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Desire Collen - One of the best experts on this subject based on the ideXlab platform.
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a dose finding clinical trial of Staphylokinase sy162 in patients with long term venous access catheter thrombotic occlusion
Journal of Thrombosis and Thrombolysis, 2007Co-Authors: Peter Verhamme, Desire Collen, Godelieve Goossens, Geert Maleux, M StasAbstract:Background We investigated the safety and efficacy of several dosing regimens of catheter-directed Staphylokinase (SY162) bolus administration for the treatment of long-term venous access catheter occlusion.
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elimination of a human t cell region in Staphylokinase by t cell screening and computer modeling
Thrombosis and Haemostasis, 2002Co-Authors: Petra A M Warmerdam, Desire Collen, Kristel Vanderlick, Petra Vandervoort, Stephane Plaisance, Kathleen Brepoels, Marc De MaeyerAbstract:Staphylokinase is a potent highly fibrin-selective thrombolytic agent, but it induces a humoral immune response in most treated patients. Staphylokinase-specific T-lymphocytes can be found in normal healthy individuals, from whom a large panel of Staphylokinase-specific T-cells were cloned. The Staphylokinase amino acid sequence 71-87 was widely recognized, as it induced proliferation of T-cell clones isolated from 90% of the donors. Computer modeling of this area, threaded as 11-mer peptides within the peptide-binding groove of the major HLA-DR alleles, indicated two putative partially overlapping binding sequences. The region-(71-87)-specific T-cell clones recognized either one or the other minimal peptide, confirming that both sequences could be functional T-cell epitopes. Furthermore, to guide the mutagenesis to eliminate T-cell reactivity, the contribution of each residue to the HLA-DR-anchoring and T-cell receptor exposure was evaluated for both binding motifs. Computer calculations combined with functional assays resulted in the design of Staphylokinase-variants, including 2 to 4 amino acid substitutions in the region 71-87. These variants were no longer recognized by the region-(71-87)-specific T-cell clones, and importantly no new Staphylokinase-variant-specific cellular immune response could be measured.
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Staphylokinase specific cell mediated immunity in humans
Journal of Immunology, 2002Co-Authors: Petra A M Warmerdam, Kristel Vanderlick, Petra Vandervoort, Heidi De Smedt, Stephane Plaisance, Marc De Maeyer, Desire CollenAbstract:Staphylokinase is a highly fibrin-specific clot-dissolving agent that constitutes a promising drug for clinical development. It is of bacterial origin, and the majority of patients develop neutralizing Ab after its administration. Several antigenic regions, recognized by these Ab, have been identified, but the underlying immunogenic features of Staphylokinase remain unknown. In this study, we show that Staphylokinase is a T cell-dependent Ag, and that an immunological memory may be acquired, even without administration of Staphylokinase. Thrombolysis with Staphylokinase provokes the proliferation of Staphylokinase-specific T lymphocytes, which remain elevated over 10 mo posttreatment. Interestingly, analysis of a large number of Staphylokinase-specific T cell clones isolated from 10 unrelated donors revealed only six distinct immunogenic regions in the molecule. Moreover, five of the six regions are recognized by T lymphocytes from several individuals, indicating that these regions are not restricted to a single HLA-DR allele. Therefore, these new insights can guide the design of variants with a lower immunogenic profile in humans.
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recombinant Staphylokinase variants with reduced antigenicity due to elimination of b lymphocyte epitopes
Blood, 2000Co-Authors: Yves Laroche, F De Cock, E Demarsin, Stephane Heymans, S Capaert, Desire CollenAbstract:Site directed mutagenesis (350 variants) of recombinant Staphylokinase (SakSTAR), a potent fibrin-selective thrombolytic agent, was undertaken in order to reduce its antigenicity while maintaining its potency. Variants with K35A, (ie, Lys[K] in position 35 substituted with Ala[A]), E65D or E65Q, K74R or K74Q, E80A+D82A, K130T, and K135R displayed increased enzymatic activity or reduced binding of human Staphylokinase-specific antibodies. Additive mutagenesis identified 8 variants with intact thrombolytic potencies, which absorbed down to less than a third of SakSTAR-specific antibodies. Intra-arterial administration in 61 patients with peripheral arterial occlusion caused no significant allergic reactions. Median neutralizing antibody titers (with 15 to 85 percentiles), expressed as microgram (μg) compound neutralized per milliliter plasma, were 4.4 (0.3 to 49) for the variants, compared with 12 (4 to 100) in 70 patients given wild-type SakSTAR ( P = .002 by Mann-Whitney rank sum test). Overt neutralizing antibody induction (more than 5 μg compound neutralized per milliliter plasma) was observed in 57 of 70 patients (81%) given wild-type SakSTAR, but only in 28 of 60 patients (47%) treated with variants ( P < .0001 by Fisher exact test). On the basis of this study, the variant SakSTAR (K35A, E65Q, K74R, D82A, S84A, T90A, E99D, T101S, E108A, K109A, K130T, K135R) (code SY155) has been selected for further clinical development.
