title: Molecular design and characterization of recombinant long half-life mutants of human tissue factor pathway inhibitor.
abstract: Tissue factor pathway inhibitor (TFPI) is a physiological inhibitor of extrinsic pathway of coagulation and has biological functions of anticoagulation and anti-inflammation. Although TFPI has been proved to be a good therapeutic agent of sepsis, inflammatory shock, and DIC, the clinical application and therapeutic effects of TFPI are impeded because of its short half-life in vivo. In order to prolong the half-life of TFPI, homology modeling and molecule docking were performed on a computer workstation principally in protein structural biology and binding characteristics between TFPI and its receptor LRP (low-density lipoprotein receptor related protein). Two recombinant long half-life human TFPI mutants coined TFPI-Mut1 and TFPI-Mut4 were designed and expressed in E.coli. In comparison with the wild-type TFPI, TFPI-Mut1 and TFPI-Mut4 presented a few of changes in spatial configuration and a decrease in relative Gibbs free energy of docking complex by 17.3% and 21.5%, respectively, as indicated by a computer simulation. After refolding and purification, anticoagulant activities, anti-TF/FVIIa and anti-FXa activities of the mutants were found to be the same as those of wide-type TFPI. The pharmacokinetics research indicated that alpha phase half-life (t1/2 alpha) of TFPI-Mut1 and TFPI-Mut4 were prolonged 1.33-fold and 1.96-fold respectively, beta phase half-life (t1/2 beta) of TFPI-Mut1 and TFPI-Mut4 were prolonged 1.62-fold and 4.22-fold respectively. These results suggested that TFPI-Mut1 and TFPI-Mut4 maintained the bioactivities of wild-type TFPI, prolonged half-life in vivo simultaneously and were expected for better clinical value and therapeutic effect.
authors.affiliation: Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, 130 Dongan Rd, Shanghai 200032, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Bai
authors.forename: Duan
authors.initials: D
authors.lastname: Ma
authors.forename: Yu-Gao
authors.initials: YG
authors.lastname: Zhang
authors.forename: Nong
authors.initials: N
authors.lastname: Zhang
authors.forename: De-Sheng
authors.initials: DS
authors.lastname: Kong
authors.forename: Hong-Shen
authors.initials: HS
authors.lastname: Guo
authors.forename: Wei
authors.initials: W
authors.lastname: Mo
authors.forename: Qi-Qun
authors.initials: QQ
authors.lastname: Tang
authors.forename: Hou-Yan
authors.initials: HY
authors.lastname: Song
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017076:Computer-Aided Design; D015195:Drug Design; D006207:Half-Life; D006801:Humans; D066298:In Vitro Techniques; D026502:LDL-Receptor Related Proteins / Q000378:metabolism; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D016296:Mutagenesis; D011487:Protein Conformation; D015202:Protein Engineering; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013816:Thermodynamics
chemical_list: D000925:Anticoagulants; D026502:LDL-Receptor Related Proteins; D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor