A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy

To develop a fast-acting clot dissolving agent, a clot-targeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein inBacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced fromB. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues. To develop a fast-acting clot dissolving agent, a clot-targeting domain derived from the Kringle-1 domain in human plasminogen was fused to the C-terminal end of staphylokinase with a linker sequence in between. Production of this fusion protein inBacillus subtilis and Pichia pastoris was examined. The Kringle domain in the fusion protein produced fromB. subtilis was improperly folded because of its complicated disulfide-bond profile, whereas the staphylokinase domain produced from P. pastoris was only partially active because of an N-linked glycosylation. A change of the glycosylation residue, Thr-30, to alanine resulted in a non-glycosylated biologically active fusion. The resulting mutein, designated SAKM3-L-K1, was overproduced in P. pastoris. Each domain in SAKM3-L-K1 was functional, and this fusion showed fibrin binding ability by binding directly to plasmin-digested clots. In vitro fibrin clot lysis in a static environment and plasma clot lysis in a flow-cell system demonstrated that the engineered fusion outperformed the non-fused staphylokinase. The time required for 50% clot lysis was reduced by 20 to 500% under different conditions. Faster clot lysis can potentially reduce the degree of damage to occluded heart tissues. tissue plasminogen activator ε-amino-N-caproic acid platelet poor plasma staphylokinase staphylokinase-linker-Kringle-1 fusion time required for 50% clot lysis matrix-assisted laser desorption ionization-time of flight mass spectrometry enzyme-linked immunosorbent assay HEPES-buffered saline Thrombolysis (1Guzman L.A. Lincoff A.M. J. Thromb. Thrombolysis. 1997; 4: 337-343Crossref PubMed Scopus (1) Google Scholar, 2Hennekens C.H. O'Donnell C.J. Ridker P.M. Marder V.J. J. Am. Coll. Cardiol. 1995; 25 Suppl. 7: S18-S22Crossref Scopus (32) Google Scholar, 3Tsikouris J.P. Tsikouris A.P. Pharmacotherapy. 2001; 21: 207-217Crossref PubMed Scopus (37) Google Scholar, 4Sinnaeve P. van de W.F. Thromb. Res. 2001; 103 Suppl. 1: S71-S79Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) is one of the well established treatments for patients with acute myocardial infarction (commonly known as heart attack). Blood clot-dissolving agents currently approved for thrombolytic therapy include tissue plasminogen activator (tPA),1 urokinase, streptokinase, and their derivatives (3Tsikouris J.P. Tsikouris A.P. Pharmacotherapy. 2001; 21: 207-217Crossref PubMed Scopus (37) Google Scholar, 4Sinnaeve P. van de W.F. Thromb. Res. 2001; 103 Suppl. 1: S71-S79Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Although the treatment can reduce mortality, several large scale clinical trials indicate that these blood clot-dissolving agents are far from ideal (4Sinnaeve P. van de W.F. Thromb. Res. 2001; 103 Suppl. 1: S71-S79Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 5The GUSTO Angiographic Investigators N. Engl. J. Med. 1993; 329: 1615-1622Crossref PubMed Scopus (1884) Google Scholar). Even with tPAs, only about 60% of the treated patients have their blood flow restored 90 min after the onset of treatment (5The GUSTO Angiographic Investigators N. Engl. J. Med. 1993; 329: 1615-1622Crossref PubMed Scopus (1884) Google Scholar). Some of the treated patients will suffer from reocclusion or bleeding complications such as hemorrhagic strokes (2Hennekens C.H. O'Donnell C.J. Ridker P.M. Marder V.J. J. Am. Coll. Cardiol. 1995; 25 Suppl. 7: S18-S22Crossref Scopus (32) Google Scholar, 3Tsikouris J.P. Tsikouris A.P. Pharmacotherapy. 2001; 21: 207-217Crossref PubMed Scopus (37) Google Scholar, 6The InTIME-II Investigators Eur. Heart J. 2000; 21: 2005-2013Crossref PubMed Scopus (182) Google Scholar). Furthermore, the reoccluded clots are usually platelet-rich and are more resistant to tPA-mediated clot lysis (7Zhu Y. Carmeliet P. Fay W.P. Circulation. 1999; 99: 3050-3055Crossref PubMed Scopus (159) Google Scholar, 8Serizawa K. Urano T. Kozima Y. Takada Y. Takada A. Thromb. Res. 1993; 71: 289-300Abstract Full Text PDF PubMed Scopus (17) Google Scholar). Hence, choices of more potent blood clot dissolving agents, which provide a rapid, complete, and sustained reperfusion with minimal side effects, are needed. Staphylokinase (SAK), a 136-amino acid protein from certain lysogenicStaphylococcus aureus strains, is a plasminogen activator and a promising blood clot-dissolving agent with clinical potency that is at least as good as tPA (9Vanderschueren S. Barrios L. Kerdsinchai P. Van den Heuvel P. Hermans L. Vrolix M. De Man F. Benit E. Muyldermans L. Collen D. Van de Werf F. Circulation. 1995; 92: 2044-2049Crossref PubMed Scopus (163) Google Scholar, 10Vanderschueren S. Dens J. Kerdsinchai P. Desmet W. Vrolix M. De Man F. Van den Heuvel P. Hermans L. Collen D. Van de Werf F. Am. Heart J. 1997; 134: 213-219Crossref PubMed Scopus (65) Google Scholar). In addition, it has some desirable features that are superior to tPA (11Collen D. Nat. Med. 1998; 4: 279-284Crossref PubMed Scopus (153) Google Scholar). Notably, SAK mediates the lysis of platelet-rich and retracted clots efficiently (12Suehiro A. Tsujioka H. Yoshimoto H. Ueda M. Higasa S. Kakishita E. Thromb. Res. 1995; 80: 135-142Abstract Full Text PDF PubMed Scopus (5) Google Scholar, 13Hauptmann J. Glusa E. Blood Coagul. Fibrinolysis. 1995; 6: 579-583Crossref PubMed Scopus (13) Google Scholar) and shows exceptional fibrin specificity (9Vanderschueren S. Barrios L. Kerdsinchai P. Van den Heuvel P. Hermans L. Vrolix M. De Man F. Benit E. Muyldermans L. Collen D. Van de Werf F. Circulation. 1995; 92: 2044-2049Crossref PubMed Scopus (163) Google Scholar, 10Vanderschueren S. Dens J. Kerdsinchai P. Desmet W. Vrolix M. De Man F. Van den Heuvel P. Hermans L. Collen D. Van de Werf F. Am. Heart J. 1997; 134: 213-219Crossref PubMed Scopus (65) Google Scholar, 14Sakharov D.V. Lijnen H.R. Rijken D.C. J. Biol. Chem. 1996; 271: 27912-27918Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 15Silence K. Collen D. Lijnen H.R. J. Biol. Chem. 1993; 268: 9811-9816Abstract Full Text PDF PubMed Google Scholar). These properties can help minimize reocclusion and bleeding complications. Much has been studied on the action of SAK in vivo. To function as the plasminogen activator, SAK first forms a complex with plasmin(ogen) (16Parry M.A. Fernandez-Catalan C. Bergner A. Huber R. Hopfner K.P. Schlott B. Guhrs K.H. Bode W. Nat. Struct. Biol. 1998; 5: 917-923Crossref PubMed Scopus (134) Google Scholar). Complex formation is followed by SAK processing in which the N-terminal peptide containing the first 10 amino acids from SAK is removed by cleaving at a twin lysine site between residues 10 and 11 (Fig. 1A). This processing step is essential for attaining an active form of SAK (17Gase A. Hartmann M. Guhrs K.H. Rocker A. Collen D. Behnke D. Schlott B. Thromb. Haemostasis. 1996; 76: 755-760Crossref PubMed Scopus (18) Google Scholar, 18Schlott B. Guhrs K.H. Hartmann M. Rocker A. Collen D. J. Biol. Chem. 1998; 273: 22346-22350Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 19Szarka S. Sihota E. Habibi H.R. Wong S.-L. Appl. Environ. 1999; PubMed Google Scholar). The complex forms a complex with of plasminogen and this plasminogen to the complex is it can by the in In the plasminogen activator complex is more resistant to K. Collen D. Lijnen H.R. J. Biol. Chem. 1993; 268: 9811-9816Abstract Full Text PDF PubMed Google Scholar). The is a plasminogen by SAK at the fibrin that to the fibrin specificity of SAK in a plasma This fibrin specificity is by the binding of SAK to plasmin(ogen) that is D.V. Lijnen H.R. Rijken D.C. J. Biol. Chem. 1996; 271: 27912-27918Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The of SAK an in clinical The in patients treated with SAK to whereas patients treated with tPA have of and in their plasma (9Vanderschueren S. Barrios L. Kerdsinchai P. Van den Heuvel P. Hermans L. Vrolix M. De Man F. Benit E. Muyldermans L. Collen D. Van de Werf F. Circulation. 