CA2327526A1 - Thrombolytic agents derived from streptokinase - Google Patents

Abstract

Structural information about the streptokinase-micro plasminogen complex has been used to identify the part of the streptokinase structure not involved in plasminogen complexation or activation. These nonessential portions can be modified to reduce antigenicity, for example, by changing the nonessential portions of streptokinose to more human-like polypeptide portions ("humanization of streptokinase"). One way this can be done is to compare the nonessential portion to a structural database of human proteins to find similar structures. Then the streptokinase nonessential structural part is replaced with the human structural part such as by genetic engineering of a mutant encoding the individual streptokinases, which is then expressed in a bacterial host such as E. coli. Alternatively, the nonessential portions can be removed or truncated to simplify streptokinase to a smaller molecule which retains plasminogen activation activity. Such smaller proteins should have reduced antigenicity and be cheaper and easier to produce. The modified streptokinases are useful in treating clotting disorders.

Description

THROMBOLYTIC AGENTS DERIVED FROM STREPTOKINASE
Background of the Invention The present invention is generally in the field of thrombolytic agents and more particularly directed to thrombolytic agents derived from S streptokina.se.
Plasminogen (SEQ ID NO:1) 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, ZISA 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 Arg56~-Va15s2 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 N0:2) and urokinase (u-PA or UK) (SEQ ID N0:3), both of which directly catalyze the hydrolysis of the activation bond. However, plasminogen can also be activated by another completely different mechanism, which requires formation of an activator complex with a molecule such as streptokinase. When streptokinase is complexed with plasminogen, plasminogen spontaneously converts into plasmin. This complexed plasmin is then able to activate free plasminogen. Plasmin on its own cannot activate plasminogen.
Streptokinase (SK) (SEQ ID N0:4) 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 mufti-domain protein (Conejero-Lara et al., Protein Science 1996; 5: 2583-2591; Medved et al., European Journal ofBiochemistry 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, Argssi-Va1562, of the substrate plasminogen, resulting in the formation of plasmin.
However, plasmin alone is not a plasminogen activator.
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) (p,Plm/~t.Plg). 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, et al., Journal ofBiological Chemistry 1988; 263: 17071-17075; Young et al., Journal of Biological Chemistry 1998; 273: 3110-3116).
Accordingly, it would be useful to have a crystalline structure of the streptokinase-micro plasmin(ogen) (SK-p,Plg) complex. Such a structure would make it possible to predict the portions of SK that complex with plasminogen and to design modified streptokinases, such as a streptokinase having less antigenicity but which is still able to complex plasminogen and lead to activation of plasminogen.
It is an object of the invention to provide a structure for streptokinase-micro plasminogen complex and identify the plasminogen complexation site and the streptokinase portions that are not essential for plasminogen complexation or activation.
It is an object of the present invention to provide streptokinase derived thrombolytic agents.
It is an object of the present invention to provide a method of making thrombolytic agents derived from streptokinase.
Summary of the Invention Structural information about the streptokinase-micro plasminogen complex has been used to identify the part of the streptokinase structure not involved in plasminogen cornplexation or activation. These nonessential portions can be modified to reduce antigenicity, for example, by changing the nonessential portions of streptokinase to more human-like polypeptide portions ("humanization of streptokinase"). One way this can be done is to compare the nonessential portion to a structural database of human proteins to find similar structures. Then the streptokinase nonessential structural part is replaced with the human structural part such as by genetic engineering of a mutant encoding the individual streptokinases, which is then expressed in a bacterial host such as E. toll. Alternatively, the nonessential portions can be removed or truncated to simplify streptokinase to a smaller molecule which retains plasminogen activation activity. Such smaller proteins should have reduced antigenicity and be cheaper and easier to produce. The modified streptokinases are useful in treating clotting disorders.

Description of the Drawings Figure 1 is a stereo view of the activation pocket of the plasmin catalytic domain. A 2.9 A resolution 2IFobsI-IF~~,~l electron density map, phased with the final refined model and contoured at 1.0 a, is superimposed S on the refined model.
Figure 2 is a stereo view of the crystal structure of the complex of human micro plasmin (p.Plm) and streptokinase. The pPlm molecule is in the middle of the complex. The a-domain of SK is at the top, left side of the complex. The (3-domain of SK is to the right side of the complex. The ~y-domain of SK is at the bottom of the complex. Only the Ca traces are shown.
Figures 3(a)- (c) illustrate ribbon diagrams of the three domains of streptokinase. Figure 3(a) is a ribbon diagram of the a-domain; Figure 3(b) is a ribbon diagram of the ~i-domain; and Figure 3(c) is a ribbon diagram of 1 S the y-domain. The domains are oriented to illustrate the similarity in their overall (3-grasp folding.
Figure 4(a) is a stereo view of the interaction between human micro plasmin and the a-domain of streptokinase. The micro plasmin molecule is at the bottom of the complex. Figure 4(b) is a stereo view of the interaction between human micro plasmin and the y-domain of streptokinase. The micro plasmin molecule is at the top of the complex. The side chains which are involved in the interaction are displayed along with the Ca backbones.
Figure 5 is a stereo view of the superposition of the catalytic domains of human plasmin and human two-chain tissue-type plasminogen activator (t-PA) (Lamba et al., Journal ofMolecularBiodogy 1996; 258: 117-135).
Selected positions are numbered accordingly. Only the Ca traces are shown.
Some commonly used loop names for trypsin-like proteases are also included (Renatus, et al., EMBO Journal 1997; 16: 4797-4805).
Figure 6(a) is modeled view of the substrate binding site of the micro plasminogen-streptokinase complex showing the molecular surface of the complex. The orientation of the complex is similar to the orientation of the complex in Figure 1. Figure 6(b) is a stereo view illustrating docking of a substrate micro-plasminogen into the substrate binding site. The enzyme micro-plasmin is to the left of the complex. The streptokinase molecule is in the center of the complex, from top to bottom. The substrate micro-plasminogen is to the right of the complex.
Figure 7 is a stereo view illustrating a putative active-zymogen form of plasminogen, compared with plasmin. The cleaved activation loop of plasmin, shown in a thicker tube, is at the center of the complex, as is the side chain of Lys69g in plasmin. The corresponding parts of the active-zymogen are towards the right. The rest of plasmin(ogen) is towards the top of the complex and possesses an active conformation around the active site, particularly the peptide segment (shown in a thicker tube) upstream of the nucleophile Ser'4'. ~i-strands in the surrounding region are shown as arrows.
The salt bridge distance between the tips of Lys69g and Asp'4° in the active-zymogen form is approximately the same as that between the free amino group of Valssz and Asp'4° in plasmin. Upstream of Lys698 is the binding site to the y-domain of streptokinase.
Figure 8 is a stereo view of the a-domain of streptokinase (SK) superimposed on staphylokinase (SAK) (SEQ ID NO:S) (Rabijns, et al., Nature Structural Biology 1997; 4: 357-360). In addition to the Ca traces, the side chains of SK Val'9 (SAK Met26) and SK G1u39 (SAK G1u46) are plotted.
Detailed Description The flexibility and multidomain nature of both SK and plasminogen have heretofore prevented the crystallization and determination of the crystal structures of plasmin(ogen), SK and the SK-Plg complex. The rapid autolysis of the SK-Plg complex renders the crystallization of the wild-type SK-Plg complex impractical.
It is known that the catalytic domain of human plasminogen can bind and be activated by SK (Shi et al., Nature Structural Biology 1990; 4: 357-360; Wang and Reich, Protein Science 1995; 4: 1758-1767). A recombinant human microplasminogen (wPlg) was constructed, containing an alanine residue substituted for the active-site serine residue. The streptokinase-microplasminogen (SK-p.Plg) complex spontaneously, but slowly, converts to a streptokinase-microplasmin (SK-p,Plm) complex. However, it does crystallize and the crystal diffracts to atomic resolution. The three-dimensional structure of the SK-p,Plm complex is disclosed herein.
1. Streptokinase Structure Streptokinase Portions Complexed With Pdasmin(ogen) Streptokinase complexation portions refers to single amino acid residues or polypeptides of streptokinase required for the complexation of plasminogen and plasmin to streptokinase and for the activation of plasminogen to plasmin.
ii. Streptokinase Portions Involved In Substrate Spec~city Streptokinase substrate specificity portions refers to single amino acid residues or polypeptides required to impart substrate specificity upon plasmin complexed to streptokinase as compared to plasmin alone.
iii. Nonessential Portions of Sireptokinase Nonessential portions as used herein refers to those portions of streptokinase that can be modified, such as by being removed or replaced, without destroying the ability of the streptokina.se to complex with plasminogen and without destroying the ability of the SK-Plm complex to activate plasminogen. Moreover, the nonessential portions include portions that can be modified, such as by being removed or replaced, or by substituting or deleting one or more of the amino acids, without destroying the ability of streptokinase to impart substrate specificity to plasmin when complexed as SK-Plm compared to plasmin alone. The modified streptokinase proteins disclosed and claimed can have one or more ofahese nonessential portions removed or replaced.

