title: Modular design of a novel chimeric protein with combined thrombin inhibitory activity and plasminogen-activating potential.
abstract: In order to design plasminogen activators with improved thrombolytic properties we sought to construct the bifunctional protein HLS-2 which combines both a plasminogen-activating and an anticoagulative activity. The chimeric protein comprises four elements: a derivative of thrombin inhibitor hirudin, a 6-amino acid spacer, the sequence of plasminogen-activator staphylokinase (Sak), and a 13-amino acid expression tag at the C-terminus. The gene of the fusion protein was obtained by SOE-PCR, cloned into pCANTAB5E, and expressed in E. coli BL21. HLS-2 was purified from periplasmatic extracts and characterized by Western blotting. Plasminogen-activation of HLS-2 and of Sak in equimolar mixtures with plasminogen showed near equivalence as measured by plasmin-mediated cleavage of chromogenic substrate S-2403. For catalytic amounts of plasminogen-activator, however, HLS-2 was less effective by a factor of 1.7. HLS-2 also inhibited both the amidolytic and the fibrinolytic activities of thrombin. Similar concentrations of either commercial HV1 (42 pmol/L) or HLS-2 (250 pmol/L) were required to halve the initial rate of thrombin reaction with fluorogenic substrate Tos-Gly-Pro-Arg-AMC, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein sufficiently strong to substitute anticoagulative comedication during fibrinolytic treatment. The results provide a rationale for further testing the efficacy of HLS-2 for the lysis of platelet-rich arterial blood clots and for the prevention of reocclusion after thrombolysis.
authors.affiliation: Abteilung fuer Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Grisebachstrasse 8, 37077 Goettingen, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Wirsching
authors.forename: Cornelia
authors.initials: C
authors.lastname: Luge
authors.forename: Andreas
authors.initials: A
authors.lastname: Schwienhorst
mesh_terms: D000577:Amides / Q000378:metabolism; D000991:Antithrombins / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects; D002384:Catalysis; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D005341:Fibrinolysin / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D006629:Hirudins / Q000235:genetics; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D010958:Plasminogen / Q000187:drug effects / Q000378:metabolism; D010960:Plasminogen Activators / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors*; D013918:Thrombin Time; D013997:Time Factors
chemical_list: D000577:Amides; D000991:Antithrombins; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010958:Plasminogen; D010960:Plasminogen Activators; D013917:Thrombin; D005341:Fibrinolysin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus