Molecular Cloning, Expression And Characterization, Pharmacodynamics Studies Of Recombinant Human Tissue Factor Pathway Inhibitor And Its Mutants

The blood coagulation pathway is assorted intrinsic and extrinsicpathway of coagulation. According to modern hematology, the extrinsic pathway ofcoagulation plays dominant role in physiological hemostasis and pathological bloodcoagulation. The extrinsic pathway of coagulation is also called tissue factor (TF)pathway which is initiated by the exposure of TF to the circulating blood. Tissuefactor pathway inhibitor (TFPI) is a physiological inhibitor of extrinsic pathway ofcoagulation. The mature full-length TFPI protein, consisting of 276 amino acidresidues, is composed of N-terminal rich in acidic amino acids, three tandem Kunitzdomains (K1, K2, and K3) and C-terminal rich in basic amino acids. Theanticoagulant activity of TFPI is exerted mostly by K1 and K2 domains. The K2domain combines with FXa directly and inhibits catalytic activity of FXa, and the K1domain combines with FVIIa/TF via TFPI/FXa and inhibits activity of FVIIa/TF. Inaddition to anticoagulant effect, TFPI possesses many other biological functionsincluding anti-inflammation, anti-angiogenesis, inhibiting growth of tumor, inhibitingproliferation and inducing apoptosis of endothelial cell, and decreasingthrombogenicity and neointimal thickness etc. So it is understood that the researchand application values of TFPI will be remarkable. Using total RNA of human placenta as a template, TFPI cDNA gene wasobtained by RT-PCR. By optimizing codon usage and expression conditions,recombinant TFPI (rTFPI) was expressed successfully in E.coli as inclusion bodiesand accounted for 10% of total bacteria protein. After refolding and purification,recombinant TFPI was obtained with purity of more than 95%, specific activity of18,440 U/mg and yield of 30 mg/L. The K3 domain and C-terminal of TFPI are positions to combine with LRP,CD14 and heparin etc. and essential for metabolism, anti-inflammatory activity,optimally anticoagulant activity and many other bioactivities. As a kind of deletionvariant containing N-terminus, K1 and K2 domains of TFPI, TFPI1-161 is an idealmolecule in the research of structure and function of TFPI and expected to play animportant role in prophylaxis and treatment of hypercoagulation and thromboticdiseases based on the long half-life and mild anticoagulant activity etc. III英文摘要 Using TFPI cDNA gene as a template, TFPI1-161 cDNA gene was amplified byPCR method. By means of screening multi-copy transformants and optimizingferment condition, recombinant TFPI1-161 was highly expressed in Pichia pastorisGS115 for the first time. The expression level was 70% of total media protein andmore than 20-fold comparing with Saccharomyces cerevisiae expression system.Recombinant TFPI1-161 appeared in two different molecular weight proteins namedTFPI1-161(24kD) and TFPI1-161(27kD) because of different glycosylation. Theirisoelectric points were 4.8 and 4.9 respectively. After refolding and purification,recombinant TFPI1-161(24kD) and TFPI1-161(27kD) were obtained with purity of morethan 95%, specific activity of 12,880 U/mg and 12,400 U/mg,yield of 350 mg/L and450 mg/L respectively. Disseminated intravascular coagulation (DIC) is a kind of common clinicalsyndrome. It is acute onset, critical state, poor prognosis and basic diseases resultingin DIC are serious and extensive. Blockading TF pathway has already been aneffective method to prevent and cure DIC because the onset of DIC is related toinitiation of TF pathway in most instances. Although it was proved that recombinantTFPI was a good curative agent of DIC, the half-life of TFPI was as short as 2min.Since TFPI was cleared away from plasma very quickly, high dosage and continuouslong time administration must to be taken. Obviously, it was inconveniences inclinical application and made a heavy economic burden for patients. Prolonginghalf-life of recombinant TFPI and improving clinical applicat...