Process for preparing tissue factor pathway inhibitor

Details Process for preparing tissue factor pathway inhibitor Process for preparing tissue factor pathway inhibitor

1994-06-28

2001-10-09

Eyler, Yvonne (Department: 1646)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S070100

Reexamination Certificate

active

06300100

ABSTRACT:

The present invention relates to a process for preparing blood coagulation inhibitor which has hitherto been known as Tissue Factor Inhibitor (TFI), Extrinsic Pathway Inhibitor (EPI) or Lipoprotein-associated Coagulation Inhibitor (LACI) and which has recently been given a unified name of Tissue Factor Pathway Inhibitor (hereinafter referred to as “TFPI”). More particularly, the present invention relates to a process for preparing an intact TFPI from a transformed animal cell wherein a DNA encoding a human TFPI is introduced.
PRIOR ART
Bleeding is one of the most serious lesions in the living body. There is a series of blood coagulation reactions for controlling the bleeding within the living body. These reactions are classified into two pathways, i.e. the intrinsic and extrinsic pathways. At present, for the initiation mechanism of a physiological coagulation reaction, the extrinsic pathway initiated by Tissue Factor (hereinafter referred to as “TF”) is considered to be more important than the intrinsic pathway [Davie, E. W., Biochem., 30, p10363 (1991)]. In case of the extrinsic pathway, TF, which is considered normally not to contact with blood, becomes in contact with blood due to vascular damage or any other causes and then it is rapidly bound with the blood coagulation factor VII(a). The resulting TF-Factor VIIa complex activates the blood coagulation Factors IX and X, leading to proteolytical activation of prothrombin to thrombin. Finally, thrombin stimulates platelets and forms fibrin clot or thrombi.
In early studies as to the control of blood coagulation initiated by TF, it was suggested that serum shows an inhibitory activity against the tissue thromboplastin (the old name of TF) [Thomas L., Bull. Johns Hopkins Hosp. 81, p26 (1947)]. It was also shown that the inhibitory activity was dependent on the presence of calcium. Based on these facts, Hjort demonstrated that this serum inhibitor targeted the TF-factor VIIa complex and required calcium [Hjort, P. F., Scand. J. Clin. Lab. Invest. 9, p1 (1957)]. In 1980's, Sanders et al. showed that Factor X is necessary for the serum inhibitor [Sanders, N. L., Blood, 66, p204 (1985)] and a density gradient centrifugation of plasma also proved that it is present in a lipoprotein fraction. The serum inhibitor and its mechanism of inhibition have been mainly elucidated by Broze et al. who purified the inhibitor from established human liver (HepG2) cell [Broze Jr., G. J., Proc. Natl. Acad. Sci. U.S.A., 84, p1886 (1987)]. The cDNA encoding TFPI is cloned from human placental &lgr;gt11 cDNA library by using anti-TFPI antibody. From this nucleotide sequence, a whole amino acid sequence was deduced and suggested the mechanism of inhibition [Wun, T C., J. Biol. Chem., 263, p6001 (1988)]. According to these data, TFPI consisted of 304 amino acids including a signal sequence of 28 amino acids and has three regions of amino acid sequence homologous to the Kunitz-type protease inhibitor. The Kunitz-type protease inhibitors were known to inhibit trypsin-like proteases, for example, soy bean trypsin inhibitor (SBTI) or aprotinin. In case of TFPI, it has been shown that, among the three Kunitz-type regions, the reaction site of the second Kunitz-type region is necessary for the inhibition of factor Xa whereas the reaction sites of the first and the second Kunitz-type regions are necessary for the inhibition of the TF-factor VIIa complex. TFPI binds and inhibits factor Xa directly and inhibits TF-factor VIIa activity in a factor Xa-dependent fashion in a reaction that appears to involve the formation of a Xa-TFPI-VIIa-TF quaternary complex. The reaction site of the third Kunitz-type region is shown to be unnecessary for the inhibition and the role of this reaction site still remains unknown [Girard, T. J., Nature, 338, p518 (1989)]. The N-terminal region of TFPI has a cluster of negatively charged (or acidic) amino acids, and the C-terminal region has a cluster of positively charged (or basic) amino acids which is important of inhibitory function.