1995; 92: 2044-2049Crossref PubMed Scopus (163) Google Scholar, 10Vanderschueren S. Dens J. Kerdsinchai P. Desmet W. Vrolix M. De Man F. Van den Heuvel P. Hermans L. Collen D. Van de Werf F. Am. Heart J. 1997; 134: 213-219Crossref PubMed Scopus (65) Google Scholar). Although SAK is a thrombolytic agent, it has fibrin binding ability by to fibrin clots only the with of to the clot lysis of SAK can by it with fibrin binding Faster clot lysis will blood flow in a more and reduce damage to heart tissues. Furthermore, SAK is required to the degree of clot the for side can to fibrin clots its Kringle at the N-terminal 1997; Google Scholar). These Kringle are amino acids some of the lysine binding N. A. S. 1993; PubMed Scopus (37) Google Scholar, K.P. A. Blood Coagul. Fibrinolysis. 5: PubMed Scopus Google Scholar, P. A. 1996; PubMed Scopus Google Scholar). plasminogen to fibrin clots clot lysis more C-terminal lysine residues are the action of a that after lysine and the from human plasminogen W. D. Eur. J. PubMed Scopus Google Scholar, N. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. B. A. 1998; PubMed Scopus Google Scholar, D. J. B. Eur. J. PubMed Scopus Google Scholar) and tPA M. 1995; PubMed Scopus Google Kringle-1 from human plasminogen has the lysine binding Kringle-1 was as the fibrin domain and fused to the C-terminal end of SAK To that domain the fusion can and that has to with its a acid linker is between these The resulting fusion and its and produced from subtilis and Pichia pastoris. clot and clot lysis of the and its derivatives In with the engineered SAKM3-L-K1 lysis of fibrin and plasma clots in is a B. S.-L. PubMed Scopus Google Scholar) for of under of subtilis for and the sequence for To the linker sequence between SAK and the Kringle-1 domain from human the sequence of the was The first from the to the of the linker The sequence was by R. S. Sihota S. Wong S.-L. 1999; PubMed Google Scholar) as The for this and The resulting was an with the was S.-L. PubMed Scopus Google Scholar) at the in the of the to The of the a sequence the C-terminal of the linker and the Kringle-1 This sequence was by a of human plasminogen as This was by T. A. at the of and of The the sequence for the C-terminal of the linker and the of the Kringle-1 The a at the end of the Kringle-1 sequence and an site at the end of the The resulting was with and and the in the of the to To the N-linked glycosylation site in was to and to and The change of to resulted in the of These by A. N. Res. PubMed Scopus Google Scholar) the in with as the The was and the was treated with and subtilis W. L. Wong S.-L. J. PubMed Scopus Google Scholar). The resulting for the by of the Each of the was as an to and These the of the of on SAK in B. of amino acids in the in the glycosylation The to the of with to or in that to are in in a The amino acids in the in the glycosylation The to the of with to or in that to are in and to of these of the sequence in the P. pastoris was as the for with as the and as the A was at the end and at the The sequence was with and to the to was to E. and for to and the with and as the or was with and pastoris to the on and and for of or with and to as the and protein a Pichia the of SAKM3-L-K1 was for at in 1999; PubMed Scopus Google Scholar). at for min and to a in 1999; PubMed Scopus Google Scholar). at in a Production of SAKM3-L-K1 was by the of the was by the at for min and to a with binding the with of the binding SAKM3-L-K1 was with acid to and of SAKM3-L-K1 by and The the binding and by SAKM3-L-K1 was at a of PubMed Scopus Google Scholar). SAKM3-L-K1 in was with the of acid and on an mass with The in the with an This was at the SAKM3-L-K1 was and at as a at 25 in the and for was as the The change binding was for the of of that was in a by the containing was to the fibrin binding ability of SAKM3-L-K1 and fibrin was on the of a the by Y. R. Habibi H.R. Wong S.