Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-11-12
2003-06-03
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S069200, C424S094640, C514S002600, C514S008100, C514S012200, C514S021800, C530S384000, C530S395000
Reexamination Certificate
active
06573071
ABSTRACT:
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is the U.S. national phase of PCT/AT98/00045, filed Feb. 27, 1998, which claims priority to Austrian Application A335/97, filed Feb. 27, 1997.
FIELD OF INVENTION
The invention relates to Factor X analogues having a modification in the region of the activation peptide, to a preparation containing the Factor X analogues according to the invention, and to a method of preparing single chain and double chain Factor X analogues.
BACKGROUND
After the blood coagulation process has been initiated, the coagulation cascade continues through sequential activation of various proenzymes (zymogens) in the blood to their active forms, the serine proteases. Among them are, inter alia, Factor XII/XIIa, Factor XI/XIa, Factor IX/IXa, Factor X/Xa, Factor VII/VIIa and prothrombin/thrombin. In their physiological state, most of these enzymes are only active if associated to a membrane surface in a complex. Ca ions are involved in many of these processes. The blood coagulation will either follow the intrinsic pathway, wherein all protein components are present in the blood, or the extrinsic pathway, wherein the cell membrane tissue factor plays a critical role. Finally, the wound will be closed by thrombin cleaving fibrinogen to fibrin.
The prothrombinase complex is responsible for activating prothrombin to thrombin. Thrombin is an important enzyme which can act as a procoagulant as well as an anticoagulant. The prothrombinase complex, in which, inter alia, Factor Va (as cofactor) and Factor Xa (as serine protease) are involved, assembles in a Ca-dependent association at the surface of phospholipids. It is discussed that Factor Xa is the catalytic component of the prothrombinase complex.
Factor X (Stuart-Prower factor) is a vitamin K-dependent coagulation glycoprotein by which the intrinsic and the extrinsic blood coagulation cascades can be activated. The primary translation product of Factor X (pre-pro-FX) has 488 amino acids and is initially synthesized by the liver or human hepatoma cells as a single-chain 75 kD precursor protein. In plasma, Factor X is largely present as a double chain molecule (Fair et al., 1984, Blood 64:194-204).
During biosynthesis, after cleavage of the presequence by a signal peptidase (between Ser23/Leu24) and of the propeptide (between Arg40/Ala41), the single chain Factor X molecule is cleaved by processing and removal of the tripeptide Arg180-Lys181-Arg182 to the double chain form consisting of the approximately 22 kD light chain and the approximately 50 kD heavy chain, which are connected via a disulfide bridge (FIGS.
1
A-
1
B). Therefore, Factor X circulates in the plasma as a double chain molecule.
During the blood coagulation process, Factor X is converted from inactive zymogen to active protease Factor Xa by limited proteolysis; wherein Factor X can be activated to Factor Xa in either of two membrane-associated complexes: in the extrinsic Factor VIIa-tissue factor complex or in the intrinsic Factor VIIIa-Factor IXa-phospholipid-Ca-complex, or “tenase complex” (Mertens et al., 1980, Biochem. J. 185:647-658). A proteolytic cleavage between amino acids Arg234/Ile235 results in the release of an activation peptide having a length of 52 amino acids from the N-terminus of the heavy chain and thus to the formation of the active enzyme, Factor Xa. The catalytic center of Factor Xa is located on the heavy chain.
Activation via the Factor VIIa-TF (extrinsic) complexresults in the formation of Factor Xa&agr; (35 kD) and Factor Xa&bgr; (31 kD), with a polypeptide of 42 (kD) forming, too, if, the Factor VIIa concentration in the complex is low. Factor Xa&agr; is formed by a cleavage at Arg234/Ile235 of the heavy chain and represents the activation of Factor X to Factor Xa. The occurence of Factor Xa&bgr; presumably results from an autocatalytic cleavage at Arg469/Gly470 in the C-terminus of the heavy chain of Factor Xa&agr; and the removal of a 4.5 kD peptide. Like Factor Xa&agr;, Factor Xa&bgr; has catalytic activity. It has been shown, however, that a plasminogen binding site is formed by the cleavage of Factor Xa&agr; to Factor Xa&bgr;, and that Factor Xa&bgr; optionally has fibrinolytic activity or is involved in fibrinolysis as a cofactor. The conversion of Factor Xa&agr; to Factor Xa&bgr;, however, is slower than the formation of thrombin, thus preventing the initiation of fibrinolysis before a blood clot is formed (Pryzdial et al., 1996, J. Biol. Chem. 271:16614-16620; Pryzdial et al., 1996, J. Biol. Chem. 271:16621-16626).
The 42 kD polypeptide results from processing in the C-terminus of the heavy chain between Arg469/Gly470 without previous processing between Arg234/Ile235. Like a Factor Xay fragment formed by proteolysis at Lys370, this intermediate has no catalytic activity (Mertens et al., 1980, Biochem. J. 185:647-658; Pryzdial et al., 1996, J. Biol. Chem. 271:16614-16620).
Intrinsic Factor X activation is catalysed by the Factor IXa-Factor VIIIa complex. The same processing products are obtained during activation, but the Factor Xa&bgr; product is obtained in larger quantity than other Factor X processing products (Jesty et al., 1974, J. Biol. Chem. 249:5614).
In vitro, Factor X can, for instance, be activated by Russell's viper venom (RVV) or trypsin (Bajaj et al., 1973, J. Biol. Chem. 248:7729-7741) or by purified physiological activators, such as FVIIa-TF complex or Factor IXa-Factor VIIIa complex (Mertens et al., 1980, Biochem. J. 185:647-658).
Most commercially available Factor X products from plasma contain a mixture of Factor Xa&agr; and Factor Xa&bgr;, because after activation of Factor X to Factor Xa mainly Factor Xa&agr; is formed, which is, in turn, cleaved to Factor Xa&bgr; in an autocatalytic process. In order to produce a uniform Factor Xa product having high structural integrity, EP 0 651 054 suggested to activate Factor X with RVV over an extended period of time so that the resulting final product substantially contains Factor Xa&bgr;. The by-products, e.g. Factor Xa&agr;, as well as the protease were subsequently removed by several chromatographic steps.
Factor X cDNA has been isolated and characterized (Leytus et al., 1984, Proc. Natl. Acad. Sci., U.S.A., 82:3699-3702; Fung et al., 1985, Proc. Natl. Acad. Sci., U.S.A., 82:3591-3595). Human Factor X has been expressed in vitro in various types of cells, such as human embryonal kidney cells or CHO cells (Rudolph et al., 1997, Prot. Expr. Purif. 10:373-378; Wolf et al., 1991, J. Biol. Chem. 266:13726-13730). However, it has been found that in the recombinant expression of human Factor X, the processing at position Arg40/Ala41is inefficient, as opposed to the situation in vivo, and that different N-termini at the light chain of Factor X are produced (Wolf et al., 1991, J. Biol. Chem. 266:13726-13730). Recombinant Factor X (rFX) was activated to rFactor Xa (rFXa) by RVV in vitro, or rFXa was expressed directly, with the activating peptide being deleted from amino acid 183 to amino acid 234 and replaced by a tripeptide in order to allow processing directly to a double chain rFXa form. About 70% of purified rFX was processed into light and heavy chain, while the remaining 30% represented single chain rFX of 75 kD. Direct expression of rFXa did result in the formation of active Factor Xa, but also of inactive intermediates. Wolf et al. (1991, J. Biol. Chem. 266:13726-13730) detected still reduced activity of recombinant Factor X, which they ascribed to the poorer ability of rFX to be activated by RVV and to the inactive protein and polypeptide populations of the single chain precursor molecule. In particular, they found high rFXa instability when expressed by recombinant cells, which they ascribed to the high rate of autoproteolysis.
In order to study the function of the C-terminal peptide of Factor Xa&agr;, Eby et al. (1992, Blood 80 (Suppl. 1):1214 A) introduced a stop codon at position Gly430 of the Factor X sequence. However, they did not find a difference between the rate of activation of Factor Xa (FXa&agr;) by &bgr;-peptide or a de
Dorner Friedrich
Eibl Johann
Fisch Andreas
Himmelspach Michele
Schlokat Uwe
Baxter Aktiengesellschaft
Low Christopher S. F.
Schnizer Holly
Townsend and Townsend and Crew L.L.P.
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