IDENTIFICATION, PRODUCTION AND USE OF STAPHYLOKINASE DERIVATIVES WITH REDUCED IMMUNOGENICITY AND/OR REDUCED CLEARANCE

Abstract: Methods for the identification, production and use of staphylokinase derivatives characterized by a reduced immunogenicity after administration in patients and that can be administered by single intravenous bolus injection. The derivatives of the invention are obtained by preparing a DNA fragment comprising at least the part of the coding sequence of staphylokinase that provides for its biological activity; performing in vitro site-directed mutagenesis on the DNA fragment to replace one or more codons for wild-type amino acids by a codon for another amino acid; cloning the mutated DNA fragment in a suitable vector; transforming or transfecting a suitable host cell with the vector; culturing the host cell under conditions suitable for expressing the DNA fragment; purifying the expressed staphylokinase derivative to homogeneity and chemically modifying substituted Cys residues with thiol-directed polyethylene glycol; preferably the DNA fragment is a 453 bp EcoRI-HindIII fragment of the plasmid pMEX602sakB, (pMEX.SakSTAR), the in vitro site-directed mutagenesis is performed by spliced overlap extension polymerase chain reaction and the mutated DNA fragment is expressed in E. coli strain TG1 or WK6. The invention also relates to pharmaceutical compositions comprising at least one of the staphylokinase derivatives according to the invention together with a suitable excipient, for treatment of arterial thrombosis.

Description: IDENTIFICATION, PRODUCTION AND USE OF STAPHYLOKINASE DERIVATIVES WITH REDUCED IMMUNO- GENICITY AND/OR REDUCED CLEARANCE The present invention relates to new staphylokinase derivatives with reduced immunogenicity which can be administered by continuous infusion or by single intravenous bolus injection, to their identification, production and use in the treatment of arterial thrombosis and to the preparation of a pharmaceutical composition for treating arterial thrombosis. More in particular the invention relates to the use of engineered staphylokinase derivatives for the preparation of a pharmaceutical composition for treating myocardial infarction. Staphylokinase, a protein produced by certain strains of Staphylococcus aureus. which was shown to have profibrinolytic properties more than 4 decades ago (1, 2) appears to constitute a potent thrombolytic agent in patients with acute myocardial infarction (3, 4). The staphylokinase gene has been cloned from the bacteriophages sakφC (5) and sak42D (6) as well as from the genomic DNA (sakSTAR) of a lysogenic Staphylococcus aureus strain (7) . The staphylokinase gene encodes a protein of 163 amino acids, with amino acid 28 corresponding to the NH 2 -terminal residue of full length mature staphylokinase (6, 8, 9) . The mature protein sequence of the wild-type variant SakSTAR (9) is represented in Figure 1. Only four nucleotide differences were found in the coding regions of the sakφC, sak42D and sakSTAR genes, one of which constituted a silent mutation (6, 8, 9). In a plasma milieu, staphylokinase is able to dissolve fibrin clots without associated fibrinogen degradation (10-12). This fibrin-specificity of staphylokinase is the result of reduced inhibition by α 2 -antiplasmin of plasmin. staphylokinase complex bound to fibrin, recycling of staphylokinase from the plasmin. staphylokinase complex following inhibition by α 2 -antiplasmin, and prevention of the conversion of circulating plasminogen. staphylokinase to plasmin. staphylokinase by α 2 -antiplasmin (13-15). In addition staphylokinase has a weak affinity for circulating but a high affinity for fibrin-bound plasminogen (16) and staphylokinase requires NH 2 -terminal processing by plasmin to display its plasminogen activating potential (17) . In several experimental animal models, staphylokinase appears to be equipotent to streptokinase for the dissolution of whole blood or plasma clots, but significantly more potent for the dissolution of platelet-rich or retracted thrombi (18, 19) . Staphylokinase is a heterologous protein and is immunogenic in man. The intrinsic immunogenicity of staphylokinase, like that of streptokinase, clearly hampers its unrestricted use. Not only will patients with preexisting high antibody titers be refractory to the thrombolytic effect of these agents, but allergic side effects and occasional life-threatening anaphylaxis may occur (20) . Because both streptokinase and staphylokinase are heterologous proteins, it is not obvious that their immunogenicity could be reduced by protein engineering. Indeed, no successful attempts to generate active low molecular weight fragments from streptokinase have been reported. In staphylokinase, deletion of the NH 2 -terminal 17 amino acids or the COOH-terminal 2 amino acids inactivates the molecule, which in addition is very sensitive to inactivation by site-specific mutagenesis (21). It is therefore the object of the present invention to provide less immunogenic variants of staphylokinase having preferably a higher specific activity and/or lower plasma clearance and/or increased thrombolytic potency. In the research that ultimately led to the present invention it was already found that the wild-type staphylokinase variant SakSTAR (9) contains three non-overlapping immunodo inant epitopes, at least two of which can be eliminated by specific site-directed mutagenesis, without inactivation of the molecule. This has been disclosed in EP-95200023.0 (22). These engineered staphylokinase variants are less reactive with antibodies elicited in patients treated with wild-type staphylokinase, and are significantly less immunogenic than wild-type staphylokinase, as demonstrated in rabbit and baboon models and in patients with peripheral arterial occlusion (22) . The present invention now relates to general methods for the identification, production and use of staphylokinase derivatives showing a reduced antigenicity and immunogenicity as compared to wild-type staphylokinase as well as for variants with selective derivatization with polyethylene glycol. The derivatives preferably have a higher specific activity and/or lower plasma clearance and/or increased thrombolytic potency. The derivatives have essentially the amino acid sequence of wild-type staphylokinase or modified versions thereof and essentially intact biological activities, but have a reduced reactivity with a panel of murine monoclonal antibodies and/or with antibodies induced in patients by treatment with wild-type SakSTAR. The polyethylene glycol substituted ("pegylated") variants have reduced plasma clearances rendering them particularly suited for use by single intravenous bolus administration. Instead of PEG other pharmaceutically acceptable macromolecules can be used. More in particular, the invention provides for staphylokinase derivatives SakSTAR(K35X,G36X,E65X,K74X, E80X,D82X,K102X,E108X,K109X,K121X,K130X, K135X,K136X, +137X) having the amino acid sequence as depicted in figure 1 in which the amino acids Lys in position 35, Gly in position 36, Glu in position 65, Lys in position 74, Glu in position 80, Asp in position 82, Lys in position 102, Glu in position 108, Lys in position 109, Lys in position 121, Lys in position 130, Lys in position 135 and/or Lys in position 136 have been replaced with other amino acids and/or in which one amino acid has been added at the COOH-terminus, thus altering the immunogenicity after administration in patients, without markedly reducing the specific activity. Further preferred embodiments of the invention are staphylokinase derivatives listed in Tables 1, 3, 4, 5, 6, 7, 8, 13, 19 and 20, having the amino acid sequence as depicted in figure 1 in which the indicated amino acids have been replaced by other amino acids thus reducing the absorption of SakSTAR-specific antibodies from plasma of patients treated with staphylokinase, without reducing the specific activity. Derivatives in which the specific activity is increased and the immunogenicity is decreased are the following: SakSTAR(K74A,E75A,R77A) , SakSTAR(K35A,E75A) , SakSTAR(E75A) , SakSTAR(E80A, D82A) , SakSTAR(E80A) , SakSTA (D82A) , SakSTAR(E75A, D82A) , SakSTAR(S34G,G36R, H43R) , SakSTAR(K35A) , SakSTAR(E80A) , SakSTAR(D82A, S84A) , SakSTAR(T90A) , SakSTAR(Y92A) , SakSTAR(K130A) , SakSTAR(V132A) , SakSTAR(S34G,G36R,H43R) , SakSTAR(G36R) , SakSTAR(H43R) , SakSTAR(G36R,K74R) , SakSTAR(K35E) , SakSTAR(K74Q) , SakSTAR(Kl30T) , SakSTAR(V132L) , SakSTAR(V132T) , SakSTAR(V132N) , SakSTAR(V132R) , SakSTAR(K130T,K135R) , SakSTAR(G36R,K130T,K135R) , SakSTAR(K74R,K130T,K135R) , SakSTAR(K74Q,K130T,K135R) , SakSTAR(G36R,K74R,K130T, K135R) , SakSTAR(G36R,K74Q,K130T,K135R) , SakSTAR(G36R, H43R,K74R,K130T,K135R) , SakSTAR(E65A,K74Q,K130T,K135R) , SakSTAR(E65Q,K74Q,K130T,K135R) , SakSTAR(K74Q,K86A, K130T,K135R) , SakSTAR(E65Q,T71S,K74Q, K130T,K135R) , SakSTAR(K74Q,K130A,K135R) , SakSTAR(E65Q,K74Q,K130A, K135R) , SakSTAR(K74Q,K130E,K135R) , SakSTAR(K74Q,K130E, V132R,K135R) , SakSTAR(E65Q,K74Q,T90A,K130A,K135R) , SakSTAR(E65Q,K74Q,N95A,K130A,K135R) , SakSTAR(E65Q,K74Q, E118A,K130A,K135R) , SakSTAR(E65Q, K74Q,N95A,E118A,K130A, K135R) , SakSTAR(N95A,K130A,K135R) , SakSTAR(E65Q, K74Q, K109A,K130,K135R) , SakSTAR(E65Q,K74Q, E108A,K109A, K130T,K135R) , SakSTAR(E65Q,K74Q,K121A,K130T,K135R) , SakSTAR(E65Q,K74Q,N95A,E118A,K130A,K135R,K136A,+137K) , SakSTAR(E80A,D82A,K130T,K135R) , SakSTAR(K74R,E80A, D82A, K130T,K135R) , SakSTAR(K74Q,E80A, D82A,K130T,K135R) , SakSTAR(K35A,K74R,E80A,D82A,K130T,K135R) , SakSTAR(E65D, K74R,E80A,D82A,K130T,K135R) , SakSTAR(E65S,K74R,E80A, D82A,K130T,K135R) , SakSTAR(S34G,G36R,K74R,K130T,K135R) , SakSTAR(E65A,K74R,E80A,D82A,K130T,K135R) , SakSTAR(E65N, K74R,E80A,D82A,K130T,K135R) , SakSTAR(E65Q,K74R,E80A, D82A,K130T,K135R) , SakSTAR(K57A, E58A, E61A,E80A, D82A, K130T,K135R) , SakSTAR(E65D,K74Q,E80A, D82A, K130T,K135R) , SakSTAR(E65Q,K74Q,E80A,D82A,K130T,K135R) , SakSTAR(K35A, E65D,K74Q,E80A,D82A,K130T,K135R) , SakSTAR(K74R,E80A,D82A, S103A,K130T,K135R) , SakSTAR(E65D,K74R,E80A, D82A,K109A, K130T,K135R) , SakSTAR(E65D,K74R,E80A,D82A,K130T, K135R,K136A) , SakSTAR(E65Q,K74Q, D82A,S84A,K130T,K135R) , SakSTAR(K35A,K74Q,E80A,D82A,K130T,K135R) , SakSTAR(K35A, E65D,K74R,E80A,D82A,K130T,K135R) . Of these SakSTAR(E65D,K74R,E80A,D82A,K130T, K135R) having the code SY19 and SakSTAR(K35A,E65Q,K74R, E80A, D82A, T90A, E99D, T101S , E108A, K109A, K130T,K135R) having the code SY161 are especially preferred. Besides the above described substitution derivatives the invention relates to derivatives having in addition an amino acid substituted with Cys. This type of substitution may result in di erization and/or increased specific activity and/or reduced clearance and/or increased thrombolytic potency. Reduced plasma clearance is in particular obtained when the derivative is substituted with polyethylene glycol. Preferred embodiments of such staphylokinase derivatives are those wherein the Cys is chemically modified with polyethylene glycol with molecular weights up to 20 kDa. In particular embodiments selected amino acids in the NH 2 -terminal region of 10 amino acids, are substituted with Cys, which is chemically modified with polyethylene glycol with molecular weights up to 20 kDa. These derivatives are characterized by a significantly reduced plasma clearance and maintained thrombolytic potency upon single intravenous bolus administration at a reduced dose. More in particular the serine in position 2 or 3 is substituted with a cystein and the cystein is chemically modified with polyethylene glycol having a molecular weight of 5, 10 or 20 kDa. Preferred embodiments of these derivatives are SY161(S3C-MP5) , SY161(S3C-P10) , SY161(S3C-P20) , SY19 (S3C-MP5) , SY19(S3C- SP5) , SY19(S2C-SP5,S3C-SP5) , SY19 (S3C-P20) , SY19 (S3C-P10) all as defined in table 20. The presence of cysteins allows the formation of dimers of two staphylokinase derivatives of the invention. The invention also relates to a method for producing the derivatives of the invention by preparing a DNA fragment comprising at least the part of the coding sequence of staphylokinase that provides for its biological activity; performing in vitro site-directed mutagenesis on the DNA fragment to replace one or more codons for wild-type amino acids by a codon for another amino acid; cloning the mutated DNA fragment in a suitable vector; transforming or transfecting a suitable host cell with the vector; culturing the host cell under conditions suitable for expressing the DNA fragment; purifying the expressed staphylokinase derivative to homogeneity and derivatizing the variant with polyethylene glycol. Preferably the DNA fragment is a 453 bp EcoRl-Hindlll fragment of the plasmid pMEX602sakB (22, 23), the in vitro site-directed mutagenesis is preferably performed by spliced overlap extension polymerase chain reaction. Such overlap extension PCR is preferably performed with Vent DNA polymerase (New England Biolabs) or Taq polymerase (Boehringer Mannheim) and with available or generated wildtype sakSTAR or sakSTAR variants as template (24) . The invention also relates to pharmaceutical compositions comprising at least one of the staphylokinase derivatives according to the invention together with a suitable excipient, for treatment of arterial thrombosis. Pharmaceutical compositions, containing less immunogenic staphylokinase variants or "pegylated" staphylokinase variants as the active ingredient, for treating arterial thrombosis in human or veterinary practice may take the form of powders or solutions and may be used for intravenous, intraarterial or parenteral administration. Such compositions may be prepared by combining (e.g. mixing, dissolving etc.) the active compound with pharmaceutically acceptable excipients of neutral character (such as aqueous or non-aqueous solvents, stabilizers, emulsifiers, detergents, additives) , and further, if necessary with dyes. Furthermore the invention relates to the use of the staphylokinase derivatives for the treatment of arterial thrombosis, in particular myocardial infarction, and to the use of staphylokinase derivatives for the preparation of a pharmaceutical composition for the treatment of arterial thrombosis, in particular myocardial infarction. In the above and the following the terms "derivatives", "mutants" and "variants" are used interchangeably. Based on the present invention other variants and improvements will be obvious for the person skilled in the art. Thus random mutagenesis is likely to generate alternative mutants with reduced immunogenicity and possibly increased functional activity, whereas deletions or substitution with other amino acids may yield additional variants with reduced immunogenicity. The present invention will be demonstrated in more detail in the following examples, that are however not intended to be limiting to the scope of the invention. In the Examples reference is made to the following figures: Pig 1. Protein sequence of wild-type staphylokinase, SakSTAR. Numbering starts with the NH2-terminal amino acid of mature full length staphylokinase. Fig 2. Time course of neutralizing activities (left panel) and specific IgG against administered agent (right panel) following intra-arterial infusion of SakSTAR (open circles, n= 9) , SakSTAR(K74A) (closed circles, n= 11) or SakSTAR(K74A,E75A, R77A) (open squares, n= 6) in patients with peripheral arterial occlusion. The data represent median values and interquartile ranges, in μg/mL. Fig 3. Protein sequence of wild-type staphylokinase, SakSTAR with indicated amino acid substititutions. squares: single amino acid substitutions; circles: combined (2 to 3) amino acid to Ala substitutions. Fig. 4. Temperature stability of SakSTAR, (A); SakSTAR(K74Q,E80A,D82A,K130T, K135R) (B) ; SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) , (C) ; and SakSTAR(K35A,E65D,K74Q,E80A,D82A, K130T,K135R) , (D) . (O) : 4°C; (•) : 20°C; (V): 37°C; (T) : 56°C; (D) : 70°C. Fig 5. Time course of neutralizing activities (left panel) and specific IgG against administered agent (right panel) following intra-arterial infusion of SakSTAR (circles, n= 6), SakSTAR(K74Q,E80A,D82A,K130T,K135R) (squares, n= 6) or SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) (triangles, n= 6) in patients with peripheral arterial occlusion. The data represent median values and 15-85 percentile ranges, in μg/mL. EXAMPLES EXAMPLE 1 Epitooe mapping of wild-type staphylokinase The epitope specificity of a panel of 15 murine MAbs (22) raised against wild-type SakSTAR was determined by real-time biospecific interaction analysis (BIA) with the BIAcore instrument (Pharmacia, Biosensor AB, Uppsala, Sweden) . The MAbs were immobilized on the surface of the Sencor Chip CM5 with the Amine Coupling Kit (Pharmacia Biosensor AB) as recommended by the manufacturer (25) . Immobilization was performed from protein solutions at a concentration of 20 μg/mL in 10 mmol/L sodium acetate at pH 5.0 at a flow rate of 5 μL/min during 6 minutes. This resulted in covalent attachment of 5,000 to 10,000 resonance unit (RU) of antibody (corresponding to 0.035 to 0.07 pmol/mm 2 ) . The SakSTAR solutions were passed by continuous flow at 20°C past the sensor surface. At least four concentrations of each analyte (range, 50 nmol/L to 50 mol/L) in 10 mmol/L HEPES, 3.4 mmol/L EDTA, 0.15 mol/L NaCl, and 0.005% Surfactant P20, pH 7.2, were injected at a flow rate of 5 μL/min during 6 minutes in the association phase. Then sample was replaced by buffer, also at a flow rate of 5 μL/min during 6 minutes. After each cycle, the surface of the sensor chip was regenerated by injection of 5 μL of 15 mmol/L HC1. Apparent association (k ass ) and apparent dissociation (k d - ss ) rate constants were derived from the sensorgrams as described in detail elsewhere (26) , and association equilibrium constants (K A ) calculated as their ratio. Determination of the equilibrium association constants for the binding of wild-type and variant SakSTAR to insolubilized MAbs (Table 1) yielded apparent association constants of 10 7 to 10 8 (mol/L) "1 , which are one to two orders of magnitude lower than the apparent association constants previously obtained for the binding of these MAbs to insolubilized wild-type SakSTAR (22) . If the MAbs instead of the SakSTAR variants are insolubilized, avidity effects of the bivalent MAbs are avoided. The present values are indeed in better agreement with known association constants of Mabs, and therefore this "reversed" procedure was used throughout the present invention. In the tables the column indicated with "Variant" states the various staphylokinase derivatives which are identified by listing between brackets the substituted amino acids in single letter symbols followed by their position number in the mature staphylokinase sequence and by the substituting amino acids in single letter symbol; the column "Exp." indicates expression levels in mg/L, and the column "Spec. Act." indicates the specific activity in Home Units as defined in example 2. Indications "17G11", "26A2" etc. refer to monoclonal antibodies binding to the indicated epitopes I, II and III as defined in reference 22. Epitope I is recognized by the antibody cluster 17G11, 26A2, 30A2, 2B12 and 3G10, whereas epitope II is recognized by the antibody cluster 18F12, 14H5, 28H4, 32B2 and 7F10, and epitope III by the antibody cluster 7H11, 25E1, 40C8, 24C4 and 1A10. Human plasma "Pool" indicates a plasma pool from initially 16 and subsequently 10 patients immunized by treatment with SakSTAR, "Subpool B" indicates a plasma pool from three patients that absorbed less than 50% of the induced antibodies with SakSTAR(K35A,E38A,K74A,E75A,R77A) and "Subpool C" indicates a plasma pool from 3 patients that absorbed >90% of the induced antibodies with SakSTAR(K35A,E38A,K74A,E75A,R77A) (22) . In tables 6, 7 and 8 an additional pool of plasma from 40 patients immunized by treatment with SakSTAR (Pool 40) was also used. EXAMPLE 2 Construction, epitope mapping with murine monoclonal antibodies and absorption with pooled plasma of immunized patients, of "alanine-to-wild-tvpe" reversal variants of "charged-cluster-to-alanine" mutants of staphylokinase 1. Introduction As stated above, wild-type staphylokinase (SakSTAR variant (9)) contains three non-overlapping immunodominant epitopes, two of which can be eliminated by specific site-directed substitution of clusters of two (K35A,E38A or E80A,D82A) or three (K74A,E75A,R77A) charged amino acids with Ala (22) . The combination mutants SakSTAR(K35A,E38A,K74A,E75A,R77A) in which Lys35, Glu38, Lys74, Glu75 and Arg77, and SakSTAR(K74A,E75A, R77A,E80A,D82A) in which Lys74, Glu75, Arg77, GluδO and Asp82 were substituted with Ala (previously identified as SakSTAR.M3.8 and SakSTAR.M8.9, respectively (22)), were found to have a reduced reactivity with murine monoclonal antibodies against two of the three immunodominant epitopes and to absorb on average only 2/3 of the neutralizing antibodies elicited in 16 patients by treatment with wild-type SakSTAR (22) . These mutants also induced less antibody formation than wild-type SakSTAR in experimental thrombolysis models in rabbits and baboons, and in patients with peripheral arterial occlusion (22) . However, their specific activities were reduced to approximately 50% of that of wild-type SakSTAR, which would be of some concern with respect to the clinical use of these compounds. In an effort to improve the activity and stability without loss of the reduced antibody recognition, the effect of a systematic reversal of one or more of these substituted amino acids to the wild-type residues was studied. Fourteen new mutants were constructed, purified and characterized in terms of specific activity, reactivity with the panel of murine monoclonal antibodies, and absorption of antibodies from plasma of patients treated with wild-type SakSTAR (Table 1) . The present example thus focusses on reversal from alanine to the wild-type residue of one or more of the seven amino acids of SakSTAR listed above i.e. K35, E38, K74, E75, R77, E80 and D82. 2. Reagents and Methods The source of all reagents used in the present study has previously been reported (22) . Restriction enzymes were purchased from Pharmacia (Uppsala, Sweden) or Boehringer Mannheim (Mannheim, Germany) . T4 DNA ligase, Klenow Fragment of E. coli DNA polymerase I and alkaline phosphatase were obtained from Boehringer Mannheim. Enzyme reactions were performed using the conditions suggested by the suppliers. Plasmid DNA was isolated using a QIAGEN-purification protocol (provided by Westburg, Leusden, The Netherlands). pMEX.602sakB (i.e. pMEX.SakSTAR) was constructed as described elsewhere (23). SakSTAR, SakSTAR(K35A,E38A) , SakSTAR(K74A,E75A,R77A) , SakSTAR(E80A,D82A) , SakSTAR(K35A,E38A,K74A,E75A,R77A) and Sak- STAR(K74A,E75A,R77A,E80A,D82A) were produced and purified as described elsewhere (22) . Transformations of E. coli were performed utilizing the calcium phosphate procedure. DNA sequencing was performed using the dideoxy chain termination reaction method and the Automated Laser fluorescent A.L.F.™ (Pharmacia). The chromogenic substrate (S2403) L-Pyroglutamyl-L-phenylalanyl- L-lysine-p-nitroanaline hydrochloride was purchased from Chromogenix (Belgium) . 125 l-labeled fibrinogen was purchased from Amersham (UK) . All other methods used in the present example have been previously described (22,27) . 3. Construction of expression plasmids The plasmids encoding SakSTAR(K35A,E38A,K74A, E75A) , SakSTAR(E38A,E75A,R77A) , SakSTAR(E38A,E75A) , SakSTAR(K35A,E75A,R77A) , SakSTAR(K35A,E75A) , SakSTAR(E80A) , SakSTAR(D82A) , SakSTAR(E75A,D82A) , SakSTAR(K74A) and SakSTAR(E75A) were constructed by the spliced overlap extension polymerase chain reaction (SOE-PCR) (24) , using Vent DNA polymerase (New England Biolabs, Leusden, The Netherlands) , and available or generated sakSTAR variants as template. Two fragments were amplified by PCR, the first one starting from the 5' end of the staphylokinase gene with primer 5 ' -CAGGAAACAGAATTCAGGAG-3 • to the region to be mutagenized (forward primer) , the second one from the same region (backward primer) to the 3 ' end of the staphylokinase gene with primer 5 ' -CAAAACAGCCAAGCTTCATTCATTCAGC-3 • . The forward and backward primers shared an overlap of around 24 bp (primers not shown) . The two purified fragments were then assembled together in a new pri erless PCR using Taq polymerase (Boehringer Mannheim) . After 7 cycles (1 min at 94°C, l min at 70°C), the extended product was reamplified by adding the 5' and 3 1 end primers (see above) to the PCR reaction and by cycling 25 times (1 min at 94°C, l min 55°C, 1 min at 72°C). The final product was purified, digested with EcoRI and Hindlll and cloned into the corresponding sites of pMEX602sakB. The plasmid encoding SakSTAR(E38A,K74A,E75A,R77A) was assembled by digestion of pMEX602sakB and pMEX.SakSTAR(K35A,E38A, K74A,E75A,R77A) with Bpml which cuts between the codons for K35 and E38 of SakSTAR, and ligation of the required fragments. The plasmid encoding SakSTAR(K35A,K74A,E75A, R77A) was constructed by digestion of pMEX.SakSTAR(K35A, E38A,K74A,E75A,R77A) and pMEX.SakSTAR(K74A,E75A,R77A) with Bpml and religation of the required fragments. The plasmids encoding SakSTAR(K35A,E38A, E75A,R77A) and SakSTAR(K35A,E38A,K74A,R77A) were constructed by two PCR using pMEX.SakSTAR(K35A,E38A,K74A,E75A,R77A) as template, followed by restriction ligation and recloning into pMEX602sakB. 4. Expression and purification of SakSTAR variants The SakSTAR variants were expressed and purified, as described below, from transformed E. coli WK6 grown either in LB medium [SakSTAR(E38A,K74A,E75A, R77A) , SakSTAR(K74A) , SakSTAR(E75A) and SakSTAR(E75A, D82A) ] , or in terrific broth (TB) (28) medium [SakSTAR(K35A,K74A,E75A,R77A) , SakSTAR(K35A,E38A,E75A, R77A) , SakSTAR(K35A,E38A,K74A,R77A) , SakSTAR(K35A, E38A,E75A), SakSTAR(E38A, E75A, R77A) , SakSTAR(E38A,E75A) , SakSTAR(K35A,E75A,R77A) , SakSTAR(K35A,E75A) , SakSTAR(E80A) , and SakSTAR(D82A) ] . For derivatives produced in LB medium, a 20 mL aliquot of an overnight saturated culture was used to inoculate a 2 L volume of LB medium containing 100 g/mL ampicillin. After 3 hours incubation at 37"C, IPTG (200 mol/L) was added to induce expression from the tac promoter. The production phase was allowed to proceed for 4 hours, after which the cells were pelleted by centrifugation at 4,000 rpm for 20 min, resuspended in 1/20 volume (100 mL) of 0.01 mol/L phosphate buffer pH 6.5 and disrupted by sonication at 0°C. Cell debris were removed by centrifugation for 20 min at 20,000 rpm and the supernatant, containing the cytosolic soluble protein fraction, was stored at -20 °C until purification. For the derivatives produced in TB medium, a 4 mL aliquot of an overnight saturated culture in LB medium was used to inoculate a 2 L culture in terrific broth containing 100 μg/mL ampicillin. The culture was grown with vigorous aeration for 20 hours at 30°C. The cells were pelleted by centrifugation, resuspended in 1/10 volume (200 L) of 0.01 mol/L phosphate buffer pH 6.5 and disrupted by sonication at 0°C. The suspension was then centrifuged for 20 min at 20,000 rpm and the supernatant was stored at -20°C until purification. Cleared cell lysates containing the SakSTAR variants were subjected to chromatography on a 1.6 x 6 cm column of SP-Sephadex, followed by chromatography on a 1.6 x 5 cm column of Q-Sepharose [variants SakSTAR(E38A,K74A,E75A,R77A) , SakSTAR(K35A,K74A,E75A, R77A) , SakSTAR(K35A,E38A, E75A,R77A), SakSTAR(K35A,E38A,K74A,R77A) and SakSTAR(K35A, E38A,K74A,E75A) ] or by chromatography on a 1.6 x 6 cm column of phenyl-Sepharose [variants Sak- STAR(E35A,E38A,R77A) , SakSTAR(E38A,E75A) , SakSTAR- (K35A,E75A,R77A) , SakSTAR(K35A,E75A) , SakSTAR(K74A) , SakSTAR(E75A) , SakSTAR(E80A) , SakSTAR(D82A) and Sak- STAR(E75A,D82A) ] . The SakSTAR containing fractions, localized by SDS-gel electrophoresis, were pooled for further analysis. 5. Physicochemical and biochemical analysis Protein concentrations were determined according to Bradford (29) . The specific activities of SakSTAR solutions were determined with a chromogenic substrate assay carried out in microtiter plates using a mixture of 80 μL SakSTAR solution and 100 μL Glu-plasminogen solution prepared as described elsewhere (30) (final concentration 0.5 μmol/L) . After incubation for 30 min at 37 °C, generated plasmin was quantitated by addition of 20 μL S2403 (final concentration 1 mmol/L) and measurement of the absorption at 405 nm. The activity was expressed in home units (HU) by comparison with an in-house standard (lot STAN5) which was assigned an activity of 100,000 HU (100 kHU) per mg protein as determined by amino acid composition (7) . SDS-PAGE was performed with the Phast System™ (Pharmacia, Uppsala, Sweden) using 10-15% gradient gels and Coomassie Brilliant blue staining. Reduction of the samples was performed by heating at 100°C for 3 min in the presence of 1% SDS and 1% dithiothreitol. The specific activities of the different SakSTAR mutants determined with the chromogenic substrate assay are summarized in Table 1. 6. Binding to murine monoclonal antibodies In agreement with previous observations (22) , SakSTAR(K74A,E75A,R77A) did not react with 4 of the 5 MAbs recognizing epitope I, whereas SakSTAR(K35A,E38A) did not react with 3 of the 5 and SakSTAR(E80A,D82A) not with 4 of the 5 Mabs recognizing epitope III. These reduced reactivities were additive in SakSTAR(K35A,E38A, K74A,E75A,R77A) and in SakSTAR(K74A,E75A,R77A,E80A, D82A) . The reduced reactivity of SakSTAR(K74A,E75A, R77A) was fully maintained in SakSTAR(K35A,E38A,K74A,E75A) and in SakSTAR(K35A, E75A,R77A) , largely in SakSTAR(K35A,E38A, E75A,R77A) , SakSTAR(E38A,E75A,R77A) , SakSTAR(E38A,E75A) and SakSTAR(E75A) , but much less in SakSTAR(K35A,E38A, K74A,R77A) and SakSTAR(K74A) , indicating that E75 is the main contributor to the binding of the 4 Mabs recognizing epitope I of SakSTAR. However, surprisingly, binding of epitope I antibodies to SakSTAR(E75A,D82A) was normal in two independent preparations from expression plasmids with confirmed DNA sequences. The reduced reactivity of the 3 MAbs of epitope III with SakSTAR(K35A,E38A) required both K35 and E38, as demonstrated with SakSTAR(E38A,K74A,E75A,R77A) and SakSTAR(K35A,K74A,E75A, R77A) , with SakSTAR(E38A,E75A) and SakSTAR(K35A,E75A) and with SakSTAR(E38A,E75A,R77A) and SakSTAR(K35A,E75A,R77A) . The reduced reactivity of the 4 MAbs of cluster III with SakSTAR(E80A,D82A) was maintained in SakSTAR(D82A) but not in SakSTAR(E80A) . 7. Absorption of antibodies, elicited in patients bv treatment with wild-type SakSTAR Plasma samples from 16 patients with acute myocardial infarction, obtained several weeks after treatment with SakSTAR (4, 31) were used. The staphylokinase-neutralizing activity in these samples was determined as follows. Increasing concentrations of wild-type or variant SakSTAR (50 μL volumes containing 0.2 to 1000 μg/mL) were added to a mixture of 300 μL citrated human plasma and 50 μL buffer or test plasma, immediately followed by addition of 100 μL of a mixture containing thrombin (50 NIH units/mL) and CaCl 2 (25 mmol/L) . The plasma clot lysis time was measured and plotted against the concentration of SakSTAR moiety. From this curve the concentration of staphylokinase moiety that produced complete clot lysis in 20 min was determined. The neutralizing activity titer was determined as the difference between the test plasma and buffer values and was expressed in μg per mL test plasma. The results of the individual patients have been reported elsewhere (22) . For the present invention, three plasma pools were made, one from 10 patients from whom sufficient residual plasma was available, one from three patients that absorbed less than 50% of the antibodies with SakSTAR(K35A,E38A, K74A, E75A,R77A) (Subpool B) and one from three patients that absorbed >90% of the antibodies with SakSTAR(K35A,E38A,K74A,E75A, R77A) (Sub- pool C) . These plasma pools were diluted (1/30 to 1/200) until their binding to SakSTAR substituted chips in the BIAcore instrument amounted to approximately 2000 RU. From this dilution a calibration curve for antibody binding was constructed using further serial two-fold dilutions. The plasma pools were absorbed for 10 min with 100 nmol/L of the SakSTAR variants, and residual binding to immobilized SakSTAR was determined. Residual binding was expressed in percent of unabsorbed plasma, using the calibration curve. The results are summarized in Table 1. Whereas wild-type SakSTAR absorbed more than 95% of the binding antibodies from pooled plasma of the 10 patients, incomplete absorption (<60%) was observed with SakSTAR(K74A,E75A,R77A) , SakSTAR(K35A,E38A,K74A, E75A,R77A) , SakSTA (E38A,K74A,E75A,R77A) , SakSTAR(K35A,K74A,E75A,R77A) , SakSTAR(K35A, E38A,K74A,R77A) , SakSTAR(K35A,E38A,K74A, E75A) , SakSTAR(K74A) and SakSTAR(K74A,E75A,R77A,E80A,D82A) but absorption was nearly complete with SakSTA (K35A,E38A) , SakSTAR(K35A,E38A,E75A,R77A) , SakSTAR(E38A,E75A,R77A) , SakSTAR(E38A,E75A) , SakSTAR(K35A,E75A,R77A) , SakSTAR(K35A,E75A) , SakSTAR(E75A) , SakSTAR(E80A, D82A) , SakSTAR(E80A) , SakSTAR(D82A) and SakSTAR(E75A, D82A) . These results, surprisingly, demonstrate that approximately 40% of the antibodies elicited in patients by treatment with wild-type SakSTAR depend on K74 for their binding (Table 1) . Absorption with pooled plasma from 3 patients from which <50% of the antibodies were absorbed with SakSTAR(K35A,E38A,K74A,E75A,R77A) (Subpool B) confirmed the predominant role of K74 for antibody recognition. As expected, absorption with pooled plasma from 3 patients from which >95% of the antibodies were absorbed with SakSTAR(K35A,E38A,K74A,E75A,R77A) (Subpool C) was nearly complete with all variants tested. EXAMPLE 3 Comparative thrombolytic efficacy and immunogenicitγ of SakSTAR(K74A.E75A.R77AΪ and SakSTAR(K74AΪ versus SakSTAR in patients with peripheral arterial occlusion 1. Purification of SakSTARfK74A.E75A.R77A) and SakSTAR(K74A) for use in vivo A 12 to 24 L culture (in 2 L batches) of the variants SakSTAR(K74A,E75A,R77A) , or of SakSTAR(K74A) was grown and IPTG-induced in LB medium supplemented with 100 μg/mL ampicillin, pelleted, resuspended, disrupted by sonication and cleared as described above. The compounds were purified by chromatography on a 5 x 20 cm column of SP-Sephadex, a 5 x 10 cm column of Q-Sepharose and/or a 5 x 13 cm column of phenyl-Sepharose using buffer systems described elsewhere (22, 23). The materials were then gel filtered on sterilized Superdex 75 to further reduce their endotoxin content. The SakSTAR variant containing fractions were pooled, the protein concentration was adjusted to 1 mg/mL and the material sterilized by filtration through a 0.22 μm Millipore filter. The methodology used to determine the biological properties of the final material required for use in vivo is described above and elsewhere (22) . 2. Materials and Methods Staphylokinase-neutralizing activity in plasma was determined as described above. Quantitation of antigen-specific IgG and IgM antibodies was performed using enzyme-linked immunosorbent assays in polystyrene microtiter plates essentialy as described previously (22) . In the IgG assays, dilution curves of affinospecific anti-SakSTAR IgG antibodies were included on each plate. These antibodies were isolated from plasma obtained from 3 patients, after thrombolytic therapy with wild-type SakSTAR, by chromatography on protein A-Sepharose and on insolubilized SakSTAR, and elution of bound antibodies with 0.1 mol/L glycine-HCl, pH 2.8. The purity of the IgG preparation was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis. In the IgM assays, titers defined as the plasma dilution giving an absorbancy at 492 nm equivalent to that of a 1/640 dilution of pooled plasma were determined and compared with the titer of baseline samples before treatment (median value 1/410, interquartile range 1/120-1/700) . 3. Thrombolytic efficacy Wild-type SakSTAR or the variants SakSTAR(K74A) or SakSTAR(K74A,E75A,R77A) were administered intra-arterially at or in the proximal end of the occlusive thrombus as a bolus of 2 mg followed by an infusion of 1 mg/hr (reduced overnight in some patients to 0.5 mg/hr) in groups of 6 to 12 patients with angiographically documented occlusion of a peripheral artery or bypass graft of less than 120 days duration. Patients were studied after giving informed consent, and the protocol was approved by the Human Studies Committee of the University of Leuven. Inclusion and exclusion criteria, conjunctive antithrombotic treatment (including continuous intravenous heparin) and the study protocol were essentially as previously described (22) . Relevant baseline characteristics of the individual patients are shown in Table 2. The majority of PAO were at the femoropopliteal level. Two iliac stent and 8 graft occlusions were included. Eight patients presented with incapacitating claudication, 5 with chronic ischemic rest pain, 7 with subacute ischemia and 7 with acute ischemia. One patient (POE) who had 2 years previously been treated with SakSTAR was included in the SakSTAR(K74A) group. This patient was not included in the statistical analyses. Table 2 also summarizes the individual treatment and outcome. Intra-arterial infusion, at a dose of 6.0 to 25 mg and a duration of 4.0 to 23 hrs, induced complete recanalization in 24 patients and partial recanalization in 3. Complementary endovascular procedures (mainly PTA) were performed in 17 patients and complementary reconstructive vascular surgery following thrombolysis in 3. No patient underwent major amputation. Early recurrence of thrombosis after the end of the angiographic procedure occurred in 4 patients. Bleeding complications were absent or limited to mild to moderate hematoma formation at the angiographic puncture sites except for 5 patients who required transfusion (data not shown) . Intracranial or visceral hemorrhage was not observed. Circulating fibrinogen, plasminogen and α 2 -antiplasmin levels remained essentially unchanged during infusion of the SakSTAR moieties (data not shown) , confirming absolute fibrin specificity of staphylokinase at the dosages used. Significant in vivo fibrin digestion occurred as evidenced by elevation of fibrin fragment D-dimer levels. Intra-arterial heparin therapy prolonged aPTT levels to a variable extent (data not shown) . 4. Antibody induction Antibody-related SakSTAR-, SakSTAR(K74A) - and SakSTAR(K74A,E75A,R77A) -neutralizing activity and an- ti-SakSTAR, anti-SakSTAR(K74A) and anti-SakSTAR(K74A, E75A,R77A) IgG, were low at baseline and during the first week after the infusion (Figure 2). From the second week on, neutralizing activity levels increased to reach median values at 3 to 4 weeks of 20 μg SakSTAR(K74A) and 2.4 μg SakSTAR(K74A,E75A,R77A) neutralized per L plasma in the patients treated with SakSTAR(K74A) and SakSTAR(K74A,E75A,R77A) , respectively, which is significantly lower than the median value of 93 μg wild-type SakSTAR neutralized per L in the patients treated with SakSTAR (p= 0.024 for differences between the three groups by Kruskal-Wallis analysis and p= 0.01 and p= 0.036, respectively, for variants vs wild-type by Mann-Whitney rank sum test) . The levels of anti-SakSTAR(K74A) and of anti-SakSTAR(K74A,E75A,R77A) IgG increased to median values at 3 to 4 weeks of 270 and 82 μg/mL plasma in patients treated with SakSTAR(K74A) and SakSTAR(K74A,E75A,R77A) respectively, which is significantly lower than the median value of 1800 μg an- ti-SakSTAR per mL plasma in the patients treated with SakSTAR ( (p= 0.024 for differences between the three groups by Kruskal-Wallis analysis and p= 0.007 and 0.05, respectively, for variants versus wild-type by Mann-Whitney rank sum test) . The titers of anti-SakSTAR(K74A) and of anti-SakSTAR(K74A,E75A,R77A) IgM increased from median baseline values of 1/460 and 1/410 to median values at 1 week of 1/510 and 1/450 in patients treated with SakSTAR(K74A) and SakSTAR(K74A,E75A,R77A) , respectively, which was not significantly different from the median values of 1/320 at baseline and 1/640 at week 1 in patients treated with SakSTAR. Corresponding values at 2 weeks were 1/590 and 1/550 in patients given SakSTAR(K74A) and SakSTAR(K74A,E75A,R77A) , not significantly different from 1/930 with SakSTAR (data not shown) . The antibodies induced by treatment with SakSTAR were completely absorbed by SakSTAR but incompletely by SakSTAR(K74A) and by SakSTAR(K74A,E75A,R77A) confirming the immunogenicity of the K74,E75,R77 epitope and the dominant role of K74 in the binding of antibodies directed against this epitope. The antibodies induced by treatment with SakSTAR(K74A) or SakSTAR(K74A,E75A,R77A) were completely absorbed by SakSTAR, by SakSTAR(K74A) and by SakSTAR(K74A,E75A,R77A) , indicating that immunization was not due to neoepitopes generated by substitution of Lys74 with Ala, but to epitopes different from the K74,E75,R77 epitope. Thus, this example illustrates that staphylokinase variants with reduced antibody induction but intact thrombolytic potency can be generated. The present experience in 26 patients treated with SakSTAR (n= 9), SakSTAR(K74A) (n= 11) and SakSTAR(K74A,E75A,R77A) (n= 6) combined with previous experience in 14 patients with SakSTAR (n= 7) and SakSTAR(K35A, E38A,K74A,E75A,R77A) (n= 7) (31) and in 24 patients with SakSTAR (32) , and with subsequent non-randomized experience in patients with SakSTAR (n= 30) with SakSTAR(K74A) (n= 12) and with SakSTAR(K74A,E75A,R77A) (n= 7) (data not shown) , allows an initial estimation of the prevalence of immunization by intra-arterial treatment with SakSTAR or variants with an altered K74,E75,R77 epitope [SakSTAR(K74A) , SakSTAR(K74A, E75A,R77A) and SakSTAR(K35A,E38A,K74A,E75A, R77A) ] . Neutralizing activity data after 2 to 4 weeks, available in 70 patients with peripheral arterial occlusion given intra-arterial SakSTAR, revealed that 56 patients (80 percent) had levels > 5 μg compound neutralized per mL plasma. Of the patients given SakSTAR(K74A) , SakSTAR(K74A,E75A, R77A) or SakSTAR(K74A,E75A,K74A,E75A, R77A) , 27 of the 43 (63 percent) had neutralizing activity levels of > 5 μg compound per mL plasma. This difference is statistically significant (p= 0.05 by Fisher's exact test) indicating that the K74,E75,R77 epitope is a major determinant of antibody induction. EXAMPLE 4 Construction, epitope mapping with murine monoclonal antibodies and absorption with pooled plasma of immunized patients, of alanine-substitution mutants of staphylokinase 1. Introduction Site-directed mutagenesis was applied to residues other than "charged amino acids" in order to identify i) additional residues belonging to epitopes I and III identified with the panel of murine Mabs and ii) amino acids determining absorption to antiserum from immunized patients. Since functional epitopes generally comprise more than one amino acid residue critical for antibody binding, identification of additional residues in these epitopes could lead to the construction of new combination derivatives displaying a lower antigenic profile, while keeping the specific activity and the temperature stability of wild-type staphylokinase. In this example, the construction and characterization of SakSTAR variants in which one or at most two amino acids (adjacent or in close vicinity) were substituted with alanine is described. The mutants described under this example are listed in Table 3. These variants were expressed in E. coli. purified and characterized in terms of specific activity, reactivity with the panel of murine monoclonal antibodies, and absorption of antibodies from plasma of patients treated with wild-type SakSTAR. 2. Reagents and Methods The source of all reagents used in the present study has previously been reported (22) , or is specified below. The template vector for mutagenesis, pMEX602sakB (i.e. pMEX.SakSTAR) , has been described elsewhere (23). Restriction and modification enzymes were purchased from New England Biolabs (Leusden, The Netherlands) , Boehringer Mannheim (Mannheim, Germany) or Pharmacia (Uppsala, Sweden) . The enzymatic reactions were performed according to the supplier recommendation. The mutagenic oUgonucleotides and primers were obtained from Eurogentec (Seraing, Belgium) . Plasmid DNA was isolated using a purification kit from Qiagen (Hilden, Germany) or the BIO 101 RPM kit (Vista, CA) , as recommended. Transformation-competent E. coli cells were prepared by the well-known calcium phosphate procedure. Nucleotide sequence determination was performed on double strand plasmid DNA with the dideoxy chain termination method, using the T7 sequencing kit (Pharmacia, Uppsala, Sweden) . Polymerase chain reactions (PCR) were performed using Taq polymerase from Boehringer Mannheim (Mannheim, Germany) or Vent polymerase (New England Biolabs, Leusden, The Netherlands) . The recombinant DNA methods required to construct the variants described in this example are well established (22, 27). 3. Construction of expression plasmids The variants SakSTAR(Y17A,F18A) , SakSTAR(F104A) , SakSTAR(F111A) , SakSTAR(Y9A) , SakSTAR(Y91A) , SakSTAR(Y92A) , SakSTAR(I87A) , SakSTAR(I106A) and SakSTAR(112OA) were constructed with the Chameleon Double-Stranded Site-Directed Mutagenesis kit from Stratagene (La Jolla, USA), using the pMEX.SakSTAR vector as template, and following instructions of the supplier. The mutagenic oUgonucleotides (not shown) were used in combination with the selection-primer LY34 5' CAAAACAGCCGAGCTTCATTCATTCAGC, which destroys the unique Hindlll site located 3 ' to the staphylokinase encoding gene in pMEX. SakSTAR and allows to counter-select the non-mutant progeny by Hindlll digestion. The deletion of the Hindlll site was in most cases correlated with the presence of the desired mutation introduced by the mutagenic oligonucleotide. The variant SakSTAR(I133A) , was constructed by performing a polymerase chain reaction on the pMEX.SakSTAR plasmid using the primer 818A located at the 5 ' end of the sakSTAR gene (5 1 CAGGAAACAGAATTCAGGAG ) and the mutagenic primer LY58 (5' TTCAGCATGCTGCAGTTATTTCTTTTCTGCAACAACCTTGG) . The amplified product (30 cycles: 30 sec at 94 °C, 30 sec at 50°C, 30 sec at 72 °C) was purified, digested with EcoRI and Pstl, and ligated into the corresponding sites of pMEXSakSTAR. The variants SakSTAR(I128A) , SakSTAR(L127A) and SakSTAR(N126V) were constructed by performing a polymerase chain reaction using the primer 818A located at the 5 ' end of the sakSTAR gene and mutagenic primers (not shown) . The amplified product (30 cycles: 1 sec at 94 °C, 1 sec at 50°C, 10 sec at 72 °C) was purified, digested with EcoRI and StyI, and ligated into the corresponding sites of pMEXSakSTAR. The variant SakSTA (F125A) was constructed by performing two consecutive PCR reactions (30 cycles: : 30 sec at 94"C, 30 sec at 50°C, 30 sec at 72°C). In the first reaction, a fragment of pMEX. SakSTAR was amplified with the primers 818A and a mutagenic primer. This amplified fragment was then used as template in a second PCR reaction with a mutagenic primer in order to further elongate the fragment downstream of the Styl site present in the sakSTAR gene (corresponding to amino acids 130-131 of SakSTAR) . The resulting product was digested with EcoRI and Styl, and ligated into the corresponding sites of pMEXSakSTAR. The plasmids encoding all the other variants listed in Table 3 were constructed by direct PCR or by the spliced overlap extension polymerase chain reaction (SOE-PCR) (24) using pMEX. SakSTAR or available plasmids encoding SakSTAR variants as template. Two fragments were amplified by PCR (30 cycles: 1 sec at 94 °C, 1 sec at 50°C, 10 sec at 72 °C), the first one starting from the 5' end (primer 818A ) of the staphylokinase gene to the region to be mutagenized (forward primer) , the second one from this same region (backward primer) to the 3 • end of the gene with primer 818D (5' CAAACAGCCAAGCTTCATTCATTCAGC) . The forward and backward primers shared an overlap of around 24 bp (primers not shown) . The two purified fragments were then assembled together in a second PCR reaction with the external primers 818A and 818D (30 cycles: 1 sec at 94 °C, 1 sec at 50°C, 10 sec at 72°C). The amplified product from this final reaction was purified, digested with EcoRI and Hindlll and ligated into the corresponding site of pMEX. SakSTAR. For each construction, the sequence of the variant was confirmed by sequencing the entire SakSTAR coding region. 4. Expression and purification of SakSTAR variants The SakSTAR variants were expressed and purified, as described below, from transformed E. coli grown in terrific broth (TB) medium (28) . A 2 to 4 mL aliquot of an overnight saturated culture in LB medium was used to inoculate a 1 to 2 L culture in terrific broth supplemented with 100 μg/mL ampicillin. The culture was incubated with vigorous aeration and at 30°C. After about 16 hours incubation, IPTG (200 μmol/L) was added to the culture to induce expression from the tac promoter. After 3 hours induction, the cells were pelleted by centrifugation at 4,000 rpm for 20 min, resuspended in 1/10 volume of 0.01 mol/L phosphate buffer pH 6-6.5 and disrupted by sonication at 0°C. The suspension was centrifuged for 20 min at 20,000 rpm and the supernatant was stored at 4°C or at -20 β C until purification. The material was purified essentially as described above (Example 2) : cleared cell lysates containing the SakSTAR variants were subjected to chromatography on a 1.6 x 5 cm column of SP-Sephadex, followed by chromatography on a 1.6 x 8 cm column of phenyl-Sepharose. The SakSTAR containing fractions, localized by SDS-gel electrophoresis, were pooled for further analysis. 5. Physicochemical and biochemical analysis Protein concentrations were determined according to Bradford (29) . SDS-PAGE was performed with the Phast SystemTM (Pharmacia, Uppsala, Sweden) using 10-15% gradient gels and Coomassie Brillant blue staining, and the specific activities of SakSTAR solutions were determined with a chromogenic substrate assay carried out in microtiter plates (as described in example 2) . The specific activity of the different SakSTAR variants are summarized in Table 3. 6. Reactivity of SakSTAR variants with a panel of murine monoclonal antibodies The methodology used to determine the reactivity of the SakSTAR variants with a panel of murine monoclonal antibodies was described in example 1 above. The results are summarized in Table 3 (the layout of this Table corresponds to the layout of Table 1, as described in example 1) . Apparent association constants at least 10-fold lower than those of wild-type staphylokinase were considered as significant and are indicated in bold type in the table. In order to obtain a comprehensive inventory of the properties of Ala-substitution variants of the SakSTAR molecule, 67 plasmids encoding variants with substitution of a single or two adjacent amino acids with Ala were constructed, expressed and purified. Together with the 35 charged residue to Ala-substitution variants previously described (22, and example 2), this analysis covers all residues in SakSTAR except Gly, Ala and Pro, as illustrated in Figure 3. Eight of the variants could not be obtained in purified form, primarily as a result of low expression levels, 11 variants were inactive, 56 had a reduced specific activity, and 27 had a maintained or increased specific activity (>100 kHU/mg) . The yields of purified material from cultures of expressed plasmids were 16 mg/L (median, 10 to 90 percentile range 4 to 41 mg/L) . SDS polyacrylamide gel electrophoresis consistently showed one main band with M r « 16,000, usually representing 95% of total protein (not shown) . Substitution of K35, N95, S103 or K135 with Ala yielded variants with specific activities of >200 kU/mg. Substitution of W66, Y73 or E75 with Ala reduced the reactivity of the variants with >3 antibodies of epitope cluster I, of H43 or V45 with Ala that with 3 antibodies from epitope cluster II and of V32, K35, D82 and K130 with Ala that with >3 antibodies of epitope cluster III. 7. Absorption of antibodies, elicited in patients by treatment with SakSTAR For the present example, the three plasma pools, as described in example 2 were used. The methodology used to evaluate the absorption with wild-type staphylokinase and with SakSTAR variants, of antibodies elicited in patients treated with SakSTAR, is described in detail in example 2. The results are summarized in Table 3. Whereas wild-type SakSTAR and most of the variants analyzed in this example absorbed more than 95% of the binding antibodies from pooled plasma of the 10 patients, incomplete absorption (<60%) was observed with SakSTAR(Y73A) , and with SakSTAR(K74A) . The predominant role of Lys74 for antibody recognition has been demonstrated previously (see example 2) . The present results indicate that Tyr73 participates to the same major epitope as Lys74, or, alternatively, that substitution at Tyr73 may indirectly induce a structural modification of the "K74-epitope". Absorption with pooled plasma from 3 patients from which >95% of the antibodies were absorbed with SakSTAR(K35A,E38A,K74A,E75A,R77A) (Subpool C, see example 2) was nearly complete with most variants tested. EXAMPLE 5 Construction, epitope mapping with murine monoclonal antibodies and absorption with pooled plasma of immunized patients, of staphylokinase variants with substitution of S34. G36 and/or H43 The natural variant Sak42D differs from SakSTAR in three amino acids and corresponds to SakSTAR(S34G, G36R,H43R) . Sak42D is characterized by reduced reactivity with some murine antibodies of epitope clusters II and III and a slightly reduced absorption of antibodies from plasma of patients treated with SakSTAR (Table 4) . Mutagenesis of these residues in SakSTAR revealed that the reduced reactivity with epitope cluster III and with immunized patient plasma could be ascribed to the G36R substitution, the H43R substitution mediated the reduced reactivity with epitope cluster II but had no effect on the reactivity with immunized patient plasma, whereas the S34A substitution had no effect. The G36R substitution could be combined with the K74R but not with the K74A substitution, without significant reduction of the specific activity (Table 4) . EXAMPLE 6 Construction, epitope mapping with murine monoclonal antibodies and absorption with pooled plasma of immunized patients, of staphylokinase variants with substitution of K35. E65. Y73. K74. E80+D82. N95. K130. V132 and/or K135 Based on the results of the alanine- substitution analysis in example 4, K35, N95 and K135 were selected for further analysis because SakSTAR(K35A) , SakSTAR(N95A) and SakSTAR(K135A) had a two-fold increased specific activity, Y73 and K74 because SakSTA (Y73A) and SakSTAR(K74A) had a markedly reduced reactivity with antibodies from epitope cluster I and diminished absorption of antibodies from plasma of patients immunized by treatment with SakSTAR, and K35, E80+D82, K130 and V132 because SakSTAR(K35A) , SakSTAR(E80A,D82A) , SakSTAR(K130A) and SakSTAR(V132A) had a reduced reactivity with antibodies from epitope cluster III. In an effort to maximize the activity/ antigenicity ratio, these amino acids were substituted with other amino acids than Ala. As summarized in Table 5, substitution of K35 with A, E or Q revealed that SakSTAR(K35A) had the most interesting properties, substitution of Y73 with F, H, L, S or W did not rescue the marked reduction in specific activity, and K74 confirmed its key role in binding of antibodies from immunized patient plasma, the best specific activity/ antigenicity ratios being obtained with SakSTAR(K74Q) and SakSTAR(K74R) . SakSTAR(E80A,D82A) was preferred over the single residue variants SakSTAR(E80A) or SakSTAR(D82A) because of its somewhat lower reactivity with immunized patient plasma. SakSTAR(N95A) could not be further improved by substitution of N95 with E, G, K or R and it was unable to confer its increased specific activity to variants containing K74A or K135R. Finally SakSTAR(Kl30A) was outperformed in terms of specific activity by SakSTAR(K130T) and SakSTAR(V132A) by SakSTAR(V132R) . EXAMPLE 7 Construction, epitope mapping with murine monoclonal antibodies and absorption with pooled plasma of immunized patients of combination variants of SakSTAR(K130T.K135R) and SakSTARfE80A.D82A.K130T.K135R) with K35A.G36R.E65X.K74X and selected other amino acids In the present and the following examples an additional plasma pool was made from 40 patients obtained several weeks after treatment with SakSTAR (Pool 40) . The original pool from 10 patients is further identified as Pool 10. The absorption of staphylokinase-specific antibodies was quantified as described above and elsewhere (22) . The SakSTAR(K130T,K135R) variant was taken as a template for additive mutagenesis because of its high specific activity with a moderate reduction of binding to antibodies of epitope cluster III and absorption of antibodies from immunized patient plasma (Table 6) . Addition of G36R, K74R, or K74Q or both to the template did not markedly reduce the specific activity, reduced the reactivity with monoclonal antibodies against epitope cluster III (G36R substitution) and decreased the absorption of antibodies from immunized patient plasma (K74R or K74Q substitution) . Combination of E65A or E65Q with K74Q in the SakSTA (K130T,K135R) template reduced the absorption of antibodies from Pool 10 and Pool 40 to around 50 and 60 percent respectively, without markedly reducing the specific activity. Addition substitution of selected amino acids in the SakSTAR(E65Q,K74Q,K130T, K135R) template did not further reduce the antibody absorption from Pool 10 or Pool 40. Surprisingly, the substitution of K136 with A and the addition of K in position 137 resulted in a marked increase in specific activity, as measured in the chromogenic substrate assay. Combination of the SakSTA (E80A,D82A) and Sak¬ STAR(K130T,K135R) templates, did not affect the specific activity and had a reduced reactivity with epitope cluster III antibodies (Table 7) . Therefore the Sak- STAR(E80A,D82A,K130T,K135R) template was selected for further mutagenesis. Addition of K74R and even more of K74Q drastically reduced the reactivity with immunized patient plasma. Finally, addition of E65D or of K35A or E65S to the SakSTAR(K74R,E80A,D82A,K130T,K135R) or Sak- STAR(K74Q,E80A,D82A,K130T, K135R) templates yielded variants with intact specific activity which only bound <45 of the antibodies of pooled immunized patient plasma and less than 15 percent of the subpool reacting for more than 50 percent with the K74,E75,R77 epitope. EXAMPLE 8 Characterization of selected variants of staphylokinase with intact specific activity and less than 50% adsorption of pooled SakSTAR specific human antibodies elicited in patients by treatment with wild-type SakSTAR 1. Introduction Twenty three of the variants constructed and characterized in the above examples combined the properties of a residual specific activity of >100 kHU/mg and <50 percent absorption with the pool of antisera obtained from 10 patients treated with wild-type SakSTAR. The results are summarized in Table 8. Results obtained with Subpool B and Subpool C and with the pool of 40 patients treated with wild-type SakSTAR are included. SakSTAR(K74Q,E80A,D82A,K130T, K135R) , SakSTAR(E65D,K74R, E80A,D82A,K130T,K135R) , SakSTAR(K35A,E65D,K74Q, E80A, D82A,K130T,K135R) and SakSTAR(E65Q,K74Q,N95A,E118A, K130A,K135R,K136A,Vl37K) were selected for further characterization. 2. Fibrinolytic properties of SakSTAR variants in human plasma in vitro The fibrinolytic and fibrinogenolytic properties of the SakSTAR variants were determined as previously described. Dose- and time-dependent lysis of 125 I-fibrin labeled human plasma clots submerged in human plasma was obtained with the selected variants (Table 9) . Spontaneous clot lysis during the experimental period was <5% (not shown) . Equi-effective concentrations of test compound (causing 50% clot lysis in 2 hrs; C 50 ) , determined graphically from plots of clot lysis at 2 hrs versus the concentration of plasminogen activator (not shown), ranged from 0.11 ± 0.01 to 0.24 ± 0.04 g/mL at which the residual fibrinogen levels ranges between 92 ± 30 and 97 ± 30 percent of baseline (Table 9) . The concentrations of compound causing 50% fibrinogen degradation in 2 hrs in human plasma in the absence of fibrin were determined graphically from dose-response curves (not shown) . These values (mean ± SD of 3 independent experiments) ranged from 14 ± 3.2 to 29 ± 3.1 μg/mL (Table 9) . Surprisingly the very high specific activity of SakSTAR(E65Q,K74Q,N95A,E118A,K130A,K135R, K136A, 137K) in the chromogenic assay was not associated with an increased thrombolytic potency in a plasma milieu. 3. Temperature stability of selected SakSTAR variants The temperature stability of preparations of SakSTAR(K74Q,E80A,D82A,K130T,K135R) , SakSTAR(E65D, K74R, E80A,D82A,K130T,K135R) and SakSTAR(K35A,E65D,K74Q,E80A, D82A,K130T,K135R) , dissolved to a concentration of 1.0 mg/mL in 0.15 mol/L NaCl, 0.01 mol/L phosphate buffer, pH 7.5 at various temperatures is illustrated in Fig. 4. At temperatures up to 37 °C, all compounds remained fully active for up at least three days. At 56 °C and 70°C the three variants were however less stable than wild-type SakSTAR. 4. Pharmacokinetic properties of SakSTAR variants following bolus injection in hamsters The pharmacokinetic parameters of the disposition of SakSTAR variants from blood were evaluated in groups of 4 hamsters following intravenous bolus injection of 100 μg/kg SakSTAR variant. SakSTAR-related antigen was assayed using the ELISA described elsewhere. The ELISA was calibrated against each of the SakSTAR variants to be quantitated. Pharmacokinetic parameters included: initial half-life (in min) , tl/2α= ln2/α; terminal half-life (in min), volume of the central (plasma) compartment (in mL) , V c = dose/(A+B); area under the curve (in μg.min.mL "1 ) , AUC= A/α + B/β; and plasma clearance (in mL.min *1 ) , Clp= dose/AUC (33) . The disposition rate of staphylokinase-related antigen from blood following bolus injection of 100 μg/kg of the selected SakSTAR variants in groups of 4 hamsters could adequately be described by a sum of two exponential terms by graphical curve peeling (results not shown) , from which the pharmacokinetic parameters summarized in Table 10 were derived. The pharmacokinetic parameters of the mutants were not markedly different from those of wild type SakSTAR. Initial plasma half-lives (tl/2(α)) ranged between 2.0 and 3.2 min and plasma clearances (Clp) between 1.6 and 4.1 mL/ in. EXAMPLE 9 Comparative thrombolytic efficacy and immunogenicity of SakSTARfK74O.E80A.D82A. K130T.K135RΪ and SakSTAR(E65D.K74R.E80A.D82A.K130T.K135R versus SakSTAR in patients with peripheral arterial occlusion 1. Purification for use in vivo Eighteen liter cultures (in 2 L batches) of the variants SakSTAR(K74Q,E80A,D82A,K130T, K135R) and SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) were grown for 20 hours in terrific broth medium (28) , supplemented with 100 μg/mL ampicillin and induced with IPTG during the last 3 hours. The cells were pelleted, resuspended in 1/10 volume of 0.01 mol/L phosphate buffer, pH 6.0, disrupted by sonication and cleared by centrifugation. The compounds were purified by chromatography on a 10 x 7 cm column of SP-Sepharose, equilibrated with 0.01 mol/L phosphate buffer, pH 6.0 and eluted with a 1 mol/L NaCl gradient (3 column volumes) . The fractions containing SakSTAR variant were pooled, solid NaCl was added to a concentration of 2.5 mol/L and the material was chromatographed on a 10 x 20 cm column of phenyl-Sepharose followed by stepwise elution with 0.01 mol/L phosphate buffer, pH 6.0. The materials were desalted on a 10 x 45 cm column of Sephadex G25, concentrated by application on a 5 x 10 cm column of SP-Sepharose with stepwise elution with 1.0 mol/L NaCl and finally gel filtered on a 6 x 60 cm column of Superdex 75 equilibrated with 0.15 m NaCl, 0.01 mol/L phosphate buffer, pH 7.5 to further reduce their endotoxin content. The SakSTAR variant containing fractions were pooled, the protein concentration was adjusted to 1 mg/mL and the material sterilized by filtration through a 0.22 m Millipore filter. The methodology used to determine specific activity, endotoxin contamination, bacterial sterility and toxicity in mice is described above and elsewhere (22) . The purity of the preparation was evaluated by SDS gel electrophoresis on 10% gels to which 40 g of compound was applied. Out of culture volumes of 18 liters of SakSTAR variant, 840 mg of SakSTAR(K74Q,E80A, D82A,K130T,K135R) with a specific activity of 140 kHU/mg and 800 mg Sak- STAR(E65D, K74R,E80A,D82A,K130T,K135R) with a specific activity of 150 were purified. The endotoxin content was <0.1 and 0.26 IU/mg. Gel filtration on HPLC revealed a single main symmetrical peak in the chromatographic range of the column, representing >98% of the eluted material (total area under the curve) (not shown) . SDS gel electrophoresis of 40 g samples revealed single main components (not shown) . Preparations sterilized by filtration proved to be sterile on 3 day testing as described elsewhere (22) . Intravenous bolus injection of SakSTAR variants in groups of 5 mice (3 mg/kg body weight) , did not provoke any acute reaction, nor reduced weight gain within 8 days, in comparison with mice given an equal amount of saline (not shown) . 2. Thrombolytic efficacy Wild-type SakSTAR or the variants SakSTAR(K74Q, E80A,D82A,K130T,K135R) or SakSTAR(E65D,K74R,E80A, D82A, K130T,K135R) were administered intra-arterially at or in the proximal end of the occlusive thrombus as a bolus of 2 mg followed by an infusion of 1 mg/hr (reduced overnight in some patients to 0.5 mg/hr) in groups of 15, 6 and 6 patients respectively with angiographically documented occlusion of a peripheral artery or bypass graft of less than 30 days duration. Patients were studied after giving informed consent, and the protocol was approved by the Human Studies Committee of the University of Leuven. Inclusion and exclusion criteria, conjunctive antithrombotic treatment (including continuous intravenous heparin) and the study protocol were essentially as previously described (22) . Relevant baseline characteristics of the individual patients and results of treatment and outcome are shown in Table 11. Intra-arterial infusion, at a dose of 3.5 to 27 g and a duration of 2 to 44 hrs, induced complete recanalization in 22 patients and partial recanalization in 5. Complementary endovascular procedures (mainly PTA) were performed in 13 patients and complementary reconstructive vascular surgery following thrombolysis in 5. One patient underwent major amputation. Bleeding complications were usually absent or limited to mild to moderate hematoma formation at the angiographic puncture sites (data not shown) . One patient, given wild-type SakSTAR suffered a non-fatal intracranial bleeding, one (BUE) a retroperitoneal hematoma and two (MAN and STRO) a gastro-intestinal bleeding. Circulating fibrinogen, plasminogen and α 2 -antiplasmin levels remained unchanged during infusion of the SakSTAR moieties (data not shown) , reflecting absolute fibrin specificity of these agents at the dosages used (data not shown) . Significant in vivo fibrin digestion occurred as evidenced by elevation of fibrin fragment D-dimer levels. Intra-arterial heparin therapy prolonged aPTT levels to a variable extent (data not shown) . 3. Antibody induction Staphylokinase-neutralizing activity in plasma and antigen-specific IgG antibodies were quantitated essentialy as described above and elsewhere (22) . Antibody-related SakSTAR-, SakSTAR(K74Q,E80A,D82A, K130T,K135R)- and SakSTAR(E65D,K74R,E80A, D82A, K130T,K135R) -neutralizing activity and anti-SakSTAR, anti-SakSTAR(K74Q,E80A,D82A,K130T,K135R) and anti-SakSTAR(E65D,K74R,E80A, D82A,K130T,K135R) IgG, were low at baseline and during the first week after the infusion (Figure 5) . From the second week on, neutralizing activity levels increased to reach median values at 3 to 4 weeks of 9 μg SakSTAR(K74Q,E80A,D82A, K130T,K135R) and 0.5 μg SakSTAR(E65D,K74R,E80A, D82A, K130T,K135R) neutralized per mL plasma in the patients treated with the corresponding moieties, respectively, as compared to median value of 24 μg wild-type SakSTAR neutralized per mL in the 15 patients treated with SakSTAR. The levels of anti-SakSTAR(K74Q,E80A,D82A, K130T,K135R) and of anti-SakSTAR(E65D,K74R,E80A, D82A,K130T,K135R) IgG increased to median values at 3 to 4 weeks of 420 and 30 μg/mL plasma in patients treated with the corresponding moieties, respectively, as compared to a median value of 590 μg anti-SakSTAR per mL plasma in the patients treated with SakSTAR (Figure 5) . The prevalence of immunization, defined as neutralizing activities in plasma after 2 to 4 weeks exceeding 5 g/ml was 3 of 6 patients (50 percent) with SakSTAR(K74Q,E80A, D82A,K130T,K135R) , 1 of 6 patients (17 percent) with SakSTAR(E65D,K74R,E80A,D82A, K130T,K135R) , as compared to 56 of 70 patients (80 percent) with SakSTAR. This difference is statistically highly significant (p= 0.01 by 2 x 3 Chi square analysis) . The antibodies induced by treatment with SakSTAR were completely absorbed by SakSTAR but incompletely by SakSTAR(K74Q,E80A,D82A,K130T,K135R) and by SakSTAR(E65D,K74R, E80A, D82A,K130T,K135R) (Table 12). Antibodies induced by treatment with Sak- STAR(K74Q,E80A,D82A,K130T,K135R) , detectable in 4 of the 6 patients, were completely (>90 percent) absorbed by SakSTAR, by SakSTAR(K74Q,E80A,D82A,K130T, K135R) and by SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) , indicating that immunization was not due to neoepitopes generated by substitution of wild-type amino acids. Antibodies induced by treatment with SakSTAR(E65D,K74R,E80A,D82A, K130T,K135R) detectable in one patient (URB) were completely absorbed with SakSTAR(K74Q,E80A,D82A, K130T,K135R) and with SakSTAR(E65D,K74Q,E80A, D82A, K130T,K135R) but incompletely (85%) with wild-type SakSTAR, suggesting that a small fraction of the induced antibodies might be directed against a neoepitope in the variant used for infusion. EXAMPLE 10 Construction and absorption with pooled plasma of immunized patients of combination variants of SakSTAR(E650. K74O.K130T.K135RΪ and other selected amino acids 1. Introduction In a final round of additive substitution mutagenesis, the SakSTAR(E65Q,K74Q,K130T, K135R) variant was taken as a template because it displayed a high specific activity with a significant reduction of absorption (to 65 percent) of antibodies from pooled immunized patient plasma (Pool 40) . The intermediate variants which were relevant for the composition of the finally selected variants are summarized in Table 13. Addition of K35A, D82A and S84A, of T90A,E99D and T101S or of E108A and K109A reduced the antibody absorption to around 50 percent, whereas the combined addition of D82A,S84A and E108A, K109A reduced it to 41 percent. Substitution of K136A combined with the addition of a Lys at the COOH terminus (-137K) increased the specific activity in a purified system but not in a plasma milieu nor in a hamster pulmonary embolism model (not shown) , and further reduced the absorption of antibodies from pooled patient plasma to 30 percent. Finally, addition of the K35A, and T90A,E99D,T101S substitutions to this template yielded a mutant with intact thrombolytic potency which only bound 24 percent of the antibodies of pooled immunized patient plasma. Based on this analysis, SakSTAR(E65Q,K74Q,D82A, S84A,E108A,K109A,K130T,K135R, K136A, 137K) , (SY118) , and SakSTAR(K35A,E65Q,K74Q, D82A, S84A,T90A,E99D,T101S, E108A,K109A,K130T,K135R,K136A,V137K) , (SY141) , were selected for further characterization. In addition, SakSTAR(K35A,E65Q,K74R,D82A,S84A,T90A,E99D,T101S, E108A,K109A,K130T,K135R,K136A,V137K) , (SY145) with a Lys in position 74, was constructed and evaluated. 2. Pharmacokinetic properties of SakSTAR variants following bolus injection in hamsters The disposition rate of staphylokinase-related antigen from blood following bolus injection of 100 μg/kg of the selected SakSTAR variants in groups of 4 hamsters could adequately be described by a sum of two exponential terms by graphical curve peeling (results not shown) . The pharmacokinetic parameters of the mutants were derived from these plasma disappearance curves not markedly different from those of wild type SakSTAR (results very similar to those of table 10, data not shown) . EXAMPLE 11 Characterization of selected variants derived from SakSTARfE650. K740.K130T.K135R) 1. Fibrinolytic properties of selected SakSTAR variants towards human plasma in vitro Dose- and time-dependent lysis of 15 I-fibrin labeled human plasma clots submerged in human plasma was obtained with the three selected variants (Table 14) . Spontaneous clot lysis during the experimental period was <5% (not shown) . Equi-effective concentrations of test compound (causing 50% clot lysis in 2 hrs; C 50 ) , determined graphically from plots of clot lysis at 2 hrs versus the concentration of plasminogen activator (not shown), ranged from 0.15 ± 0.02 to 0.19 ± 0.01 μg/ml at which no significant fibrinogen degradation occurred. The concentrations of compound causing 50% fibrinogen degradation in 2 hrs in human plasma in the absence of fibrin were determined graphically from dose-response curves (not shown) . These values (mean ± SD of 3 independent experiments) ranged from 7.0 ± 0.6 to 24 ± 3.6 μg/ml (Table 14) . 2. Temperature stability of selected SakSTAR variants The temperature stability of preparations of SakSTAR(E65Q,K74Q,D82A,S84A,E108A,K109A,K130T,K135R, K136A, 137K) , SakSTAR(K35A,E65Q,K74Q, D82A,S84A, T90A, E99D,T101S,E108A,K109A,K130T,K135R,K136A,V137K) , and SakSTAR(K35A,E65Q, K74R, D82A,S84A,T90A,E99D,T101S,E108A, K109A,K130T,K135R,K136A,V137K) dissolved to a concentration of 1.0 mg/ml in 0.15 M NaCl, 0.01 M phosphate buffer, pH 7.5 at various temperatures. At temperatures up to 37 °C, all compounds remained fully active for up to at least three days. At 56 °C and 70°C the variants were generally less stable than wild type SakSTAR (results very similar to those of Figure 4, data not shown) . EXAMPLE 12 Comparative thrombolytic efficacy and immunogenicity of SakSTAR(E650.K740. D82A.S84A.E108A.K109A.K130T.K135R. K136A.V137K) . (SY118) . SakSTAR(K35A. E650.K740. D82A. S84A. T90A.E99D.T101S.E108A.K109A.K130T.K135R.K136A. W137K) . (SY141) . and SakSTARfK35A.E65O.K74R.D82A.S84A.T90A. E99D.T101S. E108A.K109A.K130T.K135R.K136A.V137K) . (SY145) . in patients with peripheral arterial occlusion !• Large scale purification and conditioning of SakSTAR variants for use in vivo Material was purified to homogeneity out of culture volumes of 18 liters. The endotoxin content was below 2 IU/mg. Gel filtration on HPLC revealed a single main symmetrical peak in the chromatographic range of the column, representing >98% of the eluted material (total area under the curve) (not shown) . SDS gel electrophoresis of 30 μg samples revealed single main components. Preparations sterilized by filtration proved to be sterile on 3 day testing. Intravenous bolus injection of SakSTAR variants in groups of 5 mice (3 mg/kg body weight) , did not provoke any acute reaction, nor reduced weight gain within 8 days, in comparison with mice given an equal amount of saline (not shown) . Groups of 6 patients with angiographically documented peripheral arterial occlusion (PAO) were studied. Relevant baseline characteristics of the individual patients are shown in Table 15. Table 16 summarizes the individual treatment and outcome. Intra-arterial infusion, at a dose of 6 to 24 mg and a duration of 4 to 29 hrs, induced complete recanalization in most patients. Circulating fibrinogen, plasminogen and α 2 -antiplasmin levels remained essentially unchanged during infusion of the SakSTAR variants (data not shown) , reflecting absolute fibrin specificity of these agents at the dosages used. Antibody-related SakSTAR(E65Q,K74Q, D82A, S84A, E108A, K109A, K130T, K135R, K136A, V137K) - , Sak- STAR(K35A,E65Q,K74Q,D82A,S84A,T90A,E99D,T101S,E108A, K109A, K130T,K135R,K136A,V137K)- and SakSTAR(K35A,E65Q, K74R,D82A,S84A,T90A,E99D, T101S,E108A, K109A,K130T,K135R, K136A,Vl37K) -neutralizing activity, were low at baseline and during the first week after the infusion (Table 17) . From the second week on neutralizing activity levels increased to reach median values at 3 to 4 weeks of 19 μg SakSTAR(E65Q, K74Q, D82A, S84A, E108A, K109A, K130T, K135R, K136A,V137K) , (SY118) , 0.7 μg SakSTAR(K35A,E65Q,K74Q, D82A, S84A,T90A,E99D,T101S,E108A,K109A,K130T,K135R, K136A,V137K) , (SY141) , and 4.3 μg SakSTAR(K35A,E65Q,K74R, D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T,K135R, K136A, V137K) , (SY145) , neutralized per ml plasma in the patients treated with the respective compounds, which for SY141 and SY145, but not for SY118 is lower than the median value of 12 μg wild type SakSTAR neutralized per ml in 69 patients treated with wild type SakSTAR. Overt immunization (neutralizing activity at 3 to 4 weeks of 5 g compound per ml plasma) was observed in 56 of 70 patients treated with SakSTAR, in 5 of the 6 patients exposed to SakSTAR(E65Q,K74Q,D82A,S84A,E108A, K109A,K130T,K135R,K136A,V137K) , (SY118) , only in 2 of the 6 patients given SakSTAR(K35A,E65Q,K74Q,D82A,S84A,T90A, E99D, T101S,E108A,K109A,K130T,K135R,K136A, 137K) , (SY141) , and in 1 of the 3 patients given SakSTAR(K35A, E65Q,K74R,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T, K135R,K136A, V137K) , (SY145) . The results with respect to immunogenicity of the main variants studied in patients are summarized in Table 18. Clearly, variants SakSTAR(E65D,K74R,E80A,D82A, K130T,K135R) and SakSTAR(K35A,E65Q,K74Q, D82A, S84A,T90A, E99D,T101S,E108A,K109A,K130T, K135R,K136A,V137K) have a significantly reduced immunogenicity when compared to the wild type protein. EXAMPLE 13 Construction, purification and characterization of cysteine-substitution mutants of staphylokinase 1. Introduction Site-directed mutagenesis was applied to substitute exposed amino acids with single cysteine residues in order to construct i) homodimeric forms of staphylokinase, upon formation of an intermolecular disulfide bridge, and ii) polyethylene glycol-conjugated molecules (PEG-derivatives) . The aim of this example was twofold: first, the clearance can be reduced by increasing the size of the injected molecule (via dimerization or conjugation with large molecule such as PEG) and second, PEG-derivatives have also been shown to induce a reduced immunoreactivity in animal models (for review, see ref. 34) . In both cases, a prolonged half-life in vivo could help to reduce the pharmacological dose of staphylokinase in patients. This reduction could be accompanied with a reduced immunogenic reaction against the thrombolytic agent, thus enhancing its pharmacological activity as a thrombolytic agent. In this example, the construction and characterization of two SakSTAR variants in which one single amino acid was substituted with cysteine is described. The mutants described under this example are listed in Table 19. These variants were expressed in E. coli. purified and characterized in terms of specific activity, fibrinolytic properties in human plasma in vitro and pharmacokinetic properties following bolus injection in hamsters. 2. Reagents and Methods The source of all reagents used in the present study has previously been reported (22) , or is specified below. The template vector for mutagenesis, pMEX602sakB (i.e. pMEX.SakSTAR) , has been described elsewhere (23). Restriction and modification enzymes were purchased from New England Biolabs (Leusden, The Netherlands) , Boehringer Mannheim (Mannheim, Germany) or Pharmacia (Uppsala, Sweden) . The enzymatic reactions were performed according to the supplier recommendation. The mutagenic oUgonucleotides and primers were obtained from Eurogentec (Seraing, Belgium) . Plasmid DNA was isolated using a purification kit from Qiagen (Hilden, Germany) , as recommended. Transformation-competent E. coli cells were prepared by the well-known calcium phosphate procedure. Nucleotide sequence determination was performed on double strand plasmid DNA with the dideoxy chain termination method, using the T7 sequencing kit (Pharmacia, Uppsala, Sweden) . Polymerase chain reactions (PCR) were performed using Taq polymerase from Boehringer Mannheim (Mannheim, Germany) . The recombinant DNA methods required to construct the variants described in this example are well established (22, 27). 3. Construction of expression plasmids The variants SakSTAR(K102C) and SakSTAR(K109C) , were constructed by the spliced overlap extension polymerase chain reaction (SOE-PCR) (24) using pMEX.SakSTAR encoding SakSTAR as template. Two fragments were amplified by PCR (30 cycles: 1 sec at 94 °C, 1 sec at 50°C, 10 sec at 72°C), the first one starting from the 5 1 end (primer 818A) of the staphylokinase gene to the region to be mutagenized (forward primer) , the second one from this same region (backward primer) to the 3 ' end of the gene with primer 818D (5' CAAACAGCCAAGCTTCATT- CATTCAGC) . The forward and backward primers shared an overlap of around 24 bp (for the construction of K102C: TAT GAT AAG AAT TGC AAA AAA GAA GAA (backward) and TTC TTC TTT TTT GCA ATT CTT ATC ATA (forward) , for the construction of K109C: AAA AAG AAG AAA CGT GCT CTT TCC CTA (backward) and TAG GGA AAG AGC ACG TTT CTT CTT TTT (forward)). The two purified fragments were then assembled together in a second PCR reaction with the external primers 818A and 818D (30 cycles: 1 sec at 94 °C, 1 sec at 50°C, 10 sec at 72°C). The amplified product from this final reaction was purified, digested with EcoRI and Hindlll and ligated into the corresponding site of pMEX. SakSTAR. For each construction, the sequence of the variant was confirmed by sequencing the entire coding region. 4. Expression and purification of SakSTAR variants The SakSTAR variants were expressed and purified, as described below, from transformed E. coli grown in terrific broth (TB) medium (28) . A 2 to 4 mL aliquot of an overnight saturated culture in LB medium was used to inoculate a 1 to 2 L culture in terrific broth supplemented with 100 μg/mL ampicillin. The culture was incubated with vigorous aeration and at 30°C. After about 16 hours incubation, IPTG (200 μmol/L) was added to the culture to induce expression from the tac promoter. After 3 hours induction, the cells were pelleted by centrifugation at 4,000 rpm for 20 min, resuspended in 1/10 volume of 0.01 mol/L phosphate buffer pH 6-6.5 and disrupted by sonication at 0°C. The suspension was centrifuged for 20 min at 20,000 rpm and the supernatant was stored at 4°C or at -20°C until purification. The material was purified essentially as described above (Example 2) : cleared cell lysates containing the SakSTAR variants were subjected to chromatography on a 1.6 x 5 cm column of SP-Sephadex, followed by chromatography on a 1.6 x 8 cm column of phenyl-Sepharose. The SakSTAR containing fractions, localized by SDS-gel electrophoresis, were pooled for further analysis. 5. Biochemical analysis Protein concentrations were determined according to Bradford (29) . SDS-PAGE was performed with the Phast System™ (Pharmacia, Uppsala, Sweden) using 10-15% gradient gels and Coo assie Brillant blue staining, and the specific activities of SakSTAR solutions were determined with a chromogenic substrate assay carried out in microtiter plates (as described in example 2) . The specific activity of the different SakSTAR variants are summarized in Table 19. Mutant SakSTAR(K102C) was essentially monomeric as visualized by SDS-PAGE and Coomassie Brillant blue staining. Its specific activity was comparable to that of wild-type staphylokinase. In contrast, SakSTAR(K109C) showed a propensity to form dimers (> 60%) . This resulted in a markedly increased specific activity in the plasminogen-coupled chromogenic substrate assay (see Table 19) . Upon reduction with dithiothreitol (DTT) (20-fold molar excess during 1.5 hour at 37 °C) and alkylation with iodoacetamide (100-fold molar excess during 1 hour at 37 °C), the K109C dimer is converted into a stable monomer and its resulting specific activity is within the expected range towards wild-type staphylokinase (Table 19) . This result confirms that formation of homodimers is the unique determinant for this large increase in specific activity. Dimeric SakSTAR(K109C) was separated from monomeric SakSTAR(K109C) by chromatography on Source S (Pharmacia) (5 x 50mm). Loading buffer was 10 mM phosphate, pH 6.0 and dimeric SakSTAR(K109C) was eluted by a salt gradient (up to 1 M) in the same buffer. The dimeric SakSTAR(K109C) (>95% pure) containing fractions, localized by SDS-gel electrophoresis, were pooled for further analysis. 