-L. Appl. Environ. PubMed Scopus Google Scholar). of fibrin was by Y. R. Habibi H.R. Wong S.-L. Appl. Environ. PubMed Scopus Google Scholar). The fibrin was with at at different time the was removed by with SAKM3-L-K1 or SAK in containing was to the at a of at removed by SAKM3-L-K1 or SAK on the well was with SAK R. S. Sihota S. Wong S.-L. 1999; PubMed Google Scholar) followed by The of was as the to the at end was at a The was clots by human and 20 to human in HEPES-buffered saline and from after of the fibrin to the of a formation was to for at The of the clots was with and was A containing human plasminogen and of SAKM3-L-K1 or SAK in was on The in clot with time by in the at at 25 the in the min the of clot was with to SAKM3-L-K1 or of plasminogen in on the and was the which was as of clot for of SAKM3-L-K1 or SAK in and the was a some clots only with treated the Blood was by from in the of plasma was from the blood by at for min and at in plasma clot of blood an flow was on the by 1998; 92: PubMed Google Scholar). was by human and to of the plasma was to the flow to the and the and was to for at The clot was with for 20 min at 20 followed by with SAKM3-L-K1 or SAK in 20 with the flow by a The change in at at was a with a The was for SAKM3-L-K1 and was for The sequence of was to of by at the and of of SAK and of SAK and SAKM3-L-K1 followed the by S. Sihota E. Habibi H.R. Wong S.-L. Appl. Environ. 1999; PubMed Google Scholar). assay with the was as by Wong S.-L. R. S. Appl. Environ. PubMed Google Scholar). was produced as a protein in the B. subtilis an Y. R. Habibi H.R. Wong S.-L. Appl. Environ. PubMed Scopus Google as the showed that and SAK for plasminogen more of was in the with a the been to that the Kringle domain in the fusion protein was with lysine in the The a Kringle-1 domain in the fusion This is the Kringle-1 domain of in a residues are in a to form or of residues can in the formation of Kringle-1 M. L. S. A. 80: PubMed Scopus Google Scholar). human plasminogen Kringle-1 domain has been to produced efficiently from P. pastoris J. Y. J. Biol. 2000; PubMed Scopus Google it a to in P. pastoris. P. pastoris as the of the fusion protein has P. pastoris has been to SAK only in a partially active form because of an N-linked glycosylation at of the SAK M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). To glycosylation of in P. residues in that of the glycosylation site to the SAK The first and have to and whereas the SAKM3-L-K1 has to To the of these on SAK the first produced in B. subtilis Although the of these and the from B. subtilis that and SAKM3-L-K1 and of the SAK and SAKM3-L-K1 to the P. pastoris for of or SAKM3-L-K1 from of P. pastoris was in a time from to of the with with of the the of the the of of and SAKM3-L-K1 designated and and to these for known of SAKM3-L-K1 as the the of these from and to and of These by B. subtilis produced from P. pastoris as a on the of this protein a and the In and produced from P. pastoris showed a (Fig. and with to the non-glycosylated from P. pastoris and SAKM3-L-K1 produced from B. The of N-linked glycosylation with these was by with A binding was only with the P. with or SAKM3-L-K1 or to glycosylation of SAK in P. pastoris. In of plasminogen SAKM3-L-K1 produced pastoris and B. subtilis showed (Fig. and from P. on the demonstrated as by the SAKM3-L-K1 was produced at a with P. this was pastoris for To that Kringle-1 domain in SAKM3-L-K1 is functional, the from was to a of SAKM3-L-K1 on the and by This that the of SAKM3-L-K1 produced by P. pastoris lysine binding To the formation of in the Kringle-1 domain of SAKM3-L-K1, from (Fig. and and and in the or of agent by In SAKM3-L-K1 under the and because of a more with under the protein from as a with an of under the the of SAKM3-L-K1 produced by the in P. of the protein at the the for B. subtilis This that the of SAKM3-L-K1 produced pastoris forms and a whereas only a of SAKM3-L-K1 produced subtilis the This a for the in the ability