AU2012254970A1

AU2012254970A1 - Vitamin K Epoxide Recycling Polypeptide VKORC1, a Therapeutic Target of Coumarin and Their Derivatives

Info

Publication number: AU2012254970A1
Application number: AU2012254970A
Authority: AU
Inventors: Andreas Fregin; Clemens R. Muller-Reible; Johannes Oldenburg; Simone Rost; Tim-Matthias Strom
Original assignee: Baxter Healthcare SA; Baxter International Inc
Current assignee: Baxter Healthcare SA; Baxter International Inc
Priority date: 2003-10-14
Filing date: 2012-11-16
Publication date: 2012-12-13

Abstract

The invention relates to a novel polypeptide vitamin K epoxide recycling polypeptide (VKORC 1) as a target for coumarin and its derivatives. The invention further provides methods for identifying coumarin derivatives, and also claims VKORC1 polypeptides and VKORC1 nucleic acids containing a sequence abnormality associated with a VKORC1 associated deficiency such as warfarin resistance, wherein the VKORCl polypeptides and VKORC1 nucleic acids can be used for diagnosing these deficiencies. Moreover, the invention relates to methods for identifying coumarin derivatives usable in pest control of rodents.

Description

1 Related Applications This application is a divisional of Australian application No. 2009212805 filed on 26 August 2009, which is a divisional of Australian application No. 2004283246 filed on 12 October 2004, and is related to International application No. PCT/EP2004/011432 filed on 12 5 October 2004 claiming priority from US application No. 60/511,041 filed on 14 October 2003; each of which is incorporated herein by reference. Field of the Invention The invention relates to a novel polypeptide vitamin K epoxide recycling polypeptide (VKORCl) as a target for coumarin and its derivatives. The invention further provides methods 0 for identifying coumarin derivatives, and also claims VKORCl polypeptides and VKORC1 nucleic acids containing a sequence abnormality associated with aVKORCI associated deficiency such as warfarin resistance, wherein the VKORC1 polypeptides and VKORCI nucleic acids can be used for diagnosing these deficiencies. Moreover, the invention relates to methods for identifying coumarin derivatives usable in pest control of rodents. 5 Background of the invention Repression of untimely blood coagulation is the therapeutic option of choice for acute treatment and long-term prevention of thrombolic events. Among the anti-coagulants coumarins are widely used for the prevention of thrombosis such as in patients immobilized after surgery, patients having a chronic heart failure, patients having atherosclerotic vascular disease, patients 0 having a malignancy, and patients that are pregnant. Moreover, coumarins are the most widely used oral anticoagulants for the treatment and prophylaxis of thrombosis [Suttie, 1987]. Coumarins are typically derivatives of 6-hydroxycoumarin, such as 3-(acetonylbenzyl)-4 hydroxycoumarin (COUMADIN*). The coumarins target the blood coagulation cascade indirectly by inhibition of the 25 vitamin K cycle. Vitamin K is an essential cofactor for the post-translational activation by gamma carboxylation of a group of regulatory proteins, the Gla-proteins. In several metabolic pathways, some key proteins require carboxylation for proper function. The blood coagulation cascade is the best-studied example. Here, the procoagulant factors II, VII, IX and X, and the 30 anticoagulant factors protein C, protein S, protein Z are dependent on gamma-carboxylation. This post-translational modification enables the attachment of the modified proteins - in the presence of calcium - to phospholipid- bilayer membranes which is an essential step in the activation of blood coagulation [Sperling el al., 1978][Esmon et al., 1975]. Other proteins 2 requiring gamma-carboxylation are the matrix gla protein and osteocalcin, both regulators of bone metabolism [Price, 1988] and the "growth arrest specific gene", a signal transduction protein of the cell cycle[Manfioletti et al., 1993][Stitt et al., 1995]. During gamma-carboxylation, a carboxyl group is introduced into glutamate residues of 5 the target proteins by the enzyme gamma-glutamyl carboxylase (GGCX) in liver microsomes [Furie & Furie, 1988][Suttie, 1987]. The reaction requires as a cofactor stoichiometric amounts of reduced vitamin KI hydroquinone (vitamin KIH2) which is oxidized to vitamin K-2,3 epoxide [Cain et al., 1997]. The regeneration of the active cofactor is mediated by a multi protein complex termed vitamin K-2,3-epoxide reductase (VKOR) [Wallin & Martin, 1985]. 0 The same complex is targeted by the coumarin-type poisons used in rodent pest control. This "vitamin K cycle" has been characterized biochemically in great detail but the molecular components have not yet been purified to homogeneity [Guenthner et al., 19981]. Moreover, the molecular nature of coumarin activity and the molecules interacting with coumarins are still elusive. 5 It is generally appreciated in the art that although largely effective, there are a number of limitations to the use of coumarins. First of all; there are humans that are inert to coumarin treatment. The term warfarin resistance (WR) is used for individuals who maintain normal clotting factor activities despite oral anticoagulation by coumarins (OMIM Access. No. 122700). Autosomal dominant transmission has been observed in several pedigrees [O'Reilly et 0 al., 1964][O'Reilly, 1970]. Combined deficiency of all vitamin K dependent clotting factors (VKCFD) is a very rare bleeding disorder in humans of autosomal recessive inheritance with 14 cases described as yet [McMillan & Roberts, 1966][Fischer, 1966][Johnson et al., 1980][Goldsmith et al., 1982][Vicente et al., 1984][Ekelund et al., 1986][Pauli et al., 1987][Leonard, 1988][Pechlaner et al., 1992][Boneh & Bar-Ziv, 1996][Brenner et al., 25 1998][Spronk et al., 2000][Oldenburg et al., 2000]. Clinical symptoms of the disease include episodes of perinatal intracerebral hemorrhage sometimes with fatal outcome. The bleeding tendency is usually completely reversed by oral administration of vitamin K. Additional symptoms in newborns can resemble warfarin embryopathy with nasal and distal phalangeal hypoplasia and premature calcification of epiphyses[Pauli et al., 1987]. The disease may result 30 either from a defective resorption/transport of vitamin K to the liver [Prentice, 1985] or from mutations in one of the genes involved in gamma-carboxylation. In subtype 1 (VKCFD1, OMIM # 277450), mutations in the GGCX gene on chromosome 2p12 result in insufficient carboxylation of clotting factors [Brenner et al., 1998][Spronk et al., 2000 ]. There has been described a linkage of two kindreds with familial multiple coagulation factor deficiency 3 (FMFD, now re-named: VKCFD2, OMIM # 607473) to a 20 Mb interval of the pericentric region of chromosome 16p12-q21 [Fregin et al., 2002]. Patients with VKCFD2 showed significantly increased serum levels of vitamin K epoxide, thus suggesting a defect in one of the subunits of the VKOR complex. Taken together, there is evidence that there are patients that 5 display warfarin resistance. As a result, there is a need to identify novel coumarins derivatives that are effective anticoagulants for treating these patients, and methods for identifying these coumarin derivatives. The use of coumarins is associated with a risk of spontaneous bleedings, with a significant mortality rate. Moreover, the prediction of the accurate coumarin maintenance dose 0 is difficult. In the absence of the target molecule which coumarin exerts an effect on, the treatment regimen has to be established, on a patient-by-patient basis. During the time the optimum regimen is yet not established the patient either suffers from an increased risk of thrombogenesis or of an increased risk of bleeding. Therefore there is a need for a method of determining the optimal treatment regimen that is faster and saver. Further, establishing an 5 optimal treatment regimen is complicated by the fact, that there is a considerable delay between the administration of coumarins and the onset of its anticoagulant activity. Given the delayed action of coumarin and given the fact that coumarin tends to accumulate in time there is a need for coumarin derivatives that effect blood coagulation faster than the coumarins known in the art. By the same token there is also a need for coumarins that are metabolized more rapidly so 0 that accumulation of coumarin may be prevented or ameliorated and as a result the danger of overdosing is decreased or abolished. It is well appreciated that if coumarin treatment is initiated during a thrombic state, the levels of protein C and S decline, thus temporarily creating a thrombogenic potential which is usually compensated for by overlapping heparin and coumarin administration for a number of 25 days. Again, there is a need to identify the molecular target of coumarin action in order to be able to screen for novel coumarin derivatives that do not possess these limitations or at least to a lesser extend. A coumarin therapy sometimes induces skin necrosis in patients and if applied during pregnancy may cause embryopathy creating a need for novel coumarin derivatives which do not 30 cause these effects. There are a number of interactions between drugs and coumarins. Some of these drugs like Phenobarbital induce lower plasma levels of coumarins due to an increased metabolization of coumarin which is believed to be caused by the mixed-function oxidases like the cytochrome P450 mixed-function oxidases. Such interaction is of clinical relevance if the appropriate 4 regimen of e.g. Phenobarbital and coumarin has been determined and later on only administration of Phenobarbital is discontinued leading to a rise of the plasma level of coumarin which causing excessive anticoagulation. Other drugs like Amiodarone cause a delayed metabolization of coumarin leading again to excessive anticoagulation if co-administered with 5 coumarins. Since the molecules affected by coumarins are not known in the art there is a need to develop novel coumarins and tools to identify the latter in order to solve these problems. Finally, coumarins, especially warfarin, are not only used in humans but since the 1950s, coumarins have been in use as an active ingredient in rodenticidal compositions. The basis for the effectiveness of warfarin as a rodenticide lies in the fact that it is an effective anti 0 coagulant in small, multiple doses. One or two doses of the compound are seldom fatal if taken at the recommended concentration; thus the hazard of acute toxicity to man, domestic animals, and wildlife is greatly reduced. Usually the rodents begin to die after four or five daily doses of the materials, and the population is greatly reduced or eradicated in approximately three weeks. Death is caused by hemorrhages, brought about by the action of the warfarin in reducing the 5 clotting power of the blood. These hemorrhages may be external or internal and can be initiated by very slight injury or capillary damage. One of the other advantages of coumarins is that, because multiple ingestions are required to kill the rodents, they do not develop bait shyness. Beginning in 1969, rodents - particularly rats and, to a somewhat lesser extent, mice - began showing resistance to warfarin baits. The general assumption was that such resistance had a 0 genetic basis. As for the mechanism, it is the VKORCI complex mentioned above that is targeted by derivatives of warfarin in use for rodent pest control [Jackson et al., 1988]. Resistance to coumarin derivatives has arisen spontaneously in several wild rodent populations rendering the use of these drugs locally ineffective for pest control. Autosomal dominant loci for warfarin resistance have been mapped in the mouse (War) to chromosome 7 [Wallace et al., 25 1976] and in rat (Rw) to the long arm of chromosome 1 [Greavses & Ayres, 1967] [Kohn & Pelz, 1999]. Since the VKOR complex is the target of the coumarin drugs resistance is thought to be mediated by alterations in one of its protein components [Jackson, 1988]. The development of resistance in rodents has created a need for identifying the target of coumarins action which would facilitate the development of novel coumarin-derivatives for use in pest 30 control. Taken together it is an object of the present invention to provide a target molecule for coumarin and its derivatives in mammals. It is another object of the present invention to provide methods for identifying novel coumarins which solve at least one of the problems mentioned above. It is a further object of the present invention to identify polypeptides and 5 nucleic acids coding for them which cause warfarin resistance in human and non-human mammals, preferably rodents. It is also an object of the present invention to diagnose, prevent and/or treat disorders and diseases selected from diseases from warfarin resistance, familial multiple factor deficiency, a disorder or disease associated with increased blood coagulation 5 such as patients suffering from a thrombus and/or patients having an increased risk of developing a thrombus, such as an inherited increased risk of thrombogenesis, preferably an increased risk of thrombogenesis due to a surgery or due to pregnancy, and increased vascular calcification. Moreover, it is also an object of the present invention to diagnose, prevent and/or treat diseases or disorders associated with attenuated blood coagulation, such as hemophilia, 0 disorder associated decreased vascular calcification and disorders and diseases with an increased risk of bleeding. Finally, it is an object of the present invention to provide a method for identifying coumarin and its derivatives which are effective in pest control of non-human mammal and compositions for killing rodents. Summary of the invention 5 In solving the above objects a vitamin K epoxide recycling polypeptide (VKORCl) is provided, comprising or consisting of a polypeptide sequence selected from the group consisting of: (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, 17, 21, 25, and 27; 0 (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a); (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), which polypeptide sequence has VKORCl activity; and (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORCI activity. 25 Moreover, according to another aspect of the present invention there is provided a nucleic acid coding for the VKORCI polypeptide according to the invention (VKORCI nucleic acid). In addition, according to another aspect of the present invention there is provided a method of identifying a coumarin derivative which exerts an effect onto the activity of 30 VKORC 1 polypeptide according to the invention comprising the steps of: (I) providing a host cell having been introduced the VKORC1 nucleic acid or a vector containing the VKORC 1 nucleic acid; (II) expressing the VKORCI polypeptide in the host cell; 6 (III) administering a candidate coumarin derivative; (IV) determining the activity of VKORCl polypeptide (candidate activity value); (V) comparing the candidate activity value with a control activity value; and (VI) identifying the candidate coumarin derivative as a coumarin derivative exerting an 5 effect onto the activity of the VKORC1 polypeptide, provided the candidate activity value is significantly different from the control activity value. Furthermore, according to another aspect of the present invention there is provided a method of determining a VKORCI polypeptide sequence which conveys a coumarin effect exerted onto VKORC 1 activity, comprising the steps of: 0 (I) providing a cell expressing the VKORCI polypeptide according to the invention, which VKORC1 polypeptide has at least one sequence abnormality; (II) administering coumarin or a derivative thereof to the cell; (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and 5 (IV) comparing the sequence abnormality activity value with the control sequence activity value, wherein a significant deviation of the sequence abnormality activity value from to the control sequence activity value is indicative that the sequence abnormality of the VKORCI polypeptide conveys the coumarin effect exerted onto VKORC I polypeptide. 0 In another aspect of the present invention there is provided a VKORC1 polypeptide according to the invention, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORCI polypeptide. Moreover, according to another aspect of the present invention there is provided a method of diagnosing a VKORCI associated deficiency in a patient comprising the steps of: 25 (I) amplifying a DNA sample obtained from the patient or reverse transcribing a RNA sample obtained from the patient into a DNA and amplifying the DNA; and (II) analyzing the amplified DNA of step (I) to determine at least one sequence abnormality in a nucleic acid sequence coding for the VKORCl polypeptide of claim I or in an amino acid sequence of the VKORCI polypeptide; 30 wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency; preferably the sequence abnormality exerts an effect on the activity of the VKORCI polypeptide.

7 In addition, according to another aspect of the present invention there is provided a method of identifying a coumarin derivative which is toxicologically effective in warfarin resistant rodents comprising the steps of: (I) providing a warfarin-resistant rodent; 5 (II) administering a candidate coumarin derivative to the rodent; (III) determining the toxicity of the candidate coumarin derivative onto the rodent (candidate coumarin derivative toxicity value); (IV) comparing the candidate coumarin derivative toxicity value with a control coumarin toxicity value; 0 (V) identifying the candidate coumarin derivative as a rodenticidally effective coumarin derivative provided that the candidate coumarin derivative toxicity value is significantly larger than the control coumarin toxicity value. According to another aspect of the present invention, the identified coumarin derivatives can be included into a composition for killing rodents. 5 Definitions of the specific embodiments of the invention as claimed herein follow. According to a first embodiment of the invention, there is provided use of a VKORCI nucleic acid in a method for gamma-carboxylating a vitamin-K dependent polypeptide in a host cell. According to a second embodiment of the invention, there is provided use of a '0 VKORCI nucleic acid for facilitating gamma-carboxylation of a vitamin-K dependant protein in a host cell, comprising: a) introducing said VKORCl nucleic acid into the host cell; and b) expressing a VKORC1 polypeptide in the host cell. According to a third embodiment of the invention, there is provided use of a VKORC1 25 nucleic acid in a method to enzymatically convert or support the enzymatic conversion of vitamin K2, 3-epoxide to vitamin K-quinone and/or the conversion of vitamin K quinone to vitamin K hydroquinone, comprising: a) introducing said VKORCl nucleic acid into a host cell; and b) expressing a VKORC1 polypeptide in said host cell, 30 wherein said VKORCI polypeptide enzymatically converts or supports the enzymatic conversion of vitamin K2, 3-epoxide to vitamin K-quinone and/or the conversion of vitamin K quinone to vitamin K hydroquinone.

8 Brief description of the drawings Fig. 1 depicts a comparison of the candidate interval of 3cM in the genetic map containing the VKORCI gene locus in human, rat and mouse. The ideogram of human chromosome 16 is shown, the area of homozygosity in the families 1 and 2 extends from 5 16pl 1.2 to 16q 13 corresponding to approximately 25 Mb. In the right part of the figure there are homologous parts of mouse and rat-chromosomes. Synthenic genes of 16pl 1.2 and 16q 12.1 with homologous counterparts in Mus musculus (MMU) and Rattus norvegicus (RNO) are depicted. The loci for phenotype resistance to warfarin in the mouse (War) and rat (Rw) are mapped to regions homologous to 16pi 1.2. MMU: Mus musculus; RNO: Rattus norvegicus; 0 PRKCBI; Prkcb; IL4R : Interleukin4 receptor a (human); Il4ra: Interleukin4 receptor a (murin); Il4r : Interleukin4 receptor a (rat); SPS2 : Selenophosphate synthetase (human); Sps2 Selenophosphate synthetase (murin/rat); HUMMLC2B :Myosin light Chain2 (human); Mylpf: Myosin light chain 2 (murin); Myl2 : Myosin light chaun 2 (rat); SPN : Sialophorine (human); Spn : Sialophorine (murin/rat). 5 Fig. 2 displays VKORC 1 mutations in human vitamin K dependent clotting factor 2 (VKCFD2) and warfarin resistance (WR) patients. The upper part of the figure shows the segregation of the R98W mutation in two VKCFD2 families and the electropherograms of a homozygous mutant (left) compared to a control (right). The bottom part of the figure shows the heterozygous mutations of four WR patients. (85G>T, 134T>C, 172A>G, 383T>G) and a 0 Rw rat (416A>G). Fig. 3 shows a sequence alignment of VKORCI and VKORC1 like protein 1 (VKORC1L1) polypeptides. The alignment was generated with CLUSTALW and PRETTYBOX. Human (hVKORC1), mouse (mVKORCl) and rat VKORC1 (rVKORC1) and VKORC1 LI polypeptides, i.e. VKORC1 Li of human (hVKORC1 LI), mouse (mVKORC1 Li) 25 and Fugu rubripes (fVKORCiLl), share approximately 84% sequence identity within both groups and approximately 50% identity between both groups of proteins. xVKORC1 depicts the VKORC1 polypeptide sequence of Xenopus laevis, fVKORC1 the VKORCI polypeptide sequence of Fugu rubripes, and aVKORC1 the VKORC1 polypeptide sequence of Anopheles gambiae. Tree analysis allows grouping the Fugu rubripes, Xenopus laevis and Anopheles 30 gambiae proteins to the appropriate group. The locations of the predicted transmembrane domains are underlined. Residues 29, 45, 58 and 128 mutated in WR patients are conserved in all species. The arginine at position 98 mutated in the VKCFD2 patients is conserved in human, rat and mouse (plus sign).

9 Fig. 4 displays a northern blot analysis of VKORCI in fetal and adult human tissues. The upper blot depicts a northern blot of adult tissue, whereas the lower blot depicts a northern blot of fetal tissue. For more details see Example 4. The lines with fragments of the sizes 2.4, 4.4, 7.5, UND 9.5 KB indicate molecular weight markers and allow estimation of the size of the 5 all visible bands) Fig. 5 shows the subcellular location of VKORCL. For more details see example 6. To this end, COS-7 cells transiently transfected with VKORCl constructs were stained with anti calnexin (red; left column) and anti-GFP or anti-myc, respectively (green; middle column). Merged figures of the double-stained cells are shown in the right column. Both VKORCI 0 constructs (tagged with GFP or myc) co-localize with the ER specific calnexin staining. The control construct (pEGFP-N 1) shows a diffuse staining pattern throughout the cytoplasm. Fig. 6 displays a list of siRNA sequences for homo sapiens VKORC1 and primers endoding these siRNAs which can be used to express them using for example the siLentGeneTM U6 Cassette RNA Interference System. 5 Fig. 7 displays locations of siRNA targets in the coding sequence of homo sapiens vitamin K epoxide reductase complex subunit I (Hs VKORC 1), which are shown in light grey; regions which are part of two possible siRNA targets are shown in darker grey; and regions with two or more possible siRNA sequences are shown in an even darker grey. Fig. 8 provides a list of PCR primer sequences and PCR conditions for amplification of 0 Homo sapiens VKORCI and Homo sapiens VKORCI L1. Fig. 9 provides a listing of the sequences their respective SEQ ID NOs. Fig. 10 shows VKOR activities of HEK293 cells transfected with VKORCI cDNA. Values are given as percent vitamin K epoxide converted into vitamin K quinone (product/residual substrate+product). Wildtype VKORC1 activity is also defined by being 25 sensitive to warfarin (4.3 % residual activity at 80pM warfarin compared to not inhibited). Mutations Y139C and V29L leading to resistance to warfarin exhibit 69 and 11% residual activity at 80 pM warfarin respectively). All tests were run in duplicate. Untransfected and mock-transfected showed 1.49 and 0.96% activities, and were >90% inhibited by 10 pM warfarin. For further details see Example 7. 30 Fig. 11 shows the amino acid sequence of Homo sapiens vitamin K epoxide recycling polypeptide (HS_VKORCl; SEQ ID NO: 1) Fig. 12 shows the nucleic acid coding sequence of Homo sapiens vitamin K epoxide recycling polypeptide (HS_VKORCl; SEQ ID NO: 2) 10 Fig. 13 shows the result of an ARMS-PCR experiment to determine whether or not a tested rat is warfarin resistant. Wildtype rats exhibited a band at 123 bp (probe# 3351, 3133, 3137, 3142, 4724, 4684, 3138, 3162), rats homozygous to the mutation (probe# 4701) exhibited a band at 101 bp and finally, rats with the heterozygous mutation (probe# 3066, 3350, 3352, 5 3354, 3139, 3140, 4754, 3146, 3148, 3149 ) showed two bands, one at 101 and another band at 123 bp. For further details see Example 9. Detailed description of the invention In order to meet the needs for developing novel coumarin derivatives and for identifying the target of coumarin and its derivatives the vitamin K epoxide recycling polypeptide 0 (VKORC1) was cloned. This gene was previously unknown spanning a genomic region of 5126 bp and comprising three exons coding for a protein of 163 amino acids. Topology analysis suggests a highly hydrophobic protein with at least two transmembrane domains. This is compatible with the known location of the VKORC1 complex activity in ER membranes and with immunofluorescence data in COS-7 cells transfected with VKORCI constructs (Fig. 5). 5 The VKORCI gene was surprisingly identified in a mutant screen of warfarin resistant patients (for details cf. examples 1 and 2). According to the present invention, there has been surprisingly identified a gene, VKORC1, which is mutated in patients with a combined deficiency of all vitamin K dependent coagulation factors (VKCFD2) and with warfarin resistance (WR), respectively, showing that VKORC1 polypeptide contains a binding site for 0 warfarin and is a target of coumarin and its derivatives. The evidence that the mutations are causative of the two phenotypes is as follows: (i) an R98W mutation segregates with the disease in two unrelated families with VKCFD2; (ii) this arginine at position 98 is conserved in the human and in the homologous mouse and rat genes, respectively; 25 (iii) three warfarin resistant brothers share an R58G substitution; (iv) this amino acid and the other residues found mutated in two more unrelated WR patients (V29L and L 1 28R) are conserved in all species analyzed except for three bacterial genes (see Fig. 3); and (v) none of the 5 presumed mutations was found in 192 control DNA samples. 30 Moreover, homology searches in genome and protein databases have not revealed any similarities of VKORCl to any protein or peptide domain of annotated function. However, homologous genes are found in vertebrates (rat, mouse, Xenopus, Fugu), insects (Anopheles) and bacteria (Fig. 3). Surprisingly, the three mammals and Fugu each have a second VKORCl- I1 like gene of moderate similarity to the cognate gene. A number of amino acid positions within these genes are conserved throughout evolution. This is in accordance with the well established fact that gamma-carboxylation - and thus the use of vitamin K as a cofactor of this process - is an evolutionary old post-translational protein modification [Bandyopadhyay et al., 2002]. 5 A substitution of valine 29, arginine 58 leucine 128 - although dispersed over the entire VKORCl polypeptide - obviously renders the inhibition of VKORCl activity by warfarin ineffective. It can be speculated that these amino acids functionally co-operate in the tertiary structure of the VKORC1 protein 1. Taken together the mutation data in patients with two different phenotypes provide VKORC1 as the target protein, both for vitamin K and warfarin 0 binding. In one aspect of the present invention there is provided a vitamin K epoxide recycling polypeptide (VKORCI) comprising, preferably consisting of, a polypeptide sequence selected from the group consisting of: (a) a polypeptide sequence selected from the group consisting of a sequence according to 5 SEQ ID No. 1, 12, 17, 21, 25, and 27; (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a); (c) a polypeptide sequence having at least 80% homology with the polypeptide sequence defined in (a) or (b), which polypeptide sequence has VKORC 1 activity; and (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or 0 (c) having VKORCI activity. Preferably, the VKORC1 polypeptide is a target for coumarin and its derivatives in mammals. Within the meaning of the invention the term "VKORCl polypeptide" refers to the full length sequence of the VKORC1 polypeptide as defined in the preceding paragraph. The term 25 "VKORC1 polypeptide" also encompasses isolated VKORCl polypeptides and VKORC1 polypeptides that are prepared by recombinant methods, e.g. by isolation and purification from a sample, from a host cell expressing the VKORC1 polypeptide, by screening a library and by protein synthesis, all of these methods being generally known to the person skilled in the art. Preferably, the entire VKORCI polypeptide or parts thereof can be synthesized, for example, 30 with the aid of the conventional synthesis such as the Merrifield technique. More preferably, the term "VKORC I polypeptide" also encompasses polypeptides which have a sequence homology of about 80%, preferably about 90%, in particular about 95%, especially about 98% with the VKORC1 polypeptide according to one of SEQ ID No. 1, 12, 17, 21, 25, and 27, provided that such VKORC1 polypeptide has VKORC1 activity. Moreover, it is preferred that the term 12 "VKORCI polypeptide" also encompasses homologous polypeptides which originate from organisms other than human, preferably from non-human mammals such as, rodents, e.g. mouse, rats, or monkeys and pigs and other vertebrates and invertebrates, such as those amino acid sequences according to SEQ ID Nos. 12, 17, 21, 25, 27, provided that such VKORC1 5 polypeptide has VKORCI activity. It is even more preferred that the term "VKORCl polypeptide" also includes VKORCI polypeptides which are encoded by different alleles of the gene, in different individuals, in different organs of an organism or in different developmental phases, provided that such VKORC1 polypeptide has VKORC1 activity. It is further intended that the term "VKORCI polypeptide" preferably also encompasses naturally occurring or 0 synthetic mutations that exert no or only insignificant effects onto the activity of the VKORC 1 polypeptide. Other polypeptides preferably encompassed by the term "VKORC1 polypeptide" include VKORCI polypeptides that may arise from differential splicing of the VKORC1 transcript, provided that such VKORC1 polypeptide has VKORCI activity. The term "fragment of the polypeptide sequence" is intended to encompass partial 5 sequences of VKORCI polypeptides, which fragments comprise, preferably consist of at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95% of the full length sequence of the VKORC1 polypeptide. In particular, it is preferred that the fragment consists of a single contiguous sequence of the VKORCl polypeptide but it may also contain at least two, at least 0 three or at least about five different sequence portions of a VKORC1 polypeptide according to the invention which may or may not be interspaced by a heterologeous sequence or contain no extra polypeptide sequence at all. The term "sequence homology" is understood as the degree of identity (% identity) of two sequences, that in the case of polypeptides can be determined by means of for example 25 BlastP 2.0.1 and in the case of nucleic acids by means of for example BLASTN 2.014, wherein the Filter is set off and BLOSUM is 62 (Altschul et al., 1997). "VKORCl activity" within the meaning of the present invention is intended to mean the biological activity of the VKORCI polypeptide of SEQ ID No.l. More preferably, "VKORCI activity" is defined as the activity of the VKORC1 polypeptide to enzymatically convert (or 30 support the enzymatic conversion) of vitamin K2,3-epoxide to vitamin K-quinone and/or the conversion of vitamin k quinone to vitamin K hydroquinone. VKORC1 activity may be determined using an assay based on the experiments described in detail in example 7 and figure 10. Using that assay, a measured percentage of vitamin K epoxide converted into vitamin K quinone (product/substrate+product) in cells expressing a given VKORC I polypeptide, or a 13 nucleic acid molecule coding for such VKORC1 polypeptide, which raises the basal VKOR activity of HEK293 cells from about 1% (1.49 and 0.96% for untransfected and mock transfected HEK293 cells, respectively) to about 15% or more, preferably to about 18% or more, preferably to about 20% or more, most preferably to about 25% or more is considered a 5 VKORC1 activity within the meaning of the invention. The VKORC1 polypeptides according to the present invention may be produced by a method described in more detail below. Among others, the VKORC1 polypeptides are useful for identifying coumarin derivatives that avoid the problems described above. In particular they are useful for identifying coumarin derivatives, that effectively inhibit VKORC1 activity and 0 that in independent assays are tested for (1) their metabolic half life in order to identify coumarin derivatives that are metabolized faster than the coumarins known in the art, (2) their ability to cause skin necrosis to identify coumarin derivatives that do not cause skin necrosis or to a lesser extend than the coumarins known in the art, (3) coumarin derivative-drug interactions in order to identify coumarin derivatives with lesser side effects than the coumarins 5 known in the art. Moreover, the VKORC1 polypeptides according to the present invention are useful for identifying a VKORCI sequence interacting with coumarin and its derivatives, and for treating patients having a decreased or increased VKORC 1 activity relative to control levels. In another aspect the present invention relates to a VKORCI nucleic acid comprising, preferably consisting essentially of a nucleic acid sequence selected from the group consisting 0 of: (a) a nucleic acid sequence coding for the VKORCI polypeptide according to the invention; (b) a nucleic acid sequence selected from the group consisting of a sequence according to SEQ ID No. 2, 13, 18, 22, 26, and 28; (c) a nucleic acid sequence which hybridizes under stringent conditions to the nucleic acid 25 sequence defined in (a) or (b), which nucleic acid sequence codes for a polypeptide having VKORC 1 activity; (d) a nucleic acid sequence which, but for the degeneracy of the genetic code, would hybridize, preferably under stringent conditions, to the nucleic acid defined in (a), (b) or (c) and which nucleic acid sequence codes for a polypeptide having VKORCI activity; 30 and (e) a fragment of the nucleic acid sequence defined in (a), (b), (c) or (d), which fragment codes for a polypeptide having VKORCl activity. Preferably, the VKORCl nucleic acid is a target for coumarin and its derivatives in mammals.

