Cardio- and cerebrovascular diseases are leading causes of death and disability, resulting in one of the highest socio-economic burdens of any disease type. The discovery of bacterial and human plasminogen activators and their use as thrombolytic drugs have revolutionized treatment of these pathologies. Fibrin-specific agents have an advantage over non-specific factors because of lower rates of deleterious side effects. Specifically, staphylokinase (SAK) is a pharmacologically attractive indirect plasminogen activator protein of bacterial origin that forms stoichiometric noncovalent complexes with plasmin, promoting the conversion of plasminogen into plasmin. Here we report a computer-assisted re-design of the molecular surface of SAK to increase its affinity for plasmin. A set of computationally designed SAK mutants was produced recombinantly and biochemically characterized. Screening revealed a pharmacologically interesting SAK mutant with ∼7-fold enhanced affinity toward plasmin, ∼10-fold improved plasmin selectivity and moderately higher plasmin-generating efficiency in vitro. Collectively, the results obtained provide a framework for SAK engineering using computational affinity-design that could pave the way to next-generation of effective, highly selective, and less toxic thrombolytics.

Department of Biochemistry Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Czech Republic

Department of Biochemistry Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

Institute of Biophysics of the Czech Academy of Sciences Kralovopolska 135 612 65 Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Pekarska 53 656 91 Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

Neurology Department St Anne's University Hospital and Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Czech Republic

Weizmann Institute of Science Rehovot 76100 Israel

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Tillett W.S., Garner R.L. The fibrinolytic activity of hemolytic streptococci. J Exp Med. 1933;58:485–502. PubMed PMC

Sila C.A., Furlan A.J. Therapy for acute ischemic stroke: the door opens. Interpreting the NINDS rt-PA stroke study. Cleve Clin J Med. 1996;63:77–79. PubMed

Macfarlane R.G., Pilling J. Fibrinolytic activity of normal urine. Nature. 1947;159:779. PubMed

Rijken D.C., Wijngaards G., Zaal-de Jong M., Welbergen J. Purification and partial characterization of plasminogen activator from human uterine tissue. Biochim Biophys Acta. 1979;580:140–153. PubMed

Mican J., Toul M., Bednar D., Damborsky J. Structural biology and protein engineering of thrombolytics. Comput Struct Biotechnol J. 2019;17:917–938. PubMed PMC

Nikitin D., Choi S., Mican J., Toul M., Ryu W.-S., Damborsky J., et al. Development and testing of thrombolytics in stroke. J Stroke. 2021;23(1):12–36. PubMed PMC

Collen D., Lijnen H.R. The tissue-type plasminogen activator story. Arterioscler Thromb Vasc Biol. 2009;29:1151–1155. PubMed

Bivard A., Huang X., Levi C.R., Spratt N., Campbell B.C., et al. Tenecteplase in ischemic stroke offers improved recanalization: analysis of 2 trials. Neurology. 2017;89:62–67. PubMed

Yaghi S., Willey J.Z., Cucchiara B., Goldstein J.N., Gonzales N.R., Khatri P., et al. Treatment and outcome of hemorrhagic transformation after intravenous alteplase in acute ischemic stroke: A scientific statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2017;48:e343–e361. PubMed

Lack C.H. Staphylokinase; an activator of plasma protease. Nature. 1948;161:559. PubMed

Collen D. Staphylokinase: a potent, uniquely fibrin-selective thrombolytic agent. Nat Med. 1998;4:279–284. PubMed

Parry M.A., Fernandez-Catalan C., Bergner A., Huber R., Hopfner K.P., et al. The ternary microplasmin-staphylokinase-microplasmin complex is a proteinase-cofactor-substrate complex in action. Nat Struct Biol. 1998;5:917–923. PubMed

Sakharov D.V., Lijnen H.R., Rijken D.C. Interactions between staphylokinase, plasmin(ogen), and fibrin. Staphylokinase discriminates between free plasminogen and plasminogen bound to partially degraded fibrin. J Biol Chem. 1996;271:27912–27918. PubMed

Vanderschueren S., Barrios L., Kerdsinchai P., Van den Heuvel P., Hermans L., et al. A randomized trial of recombinant staphylokinase versus alteplase for coronary artery patency in acute myocardial infarction. The STAR Trial Group. Circulation. 1995 15;;92(8):2044–2049. PubMed

Armstrong P.W., Burton J., Pakola S., Molhoek P.G., Betriu A., et al. Collaborative angiographic patency trial of recombinant staphylokinase (CAPTORS II) Am Heart J. 2003;146:484–488. PubMed

Armstrong P.W., Burton J.R., Palisaitis D., Thompson C.R., Van de Werf F., et al. Collaborative angiographic patency trial of recombinant staphylokinase (CAPTORS) Am Heart J. 2000;139:820–823. PubMed

Toul M., Nikitin D., Marek M., Damborsky J., Prokop Z. Extended mechanism of the plasminogen activator staphylokinase revealed by global kinetic analysis: 1000-fold higher catalytic activity than That of clinically used alteplase. ACS Catal. 2022;12:3807–3814.

Netzer R., Listov D., Lipsh R., Dym O., Albeck S., et al. Ultrahigh specificity in a network of computationally designed protein-interaction pairs. Nat Commun. 2018;9:5286. PubMed PMC

Berman H.M., Westbrook J., Feng Z., Gilliland G., Bhat T.N., et al. The protein data bank. Nucleic Acids Res. 2000;28:235–242. PubMed PMC

The UniProt Consortium UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021;49:D480–D489. PubMed PMC

Schrödinger, LLC. The PyMOL Molecular Graphics System, Version 2.1 2015.

Singh S., Ashish D.KL. Pro(42) and Val(45) of staphylokinase modulate intermolecular interactions of His(43)-Tyr(44) pair and specificity of staphylokinase-plasmin activator complex. Febs Lett. 2012;586:653–658. PubMed

Kellogg E.H., Leaver-Fay A., Baker D. Role of conformational sampling in computing mutation-induced changes in protein structure and stability. Proteins Struct Funct Bioinform. 2011;79:830–838. PubMed PMC

Schneider C.A., Rasband W.S., Eliceiri K.W. NIH image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–675. PubMed PMC

Johnson K.A., Simpson Z.B., Blom T. Global Kinetic Explorer: a new computer program for dynamic simulation and fitting of kinetic data. Anal Biochem. 2009;387:20–29. PubMed

Johnson K.A., Simpson Z.B., Blom T. FitSpace Explorer: an algorithm to evaluate multi-dimensional parameter space in fitting kinetic data. Anal Biochem. 2009;387:30–41. PubMed

Johnson K.A. Fitting enzyme kinetic data with KinTek global kinetic explorer. Comp Methods Enzymol. 2009;467:601–626. PubMed

Harpaz D., Chen X., Francis C.W., Marder V.J., Meltzer R.S. Ultrasound enhancement of thrombolysis and reperfusion in vitro. J Am Coll Cardiol. 1993;6:1507–1511. PubMed

Prasad S., Kashyap R.S., Deopujari J.Y., Purohit H.J., Taori G.M., Daginawala H.F. Development of an in vitro model to study clot lysis activity of thrombolytic drugs. Thromb J. 2006;4:14. PubMed PMC

Thalerová S., Pešková M., Kittová P., Gulati S., Víteček J., Kubala L., et al. Effect of apixaban pretreatment on alteplase-induced thrombolysis: an in vitro study. Front Pharmacol. 2021;12 PubMed PMC

Víteček J., Vítečková Wünschová A., Thalerová S., Gulati S., Kubala L., et al. Factors influencing recombinant tissue plasminogen activator efficacy: an in vitro study. Life Sci. 2021 in submission. PubMed PMC

Diamond S.L. Engineering design of optimal strategies for blood clot dissolution. Annu Rev Biomed Eng. 1999;1:427–462. PubMed