TFPI is mainly produced in endothelial cells [Bajaj, M. S., Proc. Natl. Acad. Sci. USA, 87, p8869 (1990)] and is present at a concentration of about 100 ng/mL in plasma, among of which a percentage of TFPI free from lipoproteins is several % to several ten %. TFPI is also found in platelets in a quite small amount and is suggested to be bound with a heparin-like substance on endothelial cells of the blood vessel since TFPI was released into plasma when heparin was administered to healthy persons [Novotny, W. F., Blood, 78, p394 (1991)].
It was proved that TFPI in blood was bound with lipoproteins based on the fact that TFPI was found in a lipoprotein fraction even after a density gradient centrifugation or gel filtration thereof and the fact that TFPI purified from plasma was associated with apolipoprotein A-II under non-reducing conditions. It was also found that the lipoprotein-bound TFPI is dissociated under reducing conditions [Novotny, W. F., J. Biol. Chem., 264, p18832 (1989)]. However, the mechanism of TFPI binding with lipoproteins and the significance thereof is still unknown in detail. A concentration of lipoproteins in plasma greatly varies depending on a variety of conditions, and TFPI has been considered to be a very important factor for elucidating a relationship between the thrombus formation and the concentration of lipoproteins especially in a pathological condition such as arteriosclerosis. In recent years, it has also been studied to use TFPI as an agent for preventing reocclusion in an arteriosclerosis model [Haskel, E. J., Circulation, 84, p821 (1991)].
Since TFPI is present in blood only in a quite small amount, it has been prepared as a recombinant protein by a genetically engineering technique by many researchers. For example, it has been reported an expression of recombinant TFPI from mouse C127 cells [Day, K. C., Blood, 76, p1538 (1990)], BHK cells [Pederson, A. H., J. Biol. Chem., 265, p16786 (1990)], CHO cells, SK hepatoma cells [Wun, T C., Thromb. Haemost., 68, p54 (1992)] as a host cell. However, all of these recombinant TFPIs except for that from SK hepatoma cells were reported to apparently exhibit a lower specific activity than that of TFPI derived from plasma or TFPI on the endothelial cells of the blood vessel [Wesselschmidt, R., Blood, 79, p2004 (1992); Lindahl, A. K., Thromb. Res., 62, p607 (1991)]. This decrease in activity was mainly due to cleavage of the C-terminal basic region of the recombinant TFPI. It has been shown that the molecular species devoid of the C-terminal region is lowered in the binding activity to heparin, especially in the inhibitory efficiency to Factor Xa, and hence, the C-terminal region is physiologically very important. An intact molecular species with the C-terminal basic region and its truncated molecular species can be separated by heparin agarose chromatography or cation exchange chromatography with NaCl concentration gradient [Wesselschmidt, R., Blood, 79, p2004 (1992); Nordfang, O., Biochem., 30, p10371 (1991)], along with any purification steps, for example, affinity chromatography using an anti-TFPI antibody. A yield of the intact molecular species obtained from these cell cultures was at most 40%, normally 10% or less. The only exception is the case of using SK hepatoma cells as a host cell, which are known to produce intrinsically TFPI [Wun, T C., Thromb. Haemost., 68, p54 (1992)]. It was reported that the intact recombinant TFPI was obtained at almost 100% from the conditioned medium of the TFPI-expressing cells where an exogenous TFPI gene was introduced. These results show that, when cells other than those producing TFPI intrinsically were used as a host cell, it is very difficult to obtain the intact recombinant TFPI at a high yield. Accordingly, it has hitherto been very difficult to prepare the intact TFPI molecular species in a large amount by usi

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