title: Evaluation of a multifunctional staphylokinase variant with thrombin inhibition and antiplatelet aggregation activities produced from salt-inducible E. coli GJ1158.
abstract: Reocclusion is one of the major root causes for secondary complications that arise during thrombolytic therapy. A multifunctional staphylokinase variant SRH (staphylokinase (SAK) linked with tripeptide RGD and didecapeptide Hirulog) with antiplatelet and antithrombin activities in addition to clot specific thrombolytic function, was developed to address the reocclusion problem. We preferred to use Escherichia coli GJ1158 as the host in this study for economic production of SRH by osmotic (0.3 mol/L sodium chloride) induction, to overcome the problems associated with the yeast expression system. The therapeutic potential of SRH was evaluated in the murine model of vascular thrombosis. The SAK protein (1 mg/kg body mass) and SRH protein (1 mg/kg and 2 mg/kg) were administered intravenously to the different treatment groups. The results have shown a dose-dependent antithrombotic effect in carrageenan-induced mouse tail thrombosis. The thrombin time, activated partial thromboplastin time, and prothrombin time were significantly prolonged (p < 0.05) in the SRH-infused groups. Moreover, SRH inhibited platelet aggregation in a dose-dependent manner (p < 0.05), while the bleeding time was significantly (p < 0.05) prolonged. All of these results inferred that the osmotically produced multifunctional fusion protein SRH (SAK-RGD-Hirulog) is a promising thrombolytic agent, and one which sustained its multifunctionality in the animal models.
authors.affiliation: a Department of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur 522510, India.
authors.forename: Anmol
authors.initials: A
authors.lastname: Kumar
authors.forename: Krishna Kanth
authors.initials: KK
authors.lastname: Pulicherla
authors.forename: Candasamy
authors.initials: C
authors.lastname: Mayuren
authors.forename: Seetharam
authors.initials: S
authors.lastname: Kotra
authors.forename: Krothapalli Rajasurya Sambasiva
authors.initials: KR
authors.lastname: Rao
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects; D002351:Carrageenan; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006629:Hirudins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D008297:Male; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009842:Oligopeptides / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D010314:Partial Thromboplastin Time; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D011517:Prothrombin Time; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D012965:Sodium Chloride / Q000737:chemistry; D013918:Thrombin Time; D013927:Thrombosis / Q000097:blood / Q000139:chemically induced / Q000188:drug therapy*; D013997:Time Factors
chemical_list: D000991:Antithrombins; D006629:Hirudins; D009842:Oligopeptides; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012965:Sodium Chloride; C047981:arginyl-glycyl-aspartic acid; D002351:Carrageenan; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus; C074619:bivalirudin