6. Chemical crosslinking of cysteine mutants of SakSTAR with polyethylene glvcol The thiol group of the cysteine mutant SakSTAR(K102C) was targeted for coupling with an activated polyethylene glycol, OPSS-PEG (Shearwater Polymers Europe, Enschede, The Netherlands) . OPSS-PEG is a 5 kDa PEG molecule carrying a single activated thiol group at one end that react specifically at slightly alkaline pH with free thiols. Modification of SakSTAR(K102C) was achieved by incubating the molecule (100 μM) with a three-fold excess of SS-PEG in a 5 mM phosphate, pH 7.9 solution at room temperature. The extent of the reaction was monitored by following the release of 2-thiopyridone from OPSS-PEG at 412 nm. After reaction (about 15 min) , the excess of OPSS-PEG was removed by purifying the derivatized SakSTAR(K102C-PEG) on a 1.6 x 5 cm column of SP-Sephadex as described above (see Example 2) . The SakSTAR(K102C-PEG) containing fractions, localized by optical density at 280 nm, were pooled for further analysis. SDS-PAGE analysis and Coomassie blue staining confirmed that PEG crosslinking on SakSTAR(K102C) was quantitative. As shown in Table 19, the specific activity of the PEG-derivative was only marginally affected when compared to that of wild-type staphylokinase. 7. Fibrinolytic properties of SakSTAR variants in human plasma in vitro The fibrinolytic and fibrinogenolytic properties of SakSTAR variants were determined as previously described. Dose- and time-dependent lysis of 15 I-fibrin labeled human plasma clots submerged in human plasma was obtained with four molecules: SakSTAR(K109C) as dimer and as monomer (after reduction and alkylation with iodoacetamide) , the monomeric SakSTAR(K102C) and the PEG-derivatized SakSTAR(K102C) . Spontaneous clot lysis during the experimental period was <5% (not shown) . Equi-effective concentrations of test compound (causing 50% clot lysis in 2 hrs; C 50 ) , determined graphically from plots of clot lysis at 2 hrs versus the concentration of plasminogen activator (not shown) , were comparable to that of SakSTAR, for monomeric SakSTAR(K109C) and SakSTAR(K102C) (Table 19) . However, it was observed that the C 50 for clot lysis by dimeric SakSTAR(K109C) was only 0.12 μg/ml, which is approximately three-fold lower than for wild-type staphylokinase. In contrast, a C 50 of 0.60 μg/ml was measured for SakSTAR(K102C-PEG) , which is only two-fold higher than for wild-type staphylokinase. Thus, dimerization of SakSTAR via disulfide bridges or increasing the size of the molecule via PEG- derivatization does not preclude the fibrinolytic activity of staphylokinase. While a PEG-molecule appears to reduce the diffusion and therefore fibrinolytic potency of the derivatized staphylokinase within a fibrin clot, dimerization of staphylokinase results in a synergistic fibrinolytic effect on human fibrin clots. 8. Pharmacokinetic properties of dimeric SakSTAR(K109C) and SakSTAR(K102C-PEG) following bolus injection in hamsters The pharmacokinetic parameters of the disposition of dimeric SakSTAR(K109C) and SakSTAR(K102C-PEG) from blood were evaluated in groups of 4 hamsters following intravenous bolus injection of 100 μg/kg SakSTAR variant. SakSTAR-related antigen was assayed using the ELISA described elsewhere. The ELISA was calibrated against each of the SakSTAR variants to be quantitated. Pharmacokinetic parameters included: initial half-life (in min) , tl/2α= ln2/α; terminal half-life (in min) , tl/2B= ln2/β; volume of the central (plasma) compartment (in mL) , VC= dose/(A+B); area under the curve (in μg.min.mL "1 ) , AUC= A/α + B/β; and plasma clearance (in mL.min "1 ) , Clp= dose/AUC (32) . The disposition rate of staphylokinase-related antigen from blood following bolus injection of 100 μg/kg of the selected SakSTAR variants in groups of 4 hamsters could adequately be described by a sum of two exponential terms by graphical curve peeling (results not shown) , from which the pharmacokinetic parameters tl/2α and Clp, summarized in Table 19 were derived. The pharmacokinetic parameters of dimeric SakSTAR(K109C) and SakSTAR- (K102C-PEG) were markedly different from those of wild type SakSTAR. Initial plasma half-lives (tl/2(α)) were 3.6 and 3.0 min and plasma clearances (Clp) were 0.52 and 0.32 mL/min, for dimeric SakSTAR(K109C) and SakSTAR- (K102C-PEG) , respectively. These results may be due to the increase of the Stokes radius of SakSTAR as a result of the dimerization or crosslinking with PEG. According to size-exclusion chromatography on SuperdexδO by HPLC, dimeric SakSTAR(K109C) and SakSTAR(K102C-PEG) have apparent molecular weights of 33 kDa and 40 kDa, respectively. EXAMPLE 14 Construction, purification and characterization of cysteine-substitution mutants of variants of staphylokinase with reduced immunogenicity 1. Introduction Based on the results of example 13, additional polyethylene glycol derivatives of SakSTAR variants were constructed, purified and characterized. The least immunogenic variants SakSTAR(E65D,K74R,E80A, D82A,K130T,K135R) , (SY19) , and SakSTAR(K35A,E65Q, K74Q,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K1- 30T,K135R,K136A,V137K) , (SY141) , were used as templates, with the proviso that the COOH-terminus of the latter was reverted to the wild type sequence, S84A was replaced with E80 and K74Q replaced with K74R, yielding SakSTAR(K35A,E65Q,K74R,E80A,D82A,T90A,E99D,T101S,E108A, K109A,K130T,K135R) , (SY161) . The introduced cysteine, which functions as acceptor of the polyethylene glycol molecule was located in the amino terminal region (preferably, but not exclusively, the Ser in position number 3 of the mature staphylokinase variant) in order to be released upon activation of staphylokinase (release of the 10 NH 2 -terminal amino acids) ; finally polyethylene glycol molecules of different molecular weights (M r 5,000 to 20,000) were used, substituted with either OPSS or maleimide. The mutants described under this example are listed in Table 20. These variants were expressed in E.coli, purified and characterized in terms of specific activity, fibrinolytic properties in human plasma in vitro, pharmacokinetic properties following bolus injection in hamsters, thrombolytic properties following bolus injection in a hamster pulmonary embolism model, and absorption of antibodies from pooled immunized patient plasma (Pool 40) . 2. Reagents and Methods The source of all reagents used in the present study has previously been reported (22) , or is specified below. The template vector for mutagenesis, pMEX602sakB (i.e. pMEX.SakSTAR) , has been described elsewhere (23). Restriction and modification enzymes were purchased from New England Biolabs (Leusden, The Netherlands) , Boehringer Mannheim (Mannheim, Germany) or Pharmacia (Uppsala, Sweden) . The enzymatic reactions were performed according to the supplier recommendation. The mutagenic oUgonucleotides and primers were obtained from Eurogentec (Seraing, Belgium) . Plasmid DNA was isolated using a purification kit from Qiagen (Hilden, Germany) , as recommended. Transformation-competent E. coli cells were prepared by the well-known calcium phosphate procedure. Nucleotide sequence determination was performed on double strand plasmid DNA with the dideoxy chain termination method, using the T7 sequencing kit (Pharmacia, Uppsala, Sweden) . Polymerase chain reactions (PCR) were performed using Taq polymerase from Boehringer Mannheim (Mannheim, Germany) . The recombinant DNA methods required to construct the variants described in this example are well established (22, 27). 3. Construction of expression plasmids The variants SakSTAR(S3C,E65D,K74R,E80A,D82A, K130T,K135R) , (SY19(S3C)), SakSTAR(S2C,S3C,E65D,K74R, E80A,D82A,K130T,K135R) , (SY19 (2SC, 3SC) ) , SakSTAR(S3C, K35A,E65Q,K74Q,D82A,S84A,T90A,E99D,T101S,E108A,K109A, K130T,K135R,K136A,V137K) , (SY141 (S3C) ) , SakSTAR(S2C, S3C,K35A,E65Q,K74Q,D82A,S84A,T90A,E99D,T101S,E108A,- K109A,K130T,K135R,K136A,V137K) , (SY141 (S2C,S3C) ) , SakSTAR(S3C,K35A,E65Q,K74Q,E80A, D82A,T90A,E99D,T101S, E108A, K109A,K130T, K135R) , (SY160(S3C)) and SakSTAR(S3C,K35A, E65Q,K74R,E80A,D82A,T90A,E99D, T101S,E108A,K109A, K130T,K135R) , (SY161 (S3C) ) , were constructed by the spliced overlap extension polymerase chain reaction (SOE-PCR) (24) using pMEX. SakSTAR encoding SakSTAR as template, two fragments were amplified by PCR (30 cycles: 1 sec at 94°C, 1 sec at 50°C, 10 sec at 72°C), the first one starting from the 5' end (primer 818A) of the staphylokinase gene to the region to be mutagenized (forward primer) , the second one from this same region (backward primer) to the 3 • end of the gene with primer 818D (5 1 CAAACAGCCAAGCTTCATTCATTCAGC) . The forward and backward primers shared an overlap of around 24 bp. The two purified fragments were then assembled together in a second PCR reaction with the external primers 818A and 818D (30 cycles: 1 sec at 94 °C, 1 sec at 50°C, 10 sec at 72 °C). The amplified product from this final reaction was purified, digested with EcoRI and Hindlll and ligated into the corresponding site of pMEX. SakSTAR. For each construction, the sequence of the variant was confirmed by sequencing the entire SakSTAR coding region. 4. Expression and purification of SakSTAR variants The SakSTAR variants were expressed and purified, as described below, from transformed E. coli grown in terrific broth (TB) medium (28) . A 2 to 4 mL aliquot of an overnight saturated culture in LB medium was used to inoculate a 1 to 2 L culture in terrific broth supplemented with 100 μg/mL ampicillin. The culture was incubated with vigorous aeration and at 30 °C. After about 16 hours incubation, IPTG (200 μmol/L) was added to the culture to induce expression from the tac promoter. After 3 hours induction, the cells were pelleted by centrifugation at 4,000 rpm for 20 min, resuspended in 1/10 volume of 0.01 mol/L phosphate buffer pH 6-6.5 and disrupted by sonication at 0°C. The suspension was centrifuged for 20 min at 20,000 rpm and the supernatant was stored at 4°C or at -20 °C until purification. The material was purified essentially as described above (Example 2) : cleared cell lysates containing the SakSTAR variants were subjected to chromatography on a 1.6 x 5 cm column of SP-Sephadex, followed by chromatography on a 1.6 x 8 cm column of phenyl-Sepharose. The SakSTAR containing fractions, localized by SDS-gel electrophoresis, were pooled for further analysis. 5. Biochemical analysis Protein concentrations were determined according to Bradford (29) . SDS-PAGE was performed with the Phast System™ (Pharmacia, Uppsala, Sweden) using 10-15% gradient gels and Coomassie Brillant blue staining, and the specific activities of SakSTAR solutions were determined with a chromogenic substrate assay carried out in microtiter plates (as described in example 2) . 6. Chemical crosslinking of cysteine mutants of SakSTAR with polyethylene glycol The thiol group of the cysteine mutants was targeted for coupling with an activated polyethylene glycol, either OPSS-PEG or MAL-PEG (Shearwater Polymers Europe, Enschede, The Netherlands) . OPSS-PEG is a 5 kDa PEG molecule carrying a single activated thiol group at one end that reacts specifically at slightly alkaline pH with free thiols. MAL-PEG is a 5 kDa, 10 kDa or 20 kDa molecule carrying a maleimide group that reacts specifically with thiol groups under mild conditions in the presence of other functional groups. Modification of the variants was achieved by incubating the molecule (100 μM) with a three-fold excess of OPSS-PEG or MAL-PEG in a 5 mM phosphate, pH 7.9 solution at room temperature. After reaction (about 15 min) , the excess of OPSS-PEG or MAL-PEG was removed by purifying the derivatized SakSTAR variant on a 1.6 x 5 cm column of SP-Sephadex as described above (see Example 2) . The "pegylated" SakSTAR variant containing fractions, localized by optical densi- ty at 280 nm, were pooled for further analysis. SDS-PAGE analysis and Coomassie blue staining confirmed that PEG crosslinking was quantitative. As shown in Table 20, the specific activities of the PEG-derivatives were only marginally affected when compared to that of wild-type staphylokinase. 7. Fibrinolytic properties of SakSTAR variants in human plasma in vitro The fibrinolytic and fibrinogenolytic properties of SakSTAR variants were determined as previously described. Dose- and time-dependent lysis of 15 I-fibrin labeled human plasma clots submerged in human plasma was obtained with all molecules tested. Equi-effective concentrations of test compound (causing 50% clot lysis in 2 hrs; C 50 ) , determined graphically from plots of clot lysis at 2 hrs versus the concentration of plasminogen activator (not shown) , were comparable to or only slightly lower than that of SakSTAR (Table 20) . The C 50 for clot lysis by variants derivatized with P20 (PEG with M r 20 kDa) was about twice as high as the non-derivatized variants. Thus increasing the size of the molecule via PEG-derivatization does not markedly affect the fibrinolytic activity of staphylokinase. The PEG- olecules appear to reduce the diffusion and therefore fibrinolytic potency of the derivatized staphylokinase within a fibrin clot, but this appears to be less pronounced with variants substituted in their NH 2 -terminal region - which is released during processing of staphylokinase - than with variants substituted in the core of the molecule (cfr. Tables 19 and 20) . 8. Pharmacokinetic properties of SakSTAR variants chemically modified with polyethylene glycol following bolus injection in hamsters The pharmacokinetic parameters of the disposition of the pegylated variants from blood were evaluated in groups of 4 hamsters following intravenous bolus injection of 100 μg/kg SakSTAR variant. SakSTAR-related antigen was assayed using the ELISA described elsewhere. The ELISA was calibrated against each of the SakSTAR variants to be quantitated. Pharmacokinetic parameters included: initial half-life (in min), tl/2α = ln2/α; terminal half-life (in min), tl/2β = ln2/β; volume of the central (plasma) compartment (in mL) , VC= dose/(A+B); area under the curve (in μg.min.mL "1 ) , AUC= A/α + B/β; and plasma clearance (in mL.min "1 ), Clp= dose/AUC (32). The disposition rate of staphylokinase-related antigen from blood following bolus injection of 100 μg/kg of the selected SakSTAR variants in groups of 4 hamsters could adequately be described by a sum of two exponential terms by graphical curve peeling (results not shown) , from which the plasma clearances Clp, summarized in Table 20 were derived. The clearances of pegylated variants were markedly different from those of wild type SakSTAR and were inversely proportional to the molecular weight of the PEG molecules, with an average reduction of 5-fold with PEG 5 kDa, 10-fold with PEG 10 kDa and 30-fold with PEG 20 kDa. These results may be due to the increase of the Stokes radius of SakSTAR as a result of crosslinking with PEG. EXAMPLE 15 Comparative thrombolytic efficacy and clearance of Sak- STARfS3C-P20.E65D.K74R. E80A.D82A.K130T.K135R) . (SY19 (S3C-P20) ) . in two patients with acute myocardial infarction Large scale purification and conditioning of the SakSTAR variant for use in vivo Material was purified to homogeneity out of culture volumes of 18 liters. The endotoxin content was below 1 IU/mg. Gel filtration on HPLC revealed a single main symmetrical peak in the chromatographic range of the column, representing >98% of the eluted material (total area under the curve) (not shown) . SDS gel electrophoresis of a 30 μg sample revealed single main component. The preparation sterilized by filtration proved to be sterile on 3 day testing as described in methods. Intravenous bolus injection of the SakSTAR variant in 5 mice (3 mg/kg body weight) , did not provoke any acute reaction, nor reduced weight gain within 8 days, in comparison with mice given an equal amount of saline (not shown) . Two patients with acute myocardial infarction were given a bolus injection of 5 mg SY19(S3C-P20) . These patients had a complete recanalization of the occluded infarct-related artery as determined by coronary angiography at 90 min after the bolus injection. The material was cleared from the plasma with an initial half-life of 3 to 4 hours, as compared to 4 to 6 minutes for wild-type SakSTAR. These data confirm that pegylated variants of SakSTAR may be useful for thrombolytic therapy by single bolus injection at a reduced dose. CONCLUSION In summary, the present invention shows that staphylokinase variants with markedly reduced antibody induction but intact thrombolytic potency can be generated. This observation constitutes the first case in which a heterologous protein, with the use of protein engineering techniques, is rendered significantly