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pharmacokinetic and thrombolytic properties of cysteine linked polyethylene glycol derivatives of Staphylokinase
Blood, 2000Co-Authors: Sophie Vanwetswinkel, Desire Collen, Stephane Plaisance, I Vanlinthout, Kathleen Brepoels, Zhang Zhiyong, Ignace Lasters, Laurent Stephane JespersAbstract:Recombinant Staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH2-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-specific (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecules with molecular weights of 5000 (P5), 10 000 (P10), or 20 000 (P20). The specific activities and thrombolytic potencies in human plasma were unaltered for most variants derivatized with PEG (PEGylates), but maleimide PEG derivatives had a better temperature stability profile. In hamsters, SakSTAR was cleared at 2.2 mL/min; variants with 1 P5 molecule were cleared 2-to 5-fold; variants with 2 P5 or 1 P10 molecules were cleared 10-to 30-fold; and variants with 1 P20 molecule were cleared 35-fold slower. A bolus injection induced dose-related lysis of a plasma clot, fibrin labeled with 125 iodine (125I-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 μg/kg SakSTAR; 50 to 110 μg/kg of core-substitution derivatives with 1 P5; 25 μg/kg for NH2-terminal derivatives with 1 P5; 5 to 25 μg/kg with derivatives with 2 P5 or 1 P10; and 7 μg/kg with P20 derivatives. Core substitution with 1 or 2 P5 molecules did not significantly reduce the immunogenicity of SakSTAR in rabbits. Derivatization of Staphylokinase with a single PEG molecule allows controllable reduction of the clearance while maintaining thrombolytic potency at a reduced dose. This indicates that mono-PEGylated Staphylokinase variants may be used for single intravenous bolus injection.
D. Collen - One of the best experts on this subject based on the ideXlab platform.
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FEASIBILITY STUDY OF A LIQUID FORMULATION OF RECOMBINANT Staphylokinase FOR CORONARY ARTERY THROMBOLYSIS
Fibrinolysis and Proteolysis, 1998Co-Authors: Peter Sinnaeve, I. Roelants, F. Van De Werf, D. CollenAbstract:Summary Recombinant Staphylokinase (Sak) is a highly fibrin-selective thrombolytic agent but the optimal dose and mode of administration remain to be defined. In view of its remarkable temperature stability (>1.5 years at 4°C), the feasibility of using a liquid formulation of recombinant Staphylokinase (Sak42D variant), which had been stored for 7 months at 4°C, was tested in 10 patients with evolving acute myocardial infarction. Intravenous (i.v.) infusion over 30 min of 30 mg Sak42D induced complete coronary patency (TIMI perfusion grade 3) within 90 min in eight patients, partial patency (TIMI grade 2) in one patient and persistent occulsion in one patient, who underwent angioplasty. At 24 h TIMI grade 3 flow was observed in all patients. No major treatment-related complication occurred. Fibrinogen levels at 90 min were not significantly different from baseline whereas α 2 -antiplasmin levels were 90% of baseline. Median antibody-related Sak42D-neutralizing activity in plasma was low at baseline (0.0 μg/mL) and after 1 week (0.0 μg/mL), but increased between day 7 and 10 to 12 μg/mL. Median Anti-Sak42D IgG concentration in plasma was 7.9 μg/mL at baseline, 28 μg/mL at 1 week and 190 μg/mL at 7 to 10 days. Thus, the liquid formulation of Sak42D induces efficient coronary artery recanalization while preserving circulating fibrinogen and is in this respect indistinguishable from preparations of recombinant Staphylokinase kept frozen until use. This ready to use liquid formulation of Sak42D appears to be suitable for formal phase II dose finding studies and phase III comparative trials.
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Recombinant Staphylokinase for thrombolytic therapy
Fibrinolysis and Proteolysis, 1997Co-Authors: Steven Vanderschueren, F. Van De Werf, D. CollenAbstract:Summary Staphylokinase, a 136 amino acid bacterial protein with profibrinolytic properties which is produced in high amounts by routine recombinant DNA technology, has a unique structure, mechanism of action and fibrin-specificity. Preclinical investigations revealed attractive properties for thrombolytic therapy, including high thrombolytic potency, also towards platelet-rich thrombi, fibrin-specificity in human plasma and high sensitivity to antifibrinolytic agents. A pilot recanalization study in 10 patients with evolving transmural myocardial infarction confirmed the fibrinspecificity of recombinant Staphylokinase (Sak). In patients with peripheral arterial occlusion, the recanalization rate and speed of Sak compare favorably with figures reported for established thrombolytic agents. A disadvantage of Sak is its antigenicity. Neutralizing antibody titers were low at baseline and during the first week after administration, but increased steeply and remained elevated thereafter, precluding repeated use. Surprisingly however, the antigenicity of Sak in laboratory animals and patients could be significantly attenuated while preserving thrombolytic potential, by replacing selected clustered charged amino acids by alanine. The clinical experience to date is encouraging yet limited and will need to be expanded to determine the optimal dose and mode of administration, the optimal conjunctive therapy, the value in the treatment of other thromboembolic disorders and, ultimately, the merits in terms of safety and survival relative to established and new thrombolytics. It remains to be seen whether the immunogenicity of Sak can be reduced to the extent that repeated therapy may be feasible without jeopardizing thrombolytic efficacy and safety.