2. Design and Methods of Makin;~ the Modified Streptokinase i. Design Native streptokinase 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.
To form a humanized chimeric streptokinase, the nonessential portions are compared against a database of human proteins to identify human proteins or portions thereof which are structurally similar to the nonessential streptokinase portions. A chimeric humanized streptokinase mutant can then be made in which the nonessential portions) are replaced with the human protein portions. Alternatively, or in addition, a truncated protein can be made in which one or more nonessential portions, or one or more amino acids therein, have been removed.
Preferably, the human protein or portion thereof has a high degree of structural similarity to the streptokinase portion. However, the human portion does not have to be structurally identical to the streptokinase portion.
Preferably, the human portion does not retain any of the function of the native human protein from which it is derived. Of course, the humanized or truncated protein should retain substantially all or a substantial fraction of its ability to complex and activate plasminogen.
ii. Methods of making the modified Streptokinase The humanized or truncated proteins may be made using methods known to those of skill in the art. These include chemical synthesis, modif cations of existing proteins, and expression of humanized proteins or truncated proteins using recombinant DNA methodology. The humanized protein can be made as a single polypeptide or the human protein portion can be attached to the base streptokinase polypeptide after separate synthesis of the two component polypeptides.
Where the protein is relatively short (i.e. less than about 50 amino acids) the protein may be synthesized using standard chemical peptide 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 S 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, Ill., 1984.
Alternatively, the protein may be made by chemically modifying a native protein. Generally, this requires cleaving the native protein at one or more sites and then annealing desired polypeptides onto the newly formed termini. The desired cleaved peptides can be isolated by any protein purification technique that purifies on the basis of 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 humanized or truncated 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. Additional proteins can be made separately and then ligated to the modified streptokinase, or the polynucleotide sequence can encode the streptokinase in phase with anther protein.
DNA encoding the protein can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or S 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. Patent 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 polymerise chain reaction (PCR).
The proteins may be expressed in a variety of host cells, including E.
coli or other bacterial hosts, yeast, and various higher eukaryotic cells, such as the COS, CHO and HeLa cells lines, insect cells, and myeloma cell lines.
The recombinant protein gene is operably linked to appropriate expression control sequences for each host. The 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 mammalian cells. Cells transformed by 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 protein can be purified according to standard procedures such as ammonium sulfate precipitation, affinity columns, column chromatography, or gel electrophoresis. 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.
One of skill in the art would recognize that after chemical synthesis, biological expression, or purification, the protein may possess a conformation substantially different than the native protein. In this case, it may be necessary to denature and reduce the protein and then to cause the protein to re-fold into the preferred conformation. Methods of reducing and S denaturing the protein and inducing re-folding are well known to those of skill in the art. For example, the expressed, purified protein may be denatured in urea or guanidium chloride and renatured by slow dialysis.
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.
Methods Of Using Modified Streptokinase 1 S The modified streptokinase molecules 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 compounds are preferably administered intravenously in appropriate Garners. 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 present invention is further described by the following nonlimiting examples.
Example 1: Proteinpreparation and crystallization Recombinant streptokinase and S741A mutant of human ~,Plg were constructed. The proteins were expressed in E. coli as inclusion bodies which were washed, dissolved in 8 M urea and combined to be refolded together by rapid dilution. The 1:1 complex between SK and p,Plg was purified further using S300-chromatography. The protein sample was stored WO 99/5?251 PCT/US99/10086 at 0°C for more than two months before being used for a successful crystallization. The initial crystallization condition was determined by the use of sparse-matrix screens from Hampton Research. Crystals were grown at 20°C from sitting drops with wells containing 1.0 M sodium citrate, 0.2 M
HEPES (pH 8.0), 1 mM magnesium chloride. The protein concentration used was 40 mg/ml. Crystals appeared in about two weeks and had typical dimensions of 0.1 x 0.1 x 0.5 mm. The crystal belongs to space group P21 with cell parameters of a=80.0 A, b=125.1 A, c=86.8 A and ~i=105.4°.
One crystallographic asymmetric unit contains two SK-p,Plm complexes with VM=2.9 A3/Da (Matthews, Journal of Molecular Biology 1968; 3 3 : 491-497).
To confirm the crystal content, selected crystals were dissolved in H20 and analyzed by SDS PAGE. The results showed that the p.Plg was converted to p.Plm and SK was digested at two positions to different extents.
1 S The proteolytic cleavage in ~Plg renders the complex changed from SK-pPlg to SK-N.PIm. The low proteolytic activity in the sample may come from trace-amount protease contamination, and/or the Ser~4' to Ala mutant leaking back to the "wild-type" pPlg during the E. coli expression.
Example 2: C sry tallogr_aphic methods and data~rocessing Data collection and heavy-atom derivative screen was conducted at room temperature on a Siemens area detector. The program SAINT was used to process the data. Molecular replacement searches for the ~Plm molecules were carried out with the program MRX (Zhang and Matthews, Acta.
Crystallographica 1994; DSO, 675-686). The solution clearly showed a local two fold symmetry between the two SK-ltPlm complexes. However the overall quality of the electron density based on this molecular replacement solution alone was strongly biased by the model used and was not useful for obtaining interpretable electron density beyond the region of the search model.
The phases of the crystal structure of the SK-p,Plm complex were solved by the use of multiple isomorphous replacement (Mm) techniques, using platinum, mercury and iridium derivatives. The program packages SOLVE (Terwilliger and Berendzen, Acta. Crystallographica 1996; D52:
749-757) and MLPHARE (Otwinowsky, Proceedings of the CCP4 Study Weekend, 25-2b January, (W. Wolf, P. R. Evans and A. G. W. Leslie, eds.), pp 80-86, Daresbury Laboratory: SERC, 1991) were used to identify and refine the heavy atom parameters. The statistics of the X-ray data collection and MlZt phasing are summarized in Table 1.

x et M O~ ~h N M
' ~ C/~
~r V) b 00 v1 d' ~1 00 O o0 N
t~ 00 00 00 00 00 Ov ~ O O O O O O O ~ ~ .-~-~ O
0.i ~ l~ l~ OWE 00 l'~ Q\ E~ O ~ ~ O
O O O O O O O M O
U
O N M
Mp~pM b N
('~ 0~0 I'~ ~ ~ OM1 ~ O
O O O O O O O
~i ~ i w ~ w w. W y.., 00 '~ O
N M ~ ~ O ~O N ~. ~ ~ .-~ N
.-i .-~ Cj .-. .... r-i p M r. '~T
' O
4, v1 a1 'd' ~ O
b ~ OO V1 ~ 'd: vl ~O h ~ ~ N .C' O 'd' 00 ~O ~ ~O 00 M V1 N N ~-~ N N N ~
dp O 'C
~ v~ Ov .y NNoo~M~MN ~M~ e~ ~ r,;
A-i '~ ai vi O Q\ d' ~t Ov 00 r.r 'b at NNNNN,~!NN ~ '" ,O
ie3 M ~O O~ Ov Ow1 O~ 00 ~ ' ~ U
V1 l~- 00 ~ 00 O I~ t~ U
'd ~--i ~-~ Owe V'~
ccS ~, ,rj d' ~ ~ ~ V
V N ~ b ~ ~i, _ O .o N
No~O~t'~'N~v~'1~
~ N N N oo pv ~ 00 WO 'd' O M 00 O 00 N ~ ~ O ~ M ~ U
M ,_, M .--~ N N M N f~ ~ ~ C1. ~ U
O y 4~
O o ~ I~ tt ~O o0 VD 00 W p pip ,n O ~ ~ cUC
..., TJ
V \ cG N ~-~ I~ N N O Ov t~ ,~ ,..., ~ U
U .;~ ~ O ~ OwD V Ov O ~O ~ t~ N
=, ~'' 00 00 l~ 00 I~ 00 00 h O ~, O
~O\Od'~V~1~0~ ' V M V~ ~-~ M N OW t v~ N ~U-' w O
U ~-mt ~ M N dv N O N Ov 00 ø' ~ v ~*"' Ov CT Q\ Q\ oo Ov Ov O~ N ~
A Qi b O O ~ ~ ~ Q, a~n 01 v0 O vD N et O 'fit ~ U O
4, '.r N M M M M M M M
~ o, a) ~' .c7 p ' ~ W
'N z ~ ~ U cd b °) r '~ C! ~ ~ ~ O x p O
H ~ ~. O ~ ~ Z ,~ . ° ~; ~ oo Q, v~ w v .y U ~x x ~U V ~ ob w a. x ~ a. o, a zxx~~,xxx ~z° ~ .~ ~ .~ v The initial MIR phases at 3.0 A resolution were confirmed by the MR
solution of the p,Plm part of the complex. The phases were further improved by electron density averaging over two-fold non-crystallographic symmetry and solvent flatting. The initial mask for the electron density averaging was derived from the MR solution. The electron density improvement was carried out with the program DM (Cowtan and Main, Acta. Crystallographica 1996; D52: 43-48).
From the improved electron density map, the SK molecule was seen in three distinct domains adjacent to the region of the p,Plm molecule. The initial model of p,Plm was built using the crystal structure of chymotrypsinogen (Wang et al., Journal ofMolecular Biology 1985; 185: 595-624) as a template. The structure of SK was built directly from the electron density map. Model building was performed with the program O (Jones et al., Acta.
Crystallographica 1991; A47: I 10-119). Iterative refinement and model building were used to improve the model gradually. SigmaA-weighted maps were calculated with the program SIGMAA (Read, Acta. Crystallographica 1986; A42: 140-149) and used in the initial model building.
Refinement was carried out with the program XPLOR (Brunger et al., Science 1987; 235: 458-460) and TNT (Tronrud et al., Acta. Crystallographica 1987; A43 : 489-501 ). None-crystallographic-symmetry constrains were used throughout the refinement; in the final model, the two copies of the crystallographic independent SK-p,Plm complexes are practically identical. All of the chemically expected 250 residues of the pPlm molecule were included in the final model at 2.9 A resolution; and of the 414 residues of the SK, 322 were modeled. A representing region of the electron density map of p,Plm is shown in Figure 1. The final R factor is 20.3% over the 8.0-2.9 A resolution shell (28,600 reflections), and the free R (Brunger, Nature 1992; 355: 472-474) is 30%
(3,150 reflections). Bond and angle deviations are 0.01 A and 1.8°, respectively, as determined by XPLOR using Engh and Huber parameters (Engh and Huber, Acta. Crystallographica 1991; A47: 392-400). Structural superposition and solvent accessible surface calculation were carried out with EDPDB (Zhang and Matthews, Journal ofApplied Crystallography 1995; 28: 624-630). Figures were created by usingMOLSCRIPT (Kraulis, J. Appl. Cryst. 1991; 24: 96-950), RASTER3D (Merntt and Murphy, Acta. Cryst. 1994; DSO: 869-873) and GRASP
(Nicholls et al., Proteins 1991; 11: 281-296).
Example 3 : Overall Structure of human uPlm The crystal structure of SK-p,Plm complex was determined at 2.9 ~
resolution using X-ray crystallography. There are two SK-~,Plm complexes per crystallographic asymmetric unit, which are practically identical with each other. Figure 2 shows the Ca trace of one SK-pPlm complex. The ~Plm component of the complex contains the region from residue Alasa2 to the C-terminal residue Asn~91 of plasmin. The dimensions of the N.PIm molecule are about 40 x 45 x 50 A. Resembling the architecture of many other trypsin-like serine proteases, p.Plm consists of two domains, each of a six-stranded (3-barrel.
The C-terminus of pPlm ends with an a-helix packing against the N-terminal (3-barrel. The catalytic residues, His6o3, Aspsas~ ~d Ser~4' position confirmed the Ser to Ala substitution.
Consistent with the high sequence homology (i.e. 39% identity), the coordinate root mean square deviation (rmsd) between ~,Plm and chymotrypsin (Harel et al., Biochemistry 1991; 30: 5217-5225) is 0.7 A for 193 Ca atoms, using a 1.5 A cutoff. Compared to both chymotrypsin and chymotrypsinogen crystal structures (Harel et al., Biochemistry 1991; 30: 5217-5225; Wang et al., Journal ofMolecular Biology 1985; 185: 595-624), the active site conformation of the catalytic domain of plasmin is indeed in its enzyme form. The activation bond, Argss'-Valssz, has been cleaved as indicated by SDS PAGE and N-terminal sequencing of the crystal contents (data not shown). The new C-terminus ofthe cleaved loop, containing Pross9, Glysso~ ~d ~gs6y is mobile in the crystal and can be seen in the electron density only at a low contour level.
The newly liberated N-terminus (WGG) (amino acids 562-565 of SEQ m NO:1) enters the activation pocket that is designed precisely to fit both the main-chain atoms and the divaline side chains. Its stability and proper positioning is reinforced by the solvent inaccessible salt bridge linking the terminal amino group to the carboxylate group of Asp'4°. This salt bridge ensures that the loop immediately upstream of nucleophile Ser'41 changes its conformation to an active form. Consequently, the oxyanion hole is formed by the amide groups of residues 738-740, and the S1 specificity pocket is properly formed.
Six disulfide bonds stabilize the structure of p.Plg. Three of them, CyS58R-CyS604' Cyssso-Cys7a7~ and C s737-C s~65 y y , are within six-residue ranges of the catalytic triad residues and function to maintain the platform of the catalytic triad. Another one, CysSSa-Cys566~ w~ch is absent in many other typsin-like proteases, flanks the activation cleavage site. In the zymogen form, this disulfide bond likely constrains the conformation of the short activation loop such that the Arg561-V~562 bond is confined to be readily cleaved by plasminogen activators. The two new termini liberated by the activation cleavage are also constrained by the disulfide bond.
Immediately beneath the imidazole ring of His6o3 is residue Ala6°' which sits in a pocket perfect for its methyl group side chain. This residue was found to be mutated to a threonine residue in the plasmin of a group of patients with a predisposition of thrombosis (Ichinose et al., Proceedings of the National Academy of Sciences, U.S.A. 1991; 88: 115-119). Such an Ala-to-Thr mutation disrupts the hydrogen bond between His6°3 and Asp6a6 ~d impairs the charge delay network of the catalytic triad.
Example 4: Overall structure of streptokinase In the crystal structure of the SK-pPlm complex, SK appears as a three domain protein with several segments in the primary sequence disordered in the crystal lattice. The three domains are linked with each other by coiled coil peptides and are likely to fold independently in solution. They are denoted as a-(i-, and y-domains hereafter along the peptide chain from the N-terminus to the C-terminus (see Figure 2).
The a-domain begins at residue Asnls and ends at about residue Pro~4s.
Residues 1-11 and two extra residues (Gly-Ser) adopted from the cloning vector are disordered in the electron density map. Similarly the region of residues 70 has a lack of interpretable electron density. A proteolytic cleavage that occurs at the bond between Lyss9 and Ser6° is located in this disordered region.
The ~i-domain begins at residue Alalss and ends at residue Pro2g3. This domain also contains a cleavage site, Lys25'-Serz58, which is cleaved only in a portion of total SK-pPlm complexes as shown in our SDS PAGE analysis. No preference of SK molecules, cleaved and non-cleaved, is seen at this site in the two crystallographically independent SK-p,Plm complexes. The y-domain starts at residue Asp2g5 and becomes invisible beyond residue Arg3'2, although SDS