14 The term "VKORC1 nucleic acid" relates to RNA or DNA, which may be a single or preferably a double stranded molecule. The sequence of the VKORCI nucleic acid may further comprise at least one intron and/or one polyA sequence. The term "VKORC1 nucleic acid" may also encompass a precursor stage, for example a propolypeptide or prepropolypeptide, 5 thereof. It is also understood that untranslated sequences can be present at the 5' end and/or the 3' end of the nucleic acid, without the activity of the encoded polypeptide being significantly altered. However, the DNA region encoding the VKORC I polypeptide is particularly preferred. In eukaryotes, this region begins with the first start codon (ATG) which is located in a Kozak sequence (Kozak, 1987) and extends to the next stop codon (TAG, TGA or TAA) which is 0 located in the same reading frame as the ATG. In the case of prokaryotes, this region begins with the first AUG (or GUG) after a Shine-Dalgarno sequence and ends with the next stop codon (TAG, TGA or TAA) which is located in the same reading frame as the ATG. Moreover, the term "VKORCI nucleic acid" may also encompass sequences which exhibit at least about 70%, in particular at least about 80%, especially at least about 90%, sequence homology with 5 the sequence according to SEQ ID No. 2, 13, 18, 22, 26, and 28, preferably to the sequence according to SEQ ID No. 2, provided that the VKORCI polypeptide encoded by such nucleic acid has VKORC1 activity. In a preferred embodiment of the invention the nucleic acid comprises a nucleic acid having a sequence complementary and/or antisense to a VKORC1 nucleic acid as defined in the preceding paragraph. The VKORCl nucleic acid may also .0 comprises a non-functional mutant variant of the VKORCI nucleic acid as defined above, such a variant containing a single nucleotide polymorphism (SNP) such as the nucleic acid sequences according to SEQ ID No. 8 and 9, provided that the VKORC1 polypeptide encoded by such nucleic acid has VKORCI activity. The term "stringent hybridization conditions" is to be understood, in particular, as 25 meaning those conditions in which a hybridization takes place, for example, at 60*C in 2.5x SSC buffer followed by several washing steps at 37*C in a lower buffer concentration and remains stable. The term "fragment of the nucleic acid sequence coding for a polypeptide having VKORC1 activity" is understood to encompass nucleic acid sequence fragments comprising, 30 preferably consisting of at least about 60%, preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95% of the full length sequence coding for VKORC1 polypeptide according to the invention, preferably coding for the polypeptide according to SEQ ID No. 1, provided that the polypeptide encoded by such fragment has VKORC 1 activity. In particular, it is preferred that the fragment 15 consists of a single contiguous sequence coding for the VKORCI polypeptide but it may also contain at least two, at least three or at least about five different sequence portions, which may or may not be interspaced by a heterologeous sequence or contain no extra nucleic acid sequence at all, provided that all the sequence portions are arranged in the same reading frame. 5 It is essential to the definition of these fragments that they display VKORCI activity. The VKORCI nucleic acids can be produced by methods generally known to the skilled artisan. Nucleic acids may be prepared synthetically. Thus, the VKORC1 nucleic acids can, for example, be synthesized chemically, e.g. according to the phosphotriester method, with the aid of the DNA sequences as defined above and/or with the aid of the polypeptide sequences which 0 are likewise defined above such as the SEQ ID No. I and by referring to the genetic code (see, e.g., Uhlmann, & Peyman, 1990). Preferably the VKORC1 nucleic acids are produced by recombinant gene technology methods generally known to the person skilled in the art. Among others, the VKORC1 nucleic acids are useful (1) for identifying coumarin derivatives that avoid the problems described above, (2) for producing PCR primers, DNA and 5 RNA probes, siRNA or shRNA, and for VKORCl polypeptide, (3) for treating patients having a decreased or increased VKORC1 activity relative to control values, and (4) for identifying coumarin derivatives that may be employed for pest control of rodents, all of which are described in detail below. The present invention further provides in another aspect a method of producing a 0 VKORC1 polypeptide, preferably a polypeptide according to SEQ ID No. 1, 12, 17, 21, 25, and 27, comprising the steps of: (I) providing a host cell having been introduced the VKORC1 nucleic acid, preferably a nucleic acid according to SEQ ID No. 2, 13, 18, 22, 26, and 28, or a vector containing the VKORCI nucleic acid; 25 (II) expressing the VKORCI polypeptide in the host cell; and (III) isolating the VKORCl polypeptide from the host cell. The host cell can be any host cell as defined below. Methods for selecting and culturing the host cells and for causing the host cells to express a polypeptide are generally known to the person skilled in the art. The same is true for methods of isolating the expressed polypeptide 30 from the host cell; to this end an antibody according to the invention may be used for immunoaffinity precipitation. As an alternative the vector may contain a (poly)peptide tag that allows immunoaffinity precipitation by tag specific antibodies according to standard protocols known to the skilled worker (see also below).