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recombinant Staphylokinase variants with altered immunoreactivity iv identification of variants with reduced antibody induction but intact potency
Circulation, 1997Co-Authors: D. Collen, L Stockx, Henri Lacroix, Steven VanderschuerenAbstract:BACKGROUND: The thrombolytic potency and antibody induction of selected variants of recombinant Staphylokinase (SakSTAR), including SakSTAR(K74) with Lys74, SakSTAR(E75) with Glu75-, SakSTAR(EER) with Glu38, Glu75, and Arg77, and SakSTAR(K74ER) with Lys74, Glu75, and Arg77 replaced by Ala, were studied. METHODS AND RESULTS: In rabbits, SakSTAR(74) and SakSTAR(EER) elicited significantly less circulating neutralizing activity than SakSTAR and SakSTAR(E75) (P = .005 and P = .0002 versus SakSTAR, respectively). In baboons, SakSTAR(K74) induced significantly fewer antibodies than wild-type SakSTAR (P 10 micrograms compound neutralized/mL plasma) occurred in all 9 patients given wild-type SakSTAR, in 6 of the 11 SakSTAR(K74ER) patients (P = .038 versus SakSTAR), and in 2 of the 6 SakSTAR(K74ER) patients (P = .011 versus SakSTAR). CONCLUSIONS: SakSTAR(K74), a variant of recombinant Staphylokinase with a single substitution of Lys74 with Ala, and SakSTAR(K74), with Lys74, Glu75, and Arg77 substituted with Ala, have intact thrombolytic potencies but induce significantly less antibody formation in patients.
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recombinant Staphylokinase variants with altered immunoreactivity i construction and characterization
Circulation, 1996Co-Authors: D. Collen, H.r. Lijnen, R Bernaerts, Paul Declerck, F De Cock, E Demarsin, S Jenne, Yves Laroche, K Silence, M VerstrekenAbstract:BACKGROUND: Recombinant Staphylokinase offers promise for thrombolytic therapy in acute myocardial infarction, but it is immunogenic. Although reduced immunogenicity of heterologous proteinaceous drugs by protein engineering has not previously been reported, an attempt was made to achieve this in Staphylokinase by site-specific mutagenesis. METHODS AND RESULTS: Biospecific interaction analysis of a panel of 17 murine monoclonal antibodies against recombinant Staphylokinase (SakSTAR variant) identified three nonoverlapping immunodominant epitopes, two of which could be eliminated by substitution mutagenesis of clusters of two or three charged amino acids with alanine. Circulating anti-Staphylokinase antibodies elceted in patients by treatment with SakSTAR were incompletely (< 90%) absorbed by these mutants. Therefore, the combination variants K35A,E38A,K74A,E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A,D82A (SakSTAR.M89) were constructed, expressed in Escherichia coli, highly purified by ion-exchange and hydrophobic interaction chromatography, and characterized. These variants had specific activities that were approximately half that of SakSTAR, and they combined the reduced reactivity with the panels of monoclonal antibodies of their parent molecules. Absorption of circulating antibodies elicited in patients by treatment with SakSTAR was incomplete in 13 of 16 patients (median values, 68% and 65% with SakSTAR.M38 and SakSTAR.M89, respectively). CONCLUSIONS: SakSTAR contains three immunodominant epitopes, two of which were eliminated by site-directed mutagenesis, yielding combination mutants with relatively maintained specific activities that were not recognized by a significant fraction of the antibodies elicited in patients by treatment with wildtype SakSTAR. These mutants appear to be suitable for more detailed investigation of their thrombolytic and antigenic properties.
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Staphylokinase: fibrinolytic properties and current experience in patients with occlusive arterial thrombosis.
Verhandelingen - Koninklijke Academie voor Geneeskunde van België, 1995Co-Authors: D. Collen, H.r. Lijnen, Vanderschueren SAbstract:: Staphylokinase is a profibrinolytic agent that forms a 1:1 stoichiometric complex with plasminogen which, following conversion to plasmin, activates other plasminogen molecules to plasmin. The plasmin, Staphylokinase complex, unlike the plasmin, streptokinase complex, is rapidly inhibited by alpha 2-antiplasmin. In a plasma milieu, Staphylokinase is able to dissolve fibrin clots without associated fibrinogen degradation. This fibrin-specificity of Staphylokinase is the result of reduced inhibition by alpha 2-antiplasmin of plasmin, Staphylokinase complex bound to fibrin, recycling of Staphylokinase from the plasmin, Staphylokinase complex following inhibition by alpha 2-antiplasmin, and prevention of the conversion of plasminogen, Staphylokinase to plasmin, Staphylokinase by alpha 2-antiplasmin. 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. The feasibility of fibrin-specific coronary thrombolysis with an intravenous infusion over 30 min of 10 mg recombinant Staphylokinase was demonstrated in two small pilot studies in patients with acute myocardial infarction with angiographically confirmed total occlusion of the infarct-related coronary artery. However, neutralizing antibodies against Staphylokinase were demonstrable from the third week on in all patients. Definition of the therapeutic benefit of recombinant Staphylokinase will require more detailed dose-finding studies followed by randomized efficacy studies against other thrombolytic agents. An interim analysis after 50 patients of a randomized trial of recombinant tissue-type plasminogen activator versus Staphylokinase in patients with acute myocardial infarction revealed similar rates of coronary patency at 90 minutes but a significantly higher fibrin specificity of the latter compound.
Andrej Tarkowski - One of the best experts on this subject based on the ideXlab platform.
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Staphylococcus aureus: Staphylokinase
International Journal of Biochemistry and Cell Biology, 2006Co-Authors: Maria I. Bokarewa, Tao Jin, Andrej TarkowskiAbstract:Staphylokinase is a 136 aa long bacteriophage encoded protein expressed by lysogenic strains of Staphylococcus aureus. Present understanding of the role of Staphylokinase during bacterial infection is based on its interaction with the host proteins, α-defensins and plasminogen. α-Defensins are bactericidal peptides originating from human neutrophils. Binding of Staphylokinase to α-defensins abolishes their bactericidal properties, which makes Staphylokinase a vital tool for staphylococcal resistance to host innate immunity. Complex binding between Staphylokinase and plasminogen results in the formation of active plasmin, a broad-spectrum proteolytic enzyme facilitating bacterial penetration into the surrounding tissues. We have recently shown high levels of Staphylokinase expression in clinical isolates of skin and mucosal origin and relative low levels in isolates invading internal organs. These findings are supported by sepsis studies using isogenic S. aureus strains demonstrating increased bacterial load in the absence of Staphylokinase production. Our observations indicate that Staphylokinase favours symbiosis of staphylococci with the host that makes it an important colonization factor. © 2005 Elsevier Ltd. All rights reserved.
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staphylococcus aureus Staphylokinase
The International Journal of Biochemistry & Cell Biology, 2006Co-Authors: Maria I. Bokarewa, Andrej TarkowskiAbstract:Staphylokinase is a 136 aa long bacteriophage encoded protein expressed by lysogenic strains of Staphylococcus aureus. Present understanding of the role of Staphylokinase during bacterial infection is based on its interaction with the host proteins, α-defensins and plasminogen. α-Defensins are bactericidal peptides originating from human neutrophils. Binding of Staphylokinase to α-defensins abolishes their bactericidal properties, which makes Staphylokinase a vital tool for staphylococcal resistance to host innate immunity. Complex binding between Staphylokinase and plasminogen results in the formation of active plasmin, a broad-spectrum proteolytic enzyme facilitating bacterial penetration into the surrounding tissues. We have recently shown high levels of Staphylokinase expression in clinical isolates of skin and mucosal origin and relative low levels in isolates invading internal organs. These findings are supported by sepsis studies using isogenic S. aureus strains demonstrating increased bacterial load in the absence of Staphylokinase production. Our observations indicate that Staphylokinase favours symbiosis of staphylococci with the host that makes it an important colonization factor.
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human α defensins neutralize fibrinolytic activity exerted by Staphylokinase
Thrombosis and Haemostasis, 2004Co-Authors: Maria I. Bokarewa, Andrej TarkowskiAbstract:Defensins, cationic peptides with bacteriolytic properties, are abundantly found at inflammation sites and in human coronary vessels. Vascular occlusive diseases, such as myocardial infarction, pulmonary embolism, and peripheral arterial occlusion are presently treated by thrombolytic intervention using Staphylokinase, a plasminogen activator of bacterial origin. In this study we assessed a possible interaction between defensins and Staphylokinase, both molecules being present in an acutely ill patient. Using an ELISA-based system, we found that Staphylokinase and defensins displayed a strong and dose-dependent binding. In contrast, urokinase, another plasminogen activator of endogenous origin, displayed only minimal binding to defensins. Next, we proved that interaction between Staphylokinase and defensins led to fuctional consequences resulting in a significant decrease (p
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staphylococcus aureus resists human defensins by production of Staphylokinase a novel bacterial evasion mechanism
Journal of Immunology, 2004Co-Authors: Maria I. Bokarewa, Timothy J Foster, Jennifer Mitchell, Judy Higgins, Andrej TarkowskiAbstract:α-Defensins are peptides secreted by polymorphonuclear cells and provide antimicrobial protection mediated by disruption of the integrity of bacterial cell walls. Staphylokinase is an exoprotein produced by Staphylococcus aureus, which activates host plasminogen. In this study, we analyzed the impact of interaction between α-defensins and Staphylokinase on staphylococcal growth. We observed that Staphylokinase induced extracellular release of α-defensins from polymorphonuclear cells. Moreover, a direct binding between α-defensins and Staphylokinase was shown to result in a complex formation. The biological consequence of this interaction was an almost complete inhibition of the bactericidal effect of α-defensins. Notably, Staphylokinase with blocked plasminogen binding site still retained its ability to neutralize the bactericidal effect of α-defensins. In contrast, a single mutation of a Staphylokinase molecule at position 74, substituting lysine for alanine, resulted in a 50% reduction of its α-defensin-neutralizing properties. The bactericidal properties of α-defensins were tested in 19 S. aureus strains in vitro and in a murine model of S. aureus arthritis. Staphylococcal strains producing Staphylokinase were protected against the bactericidal effect of α-defensins. When Staphylokinase was added to Staphylokinase-negative S. aureus cultures, it almost totally abrogated the effect of α-defensins. Finally, human neutrophil peptide 2 injected intra-articularly along with bacteria alleviated joint destruction. In this study, we report a new property of Staphylokinase, its ability to induce secretion of defensins, to complex bind them and to neutralize their bactericidal effect. Staphylokinase production may therefore be responsible in vivo for defensin resistance during S. aureus infections.
H.r. Lijnen - One of the best experts on this subject based on the ideXlab platform.
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recombinant Staphylokinase variants with altered immunoreactivity i construction and characterization
Circulation, 1996Co-Authors: D. Collen, H.r. Lijnen, R Bernaerts, Paul Declerck, F De Cock, E Demarsin, S Jenne, Yves Laroche, K Silence, M VerstrekenAbstract:BACKGROUND: Recombinant Staphylokinase offers promise for thrombolytic therapy in acute myocardial infarction, but it is immunogenic. Although reduced immunogenicity of heterologous proteinaceous drugs by protein engineering has not previously been reported, an attempt was made to achieve this in Staphylokinase by site-specific mutagenesis. METHODS AND RESULTS: Biospecific interaction analysis of a panel of 17 murine monoclonal antibodies against recombinant Staphylokinase (SakSTAR variant) identified three nonoverlapping immunodominant epitopes, two of which could be eliminated by substitution mutagenesis of clusters of two or three charged amino acids with alanine. Circulating anti-Staphylokinase antibodies elceted in patients by treatment with SakSTAR were incompletely (< 90%) absorbed by these mutants. Therefore, the combination variants K35A,E38A,K74A,E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A,D82A (SakSTAR.M89) were constructed, expressed in Escherichia coli, highly purified by ion-exchange and hydrophobic interaction chromatography, and characterized. These variants had specific activities that were approximately half that of SakSTAR, and they combined the reduced reactivity with the panels of monoclonal antibodies of their parent molecules. Absorption of circulating antibodies elicited in patients by treatment with SakSTAR was incomplete in 13 of 16 patients (median values, 68% and 65% with SakSTAR.M38 and SakSTAR.M89, respectively). CONCLUSIONS: SakSTAR contains three immunodominant epitopes, two of which were eliminated by site-directed mutagenesis, yielding combination mutants with relatively maintained specific activities that were not recognized by a significant fraction of the antibodies elicited in patients by treatment with wildtype SakSTAR. These mutants appear to be suitable for more detailed investigation of their thrombolytic and antigenic properties.
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Staphylokinase: fibrinolytic properties and current experience in patients with occlusive arterial thrombosis.
Verhandelingen - Koninklijke Academie voor Geneeskunde van België, 1995Co-Authors: D. Collen, H.r. Lijnen, Vanderschueren SAbstract:: Staphylokinase is a profibrinolytic agent that forms a 1:1 stoichiometric complex with plasminogen which, following conversion to plasmin, activates other plasminogen molecules to plasmin. The plasmin, Staphylokinase complex, unlike the plasmin, streptokinase complex, is rapidly inhibited by alpha 2-antiplasmin. In a plasma milieu, Staphylokinase is able to dissolve fibrin clots without associated fibrinogen degradation. This fibrin-specificity of Staphylokinase is the result of reduced inhibition by alpha 2-antiplasmin of plasmin, Staphylokinase complex bound to fibrin, recycling of Staphylokinase from the plasmin, Staphylokinase complex following inhibition by alpha 2-antiplasmin, and prevention of the conversion of plasminogen, Staphylokinase to plasmin, Staphylokinase by alpha 2-antiplasmin. 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. The feasibility of fibrin-specific coronary thrombolysis with an intravenous infusion over 30 min of 10 mg recombinant Staphylokinase was demonstrated in two small pilot studies in patients with acute myocardial infarction with angiographically confirmed total occlusion of the infarct-related coronary artery. However, neutralizing antibodies against Staphylokinase were demonstrable from the third week on in all patients. Definition of the therapeutic benefit of recombinant Staphylokinase will require more detailed dose-finding studies followed by randomized efficacy studies against other thrombolytic agents. An interim analysis after 50 patients of a randomized trial of recombinant tissue-type plasminogen activator versus Staphylokinase in patients with acute myocardial infarction revealed similar rates of coronary patency at 90 minutes but a significantly higher fibrin specificity of the latter compound.
