title: Amino-Terminal Fusion of Epidermal Growth Factor 4,5,6 Domains of Human Thrombomodulin on Streptokinase Confers Anti-Reocclusion Characteristics along with Plasmin-Mediated Clot Specificity.
abstract: Streptokinase (SK) is a potent clot dissolver but lacks fibrin clot specificity as it activates human plasminogen (HPG) into human plasmin (HPN) throughout the system leading to increased risk of bleeding. Another major drawback associated with all thrombolytics, including tissue plasminogen activator, is the generation of transient thrombin and release of clot-bound thrombin that promotes reformation of clots. In order to obtain anti-thrombotic as well as clot-specificity properties in SK, cDNAs encoding the EGF 4,5,6 domains of human thrombomodulin were fused with that of streptokinase, either at its N- or C-termini, and expressed these in Pichia pastoris followed by purification and structural-functional characterization, including plasminogen activation, thrombin inhibition, and Protein C activation characteristics. Interestingly, the N-terminal EGF fusion construct (EGF-SK) showed plasmin-mediated plasminogen activation, whereas the C-terminal (SK-EGF) fusion construct exhibited 'spontaneous' plasminogen activation which is quite similar to SK i.e. direct activation of systemic HPG in absence of free HPN. Since HPN is normally absent in free circulation due to rapid serpin-based inactivation (such as alpha-2-antiplasmin and alpha-2-Macroglobin), but selectively present in clots, a plasmin-dependent mode of HPG activation is expected to lead to a desirable fibrin clot-specific response by the thrombolytic. Both the N- and C-terminal fusion constructs showed strong thrombin inhibition and Protein C activation properties as well, and significantly prevented re-occlusion in a specially designed assay. The EGF-SK construct exhibited fibrin clot dissolution properties with much-lowered levels of fibrinogenolysis, suggesting unmistakable promise in clot dissolver therapy with reduced hemorrhage and re-occlusion risks.
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Neeraj
authors.initials: N
authors.lastname: Maheshwari
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Satish
authors.initials: S
authors.lastname: Kantipudi
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Anand
authors.initials: A
authors.lastname: Maheshwari
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Kashika
authors.initials: K
authors.lastname: Arora
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.lastname: Vandana
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Kwatra
authors.affiliation: CSIR-Institute of Microbial Technology, Chandigarh, India.
authors.forename: Girish
authors.initials: G
authors.lastname: Sahni
mesh_terms: D005341:Fibrinolysin; D005342:Fibrinolysis; D006801:Humans; D011486:Protein C / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013300:Streptokinase / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry*; D018180:Thrombomodulin / Q000737:chemistry* / Q000235:genetics
chemical_list: D011486:Protein C; D011993:Recombinant Fusion Proteins; C546931:THBD protein, human; D018180:Thrombomodulin; D013300:Streptokinase; D013917:Thrombin; D005341:Fibrinolysin