16 In another aspect the present invention relates to a fusion protein comprising, preferably consisting essentially of, (a) the VKORCI polypeptide as defined above, preferably according to SEQ ID No.1, 12, 17, 21, 25, 27 or a polypeptide encoded by the VKORC1 nucleic acid, preferably a 5 nucleic acid according to SEQ ID No. 2, 13, 18, 22, 26, 28 and (b) a heterologeous part. This involves fusion proteins which contain the above-described VKORC1 polypeptide, with the fusion proteins themselves already being active or only becoming active after the heterologeous part has been eliminated. To this end the heterologeous part may further 0 comprise a peptide cleavable by a protease. The heterologeous part may be a proteinaceous compound, a peptide or a different compound. These fusion proteins include in particular, fusion proteins having a content of about 1-300, preferably about 1-200, particularly preferably about 1-150, in particular about 1-100, especially about 1-50 foreign amino acids constituting the heterologeous part. The heterologeous part can be located N-terminally, C-terminally and/or 5 internally relative to the VKORC1 polypeptide. Examples of such peptide sequences are prokaryotic peptide sequences which can be derived, for example, from E. coli galactosidase. Other preferred examples of peptide sequences for fusion proteins are peptides which facilitate detection of the fusion protein; examples of these are the green fluorescent protein or functional variants thereof. It is also possible to add on at least one further "polypeptide tag" 0 e.g. for the purpose of purifying the previously described VKORCI polypeptides. For example, suitable protein tags enable the fusion proteins which are to be purified, to be absorbed with high affinity to a matrix. This is then followed, for example, by stringent washing with suitable buffers without eluting the fusion proteins to any significant extent, and, subsequently, specific elution of the fusion proteins. Examples of the protein tags which are known to the skilled 25 person are a (His)6 tag, a Myc tag, a FLAG tag, a hemagglutinin tag, a glutathione transferase (GST) tag, intein having an affinity chitin-binding tag and a maltose-binding protein (MBP) tag. These protein tags can be located N-terminally, C-terminally and/or internally relative to the VKORC1 polypeptide. Fusion proteins are for example useful for the production of VKORC 1 production and subsequent isolation. Moreover, the fusion proteins may be employed 30 for detecting the localization of the expression product in the cell or the organism. In another aspect the present invention relates to a vector comprising a VKORC1 nucleic acid as defined above, preferably the VKORC1 nucleic acid according to SEQ ID No. 2. Preferably the vector is an expression vector. In order to enable the VKORC1 nucleic acids to be used according to the present invention they may be introduced into a eukaryotic or 17 prokaryotic cell by means of transfection, transformation or infection, and thereby enable the polypeptide to be expressed. The VKORC1 nucleic acid can be present as a plasmid, or as a part of a viral or non-viral vector. Particularly suitable viral vectors in this connection are: baculoviruses, vaccinia viruses, adenoviruses, adeno-associated viruses and herpes viruses. 5 Particularly suitable non-viral vectors are for example: virosomes, liposomes, cationic lipids and polylysine-conjugated DNA. The vectors can be prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors are the pGEM vectors or pUC derivatives, which are used for expression in E. coli, and examples of eukaryotic expression vectors are the vectors p426Met25 or p426GAL1 (Mumberg et al., 1994) which are used for expression in 0 Saccharomyces cerevisiae, the Baculovirus vectors, as disclosed in EP BI 0 127 839 or EP BI 0 549 721, which are used for expression in insect cells, and the vectors Rc/CMV and Rc/RSV, or SV40 vectors, which are used for expression in mammalian cells, with all these vectors being generally available. In general, the expression vectors also contain promoters which are suitable for the respective cell, such as the trp promoter for expression in E.coli (see, e.g., EP-B1-0 154 5 133), the Met 25, GAL 1 or ADH2 promoter for expression in yeasts (Russel et al, 1983; Mumberg, see above), and the baculovirus polyhedrin promoter for expression in insect cells (see, e.g., EP Bi 0 127 839). Promoters which permit constitutive, regulatable, tissue-specific, cell type-specific, cell cycle-specific or metabolism-specific expression in eukaryotic cells are suitable, for example, 0 for expression in mammalian cells. Regulatable elements in accordance with the present invention are promoters, activator sequences, enhancers, silencers and/or repressor sequences. Examples of preferred regulatable elements which permit constitutive expression in eukaryotes are promoters which are recognized by RNA polymerase III or viral promoters, CMV enhancer, CMV promoter, SV40 promoter or LTR promoters, e.g. derived from MMTV (mouse 25 mammary tumor virus; Lee et al.,1981) and other viral promoter and activator sequences which are derived from, for example, HBV, HCV, HSV, HPV, EBV, HTLV or HIV. Examples of regulatable elements which permit inducible expression in eukaryotes are the tetracycline operator in combination with an appropriate repressor (Gossen et al., 1994). The expression of VKORC1 nucleic acids preferably takes place under the control of tissue-specific promoters. 30 The expression vectors may be used for preparing a VKORCl polypeptides, DNA or RNA probes, or siRNA or shRNA, which can be used in accordance with the invention. In another preferred embodiment of the present invention the vector is a knock-out gene construct. The construction of such constructs and methods for constructing knock-out animals are known to the person skilled in the art, for example, from the US patents 5,625,122; US 18 5,698,765; US 5,583,278 and US 5,750,825. Such vectors are for example useful for generating knock-out cells and animals which in turn can be used to identify disorders and diseases associated with impaired VKORC 1 activity. "Impaired VKORCI activity" within the meaning of the invention relates to a level of 5 activity and/or expression of the VKORCI protein that is less than control level activity (as defined above) and/or expression determined in a healthy subject; the respective levels of activity may also be determined based on the assay as described in Example 7. In another aspect the present invention relates to a host cell, preferably a non-human embryonic stem cell, containing one of the aforementioned vectors, preferably an expression 0 vector or a knock-out gene construct. The host cell can be any cell suitable for expression of VKORCI polypeptides and/or VKORCI nucleic acids, preferably a HEK293-EBNA cell. Cells can be either prokaryotic or eukaryotic cells, heterologeous or autologous cells. Examples of prokaryotic cells are E. coli and examples of eukaryotic cells include primary hepatocytes cells, yeast cells, for example Saccharomyces cerevisiae or insect cells. More preferably the host cell 5 is a cell line, e.g. a COS-cell such as COS-7 cells or hepatocytes cell lines such as HepG2 cells. Moreover the host cell is preferably a non-human embryonic stem cell. Methods for selecting and culturing host cells and for causing the host cells to express a polypeptide are generally known to the person skilled in the art. Processes for the transformation of cells and/or stem cells are likewise well known to a person skilled in the art and include, for example, electroporation 0 or microinjection. The host cells of the present invention can for example be employed for methods of identifying coumarin derivatives, for producing VKORC1 polypeptides and VKORC1 nucleic acids, siRNAs and shRNAs according to the invention, and for screening new drugs such as coumarin derivatives effecting VKORCI activity and/or expression. In another aspect the present invention relates to the provision of a transgenic non 25 human mammal containing a host cell according to the invention, preferably a non-human embryonic stem cell, as defined above. Transgenic animals in general show a tissue-specifically increased expression of the VKORCl polypeptides and/or VKORC1 nucleic acids and can be used for the analysis of coagulation disorders and warfarin resistance and for development and evaluation of therapeutic strategies for such disorders. Transgenic animals may further be 30 employed in the production of VKORCI polypeptides. The polypeptide produced by the animal may for example be enriched in a body fluid of the animal. In another preferred embodiment of the present invention it is provided a transgenic non-human mammal which is transgenic for a VKORC1 polypeptide which contains at least one sequence abnormality exerting an effect on the activity of the VKORC1 polypeptide as 19 defined in detail below. Preferably the animal is transgenic for the VKORCI polypeptide according to SEQ ID No. 1, 12, 17, 21, 25, and 27, and preferably the sequence abnormality is selected from the group consisting of V29L, V45A R58G, R98W, L128R, and Y139C. These transgenic animals are for example useful (1) for testing coumarin derivatives for warfarin 5 resistance; (2) for identifying novel coumarin derivatives that are effective anticoagulants in organisms that are resistant or less susceptible to anticoagulant treatment with coumarins known in the art, such as warfarin resistance patients; and (3) as a source of cells expressing VKORC1 polypeptide and/or VKORCI nucleic acids. Moreover, these animals can be used for identifying novel coumarin derivatives. 0 Methods for the preparation of transgenic animals, in particular of transgenic mice, are likewise known to the person skilled in the art from DE 196 25 049 and US 4,736,866; US 5,625,122; US 5,698,765; US 5,583,278 and US 5,750,825 and include transgenic animals which can be produced, for example, by means of direct injection of expression vectors according to the present invention into embryos or spermatocytes or by injection of the 5 expression vectors into the pronucleus of the fertilized ovum or by means of the transfection of expression vectors into embryonic stem cells or by nuclear transfer into appropriate recipient cells (Polites & Pinkert, 1994; Doetschman, in Pinkert, 1994, supra; Wood in Pinkert, 1994, supra; Monastersky in Pinkert, 1994, supra). Within the meaning of the term "VKORCI associated deficiency" is intended to :O encompass a disorder or disease that is associated with warfarin resistance, i.e. the patient displays a reduced or abolished susceptibility to treatment with coumarin or its derivatives, preferably the warfarin resistance results from a sequence abnormality of the VKORCI polypeptide. Moreover, the term preferably also encompasses disorders or diseases associated with a level of activity and/or expression of VKORCI that differs significantly from the 25 condition in healthy patients, preferably the expression of VKORCl polypeptide and/or its activity is significantly reduced or abolished which can e.g. be determined by measuring the prothrombin time, e.g. by international normalized ration (INR) protocol. Such VKORC1 associated deficiency may be caused by a sequence abnormality in the VKORC 1 polypeptide or VKORC1 nucleic acid as described in detail below. Moreover, when the level of expression 30 and/or activity of VKORCI polypeptide or VKORC1 nucleic acid is reduced or even completely abolished, gamma-carboxylation of vitamin K dependant proteins may also be impaired as well. Thus, in this context the term "VKORC1 associated deficiency" also encompasses diseases and/or disorder selected from familial multiple factor deficiency, a disorder or disease associated with decreased blood coagulation, such as hemophilia and a 20 disorder associated decreased vascular calcification, diseases and/or disorders associated with impaired gamma-carboxylation of vitamin K dependant proteins. It is also conceivable that the level of expression and/or activity of VKORCl polypeptide will be increased relative to the condition in healthy patients. Such deficiencies 5 which are also encompassed by the term the "VKORC1 associated deficiency" may be caused by a sequence abnormality in the VKORC1 polypeptide and/or the corresponding gene. Moreover, when the level of expression and/or activity of VKORC1 polypeptide or VKORC1 nucleic acid is increased, gamma-carboxylation of vitamin K dependant proteins may be facilitated as well. Thus, in this context the term "VKORCl associated deficiency" may further 0 comprise deficiencies selected from diseases or disorders associated with increased blood coagulation including patients suffering from a thrombus and/or patients having an increased risk of developing a thrombus, preferably due to a sequence abnormality in the VKORC1 polypeptide or its gene, diseases and/or disorders associated with improved gamma carboxylation of vitamin K dependant proteins. 5 It is also possible that a sequence abnormality in the VKORCl polypeptide and/or the corresponding gene may increase the susceptibility to treatment with coumarin and its derivatives in a patient having such sequence abnormality. As a result patients undergoing coumarin treating may show very low blood coagulation values. Such disorders associated with increased susceptibility to treatment with coumarin are also intended to be encompassed by the o term "VKORCI associated deficiency". Patients carrying a VKORC1 gene having a stop mutation suffer from such deficiency associated with a increased coumarin sensitivity. In another aspect the present invention relates to a DNA or a RNA probe directed against the VKORC1 nucleic acid according to the invention, preferably against the VKORCI nucleic acid selected from according to SEQ ID No.2, 13, 18, 22, 26, and 28. A probe is a 25 nucleic acid molecule that allows detection of a VKORC1 nucleic acid it is directed against it. The probe has a sequence which hybridizes to the target sequence, i.e. the VKORCI nucleic acid. Preferably the probe allows specific detection of the VKORC1 nucleic acid, i.e. at least under stringent hybridization conditions it does not bind to molecules other than the particular VKORC1 nucleic acid. Suitable probes are, for example, DNA or RNA fragments having a 30 length of about 10 to about 492 nucleotides, preferably having a length of about 10 to about 400 nucleotides, preferably about 10 to about 250 nucleotides, in particular having a length of about 10 to about 150 nucleotides, in particular having the full length of the coding sequence, which sequence can be derived from the VKORC1 polypeptides, preferably selected from the VKORCI polypeptide according to SEQ ID No. 1, 12, 17, 21, 25, and 27 or taken directly from 21 the VKORC1 nucleic acid, preferably selected from SEQ ID No. 2, 13, 18, 22, 26, and 28. The probes may additionally contain a label suitable for direct or indirect detection such as biotin, a fluorescent label such as fluorescein or a radioactive label such as [H] 3 or other labels known to the skilled worker. Detection may be carried out by methods generally known to the skilled 5 worker including northern blotting and cDNA library blotting techniques. The construction of the probes according to the present invention is also known to the skilled worker (cf. construction of nucleic acids described above). Such probes can for example be used for diagnostic purposes and may preferably comprise or consist of the probes suitable for detection of a sequence abnormality such as those selected from the sequences according to SEQ ID NO: 0 3, 4, 5, 6, 7, 8, 9, 14, and 94 . In another aspect the present invention relates to a PCR primer, preferably a set of at least two PCR primers directed against the VKORCI nucleic acid, preferably against the VKORCI nucleic acid according to SEQ ID No. 2, 13, 18, 22, 26, and 28. Suitable primers are, for example, DNA fragments having a length of about 10 to about 100 nucleotides, preferably 5 having a length of about 15 to about 50 nucleotides, in particular having a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, preferably about 30 nucleotides. The design and synthesis of such primers is generally known to the person skilled in the art. The primers may additionally contain restriction sites, e.g. suitable for integration of the amplified sequence into vectors, or other adapters or overhang sequences, e.g. having a label as described in the 0 preceding section. For example, according to the present invention it is possible to prepare a diagnostic based on the polymerase chain reaction (PCR), suitable for detection of VKORC1 sequence abnormalities, preferably based on the assay described in Example 9 (ARMS PCR). If the amount of expressed VKORCI is to be determined, PCR primers specific for a VKORCI nucleic acid will be utilized for diagnostic or therapeutic purposes. To this end RT 25 PCR technique, preferably quantitative RT-PCR, may be carried out, wherein upon isolation of total or mRNA from the sample the RNA is reverse transcribed into cDNA and subsequently subjected to a PCR reaction using the specific primers according to the invention. This technique is well known to the skilled worker. This opens up a further possibility of obtaining the described VKORCI nucleic acids, for example by isolation from a suitable gene or cDNA 30 library, for example from a liver disorder-specific or liver specific gene bank, with the aid of a suitable primer. A preferred set of PCR primers and condition for isolating a VKORC1 nucleic acid are provided in Example 5. Examples of preferred PCR primers according to the invention directed against SEQ ID No. 2, are the primers according to SEQ ID No. 53-70 and preferred conditions for using these PCR primers are provided in Figure 8.

22 The term "sample" is intended to refer to a biomaterial comprising fetal or adult tissue or cell, preferably tissue or cells, preferably isolated or derived from heart, kidney and lung, pancreas, brain, placenta and skeletal muscle and blood, preferably from liver. The sample can be isolated from a patient or another subject by means of methods including invasive or non 5 invasive methods. Invasive methods are generally known to the skilled artisan and comprise for example isolation of the sample by means of puncturing, surgical removal of the sample from the opened body or by means of endoscopic instruments. Minimally invasive and non-invasive methods are also known to the person skilled in the art and include for example, collecting body fluids such as blood, preferably by venopuncture, or urine or feces. The term "sample" may also 0 encompass a genomic or an expression library, preferably constructed based on an sample isolated from a patient, in which case techniques for isolation of the cDNA that are generally known to the skilled worker may be used. In another aspect the present invention provides a small interfering RNA molecule (siRNA) and/or a short hairpin RNA (shRNA) directed against the VKORCl nucleic acid, 5 preferably against a sequence derived from SEQ ID No. 1, 12, 17, 21, 25, and 27, or a nucleic acid according to SEQ ID No. 2, 13, 18, 22, 26, and 28, which allows decreasing the stability of the VKORCl nucleic acid and/or inhibiting the translation of the VKORCl nucleic acid in a cell culture or in vivo. The double-stranded siRNAs mediate sequence-specific, post transcriptional silencing of a gene's expression by double-stranded RNA. The siRNAs have a 0 very specific structure: 17 to 25, preferably 19, 20, 21, 22, 23, 24, or 25 nucleotides double stranded RNAs with 2 nucleotides 3'-end overhangs. siRNAs are usually derived from longer double stranded RNA molecules by enzymatic cleavage but siRNAs can also be synthesized chemically or enzymatically outside of cells and then delivered to cells (e.g., by transfection). Thus, using siRNA or shRNA the expression of the corresponding genes in cells can be 25 decreased or even silenced both in vivo and in vitro (McManus et al. 2002). shRNAs consist of a first stem portion comprising (I) a sequence of at least 18, preferably a least 19, more preferably at least 20 nucleotides that is complementary to the mRNA sequence of a VKORCI nucleic acid, preferably a sequence complementary to the SEQ ID No. 2, 13, 18, 22, 26, and 28; and (II) a second stem portion comprising a sequence of at least 18, preferably a least 19, more 30 preferably at least 20 nucleotides that is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex stem; and (III) a loop portion that connects the two stem portions. The loop portion may comprise at least 4, preferably at least 7, more preferably at least 11 nucleotides. The siRNA or shRNAs may also be included into a vector allowing constitutive expression of the siRNA or shRNAs in the host cell upon 23 transformation of the host cell (cf. WO 03/006477). Strategies to the design of siRNA or shRNAs sequences and methods of constructing and producing these molecules are generally known to the person skilled in the art (cf. McManus et al., supra). The siRNA and shRNA molecules according to the present invention are for example useful for therapeutic regulation 5 of VKORC1 gene expression and for inclusion into methods for identifying coumarin derivatives, e.g. as a positive control for coumarin action. Preferred examples of siRNA sequences according to the invention are listed in Figure 6 and 7 and are selected from SEQ ID No. 29, 20, 33, 34, 37, 38, 41, 42, 45, 46, 47, 50. Figure 6 also provides the respective downstram primers of these siRNA molecules which can be used for integration into vectors so 0 that the siRNA may be expressed in a cell targeted for siRNA expression. As an alternative approach to silencing VKORCI activity and/or expression, the present invention provides antisense oligonucleotides directed against the VKORC1 nucleic acid as defined above, preferably according to SEQ ID No 2, 13, 18, 22, 26, and 28, preferably against a sequence derived from SEQ ID No. 1, 12, 17, 21, 25, and 27 (Zheng & Kemeny, 1995; Nellen 5 & Lichtenstein, 1993). According to another aspect of the present invention there is provided an RNA-aptamere directed against a VKORC 1 polypeptide, preferably against a sequence according to SEQ ID No. 1, which RNA-aptamere exerts an effect on the activity of the VKORCI polypeptide. RNA-aptameres are effective agonists or antagonists of proteins that are targeted by the 0 aptameres, as has been shown for the coagulation factor IXa (Rusconi et al. 2002). An aptamere according to the invention may be used as an anticoagulant by reducing VKORC 1-activity in a more fine-tuned manner than the coumarins. For testing aptameres they can be added to a VKORCI-reaction and analyzed by HPLC as described in Example 7. In another aspect of the present invention there is provided an antibody which 25 specifically recognizes and binds a VKORC1 polypeptide as defined above, preferably a VKORCI polypeptide according to SEQ ID No. 1, 12, 17, 21, 25, and 27, or a fragment of the antibody. The antibody or antibody fragment preferably is a polyclonal or a monoclonal antibody, specific for the VKORC1 polypeptides. The antibody or antibody fragment is produced according to methods generally known to the person skilled in the art by immunizing 30 a mammal, for example a rabbit, with a VKORC1 nucleic acid, or with a VKORCI polypeptide according to the invention or parts thereof having at least 6 amino acid length, preferably having at least 8 amino acid length, in particular having at least 12 amino acid length, if appropriate in the presence of, for example, Freund's adjuvant and/or aluminum hydroxide gels (see, for example, Harlow & Lane, 1998). The polyclonal antibodies formed in the animal as a 24 result of an immunological reaction can then be easily isolated from the blood according to generally known methods and purified, for example, by means of column chromatography. Monoclonal antibodies can be produced, for example, according to the known method of Winter & Milstein (1991). The antibodies according to the present invention can for example be 5 used for diagnosis of VKORCI associated deficiencies. Moreover, the antibodies may be useful for elucidating coumarin-VKORC1 interactions. Finally the antibodies may be used to isolate and and/or purify VKORCI polypeptide from a tissue or cell sample isolated from a patient. According to the present invention, the term "antibody" or "antibody fragment" is understood as also meaning antibodies or antigen-binding parts thereof prepared by genetic 0 engineering and optionally modified, such as, for example, chimeric antibodies, humanized antibodies, multifunctional antibodies, bi- or oligospecific antibodies, single-stranded antibodies, F(ab) or F(ab)2 fragments (see, for example, EP-B1-0 368 684, US 4,816,567, US 4,816,397, WO 88/01649, WO 93/06213, WO 98/24884). In another aspect the present invention provides a method of identifying a coumarin 5 derivative which exerts an effect onto the activity of VKORC1 polypeptide as defined above, preferably the VKORC1 polypeptide having the sequence selected from SEQ ID No. 1, 12, 17, 21, 25, and 27, comprising the steps of: (I) providing a host cell having been introduced the VKORCI nucleic acid or a vector containing the VKORCl nucleic acid; 0 (II) expressing the VKORCI polypeptide in the host cell; (III) administering a candidate coumarin derivative; (IV) determining the activity of VKORC 1 polypeptide (candidate activity value); (V) comparing the candidate activity value with a control activity value; and (VI) identifying the candidate coumarin derivative as a coumarin derivative exerting an 25 effect onto the activity of the VKORC1 polypeptide, provided the candidate activity value is significantly different from the control activity value. The the determined activity of VKORC1 polypeptide is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the significantly different activity value is a candidate activity value which is significantly higher than the 30 control activity value, as described in further detail above and in Example 7 and Figure 10. If essentially the same concentration of candidate coumarin derivative yields a lower percentage of vitamin K epoxide converted into vitamin K quinone (product/substrate+product) as warfarine does in this concentration, this is indicative ot the candidate coumarin derivative having a stronger inhibitory effect than warfarin, and vice versa.

25 Preferably in the method of identifying a coumarin derivative according to the present invention the control activity value is determined by a method comprising the steps of: (A) providing a host cell according to step (I); (B) expressing the VKORCI polypeptide in the host cell; and 5 (C) determining the activity of VKORC 1 polypeptide (control activity value). Even more preferably in the method of identifying a coumarin derivative according to the present invention at least one additional compound is introduced into the host cell, which compound is selected from the group consisting of vitamin K, cytochrome B5, a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or 0 for glutathion-S-transferase. Methods for introducing nucleic acids into host cells have been described in detail above. Preferably the nucleic acids are expressed under the control of a constitutively active promoter or a promoter which can be specifically activated in the host cell chosen. The methods of identifying a coumarin derivative are useful for developing novel 5 coumarin derivatives that avoid at least one of the limitations of coumarin and its derivatives known in the art. If analysis of the kinetics of blood coagulation is included as a separate assay into the determination of VKORCI polypeptide activity, the method according to the invention may be useful in identifying coumarin derivatives which mediate blood coagulation faster than coumarin and its derivatives known in the art and/or that are metabolized more rapidly so that 0 accumulation of coumarin and its derivatives may be prevented or ameliorated and as a result the danger of overdosing is substantially decreased or even abolished. Moreover, such method of identifying may be combined with other assays such that coumarin derivatives may be identified which have a stronger (weaker) effect onto VKORC1 activity and thus in turn onto the blood coagulation and which coumarin derivatives in independent assays prove (1) to be 25 metabolized more rapidly so that accumulation of coumarin may be prevented or ameliorated and as a result the danger of overdosing is substantially decreased or even abolished, (2) not to cause or to cause to lesser extend skin necrosis in patients or embryopathy if applied during pregnancy, and/or (3) to be metabolized faster or to be less more stabil and/or to be affected less by other drugs like Phenobarbital or amiodarone. The assays which are suitable to screen for 30 such properties of the coumarin derivatives and which are to be combined into a screen with the method of identifying a coumarin derivative according to the invention are generally known to the person skilled in the art. The term "coumarin" is understood as meaning 3-(acetonylbenzyl)- 4-hydroxycoumarin, i.e. COUMADIN@ or sodium warfarin.

26 The term "derivative of coumarin" is understood to encompass organic or inorganic compounds, peptides, polypeptides or complexes thereof, provided that they exert an effect onto the activity and/or expression of VKORC1 polypeptide, preferably an effect that inhibits the activity of the VKORC1 polypeptide, even more preferably a VKORC1 polypeptide 5 specific effect, i.e the coumarin derivative does not directly interact with other molecules involved in the coagulation pathway. Examples of such compounds are organic molecules that are derived from libraries of compounds, preferably those that have been analyzed for their pharmacological activity. On account of their interaction, the derivatives of coumarin can influence the activity of the VKORC1 polypeptide in vivo and/or in vitro and enter into 0 interactions of covalent or non-covalent manner with them. If the coumarin derivative is a chiral compound it is understood that "derivative of coumarin" also encompasses the respective R- and L-enantiomeril forms of the compound like those disclosed in WO 00/43003. In particular the term "derivative of coumarin" refers to compounds derived from 4 hydroxycoumarin, especially compounds derived from COUMADIN. More preferably, 5 "derivative of coumarin" also includes any coagulants which inhibits the regeneration of active vitamin K. The term "candidate coumarin derivative" is understood to encompass organic or inorganic compounds, peptides, polypeptides or complexes. Examples of such compounds are organic molecules that are derived from libraries of compounds, preferably those that have been .0 analyzed for their pharmacological activity. Preferably the term refers to compounds that are structurally related or derived from 4-hydroxycoumarin, especially compounds related or derived from COUMADIN. If the candidate coumarin derivative is a chiral compound it is understood that the respective R- and L-enantiomeric forms of the compound like those disclosed in WO 00/43003 are also encompassed by the term "candidate coumarin derivative". 25 In another aspect the present invention provides a method of determining a VKORC1 polypeptide sequence which conveys a coumarin effect exerted onto VKORC1 activity, comprising the steps of: (I) providing a cell expressing the VKORC1 polypeptide according to the invention, preferably a polypeptide according to SEQ ID NO. 1, 12, 17, 21, 25, and 27, which 30 VKORC1 polypeptide has at least one sequence abnormality, preferably a sequence abnormality selected from the group consisting of V29L, V45A, R58G, R98W, L128R and Y139C; (II) administering coumarin or a derivative thereof to the cell; 27 (III) determining the activity of the VKORCI polypeptide (sequence abnormality activity value); and (IV) comparing the sequence abnormality activity value with the control sequence activity value, 5 wherein a significant deviation of the sequence abnormality activity value from the control sequence activity value is indicative that the sequence abnormality of the VKORC 1 polypeptide conveys the coumarin effect exerted onto VKORC1 polypeptide. The activity of the VKORC1 polypeptide may be determined as described in detail above. More preferably, in the method of determining a VKORC1 polypeptide sequence the 0 control sequence activity value is determined by a method comprising the steps of: (I) providing a cell expressing the VKORCI polypeptide, preferably a polypeptide according to SEQ ID NO. 1, 12, 17, 21, 25, and 27; (II) administering coumarin or a derivative thereof to the cell; (III) determining the activity of the VKORCI polypeptide (control sequence activity value). 5 The determined VKORCI activity is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and the significantly different value is a sequence abnormality activity value which is significantly higher than the control sequence activity value. Further details are provided above and in Example 7 and Figure 10. The method may be useful in identifying VKORC 1 polypeptides that are less sensitive to coumarins. By introducing 0 VKORCI polypeptides with different sequence abnormalities this method allows identification of sites of the polypeptide which are critical for the interaction of a tested coumarin and VKORC 1. Such knowledge will for example be useful for designing new coumarins. It is particularly preferred in the method of determining a VKORCI polypeptide sequence that at least one additional compound is introduced into the cell which compound is 25 selected from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase. A further aspect of the present invention relates to a VKORC1 polypeptide as defined above, preferably to the VKORCI polypeptide according to SEQ ID No. 1, wherein the 30 VKORCI polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide. More preferably the VKORC1 polypeptide is the polypeptide according to SEQ ID No. I and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R and Y139C. In another embodiment, the invention relates to the Rattus norvegicus VKORCI polypeptide according to SEQ ID No.

28 12 having a sequence abnormality, preferably the sequence abnormality Y139C (416A>G). In another embodiment the invention relates to a Rattus norvegicus nucleic acid encoding the Rattus norvegicus VKORC1 polypeptide according to SEQ ID No. 12 having a sequence abnormality, preferably the VKORC1 nucleic acid according to SEQ ID No. 12 having a 5 sequence abnormality, preferably the 416A>G sequence abnormality. The nucleic acid VKORC1 sequence containing the abnormality is 416A>G is the nucleic acid sequence according to SEQ ID No. 14. Such VKORC1 polypeptide containing at least one sequence abnormality can be generated by methods generally known to the skilled worker, including recombinant techniques 0 and e.g. site directed mutagenesis, or by isolation the VKORC1 polypeptide having the at least one sequence abnormality from a sample obtained from a patient, preferably from a patient suffering from VKORC1 associated deficiencies. Methods for isolating proteins from a sample have been described in detail above. The VKORC1 polypeptide containing at least one sequence abnormality, which exerts 5 an effect on the activity of the VKORCl polypeptide can, for example, be used for generating antibodies binding specifically to these VKORCI polypeptides. These antibodies in turn can be utilized for diagnosing VKORCI associated deficiencies. The term "sequence abnormality" is meant to encompass additions, insertions, deletions, substitutions of at least one amino acid that result in an alteration of the VKORCI polypeptide 0 sequence, preferably of the VKORCI polypeptide sequence according to SE ID No. 1. Also encompassed are additions, insertions, deletions, substitutions of at least one nucleotide that lead to an altered amino acid sequence encoded by the VKORC1 nucleic acid sequence. Also encompassed are changes of the VKORC1 nucleic acid sequence that lead to a change in the reading frame of the nucleic acid sequence. 25 The sequence abnormalities are indicated in the single letter amino acid code with the original amino acid being placed to the left of the number indicating the number of the amino acid in the polypeptide sequence according to SEQ ID No. 1. The number to the right of the amino acid number indicates the amino acid that replaces the original amino acid. For example, the sequence abnormality V29L indicates that in position 29 of the VKORCI polypeptide of 30 SEQ ID No. I the amino acid valine (V) has been replaced by leucine (L). In case the sequence abnormality occurs in a VKORCI polypeptide other than the VKORCI polypeptide of SEQ ID No. I the number in the code refers to the sequence position according to the numbering of the amino acids in that particular other polypeptide.

29 In another aspect the present invention provides a VKORC 1 nucleic acid selected from the group consisting of: (a) a nucleic acid coding for the VKORC1 polypeptide containing at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide, 5 wherein the VKORCl polypeptide is preferably the polypeptide according to SEQ ID No. 1 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R and Y139C; (b) a nucleic acid sequence selected from the group consisting of a sequence according to SEQ ID No. 3, 4, 5, 6, and 7, 14, and 94; and 0 (c) a nucleic acid sequence which, but for the degeneracy of the genetic code, would hybridize to the nucleic acid defined in (a) or (b) and which nucleic acid sequence codes for the polypeptide containing at least one sequence abnormality as defined above. These nucleic acids are for example useful for isolating VKORCl polypeptides and nucleic acids having such sequence abnormality, for producing DNA and/or RNA probes, for 5 producing antibodies, for the construction of transgenic animals and knock-out animals, and for inclusion into screens for identifying coumarin derivatives. In a further aspect the present invention provides a vector containing the VKORC1 nucleic acid containing at least one sequence abnormality as defined above. Such vectors may be selected from the vectors described in detail above. The methods for constructing such 0 vectors are also described in detail above. Such vectors are for example useful for preparing probes described in the next paragraph, especially in a diagnostic context. In another aspect of the present invention there is provided a DNA or a RNA probe directed against VKORCl nucleic acid containing at least one sequence abnormality as defined above, preferably a nucleic acid sequence according to SEQ ID No. 3, 4, 5, 6, and 7, 14, and 94. 25 Methods of designing and producing such DNA and RNA probes have been described to a great extend above. Such probes are useful for example for detecting sequence abnormalities in the VKORC1 gene in a gene library, in an expression library, or in a sample isolated from a patient, for example in the context of diagnosis of VKORCl associated deficiencies and for site directed mutagenesis for producing VKORC1 nucleic acids containing a sequence abnormality. 30 Techniques for screening libraries are generally known to the skilled worker. In another aspect of the present invention there is provided a PCR primer directed against VKORC1 nucleic acid containing at least one sequence abnormality as defined above. Preferred PCR primers for detecting the Y139C (416A>G) sequence abnormality are the primers according to SEQ ID No. 88 to 91 (see Example 9). It is generally known to the person 30 skilled in the art to design the PCR primers such that they may be used for detecting other sequence abnormalities such as those mentioned above (V29L, V45A, R58G, R98W, L128R) for the purpose of the invention. Methods of designing and producing such PCR primers, techniques to carry out PCR amplification have been described to a great extend above, the 5 major difference being that the primers have to be designed such that only those VKORC1 nucleic acid sequences containing the sequence abnormality are specifically amplified, whereas native VKORC1 nucleic acids and other VKORC1 nucleic acid sequences that do not contain the sequence abnormality, such as the VKORCl nucleic acid of SEQ ID No. 2 remains undetected. Such primers are useful for example for detecting sequence abnormalities in the 0 VKORCl gene in a gene library, in an expression library, or in a sample isolated from a patient, for example in the context of diagnosis of VKORC1 associated deficiencies. Techniques for screening libraries are generally known to the skilled worker. In a further aspect the present invention relates to an antibody which specifically recognizes and binds the VKORC I polypeptide containing at least one sequence abnormality as 5 defined above, the VKORC1 polypeptide preferably being the polypeptide according to SEQ ID No. I and the sequence abnormality being selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C, or a fragment of the antibody. The types of antibodies encompassed, methods of constructing and producing such antibodies and fragments thereof have been described in great detail above. Such antibodies are e.g. useful for the 0 detection and isolation of VKORC1 polypeptide containing at least one sequence abnormality as defined above, especially in the context of diagnosis of VKORCI associated deficiencies and for detection of Warfarine resistance in humans and rodents such as rats. According to another aspect the present invention relates to a diagnostic comprising a compound selected from the group consisting of the VKORC 1 nucleic acid containing at least 25 one sequence abnormality, preferably at least one sequence abnormality selected from V29L, V45A, R58G, R98W, L128R, and Y139C; the DNA or the RNA probe directed against the VKORC1 nucleic acid containing at least one sequence abnormality, the PCR primer directed against the VKORC1 nucleic acid containing at least one sequence abnormality, and an antibody directed against the VKORCI polypeptide containing at least one sequence 30 abnormality; all of which have been defined above. In the case the VKORC1 associated deficiency is due to or correlated with a sequence abnormality, it is the principle of the diagnostic to be used for detection of that sequence abnormality in a probe obtained from a patient. The suitable methods for using the diagnostic according to the invention are mentioned below. Optionally the diagnostic further comprises a pharmaceutically acceptable additive 31 and/or auxiliary. Such diagnostic is useful for diagnosing VKORC1 associated deficiencies especially warfarin resistance. In on the other hand the VKORCI associated deficiency is to be diagnosed based on the detection of the level of expression of VKORCI mRNA, VKORCI cDNA or VKORCl 5 polypeptide in the sample, such levels of expression can be determined by methods generally known to the person skilled in the art. Examples of such methods for detecting the presence of a VKORC1 mRNA include RNA blot (Northern) analysis, nuclease protection, in situ hybridization, reverse transcriptase PCR (RT-PCR; including quantitative kinetic RT-PCR). cDNA and oligonucleotide microarrays are also included as such methods. An expression 0 library derived from a patient may as well be screened for the purpose of diagnosis using techniques generally known to the skilled worker. The presence of VKORC1 polypeptide can also be determined by methods generally known to the skilled worker, some of which are described below. Optionally the diagnostic further comprises a pharmaceutically acceptable additive and/or auxiliary. 5 Within the meaning of the present invention "additive" and "auxiliary" are not particularly limited and generally known to the person skilled in the art and comprise, for example, physiological saline solution, demineralized water, gelatin or glycerol-based protein stabilizing reagents. Alternatively, the VKORCI nucleic acids, probes, primers or polypeptide according to the present invention may be lyophilized for stabilization. 0 In another aspect the invention provides a method of diagnosing a VKORC 1 associated deficiency in a patient comprising the steps of: (I) amplifying a DNA sample obtained from the patient or reverse transcribing a RNA sample obtained from the patient into a DNA and amplifying the DNA; and (II) analyzing the amplified DNA of step (I) to determine at least one sequence abnormality 25 in a nucleic acid sequence coding for the VKORC1 polypeptide or in an amino acid sequence of the VKORCI polypeptide; wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency; preferably Warfarine resistance; preferably the sequence abnormality exerts an effect on the activity of the VKORCI polypeptide, preferably the 30 sequence abnormality is selected from V29L, V45A, R58G, R98W, L128R, and Y139C. Methods for obtaining samples from a patient and for isolating total RNA or mRNA are generally known to the skilled worker, some of which have been described above. Techniques for amplifying of DNA are not particularly limited and include PCR techniques which have also been described above. By the same token, techniques for reverse transcribing have been 32 mentioned above and are not particularly limited and include reverse transcription using conventional protocols and commercially available kits that usually employ reverse transcriptase and oligo dT primers. The analysis may as well be based on genomic DNA isolated from a sample obtained from a patient. 5 Upon amplification, the DNA is subjected to analysis in order to determine at least one sequence abnormality in a nucleic acid sequence coding for the VKORC1 polypeptide. Methods for analyzing the amplified DNA are not particularly limited. Preferably the amplified DNA is analyzed by a technique selected from the group consisting of PCR-based analysis, preferentially using PCR primers specific for the sequence abnormality, restriction digestion 0 analysis, and DNA sequencing analysis. In a preferred embodiment the nucleic acid carrying the sequence abnormality is coding for a VKORCI sequence having a sequence abnormality is selected from the group consisting of V29L(85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), Y139C (416 A>G). In a preferred embodiment of the method of diagnosing the amplified DNA encodes at least a partial sequence of the VKORC1 5 polypeptide according to SEQ ID No. 1. One way of determining a sequence abnormality which is associated with a VKORClassociated deficiency is provided in Example 8. The VKORCI sequences according to the invention containing the mutations 85 G>T, 134 T>C, 172 A>G, 292 C>T, and 383 T>G, are provided in SEQ ID Nos. 3 to 7 and may be used as probes for diagnosing a VKORC1 associated deficiency using hybridization technique 0 based analysis of nucleic acid samples obtained from a patient. As an alternative VKORC1 expression may be detected on the level of the VKORC1 polypeptide. Therefore, in another aspect the present invention provides a method of diagnosing a VKORC I associated deficiency in a patient comprising the steps of: (I) providing a sample obtained from the patient; and 25 (II) detecting a VKORC1 polypeptide having a sequence abnormality in the sample using the antibody directed against the VKORC1 polypeptide having a sequence abnormality as defined above, wherein the determined sequence abnormality is indicative of the patient suffering from a VKORC1 associated deficiency. Preferably the sequence abnormality is selected from the 30 group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C. Methods for obtaining samples have been described in detail already. Methods for detection of VKORC1 polypeptide having a sequence abnormality are not particularly limited, provided that the method allows specific detection of the protein carrying the sequence abnormality(ies). Examples of such methods preferably include immunohistochemical 33 detection, immunoblotting, preferably Western blotting, and ELISA of the polypeptide, particularly preferred with antibodies specific for the VKORCI polypeptide having a sequence abnormality as defined above. Analysis of sequence abnormalities on the level of the amino acid sequence is not particularly limited and may for example be carried out using VKROP1 5 antibodies which specifically recognize and bind a VKORC1 polypeptide having one or more sequence abnormalities. Preferably the methods of diagnosing are used for diagnosing diseases and/or disorder selected from warfarin resistance, familial multiple factor deficiency, a disorder or disease associated with reduced or abolished blood coagulation, such as hemophilia and a disorder 0 associated with decreased vascular calcification, diseases and/or disorders associated with impaired gamma-carboxylation of vitamin K dependant proteins. Moreover, the methods of diagnosing may also be used to diagnose disorders and diseases associated with increased blood coagulation including patients and/or patients having an increased risk of developing a thrombus due to a sequence abnormality in the VKORC1 5 polypeptide or its gene, diseases and/or disorders associated with improved gamma carboxylation of vitamin K dependant proteins. In another aspect the present invention provides a method of identifying a coumarin derivative which exerts an inhibitory effect onto the activity of a VKORC1 polypeptide having at least one sequence abnormality, comprising the steps of: 0 (I) providing a cell expressing the VKORC 1 polypeptide, preferably a polypeptide encoded by a sequence selected from the group consisting of 3, 4, 5, 6, 7, 14, and 94; (II) administering a candidate coumarin derivative to the cell; (III) determining the activity of the VKORCI polypeptide (sequence abnormality activity value); and 25 (IV) comparing the sequence abnormality activity value with the control sequence activity value, (V) identifying the candidate coumarin derivative as the coumarin derivative exerting an inhibitory effect onto the activity of a VKORCI polypeptide, if the administration of the candidate coumarin derivative results in a sequence abnormality activity value which is 30 significantly lower than the control sequence activity value. More preferably, the control sequence activity value is determined by a method comprising the steps of: (I) providing a cell expressing the VKORC1 polypeptide of claim 1, preferably a polypeptide according to SEQ ID No. 1 or 12; 34 (II) administering coumarin to the cell; (III) determining the activity of the VKORCI polypeptide (control sequence activity value). The activity of the VKORC1 polypeptide may be determined as described in detail above. And the method may be adopted from the method described in Example 7. Such method 5 of identifying a coumarin derivative is useful for identifying coumarin derivatives that can be used as anticoagulants in warfarin resistance patients. In another aspect the present invention provides a method of identifying a coumarin derivative which is toxicologically effective in warfarin-resistant rodents comprising the steps of: 0 (I) providing a warfarin-resistant rodent; (II) administering a candidate coumarin derivative to the rodent; (III) determining the toxicity of the candidate coumarin derivative onto the rodent (candidate coumarin derivative toxicity value); (IV) comparing the candidate coumarin derivative toxicity value with a control coumarin 5 toxicity value; and (V) identifying the candidate coumarin derivative as a toxicologically effective coumarin derivative, provided that the candidate coumarin derivative toxicity value is significantly larger than the control coumarin toxicity value. Preferably, the warfarin-resistant rodent is a rodent transgenic for VKORC 1 polypeptide 0 as defined above, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which causes warfarin resistance, preferably a polypeptide encoded by the sequence according to SEQ ID No. 14 or a commercially available Warfarine resistant rat or a wild catch rat having Warfarine resistance . More preferably, the VKORC1 polypeptide is the polypeptide according to SEQ ID No. 12 and the sequence abnormality is selected from the 25 group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), and Y139C (416 A>G). The sample can be any organ, tissue, body fluid or probe provided that it contains genomic DNA or mRNA from the rat to be tested. Preferably the sample is blood, tissue from tail or ear urine or feces. Further details of the method are provided in Example 9. 30 Warfarin-resistant rodents have been described (Kohn & Pelz, 1999) and may be obtained from commercial suppliers (e.g. The Federal Biological Research Center for Agriculture and Forestry, Institute for Nematology and Vertebrate Research, Toppheideweg 88, 48161 Munster, Germany). In a preferred embodiment the warfarin-resistant rodent is a rodent transgenic for VKORCI polypeptide containing at least one sequence abnormality, which 35 sequence abnormality causes warfarin resistance. More preferred, the VKORCI polypeptide containing at least one sequence abnormality is the polypeptide encoded by the nucleic acid sequence according to SEQ ID Nos. 3 to 7, and 15, and the sequence abnormality is selected from the group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 5 C>T), and L128R (383 T>G), Y139C (416 A>G). Methods for generating rodent, in particular mice transgenic for the recited VKORC 1 polypeptides have been described above. According to another aspect the invention relates to the use of PCR primers according to SEQ ID No. 88 to 91 for determining whether or not a rat has a warfarin resistance genotype in a sample obtained from a rat. 0 . The administration of coumarin and its derivatives is not particularly limited. Typically coumarin (warfarin) -containing toxicologically effective compositions are formulated as granular bait compositions containing from about 50-300 ppm, preferably about 250 ppm, of coumarin and its derivatives. The bait is typically formulated with from 0.5% to 2.5% of warfarin concentrate in a suitable binder such as corn oil. If corn oil is used as a binder, it can 5 be present in an amount of from about 0.5% to about 2% of the total composition. The binder and warfarin are then mixed in e.g. with a product based on cereal products, corn meal, rolled oats, mixed animal feeds, and similar products known in the art. The administration, i.e. the amount, formulation and frequency and duration of administration may follow standard protocols for assessing the toxicity of warfarin in rodents, more preferred follow standard 0 protocols for assessing the lethal dose 50 (LD 5 o) value for a given poison to be tested, all of which are generally known to the skilled person and described in example 9 In order for the coumarin derivative to be toxicologically effective it is desirable that multiple ingestions are required to kill the rodent so that they do not develop bait shyness. Therefore, it is preferred to repeat the administration of the candidate coumarin derivative 25 compositions a number of times. Usually the rodents begin to die after four or five daily doses of the compositions. Moreover, it may be preferred to suppress pain in the rats in order to ameliorate the suffering during the experiments by administration of pain suppressing agents generally known to the skilled worker. Inclusion of pain-suppressors in the coumarin and coumarin derivative composition may further be advantageous in order to further suppress the 30 chance that rodents develop bait shyness. Following administration the toxicity of the candidate coumarin derivative onto the rodent is determined which yields the candidate coumarin derivative toxicity value. Methods for determining the toxicity of candidate coumarin derivatives are generally known to the skilled worker and include LD 50 analysis, analysis of the blood coagulation by determining the 36 prothrombin time, e.g. by international normalized ration (INR) protocol. The determined candidate coumarin derivative toxicity value is then compared with an appropriate control coumarin toxicity value determined on the basis of subjecting a different specimen of the warfarin-resistant rodent to the same treatment but exchanging the coumarin derivative 5 administration with an administration of a standard rodent coumarin composition which the rodents are resistant for, commonly used for pest control. The same experimental conditions described above for the candidate coumarin derivative administration are used for the control. If the candidate coumarin derivative toxicity value is equal or preferably statistical significantly larger than the control coumarin toxicity value, the candidate coumarin derivative represents a 0 toxicologically effective coumarin derivative. In another aspect of the invention the invention provides a composition for killing rodents, comprising a toxicologically effective amount of the coumarin derivatives identified by the method described above. The formulation of bait compositions containing coumarin and its coumarin derivative have been described above. A typical formulation has the following 5 constituents: Ingredient %: Grain carrier 94%, Corn oil 1.0%, coumarin or coumarin derivative concentrate (0.5%) 5.0%; total 100.0%. The invention further relates to the following embodiments: A coagulant pharmaceutical composition comprising a compound selected from the group consisting of the VKORC1 polypeptide, the VKORCl nucleic acid, the fusion protein according to the invention, the vector 0 according to the invention, the host cell according to the invention, optionally combined with a pharmaceutically acceptable carrier. A method of treating a patient in need of such treatment comprising the step of administering to the patient a therapeutically effective amount of the coagulant pharmaceutical composition. The method can be used for treating a patient suffering from a VKORCI 25 associated deficiency. An anticoagulant pharmaceutical composition comprising a compound selected from the group consisting of the siRNA and/or shRNA according to the invention, the antisense RNA or DNA according to the invention, the RNA-aptamere according to the invention, the antibody according to the invention, optionally combined with a pharmaceutically acceptable carrier 30 A method of treating a patient in need or such treatment comprising the step of administering to the patient a therapeutically effective amount of the anticoagulant pharmaceutical composition. The use of a VKORCl polypeptide or a VKORC1 nucleic acid for gamma carboxylating vitamin-K dependant polypeptides. Preferably the gamma-carboxylated vitamin- 37 K dependant polypeptide is a polypeptide selected from the group consisting of blood coagulation factor II, VII, IX, X, protein C, protein S, protein Z, matrix gla protein, and osteocalcin. In a preferred embodiment the VKORCl is used in combination with at least one additional compound preferably in a cellular setting, which additional compound is selected 5 from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S transferase The invention will now be further illustrated below with the aid of the figures and examples, representing preferred embodiments and features of the invention without the 0 invention being restricted thereto. EXAMPLES Example 1: Characterization of the genomic candidate region The locus for combined deficiency of vitamin K-dependent clotting factor type 2 (VKCFD2) to the pericentromeric region of chromosome 16 between the markers D16S3131 5 and D16S419 has been mapped [Fregin et al., 2002]. This region comprises approximately 20 Mb. The genes responsible for warfarin resistance in rats (Rw) and mice (War) had been mapped to chromosome 1 [Kohn et al., 1999] and chromosome 7[Wallace, 1976][Greavses & Ayres, 1967] in close linkage to the myosin light chain 2 gene (Myl2). The human ortholog of Myl2, HUMMLC2B, is located on chromosome 16pl 1 within the VKCFD2 candidate region .0 and is part of a conserved linkage group of genes. Based on this synteny and on biochemical considerations, it is hypothesized that VKCFD2 and warfarin resistance may be due to allelic mutations in the same gene. If so, this would narrow down the critical interval in humans to a region of approximately 4.5 Mb between the interleukin 4 receptor gene (IL4R) and the integrin alpha M chain gene (ITGAM) on the short arm of chromosome 16 (Fig. 1). 25 According to the genome assembly, this region contains 141 Ensembl genes with approximately 1000 exons. Of these genes, 117 were annotated as known. Many of these genes could be excluded from further analysis because their function was well established and obviously not related to the metabolic steps of the vitamin K cycle. On the other side, genes upstream and downstream of this region are included that were regarded as functional 30 candidates into the mutation screen. Example 2: Mutation screening Using genomic DNA from two VKCFD2 and three WR subjects, a systematic mutation screen was initiated by comparative sequencing of the remaining candidate genes. Clinical data 38 of the VKCFD2 families have been described previously [Oldenburg et al., 2000]. Warfarin resistant patients were ascertained due to their abnormal response to oral warfarin administration during thrombosis treatment or prevention. Patient C and E are sporadic cases. Patient D has two brothers also suffering from warfarin resistance. Patients C and D required 5 approximately 150-250 mg warfarin per week to achieve a therapeutic range of oral anticoagulation whereas patient E did not show any response at all. All patients gave informed consent before participating. Surprisingly, missense mutations were found in a gene of unknown function in all investigated VKCFD2 and WR subjects (Fig. 2). This gene (IMAGE3455200) spans a genomic 0 region of 5126 bp and comprises three exons coding for a protein of 163 amino acids. It was named vitamin K epoxide recycling protein 1 (VKORC1). Both non-related VKCFD2 patients and their affected siblings were found to harbor the same homozygous point mutation in the third exon (292C>T) whereas the parents were found to be heterozygous. The mutation is caused by the replacement of arginine by tryptophan at amino acid residue 98 (R98W). The 5 families are of German and Lebanese origin. The haplotypes in the region of homozygosity around the mutated gene were different in both families indicating independent mutation events. In the WR patients, three different heterozygous mutations were found leading to a valine by leucine substitution (patient C: V29L), an arginine by glycine substitution (patient D: R58G) and an exchange of leucine to arginine (patient E: L128R). The R58G mutation is shared 0 by the two affected brothers of index patient D. The missense mutations were not present in 384 control chromosomes. Sequencing of the control chromosomes revealed two non-synonymous single nucleotide polymorphisms (C43C; L120L). Genome sequences and annotation were obtained from NCBI, UCSC and Ensembl. Primers for mutation screening were designed using Primer3 software integrated into a script, 25 ExonPrimer, to allow automatic primer design. For mutation screening, exons with intronic primers were amplified and amplified fragments were analyzed by direct sequencing with the BigDye Terminator Cycle sequencing kit (ABI). Primer sequences were available on request. Topology predictions were performed using TMPRED and TMHM. Example 3: Homology and protein structure 30 An orthologue of the VKORC1 gene was present in mouse (NM_178600) and the orthologues in rat and in Fugu rubripes were established by homology searches and RT-PCR (Fig. 3). The corresponding proteins share 79% to 84% identity with the human protein. Database searches did not show any homology to a known gene nor to any characterized protein domain. Topology prediction programs anticipated three transmembrane domains (TM).

39 The first TM is placed between residues 10 to 29 by all programs tested. The predictions are discordant for the second and the third TM, which are located between amino acids 100 and 150.. The PSORT II server predicted an ER membrane retention signal (KKXX or KXKXX) at position 159-163 of human VKORC1 with a probability of 67% [Jackson et al., 1990]. The 5 consensus sequence was also present in the other VKORC 1 proteins. This is in accordance with the likely location of the VKORC1 complex within the ER membrane system [Cain et al., 1997]. Tblastn searches with VKORC1 detected a homologous human (BC027734) and mouse gene (AK009497) showing 50% protein identity each. Both mRNA were wrongly predicted to 0 code for proteins that show no homology with VKORC 1. The predicted human protein starts at the third methionine. The mouse mRNA sequence is incomplete with a protein predicted in a different reading frame. The complete cDNA was established in mouse as well as in Fugu rubripes and partially in rat. These proteins were designated VKORCl like protein I (VKORC1L1). Human, mouse and rat VKORC1L1 proteins share approximately 84% identity 5 between each other and approximately 50% identity with the corresponding VKORC 1 proteins. A homologous protein was further detected in Xenopus laevis (AAH43742) and - with weaker homology (le-14) - in Anopheles gambiae (EAA06271). Tree analysis suggested that both these proteins are orthologues to the VKORC 1 gene. Example 4: Expression analysis 0 VKORCI seems to be widely expressed. The corresponding Unigene entry contains more than 100 ESTs in various tissues. The expression of VKORC1 in fetal and adult human tissues is examined by Northern blot analysis. To this end Human multiple tissue northern blots (Fetal Blot 1, Stratagene; Human 12-Lane, BD Clontech) contained 2 ig of poly(A)+-RNA. Full-length human VKORCl cDNA was radiolabeled using random primers DNA labeling 25 system (Invitrogen life technologies). and hybridized using miracleHyb High-Performance Hybridization Solution (Stratagene). A B-actin probe supplied with the multiple tissue northern blot was used for control hybridization. The highest VKORP expression levels can be observed in fetal and adult liver (Fig. 4). High expression levels were also observed in fetal heart, kidney and lung, as well as in adult 30 heart and pancreas. Fetal brain, adult placenta and skeletal muscle showed intermediate levels of expression. Minor expression levels were detected in adult brain, lung and kidney.

40 Example 5: Cloning of VKORC1 and construction of expression vectors Amplification of the complete coding sequence of VKORCl was performed from human liver and kidney cDNA (Marathon-Ready cDNA, BD Biosciences Clontech) with the following primers including cleavage sites for HindIl and EcoRI: 5 VKORCl-HindIII-F: ATTAAGCTTCACCATGGGCAGCACCTGGGGGAGCCCT (SEQ ID No. 53) VKORCl-EcoRI-R: ATTGAATTCCGTGCCTCTTAGCCTTGCCCTG (SEQ ID No. 54). The product was cloned into the pBluescript II vector (Stratagene) that was cleaved with the corresponding restriction enzymes and verified by direct sequencing. For immunocytochemistry 0 experiments, the insert was re-cloned into the mammalian expression vectors pEGFP-Nl (BD Biosciences Clontech) and pcDNA3.1/myc-His (Invitrogen). For expression studies, the VKORC1 cDNA was cloned into the pcDNA3 vector (Invitrogen) after amplification with the primers VKORCl-pcdna3-F: GGGCGGAAGCTTGAGATAATGGGCA (SEQ ID No. 92) and VKORCl-pcdna3-R: 5 GCTTGAATTCAGGGCTCAGTGC (SEQ ID No. 93). Mutagenesis was performed using the QuikChange mutagenesis Kit (Stratagene). Wildtype and mutated cDNAs were re-cloned for expression in pCEP4 (Invitrogen) using the HindIII and Xhol-sites. All constructs were verified by sequencing. Example 6: Cell culture, transient transfection and immunocytochemistry and subcellular 0 localization From biochemical fractionation experiments it is known that the VKORCI activity purifies with the microsomal membrane fraction [Cain et al., 1997]. Furthermore, the gamma glutamyl-carboxylase has been localized to the membrane of the endoplasmic reticulum by immunocytochemistry [Presnell, 2002 #31]. In order to study the subcellular localization of 25 human VKORCI GFP- and myc-epitope tagged VKORC1 fusion protein constructs were generated for transient transfection experiments of COS-7 cells. Primary antibodies against the epitope tags and fluorochrome labeled secondary antibodies were used to visualize the fusion proteins. An antibody against the ER-specific protein calnexin served as a control. To this end COS-7 cells (DSMZ, Braunschweig) were maintained in Dulbecco's modified eagle's medium 30 with 10% fetal calf serum. Cells were plated on glass cover slips in six-well plates and after 18 24 h in culture transfected with the expression vector constructs using Effectene (QIAGEN) according to the manufacturer's specifications. After 48-60 h of further culturing, the cells were washed with PBS and fixed in 70% acetone / 30% methanol at -20 0 C for 15 min. Following fixation, the cells were permeabilized in PBS, 0.1% Nonidet P-40 (SIGMA N-6507), and then 41 blocked with PBS, 2% BSA and 0.1% NP-40 at 37*C. Primary antibodies, Living Colors A.v. (Jl-8) (BD Biosciences Clontech), anti-myc antibody (Invitrogen), and anti-calnexin (SIGMA) were diluted (1:100) in the blocking solution and incubated for 45 min at 37'C. Cover slips were washed in PBS, 0.1% NP-40 for 30 min. The same incubation and washing procedures 5 were used for the secondary antibodies, i.e. anti-mouse-IgG-FITC (SIGMA) and anti-rabbit IgG F(ab')2 fragment-Cy3 (SIGMA). Cover slips were counterstained with DAPI (1:500) for I min, washed with deionized water, mounted on slides using Vectashield (Vector) and visualized using a Leica fluorescence microscope. The green immunofluorescence of the VKORC 1 fusion proteins decorated the mesh-like 0 structures of the ER within the cytoplasm and perfectly co-localized with the label of the ER marker calnexin (red) (Fig. 5). Example 7: An assay for determining enzymatic VKORC1 activity HEK293-EBNA cells (Invitrogen) were grown in MEM with 10% FCS. For each experiment, 6 x 105 cells were plated onto 94 mm Petri dishes. After 30 h at 37*C and 5% C0 2 , 5 transfection (20 pg of DNA construct per dish) was performed using the calcium phosphate method. After 40 h at 35'C, 3% CO 2 transfected cells (nearly grown to confluence) were washed in PBS, harvested and lysed in 450pl 0.25 mM imidazole, (pH = 7.6), 0.5% CHAPS. Transfection efficiency was checked by sequencing of RT-PCR products of an aliquot of cells. VKOR enzymatic activity was measured with 30 Vl of the whole-cell extracts which 0 were resuspended in 500 pl buffer A (0.25 mM imidazole, (pH = 7.6), 0.5% CHAPS). Then 20pl 125mM DTT were added with one minute incubation. Then 5pl 400 mM CaCl 2 , and Warfarin in 10 1 DMSO (final concentration 0-80pM) were added. The reaction was started by addition of 2pl vitamin K2,3-epoxide (final concentration 5pM) and incubated at 30'C for one hour. Reaction was stopped by extraction of the substrate (Vitamin K2,3-epoxide) and the 25 reaction products (Vitamin K-quinone and hydroquinone) using 1 ml 2-Propanol/Hexane (3:2, v/v); the organic supernatant was collected, dried and resolved in 50ptl methanol and analyzed with an HPLC at 254 nm. The vitamin K quinone was separated from the epoxide by HPLC on a reversed phase C- 18 column. During the extraction procedure vitamin K hydroquinone was quantitatively oxidized to the quinone form. The output of the HPLC was analyzed 30 automatically by calculating the area under the line of extinction of each peak. The percentage of conversion of substrate was estimated by setting the area of the residual substrate-peak (epoxide) plus the product-peak (quinone) as 100 percent. Measurements were run in duplicate and the activity is given as percent of substrate converted into quinone. Vitamin K 2,3-epoxide 42 was prepared by oxidation of vitamin K quinone (Sigma-Aldrich) with H 2 0 2 . Warfarin (Sigma Aldrich) was added in DMSO (< 1 Vol%). Dose-response to warfarin inhibition was measured at 5 to 80 pIM final concentration (Walin & Martin 1985). Untransfected and mock-transfected cells showed a low basal activity 5 which was warfarin sensitive. Overexpression of Wildtype VKORCl resulted in a striking stimulation of VKOR activity. Production of vitamin K quinone was 14 to 21-fold increased compared to untreated and mock-transfected cells. The activity was inhibited by warfarin in a dose-dependent manner (Figure 10). We also determined VKOR activity after transfection with mutated VKORCJ constructs 0 (Figure 10). Recombinant expression of the R98W mutation observed in the two VKCFD2 families did only slightly increase VKOR activity in HEK293 cells. Spontaneous bleeding episodes and high serum levels of vitamin K epoxide in these patients suggest that the efficiency of vitamin K recycling is also drastically decreased in vivo (Oldenburg et al. 2000). The five WR mutations showed a reduced VKOR activity ranging from 5% in the L128R 5 variant to 96% in the V29L mutation. Mutations V45A, R58G and Y139C displayed about 23%, 21% and 48% activity, respectively (Table 1). Reduced VKOR activity associated with higher vitamin K demand and death from spontaneous bleeding has been observed in heterozygous and homozygous Rw rats (Martin et al. 1979, Thijssen & Pelz 2001, Fasco et al. 1983b). Similarly, in our expression system which mimics homozygous conditions WR 0 mutations led to a lower functional efficiency of the VKOR complex. Whereas at the phenotypic level all WR variants exhibited at least partial resistance towards the anticoagulation effect of warfarin, both Wildtype and mutant proteins were sensitive to warfarin in vitro. At concentrations above 20 pM, mutations V29L and YI39C retained higher VKOR activities than the Wildtype while in mutations V45A, R58G, L128R, VKOR activity fell below the detection 25 limit (Figure 10). Example 8: A method of diagnosing a VKORC1 sequence abnormality Genomic DNA of the specimen (human patient or mammal) is isolated according to standard procedures generally known to the skilled worker. The genomic DNA of the desired Exon (1-3) of VKORCl is amplified by PCR using specific primers which can also be designed 30 by the skilled artisan. The PCR-product is then purified using e.g. SAP/Exo (shrimp alkaline phosphatase and exonuclease) under standard conditions. The purified DNA is then subjected to standard sequencing procedures such as: addition of 0.3pgl primer which is 10 pmol/pl (forward or reverse primer) to I pl of the purified 43 PCR-product; followed by addition of 8pl DTCS-Mix (Beckman-Coulter) and 10.7pI water; followed by cycle sequencing at First delay 96 0 C 60 see Danaturation 95 0 C 30 see Annealing Primer specific(55-60 0 C) 30 sec Elongation 60 0 C 4 mi After the cycle sequencing purification by precipitation follows: * ad 2 pl 100mM EDTA, 2pl 5M NaOAc (pH 4.8),l l Glycogen vortex 5 e ad von 60 1d 95 % ethanol, vortex * centrifugation at 13000 g ( 10 min) e remove supernatant * wash pellet with 180 pl 70 % Ethanol * dry pellet 0 e resolve pellet in 35 pl Sample Loading Solution (SLS) Then the probes are pipetted on a microtiter plate overlaid with a drop of paraffin-oil. Then separation in the sequencer at 4,2 V for 60-120 min follows. The raw data is analyzed and the sequences are aligned with control sequences using the CEQ 2000 XL software (Version 4.3.9, Beckman Coulter). Differences between the control sequences (preferably the genomic 5 VKORC1 nucleic acid sequence or its coding sequence according to SEQ ID NO. 2) and the sequenced DNA is indicative of the probes sequence to represent a VKORCI nucleic acid containing a sequence abnormality. Example 9: PCR-based assay for determining warfarin resistance in rats In order to determine whether or not a rat (Rattus norvegicus) is warfarin resistant, i.e. 20 whether the VKORCI coding sequence according to SEQ ID No. 13 carries a mutation Y139C (416A>G), the following assay based on ARMS-PCR was employed using rat feces as a source of rat genomic DNA. It is the principle of the assay to include into the PCR reaction (1) one PCR primer (rVKORC1-innerF) that specifically hybridizes to the DNA sequence which contains the 25 warfarin resistant mutated allele 416G and (2) another PCR primer (rVKORC1-innerR) which specifically hybridizes to the Wildtype DNA sequence which contains the Wildtype allele 416A. Moreover, these two primers are oriented in opposite direction such that they pair with one out of two additional PCR primers included into the reaction. The latter primers are located in different distances to and in opposite direction relative to the 416 site and as a result, 44 depending on whether the 416 site is mutated or not either the rVKORC 1 -innerR primer or the rVKORCI-innerF primer will anneal and the PCR reaction will result in amplified DNA of a different size which is indicative of the genotype of the rat which DNA has been analyzed. In Wildtype rats the PCR reaction will result in a band of 123 bp, whereas in rats homozygous to 5 the mutation 416G the PCR reaction will yield a band at 101 bp. Finally in rats with a heterozygous genotype, the PCR reaction will give rise to two bands, one at 101 and another band at 123 bp. The genomic DNA was isolated from the feces using standard DNA isolation procedures generally known to the skilled artisan. The following components were combined to 0 a PCR reaction: 1 l DNA (rat), 1 il 5M Betain (Sigma), 2 pmol outer-Primer-F (1 iil of a 1:50-dilution), 2 pmol outer-Primer-R (1 pl of a 1:50-dilution), 10 pmol inner-Primer-F (1 ptl of a 1:10-dilution), 10 pmol inner-Primer-R (1 V1 of a 1:10- dilution), 0,25 pI Taq/Pfu-Polymerase (1,25U Taq (Invitrogen) and 0,25 U Pfu (Stratagene)), ad 25 pl PCR-buffer (1 ml PCR-buffer contains: 100 p lOxPCR-buffer (Invitrogen), 160 pl nucleotide stem-solution (1,25 mM 5 dNTPs), 30 pl MgC1 2 , 610 pl aqua dest). The PCR conditions were: 95*C for 3 min, followed by 32 cycles of: 95'C for 20 sec, 62*C for 20 see, and 70'C for 10 sec. Finally, the reaction is incubated at 70'C for 3 min. The PCR Products were separated by gel electrophoresis on a 3,5 % TAE-Agarose-Gel with ethidium bromide (10 pl of a 1%-stem solution for every 100ml). The gelelectrophosesis was allowed to run for 30 min at 130 V. .0 The following primers were used: rVKORCl-outerF: ATC CTG AGT TCC CTG GTG TCT GTC GCT G (SEQ ID No. 88) rVKORCl-outerR: TCA GGG CTT TTT GAC CTT GTG TTC TGG C (SEQ ID No. 89) 25 416G-mutant allele-specific PCR primer "rVKORCI-innerF": TGA TTT CTG CAT TGT TTG CAT CAC CAC ATG (SEQ ID No. 90) 416A-wildtype allele-specific PCR primer "rVKORCl-innerR": CAA CAT CAG GCC CGC ATT GAT GGA AT (SEQ ID No. 91) Rats (Rattus norvegicus) with and without warfarin resistance were used for the assay. 30 The results of the PCR are shown in Figure 13. Wildtype rats exhibited a band at 123 bp, rats homozygous to the mutation exhibited a band at 101 bp and finally, rats with the heterozygous mutation showed two bands, one at 101 and another band at 123 bp. As a result, this data demonstrate, that this assay can be employed to determine whether a given rat is warfarin resistant or not. Such assays are highly versatile in order to manage pest 45 control in a given region, since knowledge of the frequency of warfarin resistant rats is critical for deciding which pesticide may be employed effectively. If in a given region there is a high prevalence of warfarin resistant rats warfarin and analogues thereof are an unsuitable means to kill the rats. If, however, the determined frequency of warfarin resistant rats is low, warfarin 5 may be effectively used to fight rodents. Definitions The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of 0 the term is required. Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