PAGE suggests that the C-terminal 40 or so residues are attached. The domain boundaries found in the crystal structure are consistent with the results from various protease mediated SK-degradations (Parrado et al., Protein Science 1996; 5: 693-704). It is also consistent with the previous observations that the N-terminal 16 residues and the C-terminal 40 residues of SK are functionally dispensable for plasminogen activation (Kim et al., Biochemical and Biophysical Research Communications 1996; 40: 939-945; Young et al., Journal of Biological Chemistry 1995; 270: 29601-29606). In fact, the N-terminal 16 residues of SK play a role in the secretion of this protein from the host cell (Pratap et al., Biochem. Biophys. Res. Commun. 1996; 227: 303-310).
There is also evidence showing that some fragments in the region of residues 45-70, which is disordered in the complex structure, exist in an inherently flexible state (Nihalani et al., Protein Science 1998; 7: 637-648).
Roughly speaking, every one of the three domains of SK belongs to the (3-grasp folding class (Murzin et al., Journal of Molecular Biology 1995; 247:
536-540), but with some noticeable differences (see Figure 3). Like a typical (i-grasp protein, the SK a-domain contains a single a-helix packing against a mixed five-stranded (3-sheet. In addition, there is a short two-stranded (3-sheet on the same side of the major (3-sheet as the a-helix. The (3-strands forming the major (3-sheet are "~i2, "(3~, "(3~, "/34, and "(3s. The topology of this ~3-sheet (Richardson et al., Journal ofMolecular Biology 1976; 102: 221-235) is (+1,-3x,-1,2x). The hydrogen bond network of the major (3-sheet is disrupted at the middle of the "~3z strand by a bulge at position 36. The a-helix is located between "~i3 and "(34 and is thus named "a3,4. Between the major ~i-sheet and the a-helix is the hydrophobic core of the a-domain. Disturbing this hydrophobic core is likely to result in a dysfunctional SK as shown by a G1y24 to His mutation (Lee et al., Biochemical and Biophysical Research Communications 1989; 165:
1085-1090). The SK (3-domain shares the same overall folding with the SK a-domain. The coordinate rmsd between the two domains is 1.7 A for 81 residues, using a 4.0 A cutoff. Some corresponding loops between the two domains, however, have different lengths. The SK y-domain contains a four-stranded major (3-sheet and a short two-stranded ~i-sheet. The major ~3-sheet has a topology similar to that of the major (3-sheet of the a-domain without "/3s.
Between Y(32 and y~33 are some coiled coil loops. The qualities of the electron density in the a- and y-domains of SK are significantly better than that of the (3-domain region. Correspondingly, the average temperature factors of the a-, ~3-, and y-domains are 43, 80, and 39 A2, respectively. These differences appear correlated with the interactions of each domain of SK with the ~Plm molecule in the complex.
Example S: Interaction between streptokinase and pPlm The SK molecule has extensive interactions with the uPlm molecule, mostly through the SK /3- and y-domains. The values of buried molecular surface area are 1,650 AZ , 950 AZ and 1,500 A2 between ~Plm and the a, ~3 and 'y-domains of SK, respectively.
The SK a-domain is located near the catalytic triad of ~,Plm. There are three major contact regions between the SK a-domain and p,Plm (see Figure 4a).
The first region contains the interaction between the major (3-sheet, particularly the strands of °'(3, and °'(i2, of SK and the loop region of residues 713-721 of p,Plm. In this contact region, Arg"9 of plasmin (SEQ ID NO:1) forms salt bridges with both G1u39 and Glu'34 of SK (SEQ ID N0:4), and it also has van de Waals interaction with SK Val'9. The uncharged alkyl group side-chain of residue 19 of SK has been shown to be important for plasminogen activation (Lee et al., Biochemistry and Molecular Biology International 1997; 41: 199-207). Arg"9 of plasminogen has also been identified as an important residue involved in the SK-Plg complex formation (Dawson and Pontin, Biochemistry 1994; 33: 12042-12047). The second contact region contains the interaction between the bulge region in the °'~i2 strand of SK and the 643-645 region of ~Plg, which is the upstream region of the catalytic residue Asp6a6. The positively charged side chain of pPlm Lys6aa also protrudes towards the C-terminus of the a-helix, °'a3,4, of SK, presuming a helix dipole-charge interaction. The third contact region is between the loop following the a-helix, aa3,4, of the SK a-domain and the 606-609 region of p,Plm. The latter is the down stream region of the catalytic residue His6os_ These close interactions between the SK a-domain and the catalytic triad of p,Plm are likely to contribute to the substrate specificity difference between plasmin and the SK-plasmin complex. The mode of interaction between the SK a-domain and N,PIm is clearly different from that of some other (i-grasp folding proteins. For example, the Ras-binding protein, C-raft, binds to its target protein, Rap 1 a, by forming an extended ~3-sheet between the edges of their existing (3-sheets (Nassar et al., Nature Structural Biology 1996; 3 : 723-729).
The SK y-domain binds to p.Plm near the activation cleavage site of plasmin (see Figure 4b). On the pPlm part, this interaction mainly involves two loop regions: p,Plm(622-628) in the so called calcium-binding loop and p,Plm(692-695) in the so called autolysis loop. The interactions include a salt-WO 99/57251 PCT/US99/1008b bridge between SK Lys3sa and p,Plm Glu6z3, hydrogen bonds (e.g. SK Glu3" and p.Plm G1n622), and hydrophobic interactions. The amino acid sequence of region 622-628 in human plasmin(ogen) is "QEVNLEP" (amino acids 622-628 of SEQ
» NO:1), and in bovine plasmin(ogen) is "NEKVREQ" (amino acids 643-649 of SEQ >D N0:6). Since this region of p,Plm is involved in the SK binding, the sequence difference shown above may provide explanation why the catalytic domain of bovine plasminogen binds with SK significantly weaker than human plasminogen does (Young et al., Journal of Biological Chemistry 1998; 273 3110-3116). On the other hand, the only close interaction of SK with the activation loop region of ~tPlm (i.e. around p,Plm(558-566)) is that between SK
Ala3az and p,plm Valsb'. Therefore human plasminogen activation by SK is unlikely to require direct contact of SK with the activation loop of plasminogen.
Furthermore, the observed C-terminus of the SK y-domain is on the side opposite to these l,~Plm binding regions. Hence it is probable that the last 40 or so residues, which are disordered in the crystal, have nothing to do with the SK-Plg complex formation.
Although there is no kringle domain present in the complex crystal, kringle domains have been shown to be involved in plasminogen activation by SK. Since the N-terminus of the catalytic domain of plasmin(ogen) is on the hemisphere opposite to the SK binding sites, the extension of kringle S from the catalytic domain is unlikely to disturb the observed interactions between SK
and p,Plm. Also, it has been shown previously (Rodriguez et al.. European Journal of Biochemistry 1995; 229: 83-90) that the complex of plasmin with the fragment SK(143-293) (i.e. the (3-domain) or SK(143-386) (i.e. the ~3- and y-domains) is very rapidly inhibited by a2-antiplasmin, whereas the complex with intact SK is resistant to inhibition. Furthermore in the case of SK(143-386), inhibition by a2-antiplasmin results in dissociation of the SK-Plm complex;
SK(143-293), in contrast, remains associated with the a2-antiplasmin-plasmin complex. The results suggest that the a2-antiplasmin-plasmin interface overlaps with the SK a-domain binding site and partially overlaps with the SK y-domain binding site on the plasmin surface, while the SK [3-domain binding site may have nothing to do with the a2-antiplasmin binding site.
Example 6: Putative substrate bindine site of the SK-Plm complex Although it is active against fibrin, plasmin alone can not convert plasminogen to plasmin. This substrate binding specificity can be explained by a comparison of the crystal structure of p,Plm with that of the catalytic domain of human t-PA (Renatus et al., EMBO Journal 1997; 16: 4797-4805}. While both are trypsin-like proteases, human t-PA has high specificity towards activation of plasminogen. The overall structures of these two domains are similar (see Figure 5). The coordinate rmsd between the catalytic domain of human two-chain t-PA (Lamba et al., Journal ofMolecular Biology 1996; 258:
117-135) and human p,Plm is 0.74 A for 177 Ca atoms, using a 1.5 A cutoff However there are many structural differences on the enzyme surface around the active site. There are at least three significant backbone differences around the active site. 1) Corresponding to plasmin(ogen) between residues 644 and 645, t-PA has a six-residue insertion with three aspartate residues in a row. This insertion in t-PA is structurally replaced by part of the SK a-domain in the SK-p,Plm complex. 2) The "autolysis loop" region of ~Plm(689-695), which contacts the SK ~y-domain, is different from the corresponding region of t-PA.
3) The region of p,Plm(711-720) is different from the corresponding region in t-PA, where it is called the "methionine loop". The pPlm conformation in this region makes it possible to have a complementary contact with the SK a-domain. 4) In the so called "37-loop" region of t-PA which interacts with the natural inhibitor, PAI-1, and is involved in fibrin specificity (Bennett et al., Journal ofBiological Chemistry 1991; 266: 5191-5201; Madison, et al., Proceedings of the National Academy of Sciences, USA 1990; 87: 3530-3533), human plasmin(ogen) is four residues shorter around residue 583. Some of these differences, if not all, are likely responsible for the substrate specificity difference between plasmin and t-PA. The SK-plasmin complex may change the substrate specificity of plasmin by compensating for some of these differences. On the other hand, it has been shown that the isolated, synthetically prepared activation loop of plasminogen can not be cleaved by plasminogen activator (Gams and Shaw, International Journal of Peptide Protein Research 1982; 20: 421-428). This observation suggests that not only the amino acid sequence of the cleavage site but also its conformation and the overall structure of the substrate zymogen contribute to the specificity of the activator.
The crystal structure of the SK-~,Plm complex shows that the complex has an opened cavity (see Figure 6a) compared with the spherical (convex) shape of the catalytic domain of plasmin(ogen). Therefore the SK-Plg complex should provide more substrate binding surface than the plasmin molecule alone can. A manual molecular model to dock a model micro plasminogen molecule into the substrate binding site of the SK-p,Plm complex is shown in Figure 6b.
In such a model, the activation bond, Arg56i-Va1562~ of the substrate (micro) plasminogen is positioned into the active site of the catalytic (micro) plasmin;
the N-terminus of substrate N.PIg is positioned to be closed to the disordered region of SK(45-70). From the bottom of the substrate binding concave, p.Plm contributes approximately 1,OSOA2 binding area, mostly from the surface of the strand, °'(32, and the a-helix, aa3,4. Since it might be modeled close to the kringle domain of the substrate plasminogen, the flexible SK(45-70) region may provide extra substrate binding surface; that would explain the observed high amity of the SK a-domain with the kringle domains of plasminogen (Young et al., Journal of Biological Chemistry 1998; 273 : 3110-3116) as well as the important role played by residues 45-51 of SK in binding with plasminogen (Nihalani et al., Protein Science 1998; 7: 637-648). For the SK ~i-domain, the major (3-sheet forms part of the wall of substrate binding concave with its helix side facing outside. The (3-strand on the rim of the ~i-sheet, ~(i2, potentially forms hydrogen bonds with the strand of residues 625-629 of substrate plasminogen. The SK ji-domain contributes ~550 A2 binding surface in total.
The SK y-domain contributes some coiled coil, around residue 330, to the substrate binding, about 150 A2 in total. Several potential salt bridges can be predicted from this hypothetical model, including Arg56~ of the substrate plasminogen (s-Plg) to Asp'35 of the catalytic plasmin (c-Phn), s-Plg Lysss' to c-Plm Glu6o6, and s-Plg Lys'S° to SK Asp'g. Lys55' of plasminogen was found to be important for plasminogen activation by t-PA (Wang and Reich, Protein Science 1995; 4: 1769-1779) and could be explained if similar binding modes were assumed for binding of t-PA to plasminogen. The Asp'35 of c-Plm, interacting with the side chain of Args6~ of s-Plg, defines the substrate specificity of the SK-Plm complex at the S 1 position.
Example 7: A possible activation mechanism of human plasmino~by SK
One of the functions of SK is to turn the zymogen plasminogen into an active "enzyme" without cleaving the peptide chain. It appears from the crystal structure of the SK-pPlm complex that it is the interaction between the SK y-domain and the catalytic domain of plasminogen that creates the enzymatic activity of human plasminogen-SK complex.
As shown by many crystal structures of trypsin(ogen)-like proteases, one characteristic of the proteases in this family is a buried salt bridge formed in the activation pocket associated with the activation. In the classical case, this salt bridge is formed between the carboxylate group of the aspartate residue that is immediately upstream of the catalytic nucleophile serine residue and the liberated amino terminus after the activation cleavage. The formation of the salt bridge reorients the aspartate residue relative to the zymogen structure and thus restructures the active site, which includes the oxyanion hole, the catalytic triad and the S 1 specificity pocket (Freer et al., Biochemistry 1970; 9: 1997-2009).
However, non-cleavage activation has also been found in some proteins in this family, including t-PA and vampire-bat plasminogen activator (v-PA) (SEQ ID
N0:7) (Renatus et al., EMBO Journal 1997; 16: 4797-4805; Renatus et al., Biochemistry 1997; 36: 13483-13493). Human t-PA has significantly high plasminogenolytic activity in its single-chain form. v-PA, on the other hand lacks the activation cleavage site. Both of them switch between the active and the inactive stages in response to environmental changes, including the presence of fibrin. In their crystal structures, complexed with high affanity inhibitors, both plasminogen activators were frozen in the "active stage". These structures demonstrate that in such an intact protein, a buried lysine residue replaces the functional role of the released amino terminus of the protease activated by the cleavage. The conformation of this lysine residue, Lys'ss (numbered as Bode &
Renatus (Bode and Renatus, Current Opinion Structural Biology 1997; 7: 865-872), unlike the newly released amino terminus which usually stays in one form, may switch between the active and the inactive stages depending on environmental conditions such as the local concentration of cofactors (Lamba et al., Journal ofMolecularBiology 1996; 258: 117-135; Nienaber et al., Biochemistry 1992; 31: 3852-3861).
A similar scenario is likely to exist in the SK-Plg complex (see Figure 7).
Based on three-dimensional structural comparison and sequence alignment, it is evident that the same lysine residue is also conserved in plasminogen and is located at position 698. When the activation loop remains intact, and the activation pocket is not occupied by the released amino terminus, the plasminogen molecule is a two-stage proenzyme which predominantly stays in its active form. The binding of SK y-domain to the "autolysis" loop region, which is upstream of the conserved lysine residue is likely to be the trigger for plasminogen to switch from its inactive form to the active form. In such an active form, Lys698 forms the critical salt bridge with Asp'4°. On the other hand, when the activation pocket is occupied by the released amino terminus, the binding of SK y-domain will have little effect on the amidolytic activity of plasmin(ogen). Some factors, which were proposed to favor the active form of single-chain t-PA and v-PA, seem to have little effect on plasminogen activation by SK. For example, the so called zymogen triad, Asp'94, His4°, and Ser32, present in trypsinogen hut not in t-PA or v-PA, was assumed to lock Asp'94, and thus the oxyanion stabilizing loop, in its inactive form. These residues are present in plasminogen as Asp'a°, Hissss~ ~d SerS'g, but they do not prohibit the formation of active-zymogen upon binding with SK.

It is believed that the SK 'y-domain binds to plasminogen to create an active-zymogen, while the binding of the SK a- and ~i-domains changes the substrate specificity of the active-zymogen. Although the detailed mechanism remains to be established, this model can be used to explain the following observations. The combination of SK(220-414) and SK( 16-251 ), but not either peptide alone, effectively activates human plasminogen (Young et al., Journal of Biological Chemistry 1998; 273: 3110-3116). An explanation to this observation might simply be that the a-domain and y-domain have different functions that compensate with each other in plasminogen activation. SK(16-251) dose-dependently enhanced the activation of plasminogen by SK(16-414) (Young et al., Journal of Biological Chemistry 1998; 273 : 3110-3116). SK or SK(16-414) can convert plasminogen to plasmin; however, plasmin alone can not convert other plasminogen to plasmin. The additional SK(16-251) peptides, on the other hand, form complexes with the newly formed plasmin molecules and modulate their substrate specificity such that new plasminogen activators are formed.
Example 8: Structure comparison between the a-domain of SK and staphylokinase Among the (3-grasp folding family (Murzin et al., Journal ofMolecular Biology 1995; 247: 536-540), staphylokinase (SAK) is another bacterial source plasminogen activator, whose crystal structure has been determined recently (Murzin et al., Journal ofMolecular Biology 1995; 247: 536-540). Like SK, staphylokinase activates human plasminogen by forming a zymogen-activator complex {Lijnen et al., Journal of Biological Chemistry 1991; 266: 11826-11832). However, SK and staphylokinase do not share detectable sequence homology. The size of staphylokinase is only one third that of streptokinase, and its binding mode and activation mechanism to human plasminogen are unknown. It is particularly interesting to find that staphylokinase shares a three-dimensional folding with the SK a-domain (see Figure 8). The coordinate rmsd between staphylokinase and the SK a-domain is 1.8 A for 91 Ca atoms, using a 4.0 A cutoff Based on the three-dimensional structural similarity between the SK oc-domain and staphylokinase, we propose that staphylokinase binds to plasminogen in the same mode as the SK oc-domain. Along this line, several observations on the staphylokinase-plasminogen interaction could be explained.
First, SAK G1u46, which corresponds to SK G1u39, was found to be important for the formation of the SAK-Plg complex (Silence et al., Journal of Biological Chemistry 1995; 270: 27192-27198). This residue would be located on the /32 strand and form a salt-bridge with Arg"9 of plasmin(ogen). SAK Met2~, which corresponds to SK Val'9, was also found to be of crucial importance for the .
activation of plasminogen by staphylokinase (Schlott et al., Biochemical and Biophysical Acta. 1994; 1204: 235-242). Since the side chain at this position has van de Waals contact with the hydrophobic portion of the Arg"9 side chain, disturbing such a contact would result in disturbing the complex contact through both van de Waals and electrostatic interactions. A few more residues of staphylokinase that were found important for the activity of the complex of SAK-Plg, including LysS°, G1u65, and Asp69, all would be located on the substrate binding surface that is similar to that we have proposed for the SK-p,Plm complex. In such a scenario, the N-terminal fragment of staphylokinase (i.e. residues 1-20), most of which are disordered in the crystal structure, would be located at a position that potentially could affect the conformation of the active site by allosteric binding. Therefore, a one domain protein like staphylokinase would perform multiple functions that are accomplished by two/three domains in SK.
The teachings of the references cited herein and referenced below are specifically incorporated herein. Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description and are intended to be encompassed by the following claims.

SEQUENCE LISTING
<110> Oklahoma Medical Research Foundation <120> Thrombolytic Agents Derived from Streptokinase <130> 5208-213 <140>
<141>
<150> 60/084,392 <151> 1998-05-06 <160> 7 <170> PatentIn Ver. 2.0 <210> 1 <211> 791 <212> PRT
<213> Homo sapiens <220>
<221> PEPTIDE
<222> (1)..(791) <223> human plasminogen <400> 1 Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asn Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn <210> 2 <211> 562 <212> PRT
<213> Homo Sapiens <220>
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<222> (1)..(562) <223> human tissue plasminogen activator <400> 2 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val Phe Val Ser Pro Ser Gln Glu Ile His Ala Arg Phe Arg Arg Gly Ala Arg Ser Tyr Gln Val Ile Cys Arg Asp Glu Lys Thr Gln Met Ile Tyr Gln Gln His Gln Ser Trp Leu Arg Pro Val Leu Arg Ser Asn Arg Val Glu Tyr Cys Trp Cys Asn Ser Gly Arg Ala Gln Cys His Ser Val Pro Val Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly Gly Thr Cys Gln Gln Ala Leu Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Glu Gly Phe Ala Gly Lys Cys Cys Glu Ile Asp Thr Arg Ala Thr Cys Tyr Glu Asp Gln Gly Ile Ser Tyr Arg Gly Thr Trp Ser Thr Ala Glu Ser Gly Ala Glu Cys Thr Asn Trp Asn Ser Ser Ala Leu Ala Gln Lys Pro Tyr Ser Gly Arg Arg Pro Asp Ala Ile Arg Leu Gly Leu Gly Asn His Asn Tyr Cys Arg Asn Pro Asp Arg Asp Ser Lys Pro Trp Cys Tyr Val Phe Lys Ala Gly Lys Tyr Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys Ser Glu Gly Asn Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg Gly Thr His Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn Ser Met Ile Leu Ile Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala Gln Ala Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Gly Asp Ala Lys Pro Trp Cys His Val Leu Lys Asn Arg Arg Leu Thr Trp Glu Tyr Cys Asp Val Pro Ser Cys Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro Gln Phe Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro Trp Gln Ala Ala Ile Phe Ala Lys His Arg Arg Ser Pro Gly Glu Arg Phe Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp Ile Leu Ser Ala Ala His Cys Phe Gln Glu Arg Phe Pro Pro His His Leu Thr Val Ile Leu Gly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu Gln Lys Phe Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asp Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser Ser Arg Cys Ala Gln Glu Ser Ser Val Val Arg Thr Val Cys Leu Pro Pro Ala Asp Leu Gln Leu Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Glu Ala Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His Val Arg Leu Tyr Pro Ser Ser Arg Cys Thr Ser Gln His Leu Leu Asn Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Gly Pro Gln Ala Asn Leu His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Leu Asn Asp Gly Arg Met Thr Leu Val Gly Ile Ile Ser Trp Gly Leu Gly Cys Gly Gln Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn Tyr Leu Asp Trp Ile Arg Asp Asn Met Arg Pro <210> 3 <211> 431 <212> PRT
<213> Homo sapiens <220>
<221> PEPTIDE
<222> (1)..(431) <223> urokinase <400> 3 Met Arg Ala Leu Leu Ala Arg Leu Leu Leu Cys Val Leu Val Val Ser Asp Ser Lys Gly Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile Asp Lys Ser Lys Thr Cys Tyr Glu Gly Asn Gly His Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr His Ala His Arg Ser Asp Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys Pro Leu Val Gln Glu Cys Met Val His Asp Cys Ala Asp Gly Lys Lys Pro Ser Ser Pro Pro Glu Glu Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly Arg Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met Lys Phe Glu Val Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His Asn Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val Val Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His Tyr Tyr Gly Ser Glu Val Thr Thr Lys Met Leu Cys Ala Ala Asp Pro Gln Trp Lys Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg Met Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu <210> 4 <211> 415 <212> PRT
<213> Streptococcus sp.
<220>
<221> PEPTIDE
<222> (1)..(415) <223> streptokinase <400> 4 Ile Ala Gly Pro Glu Trp Leu Leu Asp Arg Pro Ser Val Asn Asn Ser Gln Leu Val Val Ser Val Ala Gly Thr Val Glu Gly Thr Asn Gln Asp Ile Ser Leu Lys Phe Phe Glu Ile Asp Leu Thr Ser Arg Pro Ala His Gly Gly Lys Thr Glu Gln Gly Leu Ser Pro Lys Ser Lys Pro Phe Ala Thr Asp Ser Gly Ala Met Ser His Lys Leu Glu Lys Ala Asp Leu Leu Lys Ala Ile Gln Glu Gln Leu Ile Ala Asn Val His Ser Asn Asp Asp Tyr Phe Glu Val Ile Asp Phe Ala Ser Asp Ala Thr Ile Thr Asp Arg Asn Gly Lys Val Tyr Phe Ala Asp Lys Asp Gly Ser Val Thr Leu Pro Thr Gln Pro Val Gln Glu Phe Leu Leu Ser Gly His Val Arg Val Arg Pro Tyr Lys Glu Lys Pro Ile Gln Asn Gln Ala Lys Ser Val Asp Val Glu Tyr Thr Val Gln Phe Thr Pro Leu Asn Pro Asp Asp Asp Phe Arg Pro Gly Leu Lys Leu Thr Lys Leu Leu Lys Thr Leu Ala Ile Gly Asp Thr Ile Thr Ser Gln Glu Leu Leu Ala Gln Ala Gln Ser Ile Leu Asn Lys Asn His Pro Gly Tyr Thr Ile Tyr Glu Arg Asp Ser Ser Ile Val Thr His Asp Asn Asp Ile Phe Arg Thr Ile Leu Pro Met Asp Gln Glu Phe Thr Tyr Arg Val Lys Asn Arg Glu Gln Ala Tyr Arg Ile Asn Lys Lys Ser Gly Leu Asn Glu Glu Ile Asn Asn Thr Asp Leu Ile Ser Leu Glu Tyr Lys Tyr Val Leu Lys Lys Gly Glu Lys Pro Tyr Asp Pro Phe Asp Arg Ser His Leu Lys Leu Phe Thr Ile Lys Tyr Val Asp Val Asp Thr Asn Glu Leu Leu Lys Ser Glu Gln Leu Leu Thr Ala Ser Glu Arg Asn Leu Asp Phe Arg Asp Leu Tyr Asp Pro Arg Asp Lys Ala Lys Leu Leu Tyr Asn Asn Leu Asp Ala Phe Gly Ile Met Asp Tyr Thr Leu Thr Gly Lys Val Glu Asp Asn His Asp Asp Thr Asn Arg Ile Ile Thr Val Tyr Met Gly Lys Arg Pro Glu Gly Glu Asn Ala Ser Tyr His Leu Ala Tyr Asp Lys Asp Arg Tyr Thr Glu Glu Glu Arg Glu Val Tyr Ser Tyr Leu Arg Tyr Thr Gly Thr Pro Ile Pro Asp Asn Pro Asp Asp Lys <210> 5 <211> 136 <212> PRT
<213> Staphylococcus aureus <220>
<221> PEPTIDE
<222> (1)..(136) <223> staphylokinase <400> 5 Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala Ser Tyr Phe Glu Pro Thr Gly Pro Tyr Leu Met Val Asn Val Thr Gly Val Glu Gly Lys Glu Asn Glu Leu Leu Ser Pro His Tyr Val Glu Phe Pro Ile Lys Pro Gly Thr Thr Leu Thr Lys Glu Lys Ile Glu Tyr Tyr Val Glu Trp Ala Leu Asp Ala Thr Ala Tyr Lys Glu Phe Arg Val Val Glu Leu Asp Pro Ser Ala Lys Ile Glu Val Thr Tyr Tyr Asp Lys Asn Lys Lys Lys Glu Glu Thr Lys Ser Phe Pro Ile Thr Glu Lys Gly Phe Val Val Pro Asp Leu Ser Glu His Ile Lys Asn Pro Gly Phe Asn Leu Ile Thr Lys Val Val Ile Glu Lys Lys <210> 6 <211> 812 <212> PRT
<213> Bos taurus <220>
<221> PEPTIDE
<222> (1)..(812) <223> bovine plasminogen <400> 6 Met Leu Pro Ala Ser Pro Lys Met Glu His Lys Ala Val Val Phe Leu Leu Leu Leu Phe Leu Lys Ser Gly Leu Gly Asp Leu Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Leu Ser Leu Ser Arg Lys Asn Leu Ala Gly Arg Ser Val Glu Asp Cys Ala Ala Lys Cys Glu Glu Glu Thr Asp Phe Val Cys Arg Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val Val Met Ala Glu Asn Ser Lys Asn Thr Pro Val Phe Arg Met Arg Asp Val Ile Leu Tyr Glu Lys Arg Ile Tyr Leu Leu Glu Cys Lys Thr Gly Asn Gly Gln Thr Tyr Arg Gly Thr Thr Ala Glu Thr Lys Ser Gly Val Thr Cys Gln Lys Trp Ser Ala Thr Ser Pro His Val Pro Lys Phe Ser Pro Glu Lys Phe Pro Leu Ala Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asp Ile Pro Glu Cys Glu Asp Lys Cys Met His Cys Ser Gly Glu Asn Tyr Glu Gly Lys Ile Ala Lys Thr Met Ser Gly Arg Asp Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr Asp Pro Gln Lys Arg Trp Glu Phe Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Lys Tyr Gln Cys Leu Lys Gly Thr Gly Lys Asn Tyr Gly Gly Thr Val Ala Val Thr Glu Ser Gly His Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asn Gly Glu Lys Ala Pro Trp Cys Tyr Thr Thr Asn Ser Glu Val Arg Trp Glu Tyr Cys Thr Ile Pro Ser Cys Glu Ser Ser Pro Leu Ser Thr Glu Arg Met Asp Val Pro Val Pro Pro Glu Gln Thr Pro Val Pro Gln Asp Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Arg Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His Leu Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Ser Pro Trp Cys Tyr Thr Thr Asp Pro Arg Val Arg Trp Glu Phe Cys Asn Leu Lys Lys Cys Ser Glu Thr Pro Glu Gln Val Pro Ala Ala Pro Gln Ala Pro Gly Val Glu Asn Pro Pro Glu Ala Asp Cys Met Ile Gly Thr Gly Lys Ser Tyr Arg Gly Lys Lys Ala Thr Thr Val Ala Gly Val Pro Cys Gln Glu Trp Ala Ala Gln Glu Pro His Gln His Ser Ile Phe Thr Pro Glu Thr Asn Pro Gln Ser Gly Leu Glu Arg Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Asn Gly Pro Trp Cys Tyr Thr Met Asn Pro Arg Lys Pro Phe Asp Tyr Cys Asp Val Pro Gln Cys Glu Ser Ser Phe Asp Cys Gly Lys Pro Lys Val Glu Pro Lys Lys Cys Ser Gly Arg Ile Val Gly Gly Cys Val Ser Lys Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Arg Ser Ser Arg His Phe Cys Gly Gly Thr Leu Ile Ser Pro Lys Trp Val Leu Thr Ala Ala His Cys Leu Asp Asn Ile Leu Ala Leu Ser Phe Tyr Lys Val Ile Leu Gly Ala His Asn Glu Lys Val Arg Glu Gln Ser Val Gln Glu Ile Pro Val Ser Arg Leu Phe Arg Glu Pro Ser Gln Ala Asp Ile Ala Leu Leu Lys Leu Ser Arg Pro Ala Ile Ile Thr Lys Glu Val Ile Pro Ala Cys Leu Pro Pro Pro Asn Tyr Met Val Ala Ala Arg Thr Glu Cys Tyr Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Glu Gly Leu Leu Lys Glu Ala His Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Asn Glu Tyr Leu Asp Gly Arg Val Lys Pro Thr Glu Leu Cys Ala Gly His Leu Ile Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Pro Tyr Val Pro Trp Ile Glu Glu Thr Met Arg Arg Asn <210> 7 <211> 265 <212> PRT
<213> Desmodus rotundus <220>
<221> PEPTIDE
<222> (1)..(265) <223> vampire bat saliva plasminogen activator <400> 7 Thr Cys Gly Leu Arg Lys Tyr Lys Glu Pro Gln Leu His Ser Thr Gly Gly Leu Phe Thr Asp Ile Thr Ser His Pro Trp Gln Ala Ala Ile Phe Ala Gln Asn Arg Arg Ser Ser Gly Glu Arg Phe Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp Val Leu Thr Ala Ala His Cys Phe Gln Glu Ser Tyr Leu Pro Asp Gln Leu Lys Val Val Leu Gly Arg Thr Tyr Arg Val Lys Pro Gly Glu Glu Glu Gln Thr Phe Lys Val Lys Lys Tyr Ile Val His Lys Glu Phe Asp Asp Asp Thr Tyr Asn Asn Asp Ile Ala Leu Leu Gln Leu Lys Ser Asp Ser Pro Gln Cys Ala Gln Glu Ser Asp Ser Val Arg Ala Ile Cys Leu Pro Glu Ala Asn Leu Gln Leu Pro Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Lys Ser Ser Ser Pro Phe Tyr Ser Glu Gln Leu Lys Glu Gly His Val Arg Leu Tyr Pro Ser Ser Arg Cys Ala Pro Lys Phe Leu Phe Asn Lys Thr Val Thr Asn Asn Met Leu Cys Ala Gly Asp Thr Arg Ser Gly Glu Ile Tyr Pro Asn Val His Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Met Asn Asp Asn His Met Thr Leu Leu Gly Ile Ile Ser Trp Gly Val Gly Cys Gly Glu Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn Tyr Leu Gly Trp Ile Arg Asp Asn Met His Leu

Claims (6)

Claims

1. A thrombolytic agent comprising streptokinase wherein at least one nonessential portion has been replaced with a structurally similar polypeptide from a human protein.

2. The thrombolytic agent of claim 2 wherein the nonessential portion is a portion of streptokinase selected from the group consisting of a portion which does not function in plasminogen complexation, a portion which does not function in plasminogen activation, and a portion which does not function in substrate specificity.

3. A method of forming a thrombolytic agent comprising the steps:
determining a nonessential portion of streptokinase; and replacing the nonessential portion with a portion of a structurally similar human protein.

4. The method of claim 3 wherein the nonessential portion is a portion of streptokinase selected from the group consisting of a portion which does not function in plasminogen complexation, a portion which does not function in plasminogen activation, and a portion which does not function in substrate specificity.

5. A method of treating blood clot disorders comprising administration of a streptokinase wherein at least one nonessential portion has been modified.

6. The method of claim 8 wherein the nonessential portion is a portion of streptokinase selected from the group consisting of a portion which does not function in plasminogen complexation, a portion which does not function in plasminogen activation, and a portion which does not function in substrate specificity.