Methods for detecting human tissue factor inhibitor

Details Methods for detecting human tissue factor inhibitor Methods for detecting human tissue factor inhibitor

2000-07-28

2003-03-18

Mertz, Prema (Department: 1646)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C436S536000, C436S538000, C436S545000

Reexamination Certificate

active

06534276

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to a coagulation inhibitor known as tissue factor inhibitor (TFI) and alternatively as lipoprotein associated coagulation inhibitor (LACI). More particularly, the invention relates to a cDNA clone representing essentially the full size TFI.
The coagulation cascade that occurs in mammalian blood comprises two distinct systems—the so-called intrinsic and extrinsic systems. The latter system is activated by exposure of blood to tissue thromboplastin (Factor III), hereinafter referred to as tissue factor (TF). Tissue factor is a lipoprotein that arises in the plasma membrane of many cell types and in which the brain and lung are particularly rich. Upon coming into contact with TF, plasma Factor VII or its activated form, Factor VII
a
, forms a calcium-dependent complex with TF and then proteolytically activates Factor X to Factor X
a
, and Factor IX to Factor IX
a
.
Early studies concerning the regulation of TF-initiated coagulation showed that incubation of TF (in crude tissue thromboplastin preparations) with serum inhibited its activity in vitro and prevented its lethal effect when it was infused into mice. Extensive studies by Hjort,
Scand. J. Clin. Lab. Invest
. 9, Suppl. 27, 76-97 (1957), confirmed and extended previous work in the area, and led to the conclusion that an inhibitory moiety in serum recognized the Factor VII-TF complex. Consistent with this hypothesis are the facts that the inhibition of TF that occurs in plasma requires the presence of Ca
2+
(which is also necessary for the binding of Factor VII/VII
a
to TF) and that inhibition can be prevented and/or reversed by chelation of divalent cations with EDTA. More recent investigations have shown that not only Factor VII
a
but also catalytically active Factor X
a
and an additional factor are required for the generation of TF inhibition in plasma or serum. See Broze and Miletich,
Blood
69, 150-155 (1987), and Sanders et al.,
Ibid
., 66, 204-212 (1985). This additional factor, defined herein as tissue factor inhibitor (TFI), and alternatively as lipoprotein associated coagulation inhibitor (LACI), is present in barium-absorbed plasma and appears to be associated with lipoproteins, since TFI functional activity segregates with the lipoprotein fraction that floats when serum is centrifuged at a density of 1.21 g/cm
3
. According to Broze and Miletich, supra, and
Proc. Natl. Acad. Sci. USA
84, 1886-1890 (1987), HepG2 cells (a human hepatoma cell line) secrete an inhibitory moiety with the same characteristics as the TFI present in plasma.
In copending application Ser. No. 77,366, filed Jul. 23, 1987, a purified tissue factor inhibitor (TFI) is disclosed which was secreted from HepG2 cells. It was found to exist in two forms, a TFI
1
, migrating at about 37-40,000 daltons and a TFI
2
at about 25-26,000 daltons, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). A partial N-terminal amino acid sequence for the TFI was assigned as:
1
X-X-Glu-Glu-Asp-Glu-Glu-His-Thr-Ile-Ile-Thr-Asp-