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interaction between Staphylokinase plasmin ogen and alpha 2 antiplasmin recycling of Staphylokinase after neutralization of the plasmin Staphylokinase complex by alpha 2 antiplasmin
Journal of Biological Chemistry, 1993Co-Authors: K Silence, D. Collen, H.r. LijnenAbstract:Abstract Although the plasminogen activating equimolar complex of Staphylokinase (STA) with human plasmin is very rapidly inhibited by alpha 2-antiplasmin, STA is a potent fibrinolytic agent in a human plasma milieu which contains 1 microM alpha 2-antiplasmin. In the present study, it was found that the complex of plasmin with recombinant STA (STAR), after neutralization with alpha 2-antiplasmin, retained the full plasminogen activating potential of STAR when added to a plasminogen solution (93 +/- 5% residual activity). When added to human plasma containing a 125I-fibrin-labeled plasma clot, equi-effective concentrations (causing 50% lysis in 2 h) were 17 +/- 3.0, 13 +/- 1.0, and 20 +/- 1.0 nM for STAR, equimolar plasmin-STAR mixtures, and plasmin-STAR mixtures neutralized by alpha 2-antiplasmin, respectively. Gel filtration of mixtures of plasmin(ogen) and STAR revealed elution as plasmin-STAR complex (Mr approximately 100,000), whereas after addition of alpha 2-antiplasmin, STAR eluted with an apparent Mr of 20,000. When mixtures of plasmin and STAR were adsorbed to lysine-Sepharose, STAR adsorbed quantitatively (96 +/- 1%) to the gel, whereas it was nearly quantitatively recovered in the unbound fraction (92 +/- 4%) after addition of alpha 2-antiplasmin to the mixture. Scatchard analysis of the binding of STAR to plasmin-Sepharose yielded a dissociation constant of 55 nM, whereas no specific binding of STAR to plasmin-alpha 2-antiplasmin-Sepharose could be demonstrated. These findings indicate that, both in purified systems and in a human plasma milieu containing a 125I-fibrin-labeled plasma clot, neutralization of the plasmin-STAR complex by alpha 2-antiplasmin results in dissociation of functionally active STAR from the complex and recycling of STAR to other plasminogen molecules. This dissociation-recycling process may explain the high fibrinolytic potency of STAR in a plasma milieu in the presence of high concentrations of alpha 2-antiplasmin.
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isolation and characterisation of natural and recombinant Staphylokinase
Fibrinolysis and Proteolysis, 1992Co-Authors: D. Collen, E Demarsin, K Silence, H.r. LijnenAbstract:Abstract Staphylokinase (STA), a Mr 18000 protein produced by Staphylococcus aureus has profibrinolytic properties (Lack CH, Nature 161, 559–560, 1948), but its potential for thrombolytic therapy has not been thoroughly investigated. Therefore we have elaborated procedures for the large scale production of natural (STAN) and recombinant (STAR) Staphylokinase. A strain of Staphylococcus aureus (strain no. 23), selected from 100 consecutive cultures at our Bacteriology Laboratory, was found to secrete up to 300 μg Staphylokinase per litre culture broth within 18h. The material, purified by batch adsorption on SP-Sephadex and chromatography on insolubilised active site-blocked plasmin with a yield of approximately 40 μg per l, migrated as a single band on SDS gel electrophoresis with Mr ≈ 16 500 and had NHZ-terminal sequence Lys-Gly-Asp-AspAla-. Restriction enzyme digested genomic DNA from this strain was hybridised with a degenerate 14-mer deoxyoligonucleotide encoding the NH2-terminal sequence, hybridising fragments were isolated and ligated into pUC19, competent E. coli cells were transformed, recombinant clones isolated and culture medium assayed for STA activity. A clone (subclone 159-2) transformed with recombinant pUC19 containing a 2.9 kb insert obtained by HindIII restriction enzyme digestion was found to secrete STAR without further manipulation (up to 20 mg/l into the culture broth and 30 mg/l into the periplasmic space). The material, purified by chromatography on insolubilised active-site blocked plasmin with a yield of 50 and 25%, respectively, contained three STAR variants with NH2-terminal sequence Ser-Ser-Ser-Phe-Asp-Lys-Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala- (STA-M), with Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala- (STA-Δ6) and with Lys-Gly-Asp-Asp-Ala- (STA-Δ10). Chromatography on CM-Sephadex yielded two peaks, STA-CM-I containing STA-Δ10 and STA-CM-11 containing STA-M and STA-Δ6. The specific activities of STAN, of STA-Δ10 obtained from STAR and of mixtures of STA-M and STA-Δ6 from STAR were indistinguishable by clot lysis and plasminogen coupled chromogenic substrate assays. Thus, highly purified preparations of natural (STAN) and recombinant (STAR) Staphylokinase can be obtained by simple and straightforward techniques in sufficiently large amounts to allow detailed investigation of their biochemical and thrombolytic properties.