less immunogenic in man without reducing its biological activity. In addition, the present invention shows that selective chemical modification of staphylokinase or its variants with polyethylene glycol of varying molecular weights is feasible, resulting in a reduction of the plasma clearance proportional to the molecular weight. In the preferred embodiment an amino acid in the NH 2 -terminal region of staphylokinase, the portion that is removed by processing, is substituted with Cys and the introduced thiol group is chemically modified with OPSS-PEG or MAL-PEG. This results in homogeneous products which, upon single intravenous bolus injection in experimental animals and in patients have a maintained thrombolytic potency at markedly reduced doses. Table 1: Alanine-to-wild-type" reversal variants of "charged-cluster-to-alanine" mutants of SakSTAR: Association constants (KA X 10^ mol L'l) for the binding to insolubilized murine monoclonal antibodies (Mabs), and absorption (percent) of antibodies of immunized patient plasma Table 2: Baseline characteristics and treatment outcome of the patients with peripheral arterial occlusion treated with SakSTAR, SakSTAR (K74A) or SakSTAR(K74A,E75A,R77A) Compound Gender Age Clinical Locus of Age of Length of Recanalization by Total dose ol Total duration Additional therapy Patient Id (yrs) ischemia occlusion occlusion occlusion thrombolysis thrombolytic agent of infusion (hrs) (days) (cm) (mg) SakSTAR MEE F 67 Rest pain Lett SFA 30 8 complete 7 0 5 0 PTA FOR M 68 Claudication Left IA (stent) 14 18 complete 6 5 4 5 PTA + stent DAN M 73 Claudication Right SFA 30 6 complete 7 5 5 5 PTA BER F 63 Rest pain Left FT graft 18 55 complete 18 28 PTA DAM F 43 Acute Left brachial and 2 7 complete 19 17 PTA + stent radial artery TOR M 68 Claudication Right SFA (popliteal 50 12 complete 6 0 4 0 PTA + femoropopliteal bypass C 5 aneurysm) graft d CLA M 7 4 Acute Left PA 1 5 20 complete 9 0 7 0 - 53 MAN M 65 Acute Left EIA (stent) 4 20 complete 6 5 4 5 (amputation left digit V) H MAT M 64 Subacute Right FP graft 3 45 complete 8 0 6 0 (-) H Mean ± SEM 65 ± 3 0 17 ± 5 6 21 ± 5 8 9 7 ± 1 7 9 1 ± 2 7 C SakSTAR(K74A) H LIE M 70 Subacute Right FF graft 10 48 complete 1 1 9 0 PTA W ENG M 50 Claudication Right SFA 28 10 complete 12 10 PTA Λ cox F 48 Claudication Right PA graft 25 7 partial 15 15 PTA H MAN F 68 Claudication Right SFA >120 9 complete 9 0 7 0 PTA W VHE M 47 Acute Right IF graft 10 54 complete 18 16 S iurgical graft revision W MUL F 51 Acute Right IF and FP graft 1 63 complete 16 20 PTA H BUR F 67 Rest pain Right TF trunc 90 38 partial 18 21 - NIJ F 60 Rest pain Left AF graft 23 78 complete 15 21 - POE* M 49 Subacute Right TF trunc 2 30 partial 60 40 rt-PA, surgical graft lengthening VBE M 39 Subacute Right BA (embolism) 20 28 complete 18 23 Stent πght SC artery, first πb f w resection SME F 50 Subacute TF trunc 18 32 complete 21 19 None ©\ WOL M 67 Subacute Right PA 4 25 complete 16 22 - Mean ± SEM 56 ± 3 0 23 ± 9 2 35 ± 6 4 15 ± 1 2 16 ± 1 9 SakSTAR(K74A,E75A,R77A) JAC F 65 Acute Right BA and UA 0 3 5 complete 14 12 - MAE M 74 Rest pain Left SFA 10 50 complete 90 7 0 PTA CRA F 52 Claudication Right IA and FA 14 28 complete 25 23 PTA + stent artery VDB M 68 Claudication Left SFA 90 12 complete 9 0 7 0 PTA DUN M 71 Subacute Left SFA 14 6 complete 90 70 PTA DEL M 59 Acute Right FT graft 3 42 complete 9 0 7 0 PTA Mean ± SEM 65±3 3 22±14 24±7 8 13 ± 2.6 11 ± 2.6 AF aortofemoral; BA brachial artery , CIA common iliac artery, FF femorofibular; FP- femoropopliteal; FT: femorotibial, IA iliac artery, IF- lhofemoral; PA: popliteal artery , PTA. percutaneous transluminal aπgioplasty, SFA su perficial femoral artery, TF- tibtofϊbular, UA ulnar artery *Prevιous treatment with SakSTAR in 1994 Table 3: Alanine-substitution variants of SakSTAR: Association constants (K A x lO'mol/L' 1 ) for binding to insolubUized murine monoclonal antibodies (Mab) and absorption Table 3 - cont'd: Alanine-substitution variants of SakSTAR: Association constants (K A x lO'mol/L "1 ) for binding to insolubUized murine monoclonal antibodies (Mab) and Alanine-substitution variants of SakSTAR: Association constants (K A x 10 mol/L " ') for binding to insolubilized murine monoclonal antibodies (Mab) and Table 4: Mutagenesis of S34, G36 and H43: Association constants (K A x lO'mol/L' 1 ) for binding to insolubilized murine monoclonal antibodies (Mab) and absorption (percent) of antibodies of immunized patient plasma murine MAbs Exp Spec Epitope cluster 1 Epitope cluster 11 Epitope cluster III SakSIAR patient plasma Act (mg/L) (kU/mg) 17G1 6A2 30Ai 2BI2 3CI0 isFi: I4H5 28H4 32B2 7F10 7H11 !5EI 408 24C4 1A10 Pool Subpool B Subpool l_ SakSTAR 120 93 13 29 78 II 35" 74 19 77 24 40 14 54 29 Oi 95 95 95 Sal_STAR(S34G.G36R.H43R) 120 10 14 33 75 II <0.1 <0.1 <0.1 20 27 <0.1 <0.1 <01 0.15 17 87 76 75 SakSTAR(S14A) 29 110 17 24 46 95 11 28 11 22 15 29 31 88 38 20 02 95 95 9.1 OO c SakSTARlG16A) 14 72 15 98 15 57 65 52 42 17 92 14 <0.1 <0.1 09 50 10 86 81 78 o wo SakSTAR(G36E) 12 66 11 87 14 48 47 12 28 61 76 10 <0.1 <0.1 <0.I 34 11 89 83 72 H SakSTAR(G16K) 14 88 99 21 11 81 98 21 19 11 15 30 <0.1 <0.1 <0.1 26 12 88 80 69 H c SakSrAR(G16L) 15 92 16 II 18 61 61 16 14 64 12 13 <0.1 <0.1 <0.1 06 11 91 85 72 = SakSTAR(G16N) 10 91 87 10 16 61 62 11 31 78 79 18 <0.1 <0.1 10 03 05 86 80 75 o SakSTAR(G16Ql 21 92 10 12 18 67 65 21 18 75 73 15 <0.1 <0.1 <0.1 0.1 04 87 84 71 c SakSTAR(G36R) 45 100 11 24 31 10 10 27 46 14 20 34 <0.1 <0.l t <0.1 31 12 89 81 70 W SakSTAR(H41A) 17 H 69 15 28 97 18 76 <0.1 <0.1 <0.l 91 15 20 21 78 72 16 95 95 95 SakSTAR(H43R) 43 120 15 11 27 76 11 <0.1 <0.1 <0.1 13 64 07 18 67 57 14 95 95 95 SakSTAR(S14GG36R) 45 90 11 12 23 48 42 13 83 24 91 19 <0.1 <0.1 <0.l <0.1 06 92 83 69 w SakSTAR(S14G G36R H43R K74AI 1 12 12 47 38 74 94 <0.1 0.1 0.6 25 23 <0.1 <0.1 0.5 08 17 67 56 81 SakSTAR(S14G G16R K74A) 15 26 40 21 <0.l 18 06 10 22 15 13 18 <0.1 <0.1 <0.1 OJ 22 59 28 68 SakSTAR(K35G G36R H43D) 12 6 18 15 16 20 89 <0.1 <0.1 <0.1 71 13 <0.1 <0.l <0.1 <0.1 09 82 75 72 SakSTAR(G16R.K74A) 40 35 19 70 0.2 43 20 53 27 28 19 44 <0.1 <0.l <0.1 12 10 48 33 58 SakSTAR(G36R.K74R) 68 150 47 17 18 11 80 16 60 64 30 16 <0.1 <0.l <0.1 0.2 08 81 54 71 SakSTARlG36R K74A N95A1 II 25 61 29 29 24 <0.l 11 57 12 53 41 <0.l <0.1 <oι <0.1 09 53 32 61 SakSTAR(G36R K74A KI35R) 20 33 58 14 <0.1 17 0.7 26 16 14 17 12 <0.1 <0.1 <0.l 04 05 64 32 68 SakSTAR(G36R K74R K115R) 48 75 61 17 58 10 33 31 14 1.1 57 23 <0.1 <0.1 <0.1 0J 08 77 49 68 Table 5: Mutagenesis of K35, Y73, K74, E80/D82, N95, K130, V132 and K135: Association constants (K A x 10 7 mol/L ') for binding to insolubilized murine monoclonal antibodies (Mab) and absorption (percent) of antibodies of immunized patient plasma s Abs Lxp Spet. Epitope cluster I Epitope cluster II Epitope cluster 111 SakSTAK patient plasma Act (mg/L) (kU/mg) 17GII 26A2 — 10A22BI2 3c!IO I8FIZ I4H5 28H4 32B2 7FI0 7H11 25E1 4I 24« 1AI0 Tool Subpool B S ool r SalsTAR τw T T5 — IT TS — 7τ — rc — r. — TΓ Tδ- T3 T9 W -9T —9 SakSTAR(S34G G36R H43R) <01 <01 <01 20 27 <oι <01 015 17 87 76 75 SakSTAR(K35A) 230 64 14 11 80 74 II 12 II 26 <01 I 1 02 17 08 91 95 CO <=. SakSTAR(K35E) 160 48 10 06 27 27 57 91 48 10 <01 <01 01 15 04 95 95 92 W oo SakSTAR(K15Ql 69 32 95 13 52 54 93 12 05 17 01 19 10 95 95 95 H S»kSTAR(V7 A) <5 95 <01 <01 <01 48 71 11 89 20 05 90 25 14 08 63 44 91 c SakSTAR! Y71F) II 79 12 13 21 80 15 19 15 26 I 6 62 35 72 14 93 95 95 = SakSTAR(Y73Hl <5 73 15 <0 I <01 14 75 17 42 43 35 68 59 97 13 76 65 95 SakSTAR(Y71L) <5 II <01 <01 <01 <01 19 26 53 34 42 71 42 34 10 81 60 94 H w SakSTAR(Y71S) <5 70 1« 059 06 10 81 14 21 12 22 67 II II 07 86 69 95 w SakSTARCl71 l H 27 48 90 46 46 11 84 45 II 80 27 29 50 30 38 I 1 73 53 91 SakSTAR! K74A) 69 44 27 02 22 11 17 52 14 76 22 20 68 11 18 09 64 58 95 SakSTAR( 74E) <5 22 06 •=01 07 01 14 18 75 91 12 20 30 12 06 10 81 43 90 r w SakSTAR(K74Nl 19 29 47 I I 33 17 10 16 48 13 15 19 40 18 14 09 63 46 95 » SakSTAR! 74Q) 110 53 58 01 25 I I 24 59 12 54 I I 20 62 23 20 04 70 62 94 SakSTAR(K74R) 150 21 75 20 41 42 24 69 80 83 13 22 78 32 21 05 75 70 95 Sa STAR(E80AJ)82A) 130 71 12 21 65 59 79 61 84 78 1 <0I <01 <01 <01 04 89 81 92 SakSTAR(E80A) 160 II II 33 79 10 35 74 17 86 2 I <01 16 16 <0I 17 94 91 95 SakSTAR(D82A) 160 17 12 48 73 II 31 78 17 12 27 <01 02 <01 <01 21 95 91 95 SakSTAR(N95A) 260 10 40 10 II 50 11 14 49 21 17 71 47 29 08 95 94 95 SakSTAR(N95E) 79 85 11 52 54 27 10 52 I I 05 40 17 18 06 95 92 95 SakSTAR(N95G) 160 4 I II 15 68 76 36 33 36 15 07 58 27 27 09 95 90 95 SakSTAR! N95K) 180 95 14 32 90 II 50 18 48 25 I 6 81 29 48 I I 95 95 95 SakSTAR(N95Rl Table 5 - cont'd: Mutagenesis of K35, Y73, K74, E80/D82, N95, K130, V132 and K135: Association constants (K A x lO'moIL') for binding to insolubilized murine monoclonal antibodies (Mab) and absorption (percent) of antibodies of immunized patient plasma munne MAbs Van int Exp Spec Epitope cluster 1 Epitope cluster 11 Epitope cluster III SaleSTAI. patient plasma Act (mg/LI (kU/mg) ncn 26A2 30A2 2BI2 3GI0 IBFI2 14H5 28H4 32B2 7FI0 7H1I 25E1 40C8 24C4 IAI0 Pool Subpool B Subpool C 3 d w S_kSTΛR(K130Λ) 280 51 12 12 64 35 22 67 11 15 27 <01 <0I 41 09 06 92 74 71 o H SakSTARlKIIOTl 290 78 14 11 80 56 41 54 97 17 27 <01 <01 14 05 05 92 84 82 H S»kSTΛR(V132A) 102 130 42 15 26 92 II 31 12 30 19 21 21 36 <01 26 04 95 95 95 ej H SakSTAR(VI32L) 126 120 48 14 23 80 97 67 10 48 20 25 20 11 <01 48 04 95 95 95 W SakSTAR! V 132T) 78 140 45 13 24 78 90 39 10 25 16 20 11 20 <01 <01 04 95 95 95 oo W SakSTAR(V132N) 16 ISO 45 II 1 70 72 21 17 20 15 20 15 1J <01 42 07 95 95 95 w w SakSTAR(VI12R) 76 230 54 12 08 11 12 21 55 78 48 11 21 14 <01 04 04 95 95 95 H S«kSTAR(KI35A) 54 410 52 12 II 79 II 20 II 11 18 20 16 69 37 19 09 95 95 95 SakSTAR(KI35Fl 68 64 39 61 10 61 41 13 45 84 16 21 16 58 19 15 09 95 95 95 r SakSTAR! KI15R) 54 230 40 14 | 4 91 50 11 81 11 21 19 24 24 26 21 05 95 95 95 w SakSTAR(K15A 74A) 20 130 NT 84 50 95 Os SakSTAR(Y73A K74A) 24 <5 18 <01 <01 <01 <01 19 67 23 99 32 27 11 40 16 l l 47 28 87 SakSTAR! Y71FK74A) 21 6 17 67 <0I 21 <0I 42 19 21 20 49 19 28 66 21 08 51 34 90 SakSTAR(l60A K74A N95A) 84 53 27 <01 25 53 17 72 96 34 23 21 62 27 29 08 60 47 95 SakSTAR(N95A KI15R) 120 240 49 13 19 90 99 18 12 15 25 15 18 59 16 17 08 95 95 95 SakSTAR(K130TKI15R) 15 280 17 10 16 72 80 13 77 45 37 16 <01 <01 24 04 06 89 60 71 _ OS V) φ r. α. o <D VI c © o o 05 3 (0 H — Table 7: Combination mutants of SakSTAR(E80A,D82A,K130T,K135R) with K35A, G36R, E65X, K74X, and selected other amino acids Table 7 - cont'd: Combination mutants of _ sakSTAR(EI ΪOA, 1)82, l,Kl 30T ,K135R) ι with K35A, G36R, E65X, K74X, a nd s elected other amino acid S Table 8; SakSTAR variants with intact specific activity (≥ 100 kHU/mg) and ≤50 percent absorption of human antibodies elicited by treatment wit wild-type SakSTAR CΛ cl W CΛ Table 8 • cont'd: SakSTAR variants with intact specific activity (≥ 100 kHU/mg) and ≤S percent absorption of human antibodies elicited b H treatment with wild-type SakSTAR H cl Variant Spec. Act. SakSTAR patient plasma (kU/mg) Pool 10 Subpool B Subpool Pool 40 Code C CΛ SakSTAR(E65Q,K74Q,N95A,E118A,K130A,K135R,V137A) 120 45 30 74 6b SY93 S SakSTAR(E65Q,K74Q,N95A,E1 18A,K130A,K135R,K136A,V l37K) 1,400 37 16 70 54 SY94 H SakSTAR(E65Q,K74Q,D82A,S84A,E108A,K109A,K130A,K135R) 110 46 26 63 41 SY95 Antibody absorption <60 percent of wild-type SakSTAR are represented in bold type; ≥ 100,000 HU/mg represented in bold type. Table 9: Fibrinolytic properties of selected SakSTAR variants in human plasma in vitro CΛ cl Compound Fibrinolytic potency Residual fibrinogen Fibrinogenolytic W CΛ (C50 in μg/mL) at C50 potency Code H (% of baseline) (C50 in μg/mL) H cl H SakSTAR 0.18 ± 0.01 93 ± 3.5 24 ± 3.6 W CΛ SakSTAR(K74Q,E80A,D82A,K130T,K135R) 0.15 + 0.01 97 ± 3.0 14 ± 3.2 SY15 m w SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) 0.24 ± 0.04 94 ± 10 29 ± 3.1 SY19 w H SakSTAR(K35A,E65D,K74Q,E80A,D82A,K130T,K135R) 0.11 ± 0.01 92 ± 3.0 20 ± 2.0 SY46 SakSTAR(E65Q,K74Q,N95A,E1 18A,K__30A.K135R,K136A,V l37K) 0.13 91 SY93 w The data represent mean ± SD of 3 experiments. C, 0 : amount of wild type or variant SakSTAR required for 50% clot lysis or 50% fibrinogen breakdown in 2 hrs. d Table 10: Pharmacokinetic parameters of the disposition of staphylokinase-related antigen from plasma following bolus injection of SakSTAR W „ v vaarriiaannttss C (11000 μ llαg//kkpg) i inn h haammsstteerrss. H Variant Co A B tl/2(α) tl/2(β) Vc AUC Cl p (H (μg/mL) (μg/mL) (μg/mL) (min) (min) ( mL) (μg.miπ.mL- 1 ) (mL.min" 1 ) H H SakSTAR OS±Ol Oό±O.l 0.2±0.0 2.8 7.0 13 ± 1.0 4.6 ± 0.4 2.2 ± 0.2 CΛ M SakSTAR(K74Q,E80A,D82A,K130T, I35R) 05±01 04 + 01 0.1 ±0.0 2.0 10 20±2.2 25±0.3 41 ± 0.5 w W SakSTAR(E65D, 74R,E80A,D82A, 130T, 135R) 06±()0 05±00 O.l±O.O 2.0 10 16 ± 1.1 2.8 ± 0.2 37±03 'j SakSTAR(K35A,E65DK74Q,E80A,D82A,K130T,K135R) I I ±01 l.O±Ol O.l±O.O 2.0 24 9.6 ±0.7 6.4 ±0.5 1.6 ±0.1 c j~* Data are mean ± SEM of 4 experiments. Table 11: Baseline characteristics and treatment outcome of the patients with peripheral arterial occlusion treated with SakSTAR, SakSTAR(K74Q,E80A,D82A,K130T,K135R) or SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) Compound Clinical Locus of Age of Length of Recanalization Total dose of Total duration Additional therapy Patient Id Gender Age ischemia occlusion occlusion occlusion by thrombolytic of infusion (yrs) (days} (cm) thrombolysis agent (mg) (hrs) SakSTAR PUT M 66 Subacute I -emoro-femoral graft 6 5 Complete 2 23 Stenting left IF artery VERM M 73 Acute Right PA 2 6 Partial 13 23 Right upper leg amputation GEIV V 63 Restpain Left SFA 10 5 Complete 8 7 PTA POL M 46 Subacute Right SFA 30 50 Partial 22 29 Lumbal sympathectomie BUE F Η Claudication Right AF graft 1 15 Complete 10 13 Desobstruction VIJ F 75 Subacute Left FT graft 2 34 Complete 7 10 PTA C aΛ REN M 4 8 Restpain Right IF graft 4 20 Complete 6 5 5 Lett AF graft W COR V 78 Acute Left AFS 14 9 Complete 4 3 PTA MAN M 67 Restpain Left tibial artery 1 6 Partial 6 5 - H STRA M 66 Claudication Right FP graft 14 16 Complete 19 26 - 3 VANH M 38 Acute Left radial artery 4 1 Complete 6 5 - VANW F 57 Acute Right FP graft I 25 Complete 20 24 New πght FP graft BRA M 57 Acute Left FT graft 1 30 Complete 25 43 - 3 DON M 60 Claudication Left FT graft 1 20 Complete 13 19 PTA + stenting CAM M 77 Restpain Right SFA graft 8 30 Complete 27 44 FF graft CΛ Mean + SEM 62 ± 3 1 66 ± 2 1 18 ± 35 13 ± 2 1 19 ± 3 5 ω SakSTAR(K74Q,E80A,D82A,K130T,K135R) H 1MB M 66 Claudication Left SFA 30 5 Complete 24 24 PTA AZY M 44 Subacute Right C I A 7 8 Complete 18 23 Stenting VIN M 51 Acute Right E I A 5 70 Complete 24 30 - STRO M 53 Claudication Left FP junction 14 5 Partial 3 5 2 Aspiration thrombectomy, PTA VERG M 62 Restpain Left SFA 20 6 Complete 19 25 FP bypass GIE M 76 Acute Right FP bypass 2 15 Complete 8 5 17 - 9s Mean ± SEM 59 ± 4 7 13 ± 4 3 18 ± 10 16 ± 3 4 20 ± 4 0 SakSTAR ( E65D,K74R,E80A,D82A,K130T,K135R) URB M 57 Subacute Right E I A Complete Pseudo aneurysm, πght AF graft revision COM M S9 Reslpain Right AF graft 7 65 Complete 16 22 MAC M 70 Restpain Left antcπor tibial artery 7 15 Complete 12 14 DEW F 76 Restpain SFA 21 6 Complete 6 4 VAI F 65 Subacute Left PA 25 10 Partial 8 6 Aspiration thrombectomy FIL M 76 Claudication Right SFA 28 Complete 24 31 PTA Mean + SEM 67 + 34 15 ±43 I9 ± 94 12 ± 28 14±44 AF aortofemoral CABG coronary artery bypass graft CAD coronary artery disease. CIA common iliac artery. COPD chronic obstructive pulmonary disease, DM diabetes mellltus. EIA, external iliac artery. FF femorofibular. hfi 0rOf>OP FT femorollblal ,A ll ac aπe ry IF iliofemoral occl occlusion PA popliteal artery, PTA percutaneous transluminal angioplasty, SFA superficial femoral artery. TA tibial artery. TF tibioflbular SC Table 12: Absorption with SakSTAR variants of antibodies elicited with SakSTAR variants in patients with peripheral arterial occlusion CΛ cl Insolubilized compound W Treatment Absorbant SakSTAR SakSTAR(K74Q,E80A.D82A, l 30T.K135R) SakSTAR(E65D.K74R.E80A.D82A.K 130T.K135R) CΛ I H--. SakSTAR (Pool 40) H SakSTAR 95 cl SakSTAR(K74Q,E80A,D82A,Kl 30T.K 135R) 8 H W SalSTAR(E65D,K74R,E80A,D82A, 130T.K 135R) 57 !