation of plasmin. A native plasminogen molecule contains at least seven structural domains, including the N-terminal 77-residue pre-activation peptide, five 'kringles' and a C-terminal trypsinogen-like zymogen domain (Sottrup- Jensen et al., Program of Chemical Fibrinolysis Thrombolysis 1978; 3: 191- 209). An isolated catalytic domain of plasmin(ogen) is called micro- plasmin(ogen) (μPlm/μPlg). Microplasminogen corresponds to amino acid residues 542 to 791 of human plasminogen (SEQ ID NO:l) (Wang et al., Science 1998; 281: 1662-1665). Human plasminogen contains 24 disulfide bonds. Human plasminogen is glycosylated at two positions that are located within the third kringle and between the third and fourth kringles, respectively. Many isolated SK and plasminogen fragments, obtained via proteolytic reaction or recombinant methods, have been used to identify the regions involved in the interaction between the two molecules (Rodriguez et al., European Journal of Biochemistry 1995; 229: 83-90; Shi and Wu, Journal of Biological Chemistry 1988; 263: 17071-17075; Young et al., Journal of Biological Chemistry 1998; 273: 3110-3116). While streptokinase can be used as a thrombolytic agent, it can induce formation of anti-streptokinase antibodies following administration of a single dose. Subsequent doses are then attacked by these anti-streptokinase antibodies, making subsequent doses ineffective. Accordingly, it would be useful to have other plasminogen activating agents. It is an object of the invention to identify the plasrninogen complexation site of naturally occurring plasminogen activators. It is an object of the present invention to provide a new plasminogen activator based upon the plasminogen activation/ recognition site of plasminogen binding proteins. Summary of the Invention Review of sequence homologies of several plasminogen activators and chyrnotrypsin has identified the part of these proteins that is involved in plasminogen complexation or activation. This portion includes a six amino acid peptide which can be used as a plasminogen activator. This peptide is particularly useful when inserted into a plasminogen or plasmin sequence, especially when inserted between the amino acid residues corresponding to amino acid residues 644 and 645 of full length human pl-isrmnogen (see Figures 1A-1C). Brief Description of the Drawings Figures 1A-1C illustrate the sequence alignments between the catalytic units of human plasminogen (h-Plg) (amino acids 542-791 of SEQ ID NO:l), bovine plasminogen (b-Plg) (amino acids 565-812 of SEQ ID NO:7), human tissue plasminogen activator (t-PA) (amino acids 297-562 of SEQ ID NO:2), vampire bat (Desmodus rotundus) plasrninogen activator (b-P A) (DSPA in the text) (SEQ ID NO:4) and bovine chymotrypsinogen (Chm) (SEQ ID NO:8). The RECTIFIED SHEET (RULE 91) ISA/EP The six-residue loop unique for t-PA and b-PA is marked with six downward pointed arrows (between the El and FI regions). Figure 2 is a schematic presentation of the modification of expression plasmid for micro-plasniinogen[+6]. PI, P2, P3 and P4 are four primers used in PCR based reaction to change the cDNA sequence of n_ cro-plasminogen to micro-plasrninogen[+6] . Detailed Description of the Invention The crystal structure of streptolrinase-nύcroplasmin (the catalytic unit of plasmin) complex (SK-^-Pm complex) has been determined at 2.9 A resolution (Wang et al., Science 1998; 281:1662-1665). This structure was used to explain why SK-μPm complex, TP A and DSPA can activate plasminogen but plasmin can not. The alpha subunit of streptokinase in the SK-/-Pm complex provides recognition for interaction with human plasminogen substrate (Wang et al., Scien e 1998; 281:1662-1665). TPA and DSPA each have a six-residue loop near their active site which occupies about the same spatial position as the alpha subunit of streptokinase. The sequence of this loop is Asp-Asp-Asp-Thr-Tyr-Asp in TPA (amino acids 399-404 in SEQ ID NO:2) and Asp-Asp-Asp-Thr-Tyr-Asn in DSPA (amino acids 103-108 in SEQ ID NO:4). The alignment of sequence homology among human plasminogen, bovine plasminogen, TPA and DSPA shows that this loop is present in TPA and DSPA but absent in two plasminogens (Figures 1 A-1C, see six arrows pointing downward). This six-residue peptide provides recognition for human plasminogen substrate. As shown in Figures 1 A-1C, human plasmin does not have a similar six-residue loop and can not activate plasminogen. The position of the peptide in the other proteins corresponds to a position in plasminogen of between Arg- 644 and Lys-645 of human plasminogen. Recombinant D A technology was used to insert the loop sequence Asp-Asp-Asp-Thr-Tyr-Asp (amino acids 399- 404 in SEQ ID NO:2) in micro-plasminogen between residues 644 and 645 (using the full plasminogen numbering). RECTIFIED SHEET (RULE 91) ISA EP The modified nύcro-plasminogen (called micro-plasminogen [+6]) was activated by urokinase to micro-plasmin[+6], which was tested for its ability to activate human rmcro-plasminogen to micro-plasmin. 