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Comparative fibrinolytic and fibrinogenolytic properties of Staphylokinase and streptokinase in plasma of different species in vitro
Fibrinolysis and Proteolysis, 1992Co-Authors: H.r. Lijnen, F De Cock, Osamu Matsuo, D. CollenAbstract:Abstract The comparative fibrinolytic and fibrinogenolytic properties of Staphylokinase and streptokinase were studied in plasma of the human, baboon, rabbit, hamster, rat and dog species in vitro. Equipotent concentrations of Staphylokinase and streptokinase were determined as the concentrations that caused 50% lysis in 2 h (C 50 ) of 60 μl 125 I-fibrin labelled autologous or human plasma clots submersed in 250 μl citrated plasma. In addition, residual fibrinogen levels at C 50 were determined. In human plasma, Staphylokinase was 4-fold more potent than streptokinase (C 50 of 17 and 68 nM respectively) and was more fibrin-specific (residual fibrinogen level 95 and 50 of 32 nM, 27 nM and 36 nM vs. 370 nM, 270 nM and >450 nM respectively) or human plasma clots submersed in autologous plasma (C 50 of 24nM, 24nM and 17nM vs. 100nM, 87nM and 10nM respectively). Residual fibrinogen levels at C 50 in the autologous systems were ⩾80% of baseline. The autologous rat system was very resistant to lysis with both Staphylokinase and streptokinase (less than 5% clot lysis at concentrations of 500 nM), whereas in the dog system Staphylokinase appeared to be 60-fold more potent than streptokinase (C 50 of 7 nM and 430 nM respectively). These results indicate that the plasma fibrinolytic systems of baboons, rabbits and hamsters react comparably to the human system in terms of their fibrinolytic and fibrinogenolytic response to Staphylokinase. The rat system appears to be more resistant and the dog system more sensitive.
Bernhard Schlott - One of the best experts on this subject based on the ideXlab platform.
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the ternary microplasmin Staphylokinase microplasmin complex is a proteinase cofactor substrate complex in action
Nature Structural & Molecular Biology, 1998Co-Authors: Marina A A Parry, Karlheinz Guhrs, Bernhard Schlott, Carlos Fernandezcatalan, Andreas Bergner, Robert Huber, Karlpeter Hopfner, Wolfram BodeAbstract:The serine proteinase plasmin is the key fibrinolytic enzyme that dissolves blood clots and also promotes cell migration and tissue remodeling. Here, we report the 2.65 A crystal structure of a ternary complex of microplasmin–Staphylokinase bound to a second microplasmin. The Staphylokinase 'cofactor' does not affect the active-site geometry of the plasmin 'enzyme', but instead modifies its subsite specificity by providing additional docking sites for enhanced presentation of the plasminogen 'substrate' to the 'enzymes's' active site. The activation loop of the plasmin 'substrate', cleaved in these crystals, can be reconstructed to show how it runs across the active site of the plasmin 'enzyme' prior to activation cleavage. This is the first experimental structure of a productive proteinase–cofactor–macromolecular substrate complex. Furthermore, it provides a template for the design of improved plasminogen activators and plasmin inhibitors with considerable therapeutical potential.
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Nuclear magnetic resonance solution structure of the plasminogen-activator protein Staphylokinase.
Biochemistry, 1998Co-Authors: Oliver Ohlenschläger, Karlheinz Guhrs, Bernhard Schlott, Ramadurai Ramachandran, Larry R. BrownAbstract:: Staphylokinase, a 15.5 kDa protein from Staphylococcus aureus, is a plasminogen activator which is currently undergoing clinical trials for the therapy of myocardial infarction and peripheral thrombosis. The three-dimensional (3D) NMR solution structure has been determined by multidimensional heteronuclear NMR spectroscopy on uniformly 15N- and 15N,13C-labeled samples of Staphylokinase. Structural constraints were obtained from 82 3JHNH alpha as well as 22 3JNH beta scalar coupling constants and 2345 NOE cross-peaks, derived from 15N-edited and 13C-edited 3D NOE spectra. NOE cross-peak assignments were confirmed by analysis of ?15N,13C?-edited and ?13C,13C?-edited 4D NOE spectra. The structure is presented as a family of 20 conformers which show an average rmsd of 1.02 +/- 0.15 A from the mean structure for the backbone atoms. The tertiary structure of Staphylokinase shows a well-defined global structure consisting of a central 13-residue alpha-helix flanked by a two-stranded beta-sheet, both of which are located above a five-stranded beta-sheet. Two of the connecting loops exhibit a higher conformational heterogeneity. Overall, Staphylokinase shows a strong asymmetry of hydrophilic and hydrophobic surfaces. The N-terminal sequence, including Lys10 which is the site of the initial proteolytic cleavage during activation of plasminogen, folds back onto the protein core, thereby shielding amino acids with functional importance in the plasminogen activation process. From a comparison of the structure with mutational studies, a binding region for plasminogen is proposed.