of SAKM3-L-K1 produced by to to To that fusion of SAK and Kringle-1 in SAKM3-L-K1 with the function of SAKM3-L-K1 from P. pastoris was on a and for in with of the of plasminogen by SAKM3-L-K1 was to that by To the of the Kringle-1 domain in SAKM3-L-K1, the of this domain in SAKM3-L-K1 to a lysine was by A of binding of to the Kringle-1 domain of SAKM3-L-K1 was in The binding that resulted from of such is in The properties of the in this a of of a of and a of on the of for the between SAKM3-L-K1 and was to a the binding of Kringle-1 domain and its the N-terminal peptide of human plasminogen to in of the has been to by W. D. Eur. J. PubMed Scopus Google by N. PubMed Scopus Google and by M. M. J. PubMed Scopus Google Scholar). with SAKM3-L-K1 is the of the the mass of SAKM3-L-K1 on from that SAKM3-L-K1 was to N-terminal and mass spectrometry of the first amino acid residues from SAKM3-L-K1 with the SAK sequence (Fig. 1A). the peptide was by the P. from mass spectrometry the mass of SAKM3-L-K1 to which is in with the This the of N-linked glycosylation in SAKM3-L-K1 the mass of the N-linked the SAKM3-L-K1 fusion is to a fibrin to SAK the Kringle-1 it is essential that the Kringle-1 domain in SAKM3-L-K1 in the of the of this fusion in clot lysis will The C-terminal residues in SAK are known to lysine (Fig. 1A). a SAK or its at this domain to SAK will To the of SAKM3-L-K1 in the of SAKM3-L-K1 was with plasminogen at a and the of SAKM3-L-K1 was at different time by under or in min after SAKM3-L-K1 with plasminogen SAKM3-L-K1 was from the form with an mass of to a form This change in the of the first 10 amino acid residues (Fig. of SAKM3-L-K1 by the of the SAKM3-L-K1 showed in the which to that of SAK after SAKM3-L-K1 been with plasminogen for (Fig. This that the twin lysine site at the C-terminal end of of a of under the shows that plasminogen was to with the of a and a 20 min after with SAKM3-L-K1 (Fig. of an with that the SAKM3-L-K1 for at least an in the of The was on SAKM3-L-K1 with plasminogen at a A with an mass of was after 20 min and The of this a of of SAKM3-L-K1, to the N-terminal of the Kringle-1 lysine residues are for binding of the Kringle-1 fibrin on the in the to partially for the binding Hence, was to the fibrin with time to the for Kringle-1 shows a the between SAKM3-L-K1 and SAK in their ability to to Although SAK showed only a fibrin binding for the time SAKM3-L-K1 to the The fibrin to at least for binding to of C-terminal lysine residues and of the fibrin to SAKM3-L-K1 to the binding of SAKM3-L-K1 to the partially In this at the from 20 to min of fibrin the of SAKM3-L-K1 was that of the fibrin binding ability by SAKM3-L-K1 to a clot lysis was in a fibrin clot lysis assay an under different conditions. the first of the thrombolytic agents removed from the clots the clot lysis shows the in clot that clot lysis by SAK clot lysis with a and the time required for 50% clot lysis was that by SAKM3-L-K1 with different of SAKM3-L-K1 or by SAK was that by SAK in is to patients by a and the in of SAK in human is only min D. P. E. H. De M. L. Y. Van de Werf F. Circulation. 2000; PubMed Scopus Google the SAK is to from the is To this clot lysis was under the in which agent or was removed by the clot min after with the clots. The between SAK and SAKM3-L-K1 was more (Fig. SAK shows that on the in the SAK time to 50% clot the for SAK was required to the of lysis by the agents the lysis or the of clot lysis by SAKM3-L-K1 was at of thrombolytic agent of the time required to 50% of the fibrin clot with different of SAK or of of of of clots after min of with plasminogen and or the and from clots for of SAK or of of SAKM3-L-K1 in a The clots after min of with plasminogen and or the and from clots for of SAK or To more the clot lysis was human plasma under a flow plasma can plasminogen to the clot This can clot lysis D.V. Rijken D.C. Thromb. Haemostasis. 2000; PubMed Scopus Google Scholar). human plasma has a at was at which human plasma has to a more of clot shows that SAK was as the thrombolytic agent, a of min clot lysis was In clot lysis after the of on the of was min for SAK and min for SAKM3-L-K1 was at least more SAK in plasma clot in this the of non-glycosylated fusion protein in P. pastoris. of SAK in P. pastoris has been to in a SAK with plasminogen activator M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the is to in the of that the complex is in plasminogen residues in the glycosylation in this and that is a for the function of of with or reduced the of SAK F. J. and S.-L. with B. A (16Parry M.A. Fernandez-Catalan C. Bergner A. Huber R. Hopfner K.P. Schlott B. Guhrs K.H. Bode W. Nat. Struct. Biol. 1998; 5: 917-923Crossref PubMed Scopus (134) Google Scholar) of the complex of plasminogen activator that of SAK forms and with and of the one in the active plasminogen activator has with which has been to for SAK function B. Hartmann M. E. A. S. Collen D. Lijnen H.R. PubMed Scopus Google Scholar). some or of these the reduced of SAK in and In of with is well in SAK and in the of large of the SAKM3-L-K1 pastoris for and of thrombolytic therapy on the and of blood flow in occluded blood to one of the thrombolytic agents on min to blood flow under an treatment Furthermore, only about 60% of the treated patients have blood flow restored 90 min after the of more potent and thrombolytic agents that can the clot lysis and have a reperfusion In this by staphylokinase with clot binding the of this potent thrombolytic agent can In with SAKM3-L-K1 resulted in a in for plasma clot lysis in the flow-cell or a in for clot lysis in a static fibrin clot lysis The of clot lysis by SAKM3-L-K1 to the of the of this plasminogen activator to the SAK has fibrin binding the of SAK on the of in the fibrin binding (Fig. SAK to the fibrin of in the clot the of SAK is to or 20 of SAK in the binding In the of SAKM3-L-K1 on clots can 20 more that of SAK under the conditions. This was was in this (Fig. The of SAKM3-L-K1 can of SAKM3-L-K1 with the plasmin(ogen) to form plasminogen or to more plasmin(ogen) from plasma to the clot plasminogen is to clot and is to K. Collen D. Lijnen H.R. J. Biol. Chem. 1993; 268: 9811-9816Abstract Full Text PDF PubMed Google Scholar). Although of to tPA or has been to the potency of these agents E. T. PubMed Scopus Google Scholar, Bode C. E. PubMed Scopus Google Scholar, M. Lijnen H.R. Van B. De F. Collen D. Eur. J. PubMed Scopus Google a that of domain from tPA to SAK to clot lysis under in vitro J. E. W. T. Thromb. Haemostasis. 2001; Suppl. Scholar). These SAK are and The to clot lysis by these to the of the Kringle domain at the N-terminal end of SAK and the of Kringle from tPA as the fibrin has to SAK by cleaving the N-terminal peptide at the twin lysine site (Fig. domain fused to the N-terminal end of the SAK fusion can potentially by the plasminogen This reduce the clot-targeting of the fusion This is in of SAKM3-L-K1 by the Kringle domain in the C-terminal end of SAK and the of of the fusion protein in the of (Fig. Furthermore, Kringle from tPA has to lysine and to the Kringle-1 domain from human plasminogen M. 1995; PubMed Scopus Google Scholar). The can to a of the complex and the of a Kringle domain can the of SAK has been C. Schlott B. Hartmann M. Guhrs K.H. Glusa E. PubMed Scopus Google Scholar). the first of to that the of SAK can by a fibrin The of the Kringle-1 domain from human plasminogen as a domain in the of the SAK fusion this domain from a human it a protein to SAK has been demonstrated to in This has been by the of the of SAK with Y. S. S. De F. E. Collen D. 2000; PubMed Google Scholar) and the of in a to the of staphylokinase D. P. E. H. De M. L. Y. Van de Werf F. Circulation. 2000; PubMed Scopus Google Scholar). The of these SAK in with the Kringle-1 domain from human plasminogen the to develop potent and fast-acting thrombolytic agents with S. and S. Sihota for the of for the of T. A. at of for the of the human plasminogen and R. L. and S. at of for of the

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