1. The Plasminogen Recognition/ Activation Peptide The sequence of the recognition/ activation loop is Asp-Asp-Asp-Thr- Tyr-Asp (amino acids 399-404 in SEQ ID NO:2) in TPA and Asp-Asp-Asp- Thr-Tyr-Asn (amino acids 103-108 in SEQ ID NO:4) in DSPA. It is expected that other structurally similar amino acid sequences may also function as similar recognition/ activation peptides. Preferred peptides of this type correspond to the region between the El and FI regions in a plasminogen activator. These regions can be identified in plasminogen activators by, for example, aligning the plasrninogen activators as illustrated in Figures lA-lC. 2. Design and Methods of Making Plasminogen Activators Incorporating the Plasminogen Recognition/ Activation Peptide Plasminogen activators incorporating the plasminogen recognition/ activation peptide may be polypeptides of various structures. For example, native plasminogen may be modified to include the sequence. The use of naturally occurring proteins, and particularly plasminogen, is advantageous because other portions of the protein that may be necessary for function are present. Preferred plasminogen activator sequences for incorporating the plasminogen recognition/ activation peptide are human plasminogen sequence comprising amino acid residues 562 to 791 of human plasminogen (SEQ ID NO: 1) and human plasminogen sequence comprising amino acid residues 542 to 791 of human plasminogen. However, the peptide may also be used alone, preferably in a pharmaceutically acceptable carrier, or attached to another carrier protein. Polypeptide plasminogen activators incorporating the plasrninogen recognition/ activation peptide may be made using methods known to those of skill in the art. These include chemical synthesis, modifications of existing proteins, and expression of proteins using recombinant DNA methodology. Where the protein is relatively short (i.e. less than about 50 amino acids) the protein may be synthesized using standard chemical peptide RECTIFIED SHEET (RULE 91) ISA/EP synthesis techniques. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential additional of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the proteins described herein Chemical synthesis produces a single stranded oligonucleotide This may be converted into a double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template One of skill would recognize that while current methods for chemical synthesis of DNA are limited to preparing sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp 3- 284 in The Peptides. Analysis. Synthesis. Biology Vol 2 Special Methods in Peptide Synthesis. Part A Merrifield, et al , J. Am. Chem. Soc. 1963 85 2149-2156, and Stewart et al , Solid Phase Peptide Synthesis. 2nd ed Pierce Chem Co Rockford, 111 , 1984. Alternatively, the protein may be made by chemically modifying a native protein Generally, this requires inserting the recognition/ activation peptide into the native protein This may be accomplished chemically or enzymatically using, for example, a peptidase Any peptide fragments resulting from hydrolysis can be isolated by any protein purification technique that selects for size (e g by size exclusion chromatography or electrophoresis) Alternatively, various sites in the protein may be protected from hydrolysis by chemical modification of the amino acid side chains which may interfere with enzyme binding, or by chemical blocking of the vulnerable groups participating in the peptide bond In the preferred embodiment, the proteins will be synthesized using recombinant methodology Generally, this involves creating a polynucleotide sequence that encodes the protein, placing the polynucleotide in an expression cassette under the control of a suitable expression promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein DNA encoding a protein can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 1979; 68: 90-99; the phosphodiester method of Brown et al., Meth. Enzymol. 1979; 68: 109-151; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 1981: 22: 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066. Alternatively, partial length sequences may be cloned and the appropriate partial length sequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence. In a preferred embodiment, DNA encoding the protein will be produced using DNA amplification methods, for example polymerase chain reaction (PCR). The proteins may be expressed in a variety of host cells, preferably E. coli or other bacterial hosts. Plasmids encoding the protein can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for yeast or mammalian cells. Cells transformed with the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes. Once expressed, the recombinant protein can be purified using standard procedures, such as ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used as desired. To determine which proteins are preferred, the proteins should be assayed for biological activity. Such assays, well known to those of skill in the art, generally fall into two categories; those that measure the binding affinity of the protein to a particular target, and those that measure the biological activity of the protein. 