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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.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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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.
 
Creator Maheshwari, Neeraj
Kantipudi, Satish
Maheshwari, Anand
Arora, Kashika
Vandana, .
Kwatra, Neha
Sahni, Girish
 
Subject QR Microbiology
 
Description 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.
 
Publisher Public Library of Science
 
Date 2016
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/1887/1/52.pdf
Maheshwari, Neeraj and Kantipudi, Satish and Maheshwari, Anand and Arora, Kashika and Vandana, . and Kwatra, Neha and Sahni, Girish (2016) 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. PloS one, 11 (3). e0150315. ISSN 1932-6203
 
Relation http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150315
http://crdd.osdd.net/open/1887/