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functional significance of nh2 and cooh terminal regions of Staphylokinase in plasminogen activation
Thrombosis and Haemostasis, 1996Co-Authors: Ariane Gase, Desire Collen, Detlev Behnke, Karlheinz Guhrs, Manfred Hartmann, Anja Rocker, Bernhard SchlottAbstract:: Structure/function relationships in the activation of plasminogen with Staphylokinase were studied using mutants of recombinant Staphylokinase (Sak42D). Deletion of up to 10 NH2-terminal amino acids (Sak42D delta N10) did not affect plasminogen activation, but removal of 11 amino acids completely abolished the ability to activate plasminogen. Elimination of potential plasmin cleavage sites in the NH2-terminal region yielding mutants Sak42D(K8H,K10H,K11H) and Sak42D(K6H,K8H,K11H) did not alter the rate of the exposure of a proteolytically active site (amidolytic activity) in equimolar mixtures with plasminogen, but destroyed the plasminogen activator properties of these muteins. Deleting two residues following the preferred processing site at position 10 (Sak42 delta (K11,G12)) resulted in a mutein also inactive in plasminogen activation. Removal of the COOH-terminal Lys136, yielding Sak42D delta C1, or of Lys135 and Lys136 in Sak42D delta C2 resulted in proteins with strongly reduced plasminogen activation capacity. In contrast, substitution of Lys135 and Lys136 with Ala in Sak42D(K135A,K136A) did not affect activation. Cyanogen bromide cleavage of Sak42D(M26L,E61M,D82E) produced a 61 amino acid NH2-terminal and a 65 amino acid COOH-terminal fragment which did not activate plasminogen, but bound to plasminogen with affinity constants Ka of 4.0 x 10(5) M-1 and 1.4 x 10(7) M-1, respectively (as compared to a Ka of 1.1 x 10(8) M-1 for Sak42D). These results indicate that Lys11 and the COOH-terminal region of Staphylokinase play a key role in the activation of plasminogen.
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characterization of the interaction between plasminogen and Staphylokinase
FEBS Journal, 1994Co-Authors: Roger H Lijnen, F De Cock, B Van Hoef, Bernhard Schlott, Desire CollenAbstract:Binding parameters [association (ka) and dissociation (kd) rate constants, and affinity constants (Ka=ka/kd)] for the interaction between recombinant Staphylokinase (SakSTAR) and plasmin(ogen) were determined by real-time biospecific interaction analysis. The Ka value for binding of SakSTAR to native human Glu-plasminogen was 0.93×108M-1 as compared to 2.0×108M-1 and 1.6×108M-1, respectively, for the binding to [S741A]recombinant plasminogen or Lys-[S741A]recombinant plasminogen (intact or proteolytically degraded plasminogen with the active site Ser741 replaced by alanine). Binding of SakSTAR to active plasmin or to active-site blocked plasmin occurred with Ka, values of 4.0×108M-1 and 8.4×108M--1, respectively, whereas active-site blocked LMM-plasmin (a plasmin derivative lacking kringles 1– 4) and the plasmin B-chain bound with Ka values of 1.0×108M-1 and 0.49×108M-1, respectively. Lysine-binding site I (a plasminogen derivative consisting of kringles 1–3) and lysine-binding site II (a plasminogen derivative consisting of kringle 4) bound with much lower affinity (Ka values of 1.2×105M-1 and 2.9×105M-1, respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative Ka values. The Ka values for binding of the plasmin-SakSTAR complex to streptokinase and binding of the plasmin-streptokinase complex to SakSTAR, were, respectively, 44-fold and 30-fold lower than the values for free plasmin. The Ka for binding of plasminogen to the inactive mutants [M26R]Sak42D or [M26A]Sak42D (site-specific mutagenesis of Met26 to arginine or alanine) were 10–20-fold lower than that of native Staphylokinase. These results indicate that: (a) the affinity of Staphylokinase for Glu-plasminogen and Lysplasminogen is comparable; (b) the active site in the plasmin molecule is not required for binding; (c) kringle structures 1– 4 of plasminogen do not contribute significantly to plasminogen binding of Staphylokinase; (d) Met26 in Staphylokinase is important for its high-affinity binding to plasminogen; (e) the binding sites on plasmin for Staphylokinase and streptokinase overlap at least partially.
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high yield production and purification of recombinant Staphylokinase for thrombolytic therapy
Nature Biotechnology, 1994Co-Authors: Bernhard Schlott, Steven Vanderschueren, Desire Collen, Frans Van De Werf, Karlheinz Guhrs, Manfred Hartmann, Eckhard Birchhirschfeid, Hansdieter Pohl, Armand Michoel, Detlev BehnkeAbstract:Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR Staphylokinase genes were replaced by an ATG start codon and which express Staphylokinase under the control of a tac promoter and two Shine–Dalgarno sequences in tandem. Induction of transfected E. coli TGI cells in a bacterial fermentor produced intracellular Staphylokinase representing 10 to 15% of total cell protein, Gram quantities of highly purified recombinant Staphylokinase were obtained from cytosol fractions by chromatography, at room temperature, on SP–Sepharose and on phehyl–Sepharose columns, with yields of 50 to 70 percent. The material, at a dose of 4 mg/kg, did not produce acute reactions or affect body weight in mice. Intravenous administration of 10 mg SakSTAR over 30 minutes in five patients with acute myocardial infarction induced complete coronary artery recanaiization, without associated fibrinogen degradation. However, neutralizing antibodies appeared in the plasma of all patients within 12 to 20 days. Thus, the present expression and purification method for recombinant Staphylokinase yields large amounts of highly purified mature protein (approximately 200 mg per liter fermentation broth) suitable for a more detailed clinical investigation of its potential as a thrombolytic agent.