3. Methods of Using Plasminogen Activators Incorporating the Plasminogen Recognition/ Activation Peptide Plasminogen activators including the plasminogen recognition/ activation peptide can be administered for treatment of blood clot disorders, such as in heart attacks, as known in the art for administration of native streptokinase and tissue-type plasminogen activator (t-PA) and urokinase (u- PA or UK). The plasminogen activator is also useful for applications in which present plasminogen activators are used. The compounds are preferably administered intravenously in appropriate carriers. The appropriate dosages will depend upon the route of administration and the treatment indicated, and can be readily determined by one skilled in the art. Dosages are generally initiated at lower levels and increased until desired effects are achieved. The modified protein is preferably administered in a pharmaceutically acceptable vehicle. Suitable pharmaceutical vehicles are known to those skilled in the art. For parenteral administration, the compound will usually be dissolved or suspended in sterile water or saline. The present invention is further described by the following nonlimiting examples. Example 1; Production of recombinant micro-plasminogenf+61 The 6 amino acid sequence, DDDTYD (amino acids 399-404 of SEQ ID NO: 2), was inserted into the μPg sequence between R644 and K645 (Pig sequence number). This was done by modifying the / Pg expression plasmid, pETl 1- Pg, using a recombination PCR method. Two PCR (PCR-1 and PCR- 2) were performed first using pETl 1-μPg as template and primers P1/P2 and P3/P4 (PI (SEQ ID NO:9): CATATGGCGGCCCCTTCATTTGATTG; P2 (SEQ ID NO: 10): ATCTTTgtcgtaagtgtcatcatcTCGTGTGGGCTCCAAGAACAG; P3 (SEQ ID NO: 11): gatgatgacacttacgacAAAGATATTGCCTTGCTAAAGCTAAGC; P4 (SEQ ID NO: 12): GTATCACGAGGCCCTTTCGTC). The third PCR was then performed with PCR-1 and PCR-2 as template, and primers P1/P4. A schematic presentation of these primer positions in the plasmid is shown in Fig. 2. The resulting PCR product, which is about 1.1 Kb, was cut with Stu I/Hind HI, and the 0.86 Kb fragment was cloned into the Stu I/Hnd HI cut of vector pET 11 -μPg. The resulting plasmid pET 11 -μPg-D6 was transformed into E. coli BL21(DE3), and protein was expressed as inclusion bodies, which were purified using standard procedures. The purified inclusion bodies were dissolved in 8 M urea, and the protein was refolded by dialysis. The refolded protein was partially purified using Sephacryl S-300 gel filtration and FPLC Resource Q. Example 2: Properties of the recombinant micro-plasminogen 1+61 Plasmin activity was measured. The chromogenic substrate, KABI S- 2251, was dissolved in 20 mM Tris/IM NaCl, pH 7.5 (Buffer B). Native streptokinase (50000 IU/ml) was dissolved in 50 mM Tris/HCI, pH 7.5 (Buffer A). Partially purified recombinant micro-plasminogen (0.5 mg/ml) was in 20 mM Tris/HCL, pH 8. The modified μPg, micro-plasminogen[+6], was in 20 mM Tris/HCI, 0.4 M urea, pH 8.0. Micro-plasminogen (substrate) was incubated with SK in buffer A at 37 °C, to activate the micro-plasminogen before the chromogenic substrate was added. The reaction was continued for 5 to 10 min, and then was stopped by adding 0.2 M acetic acid. Reactions were performed in 96-well microtiter plates and absorption at 405 nm was recorded. The results are shown in Table I, where μPg[+6] is micro-plasminogen[+6] and μPg is micro-plasminogen. Table I. The activity of micro-plasminogen [+6] Sample A™ x 1000 1. μPg[+6] 5 2. μPg[+6] + μPg 57 3. μPg 40 4. μPg[+6] + SK 5 5. SK + μPg 76 6. Preincubation ofμPg[+6] & SK then uPg 167 The results show that micro-plasminogen[+6] has no activity (Table I, line 1) and micro-plasminogen[+6] can not activate plasminogen (Table I, line 2 as compared to Table I, line 3). Micro-plasminogen[+6] can not be activated by SK (Table I, line 4). However, when micro-plasminogen[+6] was pre- incubated with SK, the complex or activated micro-plasmin[+6] can activate plasminogen (Table 1-6 as compared to Table I, line 5). The results indicate that the properties of micro-plasminogen[+6] are different from those of micro-plasminogen. The activity of plasminogen activation shown in Table I, line 6, can come either from micro-plasmin[+6] or from its complexation with streptokinase. Example 3: Demonstrate that micro-plasminf+61 is a plasminogen activator To assess if micro-plasmin[+6] itself is a plasminogen activator, urokinase (UK) was used to activate micro-plasminogen[+6]. The results are shown in Table II, where UK is urokinase, μPg is micro-plasminogen, and μPg[+6] is micro-plasminogen[+6]. The data are optical density of hydrolysis ofKABI S-2251. Table II. Plasminogen activation by urokinase treated micro-plasminogen [+6] Table H, line 1, shows the plasminogen activation activity of UK at different dilutions. Table 13, line 2, shows the activity of the urokinase- micro- plasminogen [+6] complex, which was pre-incubated to convert micro- plasminogen[+6] to micro-plasmin[+6] prior to the addition of the micro- plasminogen substrate, at the same dilutions as in line 1. The plasmin activity of complex was twice that of urokinase at all dilutions. These results clearly show that micro-plasminogen can be activated into a plasminogen activator. The results demonstrate that the six-residue loop can convert micro- plasmin to an activator of human micro-plasminogen. Since the essential catalytic properties and specificities of human plasminogen and plasmin are represented in the micro-plasminogen and micro-plasmin, it is predictable that the same modification in the full sized human plasminogen will convert it to an activator for the full size plasminogen. These data demonstrate the creation of a new class of plasminogen activator based on the modification by addition of a loop.

Claims: What is claimed is: 1. A polypeptide comprising a peptide fragment often amino acids or less for recognition or activation of plasminogen, wherein the polypeptide has plasminogen activator activity or can be activated by a plasminogen activator to have plasminogen activator activity. 2. The polypeptide of claiml wherein the peptide fragment corresponds to the region between the El and FI regions in a plasminogen activator. 3. The polypeptide of claim 1 wherein the peptide fragment consists of the sequence Asp-Asp-Asp-Thr-Tyr-Asp (amino acids 399-404 of SEQ ID NO:2) or Asp-Asp-Asp-Thr-Tyr-Asn (amino acids 103-108 of SEQ ID NO:4). 4. The polypeptide of claim 1 further comprising a human plasminogen sequence. 5. The polypeptide of claim 4 wherein the peptide fragment consists of the sequence Asp-Asp-Asp-Thr-Tyr-Asp (amino acids 399-404 of SEQ ID NO:2) or Asp-Asp-Asp-Thr-Tyr-Asn (amino acids 103-108 of SEQ ID NO:4). 6. The polypeptide of claim 5 wherein the peptide fragment is inserted into the human plasminogen sequence between the amino acid residues corresponding to amino acid residues 644 and 645 of full length human plasminogen. 7. The polypeptide of claim 6 wherein the human plasminogen sequence comprises amino acid residues 562 to 791 of human plasminogen. 8. The polypeptide of claim 7 wherein the human plasminogen sequence comprises amino acid residues 542 to 791 of human plasminogen. 9. A thrombolytic agent comprising a polypeptide wherein the polypeptide comprises a peptide fragment often amino acids or less for recognition or activation of plasminogen, wherein the polypeptide has plasminogen activator activity or can be activated by a plasminogen activator to have plasminogen activator activity. 10. The thrombolytic agent of claim 9 wherein the peptide fragment corresponds to the region between the El and FI regions in a plasminogen activator. 11. The thrombolytic agent of claim 9 wherein the peptide fragment consists of the sequence Asp-Asp-Asp-Thr-Tyr-Asp (amino acids 399-404 of SEQ ID NO:2) or Asp-Asp-Asp-Thr-Tyr-Asn (amino acids 103- 108 ofSEQ ID NO:4). 12. The thrombolytic agent of claim 9 wherein the polypeptide further comprises a human plasminogen sequence. 13. The thrombolytic agent of claim 12 wherein the peptide fragment consists of the sequence Asp-Asp-Asp-Thr-Tyr-Asp (amino acids 399-404 of SEQ ID NO:2) or Asp-Asp-Asp-Thr-Tyr-Asn (amino acids 103- 108 ofSEQ ID NO:4). 14. The thrombolytic agent of claim 13 wherein the peptide fragment is inserted into the human plasminogen sequence between the amino acid residues corresponding to amino acid residues 644 and 645 of full length human plasminogen. 15. The thrombolytic agent of claim 14 wherein the human plasminogen sequence comprises amino acid residues 562 to 791 of human plasminogen. 16. The thrombolytic agent of claim 15 wherein the human plasminogen sequence comprises amino acid residues 542 to 791 of human plasminogen.

Inventors: LIN XINLI, TANG JORDAN J N, ZHANG XUEJUN C, LIN, XINLI, TANG, JORDAN, J., N., ZHANG, XUEJUN, C.
Applicants: OKLAHOMA MED RES FOUND, OKLAHOMA MEDICAL RESEARCH FOUNDATION
Application year: 1999
Main classification: C12N9
IPC codes: A61K38, C07K7, C12N9