     15 16
Thr-Glu-Leu-Pro-Pro-Leu-Lys-Leu-Met-His-Ser-Phe-

       27
(Phe)-Ala
wherein X-X had not been determined. The disclosure of said application is incorporated herein by reference.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, the complete coding sequence of a cDNA clone representing essentially the full size tissue factor inhibitor (TFI) has been developed.
Initially, human placental and fetal liver &lgr;gt11 CDNA libraries were screened with a rabbit polyclonal antiserum raised against a purified TFI. Immunologically positive clones were further screened for
125
I-Factor X
a
binding activity. Seven clones were obtained which were immunologically and functionally active. The longest clone, placental-derived &lgr;P9, was 1.4 kilobases (kb) long while the other six were 1.0 kb in length. Partial DNA sequencing showed the 1.0 kb clones to have sequences identical to part of the longer 1.4 kb clone. Nucleotide sequence analysis showed that &lgr;P9 consisted of a 1432 basepair (bp) cDNA insert that includes a 5′-noncoding region of 133 bp, an open reading frame of 912 bp, a stop codon, and a 3′-noncoding region of 384 bp.
The cDNA sequence encodes a 31,950 Dalton protein of 276 amino acids which includes 18 cysteines and 7 methionines. The translated amino acid sequence shows that a signal peptide of about 28 amino acids precedes the mature TFI protein. It will be understood that the “mature” TFI is defined to include both TFI and methionyl TFI by virtue of the ATG translational codon in the &lgr;P9 clone described herein.
There are three potential N-linked glycosylation sites in the TFI protein with the sequence Asn-X-Ser/Thr, wherein X can be any of the common 20 amino acids. These sites are at amino acid positions Asn 145, Asn 195, and Asn 256, when the first methionine after the 5′-noncoding region is assigned amino acid position +1.
The translated amino acid sequence of TFI shows several discernible domains, including a highly negatively charged N-terminal, a highly positively charged carboxy-terminal, and an intervening portion consisting of 3 homologous domains with sequences typical of Kunitz-type enzyme inhibitors. Based on a homology study, TFI appears to be a member of the basic protease inhibitor gene superfamily.
The original source of the protein material for developing the cDNA clone &lgr;P9 was human placental tissue. Such tissue is widely available after delivery by conventional surgical procedures. The &lgr;gt11 (lac5 nin5 c1857 S100) used herein is a well-known and commonly available lambda phage expression vector. Its construction and restriction endonuclease map is described by Young and Davis,
Proc. Natl. Acad. Sci. USA
80, 1194-1198 (1983).
Northern blot analysis showed that the following liver-derived cell lines:
Chang liver, HepG2 hepatoma, and SK-HEP-1 hepatoma, all contained 2 major species of mRNA (1.4 and 4.4 kb) which hybridized with the TFI cDNA.
The cloning of the cDNA for TFI and 5 development of its entire protein sequence and structural domains as disclosed herein permits detailed structure-functional analyses and provides a foundation for study of its biosynthetic regulations. The invention thus is important to medical science in the study of the coagulation cascade with respect to agents which are able to inhibit Factor X
a
and Factor VII
a
/TF enzymatic complex.


REFERENCES:
U.S. patent application Ser. No. 07/077,366, Broze, filed Jul. 23, 1987.
Hjort, Scan. J. Clin. Lab. Invest., 9(suppl. 27), p. 76-97, (1957).
Broze et al., Blood, 69, 150-155, (1987).
Sanders et al., Blood, 66, 204-212 (1985).
Broze et al., Proc. Natl. Acad. Sci. USA 84, 1886-1890 (1987).
Proudfoot et al., Nature, 252, 359-362 (1981).
Von Heijne, Eur. J. Biochem. 123, 17-21 (1983).
Von Heijne, J. Mol. Biol. 184, 99-105 (1985).
Kozak, Cell, 44, 283-292, (1986).
Henikoff, Gene, 28, 351-359 (1984).
Lipman & Pearson, Science 227, 1435-1441 (1985).
Sanger et al., Proc. Natl. Acad. Sci. USA 70(4), 1209-1213 (1973).
Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1982).
Thomas, Methods Enzymol. 100, 255-266 (1983).
Lizardi & Engelberg, Anal. Biochem. 98, 116-122 (1979).
Wun & Kretzmer, FEBS Lett. 1, 11-16 (1987).
Kidera et al., J. Prot. Chem. 4, 23-55 (1985).
Young & Davis, Proc. Natl. Acad. Sci. USA, 80, 1194-1198 (1983).

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