Λ a SakSTAR(K74Q,E80A,D82A,K130T,K135R) (Imb , Vin , Ver . Gie ) w w SakSTAR 94 95 95 H SakSTAR( 7 4 Q,E80A,D82A, 130T.K 135R) 91 93 89 SalSTAR(E65D.K74R,E80A,D82A, 130T.K 135R) 92 94 94 r SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) (Urb.) w SakSTAR 90 88 85 SakSTAR(K74Q,E80A,D82A,K 130T.K 135R) 94 95 94 SalSTAR(E 6 5D, 74R,E80A,D82A,K 130T.K 135R) 94 95 94 Data represent median values of the percent absorption with 250 nM absorbant, measured by residual binding to insolubilized compound. Table 13: Additive substitution mutagenesis of SakSTAR(E65Q,K74Q,K130T, K135R) with selected other amino acids CΛ (0 53 CΛ H cl (Λ a w H r w Spec. Act. > 100 kU/mg is represented in bold type. Absorption of antibodies (in percent) from pooled immunized patient plasma; values <60% are ON represented in bold type. CΛ c Table 14: Fibrinolytic properties of SakSTAR variants in human plasma in vitro W CΛ H Compound Fibrinolytic Residual fibrinogen Fibrinogenolytic Code potency at C50 potency (C50 in μg/ml) (% of baseline) (C50 in μg/ml) cl H W CΛ W SakSTAR 0.18 ±0.01 93 ± 3.5 24 ±3.6 w w SakSTAR(E65Q,K74Q,D82A,S84A,El 08A.K 109A. 130T. 135R. 13όA, 137K) 0.15 ±0.02 90 ± 5.0 14± 1.0 SY118 H SakSTAR(K35A.E65Q.K74Q,D82A,S84A;T90A.E99D,T101S,E108A.K109A,K130T,K135R,K136A,Vl37K) 0.17 ±0.01 87 ± 3.0 7 ±0.6 SY141 r SakSTAR(K35A.E65Q. 74R.D82A.S8 4 A;T90A.E99D.T101S.E108A,K109A.K130T.K13 5 R,K136A,V| 3 7K) 0.19±0.01 82 ±3.0 7 ±0.9 SY145 w κ» The data represent mean ± SD of 3 experiments. C50: amount of wild type or variant SakSTAR required for 50% clot lysis or 50% fibrinogen breakdown in the absence of fibrin in 2 hrs. Table IS: Characteristics of the patients with peripheral arterial occlusion treated with SakSTAR(E65Q,K74Q,D82A,S84A,E108A,K109A,K130T,K135R, K136A,V137K), SakSTAR(K35A,E65Q,K74Q,D82A,S84A,T90A.E99D,T101S,E108A,K109A,K130T,K135R,K136A > V 137K) or SakSTAR(K35A,E65Q > K74R,D82A,S84A,T90A,E99D,T101S, E108A,K109A,K130T,K135R,K136A,V137K) Compound Gen Age Clinical Risk factors Current Locus of Age of Length of Patient Id der (yrs) ischemia Relevant history Smoking occlusion occlusion occlusion (cm) (days) SakSTAR(E65Q,K74Q,D82A,S84A,E108A,K109A,K130T,K135R,K136A,Vl37K) (SY118) VCL M 69 Acute Hypertension, ischemic heart disease, Left AF graft 10 14 ABF graft REN M 76 Subacute Hypercholesterolemia Right PA 18 14 CΛ r HOL M 69 Acute Hypertension, hypercholesterolemia, Right FT bypass 16 3 0 right bypass 53 CΛ PAR M 79 Pain, swelling Left popliteal to communal femoral vein i 50 8 0 H FRA M 60 Subacute Ischemic heart disease, left FP graft Left FP graft 30 14 MAC V 73 Acute Hypertension, ABF graft Left branch ABF graft 2 0 10 d Mean ± SEM 71 ± 2 7 21 ± 6 9 l l ± 1 8 H SakSTAR(K35A,E65Q,K74Q,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T,K135R,K136A,Vl37K) (SY141) W VERH 52 Claudication Hypertension, hypercholesterolemia, Right IA 14 12 !Λ w right IF endoprothesis DUB M 54 Claudication Hypertension, stenting left, right IA Right EIA w 30 18 w VAP M 46 Claudication Hypertension, hypercholesterolemia, Aortabifurcation 21 25 H stenting left + right IA YN M 43 Claudication CAD, hypercholesterolemia, stenting left Left FP graft 5 0 30 g FP graft r HOR M 57 Acute Hypertension, left FP graft Left CIA, left FP graft 7 0 >60 w AND M 75 Acute Diabetes, hypertension, cardiac valve Left SF artery 3 0 10 replacements Mean ± SEM 55 ± 4 6 13 ± 4 3 19 ± 3 5 SakSTAR(K35A,E65Q,K74R,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T,K135R,K136A,Vl37K) (SY145) LIN F 48 Restpain Hypertension, ischemic heart disease Right SF artery 7 0 16 DEL M 68 Claudication Hypertension Left PA 21 6 0 LAM M 7 1 Acute FP graft Right FP graft 7 0 26 BAS M 64 Acute ABF graft, ischemic heart disease, Right SFA 3 0 6 0 hypertension TOU M 68 Acute Ischemic heart disease, hypertension Right PA 1 ,0 6 0 Mean ± SEM 64 ± 4 1 7 8 ± 3 5 12 ± 40 ABF Aortobifemoral, AF aortofemoral, CABG coronary artery bypass grafting, CAD, coronary artery disease, CIA common iliac artery, COPD chronic obstructive pulmonary disease, DM diabetes mellitus, EIA external iliac artery, FF femorofibular, FP femoropopliteal, FT fe orotibial, IA iliac artery, IF i l i ofemoral, PA popliteal artery, SFA superficial femoral artery, TF tibiofibular, AMI acute myocardial infarction lab Jώ: Treatment and outcome in patients with peripheral arterial occlusion, treated with SakSTAR(E65Q,K74Q,D82A,S84A,E108A,K109A, K130T,K135R,K136A,V137K), SakSTAR(K35A,E65Q,K74Q,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T,K135R,K136A, V137K) or SakSTAR(K35A,E65Q,K74R,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T, K135R,K136A,Vl37K) Compound Recanalization by Total dose of Total duration of Additional therapy Complications and remarks Patient thrombolysis thrombolytic agent infusion (hrs) Id (JUS ) CΛ SakSTAR(E65Q,K74Q,D82A,S84A,E108A,K109A,K130T,K135R,K136A,V137K) (SY118) VCL Complete 17 22 None Puncture site hematoma C ≡Λ REN Complete 24 22 None Puncture site hematoma HOL Complete 14 9 0 None None PAR Small subdural hematoma =. Partial (normal 10 6 0 C< patency with H residual thrombi W after first control) 24 22 New FP graft None CΛ FRA Complete MAC Complete 8 0 6 0 None Brain stem hemorrhage, death Mean ± SEM 16 ± 2 8 15 ± 34 W SakSTAR(K35A,E65Q,K74Q,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T,K135R,K136A,Vl37K) (SY141) H VERH Complete 15 18 Femorofemoral cross over DUB Complete 60 40 VAP Complete 14 22 RIA-stenting, bilateral IA stenting Puncture sue hematoma WYN Complete 23 29 FP graft revision None HOR Complete 10 8 0 None None AND Complete 13 17 None Retropeπtoneal hematoma, died due to septicemia OS Mean ± SEM 14 ± 2 3 16 ± 3 7 SalkSTAR(K35A,E65Q,K74R,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T,K135R,K136A,Vl37K) (SY145) LIN Complete 14 24 None Retropeπtoneal hematoma, Hemonragies DEL Complete 70 5 0 None None LAM Complete 23 30 None Puncture site hematoma BAS Complete None TOU Mean ± SEM 15 + 46 20 + 7 5 PTA, percutaneous translummal angioplasty, IF tliofemoral, FT femorotibial, FP femoropopliteal Table 17: Neutralizing antibody activity before and after administration of SakSTAR(E65Q,K74Q,D82A,S84A,E108A,K109A,K130T,K135R,K136A, V137K), SakSTAR(K35A,E65Q,K74Q,D82A,S84A ) T90A,E99D,T101S,E108A,K109A,K130T,K135R,K136A,Vl37K) or SakSTAR(K35A, E65Q.K74R, D82A, S84A,T90A,E99D,T101S,E108A,K109A,K130T,K135R,K136A,V137K) in patients with peripheral arterial occlusion Compound Neutralizing antibody activity (μg/ml) Patient Id. Before 3 weeks 4 weeks SakSTAR(E65Q,K74Q,D82A,S84A,E108A,K109A,K130T,K135R,K136A,Vl37K) (SY118) VCL 0.2 46 50 r /3 REN 0.1 1.6 0.9 cl HOL 0.2 22 18 S PAR 0.1 19 15 H FRA 1.2 15 39 MAC 0O - - C Median 0.15 19 18 H W CΛ SakSTAR(K35A,E65Q,K74Q,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T, K135R,K136A,Vl37K) (SY141) H VERH 0.2 0.3 0.2 2 DUB 0.2 4.3 2.0 _l VAP 0.0 0.0 0.0 !0 WYN 0.2 10 5.6 Cl HOR 0.2 0.1 0.0 g AND 0.8 - 100 Median 0.2 0.3 1.1 SakSTAR(K35A,E65Q,K74R,D82A,S84A,T90A,E99D,T101S,E108A,K109A,K130T, K135R,K136A,Vl37K) (SY145) LIN 0.0 5.5 3.8 DEL 0.2 1.6 1.9 LAM 0.2 164 190 BAS 0.1 TOU Median OΪ5 51 Ϊ8 Table 18: Immunogenicity of SakSTAR variants in patients with peripheral arterial occlusion CΛ cl 53 CΛ n Neutralizing activity Specific IgG Code H (μg/ml) >5 μg/ml (μg/ml) _* SakSTAR 69 12(4-100) 56 380(81- 1850) W SakSTAR(K74Q,E80A,D82A,K130T,K135R) 6 90(01-23) 3 420(31-730) SY15 CΛ W SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) 18 15(02-70) 5 30(24- 100) SY19 jU SakSTAR(E65Q.K74Q,D82A,S84A,E108A,K109A,K130T, l35R,K136A,V137K) 6 27(17-49) 5 2000(1300-3600) SY118 SakSTAR( 35A.E65Q,K74Q,D82A,S84A,T90A,E99D,T101S,EI08A,K109A,K130T,K135R,K136A,V137 6 07(01-43) 2 77(51-510) SY141 c5 C SakSTAR(K35A,E65Q,K74R,D82A,S84A,T90A,E99D,T101S,E108A,K109A, 130T,K135R,K136A,Vl37K 3 47 1 - SY145 > _ ON Data represent median and 15-85 percentile range ON Table 20: Cysteine-substitution variants of SakSTAR with reduced immunogenicity, substituted with maleimide-polyethylene glycol Fibrinolytic potency Code Specific Human Hamsters Clp Antibody activity plasma bolus (ml/mm) absorption (kU/mg) (C so μg/ml) (C !0 μg/kg) P40 ( ) CΛ SakSTAR 1 30 0 23 120 2 2 95 W CΛ H SY19 SakSTAR(E65D K74R E80A D82A K130T K135R) 1 4 0 0 2 4 3 7 57 cj SY19(S3C SP5)- SakSTAR(S3C-SP5 E65D K74R E80A D82A K130T K135R) 51 0 37 42 0 45 58 H W SY19(S3C MP5)- SakSTAR(S3C-MP5 E65D K74R E80A D82A K130T K135R) 20 0 65 0 28 50 CΛ SY19(S2C SP5 S3C SP5) SakSTAR(S2C-SP5,S3C-SP5 E65D K74R E80A D82A K130T K135R) 4 3 0 42 20 0 1 5 57 n w SYI 9(S3C P20) SakSTAR(S3C-P20 E65D K74R E80A D82A K130T K135R) 60 0 70 1 8 0 065 57 C w SY19(S3C P10) SakSTAR(S3C-P10 E65D K74R E80A D82A K130T K135R) 1 7 0 56 20 0 19 5 1 H SY141 SakSTAR(K35A E65Q K74Q D82A S84A T90A E99D T101 S E108A K109A K130T K135R K136A V137K) 3 700 0 1 9 0 95 24 r w SY141 (S3C SP5) SakSTAR(S3C-SP5 K35A E65Q K74Q D82A S84A T90A E99D T101 S E108A K109A K130T K135R K136A V1 37K) 1 200 0 24 1 2 1 8 SY141(S2C SP5 S3C SP5) SakSTAR(S2C-SP5 S3C-SP5 K35A E65Q K74Q D82A S84A T90A E99D T101 S E108A K109A K130T K135R K136A V1 37K) 1 4 00 0 28 1 8 SY160(S3C P20) SakSTAR(S3C-P20 K35A E65Q K74Q E80A D82A T90A E99D T101S E108A K109A K130T K135R) 65 0 33 6 0 08 32 SY161(S3C MP5) SakSTAR(S3C MP5 K35A E65Q K74R E80A D82A T90A E99D T101S E108A K109A K130T K135R) 71 0 36 1 5 0 56 35 SY161 (S3C P10) SakSTAR(S3C-P10 K35A E65Q K74R E80A D82A T90A E99D T101S E108A K109A K130T K135R) 66 0 40 9 0 1 5 38 SY161 (S3C P20) SakSTAR(S3C P20 K35A E65Q K74R E80A D82A T90A E99D T101 S E108A K109A K130T K135R) 1 55 0 32 8 0 04 44 *SP5 OPSS PEG 5 kDa MP5 AL PEG 5 kDa PIO MAL-PEG 10 kDa, P20 MAL PEG 20 kDa REFERENCES 1. 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Claims: CLAIMS 1. Staphylokinase derivatives showing a reduced immunogenicity as compared to wild-type staphylokinase, after administration to patients with arterial thrombosis. 2. Staphylokinase derivatives as claimed in claim 1 having essentially the amino acid sequence as depicted in figure 1 in which one or more amino acids have been replaced by another amino acid thus reducing the reactivity with a panel of murine monoclonal antibodies. 3. Staphylokinase derivatives as claimed in claim 1 having essentially the amino acid sequence as depicted in figure 1 in which one or more amino acids have been replaced by another amino acid thus reducing the absorption of SakSTAR-specific antibodies from plasma of patients treated with staphylokinase. 4. Staphylokinase derivatives as claimed in claim 1 having essentially the amino acid sequence as depicted in figure 1 in which one or more amino acids have been replaced by other amino acids, without reducing the specific activity by more than 50 percent. 5. Staphylokinase derivatives SakSTAR(K35X, G36X,E65X,K74X,E80X,D82X,K102X,E108X,K109X,K121X,K130X, K135X,K136X,+137X) having the amino acid sequence as depicted in figure 1 in which one or more of the amino acids Lys in position 35, Gly in position 36, Glu in position 65, Lys in position 74, Glu in position 80, Asp in position 82, Lys in position 102, Glu in position 108, Lys in position 109, Lys in position 121, Lys in position 130, Lys in position 135 and/or Lys in position 136 have been replaced with other amino acids and/or in which one amino acid has been added at the COOH-terminus, thus altering the immunogenicity after administration in patients, without markedly reducing the specific activity. 6. Staphylokinase derivatives listed in Tables 1, 3, 4, 5, 6, 7, 8, 13, 19 and 20, having the amino acid sequence as depicted in figure 1 in which the indicated amino acids have been replaced by other amino acids thus reducing the absorption of SakSTAR-specific antibodies from plasma of patients treated with staphylokinase, without reducing the specific activity. 7. Staphylokinase derivative as claimed in claims 1-6 selected from the group consisting of SakSTAR(K74A,E75A,R77A) , SakSTAR(K35A,E75A) , SakSTAR(E75A) , SakSTAR(E80A,D82A) , SakSTAR(E8OA) , SakSTAR(D82A) , SakSTAR(E75A,D82A) , SakSTAR(S34G, G36R, H43R) , SakSTAR(K35A) , SakSTAR(D82A) , SakSTAR(D82A, S84A) , SakSTAR(T90A) , SakSTAR(Y92A) , SakSTAR(K13OA) , SakSTAR(V132A) , SakSTAR(S34G,G36R,H43R) , SakSTAR(G36R) , SakSTAR(H43R) , SakSTAR(G36R,K74R) , SakSTAR(K35E) , SakSTAR(K74Q) , SakSTAR(Kl30T) , SakSTAR(V132L) , SakSTAR(V132T) , SakSTAR(V132N) , SakSTAR(V132R) , SakSTAR(K130T,K135R) , SakSTAR(G36R,K130T,K135R) , SakSTAR(K74R,K130T,K135R) , SakSTAR(K74Q,K130T,K135R) , SakSTAR(G36R,K74R,K130T,K135R) , SakSTAR(G36R,K74Q, K130T,K135R) , SakSTAR(G36R,H43R,K74R,K130T,K135R) , SakSTAR(E65A,K74Q,K130T,K135R) , SakSTAR(E65Q,K74Q, K130T,K135R) , SakSTAR(K74Q,K86A,K130T,K135R) , SakSTAR(E65Q,T71S,K74Q,K130T,K135R) , SakSTAR(K74Q, K130A,K135R) , SakSTAR(E65Q,K74Q,K130A,K135R) , SakSTAR(K74Q,K130E,K135R) , SakSTAR(K74Q,K130E, V132R,K135R) , SakSTAR(E65Q,K74Q,T90A,K130A,K135R) , SakSTAR(E65Q,K74Q,N95A,K130A,K135R) , SakSTAR(E65Q, K74Q, E118A,K130A,K135R) , SakSTAR(E65Q,K74Q,N95A,E118A,K130A, K135R) , SakSTAR(N95A, K130A, K135R) , SakSTAR(E65Q,K74Q, K109A,K130,K135R) , SakSTAR(E65Q,K74Q,E108A,K109A, K130T,K135R) , SakSTAR(E65Q,K74Q,K121A,K130T, K135R) , SakSTAR(E65Q,K74Q,N95A,E118A,K130A,K135R,K136A,+137K) , SakSTAR(E80A,D82A,K130T,K135R) , SakSTAR(K74R,E80A,D82A, K130T,K135R) , SakSTAR(K74Q,E80A,D82A,K130T,K135R) , SakSTAR(K35A,K74R,E80A,D82A,K130T,K135R) , SakSTAR(E65D, K74R,E80A,D82A,K130T,K135R) , SakSTAR(E65S,K74R,E80A, D82A,K130T,K135R) , SakSTAR(S34G,G36R,K74R,K130T, K135R) , SakSTAR(E65A,K74R,E80A,D82A,K130T,K135R) , SakSTAR(E65N, K74R,E80A,D82A,K130T,K135R) , SakSTAR(E65Q,K74R,E80A, D82A,K130T,K135R) , SakSTAR(K57A, E58A, E61A, E80A, D82A, K130T,K135R) , SakSTAR(E65D,K74Q,E80A, D82A,K130T, K135R) , SakSTAR(E65Q,K74Q,E80A,D82A,K130T,K135R) , SakSTAR(K35A, E65D,K74Q,E80A,D82A,K130T,K135R) , SakSTAR(K74R, E80A, D82A, S103A,K130T,K135R) , SakSTAR(E65D,K74R,E80A, D82A, K109A, K130T,K135R) , SakSTAR(E65D,K74R,E80A, D82A,K130T, K135R,K136A) , SakSTAR(E65Q, K74Q, D82A,S84A,K130T, K135R) , SakSTAR(K35A,K74Q,E80A,D82A,K130T,K135R) , SakSTAR(K35A, E65D,K74R,E80A,D82A,K130T,K135R) . 8. SakSTAR(E65D,K74R,E80A,D82A,K130T,K135R) having the code SY19. 9. SakSTAR(K35A,E65Q,K74R,E80A,D82A,T90A,E99D, T101S,E108A,K109A,K130T,K135R) having the code SY161. 10. Staphylokinase derivatives as claimed in claims 1-9 having an amino acid substituted with Cys, resulting in dimerization and/or increased specific activity and/or reduced clearance and/or increased thrombolytic potency. 11. Staphylokinase derivatives as claimed in claims 1-10 with polyethylene glycol substitution, characterized by a maintained specific activity and a significantly reduced plasma clearance. 12. Staphylokinase derivatives as claimed in claim 10 wherein the Cys is chemically modified with polyethylene glycol with molecular weights up to 20 kDa. 13. Staphylokinase derivatives as claimed in claim 12 wherein selected amino acids in the NH 2 -terminal region of 10 amino acids, are substituted with Cys, which is chemically modified with polyethylene glycol with molecular weights up to 20 kDa, which derivatives are characterized by a significantly reduced plasma clearance and maintained thrombolytic potency upon single intravenous bolus administration at a reduced dose. 14. Staphylokinase derivative as claimed in claim 13, wherein the serine in position 2 or 3 is substituted with a cystein and the cystein is chemically modified with polyethylene glycol having a molecular weight of 5, 10 or 20 kDa. 15. Staphylokinase derivative as claimed in claim 14, which derivative is SY161(S3C-MP5) as defined in table 20. 16. Staphylokinase derivative as claimed in claim 14, which derivative is SY161(S3C-P10) as defined in table 20. 17. Staphylokinase derivative as claimed in claim 14, which derivative is SY161 (S3C-P20) as defined in table 20. 18. Staphylokinase derivative as claimed in claim 14, which derivative is SY19(S3C-MP5) as defined in table 20. 19. Staphylokinase derivative as claimed in claim 14, which derivative is SY19(S3C-SP5) as defined in table 20. 20. Staphylokinase derivative as claimed in claim 14, which derivative is SY19(S2C-SP5,S3C-SP5) as defined in table 20. 21. Staphylokinase derivative as claimed in claim 14, which derivative is SY19(S3C-P20) as defined in table 20. 22. Staphylokinase derivative as claimed in claim 14, which derivative is SY19(S3C-P10) as defined in table 20. 23. Dimer of two staphylokinase derivatives as claimed in claim 10. 24. Method for producing the staphylokinase derivatives as claimed in claims 1 to 10, comprising the steps of: a. preparing a DNA fragment comprising at least the part of the coding sequence of staphylokinase that provides for its biological activity; b. performing in vitro site-directed mutagenesis on the DNA fragment to replace one or more RECTIFIED SHEET (RULE 91) codons for wild-type amino acids by a codon for another amino acid; c. cloning the mutated DNA fragment in a suitable vector; d. transforming or transfecting a suitable host cell with the vector; and e. culturing the host cell under conditions suitable for expressing the DNA fragment. 25. Method as claimed in claim 24, wherein the DNA fragment is a 453 bp EcoRI-Hindlll fragment of the plasmid pMEX602sakB, the in vitro site-directed mutagenesis is performed and the mutated DNA fragment is expressed in E. coli. 26. Pharmaceutical composition comprising at least one of the staphylokinase derivatives as claimed in claims 1 to 23 together with a suitable excipient. 27. Pharmaceutical composition as claimed in claim 26 for treating arterial thrombosis. RECTIFIED SHEET (RULE 91)

Inventors: COLLEN DESIRE JOSE, COLLEN, DESIRE, JOSE
Applicants: COLLEN DESIRE JOSE, LEUVEN RES & DEV VZW, COLLEN, DESIRE, JOSE, LEUVEN RESEARCH & DEVELOPMENT VZW
Application year: 1999
Main classification: C12N15
IPC codes: A61K38, A61P7, A61P9, C07K14, C12N1, C12N9, C12N15