Rottenberger Z., Komorowicz E., Szabó L., Bóta A., Varga Z., et al. Lytic and mechanical stability of clots composed of fibrin and blood vessel wall components. J Thromb Haemost. 2013;11:529–538. PubMed PMC

Marcos-Contreras O.A., Ganguly K., Yamamoto A., Shlansky-Goldberg R., Cines D.B., Muzykantov V.R., et al. Clot penetration and retention by plasminogen activators promote fibrinolysis. Biochem Pharmacol. 2013;85:216–222. PubMed

Longstaff C. Measuring fibrinolysis: from research to routine diagnostic assays. J Thromb Haemost. 2018;16:652–662. PubMed PMC

Longstaff C., Varjú I., Sótonyi P., Szabó L., Krumrey M., et al. Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones. J Biol Chem. 2013;288:6946–6956. PubMed PMC

Morrow G.B., Whyte C.S., Mutch N.J. Functional plasminogen activator inhibitor 1 is retained on the activated platelet membrane following platelet activation. Haematologica. 2020;105:2824–2833. PubMed PMC

Acheampong P., Ford G.A. Pharmacokinetics of alteplase in the treatment of ischaemic stroke. Expert Opin Drug Metab Toxicol. 2012;8:271–281. PubMed

Elnager A., Abdullah W.Z., Hassan R., Idris Z., Arfah N.W., Sulaiman S.A., et al. In vitro whole blood clot lysis for fibrinolytic activity study using D-dimer and confocal microscopy. Adv Hematol. 2014;2014 PubMed PMC

Lijnen H.R., De Cock F., Van Hoef B., Schlott B., Collen D. Characterization of the interaction between plasminogen and staphylokinase. Eur J Biochem. 1994;224(1):143–149. PubMed

Schlott B., Hartmann M., Gührs K.H., Birch-Hirschfeld E., Gase A., Vettermann S., et al. Functional properties of recombinant staphylokinase variants obtained by site-specific mutagenesis of methionine-26. Biochim Biophys Acta. 1994;1204(2):235–242. PubMed

Dornberger U., Fandrei D., Backmann J., Hübner W., Rahmelow K., Gührs K.H., et al. A correlation between thermal stability and structural features of staphylokinase and selected mutants: a Fourier-transform infrared study. Biochim Biophys Acta. 1996;1294(2):168–176. PubMed

Dahiya M, Singh S, Rajamohan G, Sethi D, Dikshit A. Intermolecular interactions in staphylokinase-plasmin(ogen) bimolecular complex: function of His43 and Tyr44 FEBS Lett 2011; 585:1814. PubMed

Lee K.S., Yang J., Niu J., Ng C.J., Wagner K.M., et al. Drug-target residence time affects in vivo target occupancy through multiple pathways. ACS Cent Sci. 2019;5:1614–1624. PubMed PMC

Copeland R.A. The drug–target residence time model: a 10-year retrospective. Nat Rev Drug Discov. 2016;15:87–95. PubMed

Leach J.K., Patterson E., O’Rear E.A. Distributed intraclot thrombolysis: mechanism of accelerated thrombolysis with encapsulated plasminogen activators. J Thromb Haemost. 2004;2:1548–1555. PubMed

Alexandrov A., Grotta J. Arterial reocclusion in stroke patients treated with intravenous tissue plasminogen activator. Neurology. 2002;59(6):862–867. PubMed

Knuttinen M.G., Emmanuel N., Isa F., Rogers A.W., Gaba R.C., Bui J.T., et al. Review of pharmacology and physiology in thrombolysis interventions. Semin Intervent Radiol. 2010;27:374–383. PubMed PMC

Baruah D.B., Dash R.N., Chaudhari M.R., Kadam S.S. Plasminogen activators: a comparison. Vascul Pharmacol. 2006;44:1–9. PubMed

Nedaeinia R., Faraji H., Javanmard S.H., Ferns G.S., Ghayour-Mobarhan M., et al. Bacterial staphylokinase as a promising third-generation drug in the treatment for vascular occlusion. Mol Biol Rep. 2020;47:819–841. PubMed

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