46 List of References Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Bandyopadhyay, P.K., Garrett, J.E., Shetty, R.P., Keate, T., Walker, C.S., and Olivera, B.M. (2002). gamma -Glutamyl carboxylation: An extracellular posttranslational modification that antedates the divergence of molluscs, arthropods, and chordates, Proc Natl Acad Sci U S A 99, 1264-1269. Boneh, A., and Bar-Ziv, J. (1996). Hereditary deficiency of vitamin K-dependent coagulation factors with skeletal abnormalities, Am J Med Genet 65, 241-243. Brenner, B., Sanchez-Vega, B., Wu, S.M., Lanir, N., Stafford, D.W., and Solera, J. (1998). A missense mutation in gamma-glutamyl carboxylase gene causes combined deficiency of all vitamin K-dependent blood coagulation factors, Blood 92, 4554-4559. Cain, D., Hutson, S.M., and Wallin, R. (1997). Assembly of the warfarin-sensitive vitamin K 2,3-epoxide reductase enzyme complex in the endoplasmic reticulum membrane, J Biol Chem 272, 29068-29075. Chen, C.A. and Okayama, H. (1988). Calcium phosphate-mediated transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6, 632-638. Dockal, M., Carter, D.C., and Ruker, F. (1999). The three recombinant domains of human serum albumin. Structural characterization and ligand binding properties, J Biol Chem 274, 29303-29310. Dockal, M., Chang, M., Carter, D.C., and Ruker, F. (2000). Five recombinant fragments of human serum albumin-tools for the characterization of the warfarin binding site, Protein Sci 9, 1455-1465. Doetschman, Gene Transfer in Embryonic Stem Cells, page 115 to 146 in Pinkert, 1994. Ekelund, H., Lindeberg, L., and Wranne, L. (1986). Combined deficiency of coagulation factors II, VII, IX, and X: a case of probable congenital origin, Pediatr Hematol Oncol 3, 187-193. Esmon, C.T., Suttie, J.W., and Jackson, C.M. (1975). The functional significance of vitamin K action. Difference in phospholipid binding between normal and abnormal prothrombin, J Biol Chem 250, 4095-4099. Fasco, M.J., Principe, L.M., Walsh, W.A., and Friedman, P.A. (1983). Warfarin inhibition of vitamin K 2,3-epoxide reductase in rat liver microsomes, Biochemistry 22, 5655-5660.

47 Fasco, M.J., Preusch, P.C., Hildebrandt, E. & Suttie, J.W. (1983b). Formation of hydroxyvitamin K by vitamin K epoxide reductase of warfarin-resistant rats. J Biol Chem 258, 4372-4380. Fischer, M., and E., Z. (1966). Kongenitaler Mangel der Faktoren II, VIII und X, Zeitschrift flr Kinderheilkunde 95, 309-323. Fregin, A., Rost, S., Wolz, W., Krebsova, A., Muller, C.R., and Oldenburg, J. (2002). Homozygosity mapping of a second gene locus for hereditary combined deficiency of vitamin K-dependent clotting factors to the centromeric region of chromosome 16, Blood 100, 3229 3232. Furie, B., and Furie, B.C. (1988). The molecular basis of blood coagulation, Cell 53, 505-518. Goldsmith, G.H., Jr., Pence, R.E., Ratnoff, O.D., Adelstein, D.J., and Furie, B. (1982). Studies on a family with combined functional deficiencies of vitamin K- dependent coagulation factors, J Clin Invest 69, 1253-1260. Gossen M. et al. (1994) Curr. Opin. Biotechnol. 5, 516-20. Greavses, J.H., and Ayres, P. (1967). Heritable resistance to warfarin in rats, Nature 215, 877 878. Guenthner, T.M., Cai, D., and Wallin, R. (1998). Co-purification of microsomal epoxide hydrolase with the warfarin- sensitive vitamin KI oxide reductase of the vitamin K cycle, Biochem Pharmacol 55, 169-175. Harlow & Lane, 1998, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, USA, Chapter 5, pp. 53-135. Jackson, M.R., Nilsson, T., and Peterson, P.A. (1990). Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum, Embo J 9, 3153-3162. Jackson, W.B., Ashton, A.D., and Delventhal, K. (1988). Overview of anticoagulant rodenticide usage and resistance. In Current advances in vitamin K research, J. W. Suttie, ed. (New York, Elsivier), pp. 381-397. Johnson, C.A., Chung, K.S., McGrath, K.M., Bean, P.E., and Roberts, H.R. (1980). Characterization of a variant prothrombin in a patient congenitally deficient in factors II, VII, IX and X, Br J Haematol 44, 461-469.

48 Kohn, M.H., and Pelz, H.J. (1999). Genomic assignment of the warfarin resistance locus, Rw, in the rat, Mamm Genome 10, 696-698. Kozak, 1987, Nucleic. Acids Res. 15: 8125-48. Lee et al. (1981) Nature 214, 228-232. Leonard, C.O. (1988). Vitamin K responsive bleeding disorder: a genocopy of the warfarin embryopathy, Proceedings of the Greenwood Genetic Center 7, 165-166. Manfioletti, G., Brancolini, C., Avanzi, G., and Schneider, C. (1993). The protein encoded by a growth arrest-specific gene (gas6) is a new member of the vitamin K-dependent proteins related to protein S, a negative coregulator in the blood coagulation cascade, Mol Cell Biol 13, 4976 4985. Martin, A.D., Steed, L.C., Redfern, R., Gill, J.E. & Huson, L.W. Warfarin-resistance genotype determination in the Norway rat, Rattus norvegicus. Laboratory Animals, 209-214 (1979). McManus et al. 2002, Nature Reviews 3: 737-747, Gene Silencing in Mammals by small interfering RNAs. McMillan, C.W., and Roberts, H.R. (1966). Congenital combined deficiency of coagulation factors II, VII, IX and X. Report of a case, N Engl J Med 274, 1313-1315. Mutero, A., Pralavorio, M., Bride, J.M., and Fournier, D. (1994). Resistance-associated point mutations in insecticide-insensitive acetylcholinesterase, Proc Natl Acad Sci USA 91, 5922 5926. Monastersky, Gene Transfer Technology; Alternative Techniques and Applications, page 177 to 220 in Pinkert, 1994, supra. Mumberg et al. (1994) Nucl. Acids Res., 22, 5767 5768. Nellen and Lichtenstein, 1993, Trends Biochem. Sci. 18: 419-23; Stein, 1992, Leukemia 6: 967-74. Oldenburg, J., von Brederlow, B., Fregin, A., Rost, S., Wolz, W., Eberl, W., Eber, S., Lenz, E., Schwaab, R., Brackmann, H.H., et al. (2000). Congenital deficiency of vitamin K dependent coagulation factors in two families presents as a genetic defect of the vitamin K-epoxide reductase-complex, Thromb Haemost 84, 937-941. O'Reilly, R.A. (1970). The second reported kindred with hereditary resistance to oral anticoagulant drugs, N Engl J Med 282, 1448-145 1.

49 O'Reilly, R.A., Aggeler, P.M., Silvija Hoag, M., Leong, L.S., and Kropatkin, M.L. (1964). Hereditary transmission of exceptional resistance to coumarin anticoagulant drugs: the first reported kindred, N EngI J Med 271, 809-815. Pauli, R.M., Lian, J.B., Mosher, D.F., and Suttie, J.W. (1987). Association of congenital deficiency of multiple vitamin K-dependent coagulation factors and the phenotype of the warfarin embryopathy: clues to the mechanism of teratogenicity of coumarin derivatives, Am J Hum Genet 41, 566-583. Pechlaner, C., Vogel, W., Erhart, R., Pumpel, E., and Kunz, F. (1992). A new case of combined deficiency of vitamin K dependent coagulation factors, Thromb Haemost 68, 617. Petersen, C.E., Ha, C.E., Curry, S., and Bhagavan, N.V. (2002). Probing the structure of the warfarin-binding site on human serum albumin using site-directed mutagenesis, Proteins 47, 116-125. Prentice, C.R. (1985). Acquired coagulation disorders, Clin Haematol 14, 413-442. Presnell, S.R., and Stafford, D.W. (2002). The vitamin K-dependent carboxylase, Thromb Haemost 87, 937-946. Polites and Pinkert, DNA Microinjection and Transgenic Animal Production, page 15 to 68 in Pinkert, 1994, Transgenic animal technology: a laboratory handbook, Academic Press, London, UK. Price, P.A. (1988). Role of vitamin-K-dependent proteins in bone metabolism, Annu Rev Nutr 8, 565-583. Russel et al. (1983), J. Biol. Chem. 258, 2674-2682. Sperling, R., Furie, B.C., Blumenstein, M., Keyt, B., and Furie, B. (1978). Metal binding properties of gamma-carboxyglutamic acid. Implications for the vitamin K-dependent blood coagulation proteins, J Biol Chem 253, 3898-3906. Spronk, H.M., Farah, R.A., Buchanan, G.R., Vermeer, C., and Soute, B.A. (2000). Novel mutation in the gamma-glutamyl carboxylase gene resulting in congenital combined deficiency of all vitamin K-dependent blood coagulation factors, Blood 96, 3650-3652. Stitt, T.N., Conn, G., Gore, M., Lai, C., Bruno, J., Radziejewski, C., Mattsson, K., Fisher, J., Gies, D.R., Jones, P.F., and et al. (1995). The anticoagulation factor protein S and its relative, Gas6, are ligands for the Tyro 3/Axl family of receptor tyrosine kinases, Cell 80, 661-670.

50 Suttie, J.W. (1987). The biochemical basis of warfarin therapy, Adv Exp Med Biol 214, 3-16. Thijssen, H.H. & Pelz, H.J. in Advances in vertebrate pest management (eds. Pelz, H.J., Cowan, D.P. & Feare, C.J.) 181-192 (Filander-Verlag, Furth, 2001). Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584, No. 4. Vicente, V., Maia, R., Alberca, I., Tamagnini, G.P., and Lopez Borrasca, A. (1984). Congenital deficiency of vitamin K-dependent coagulation factors and protein C, Thromb Haemost 51, 343-346. Wallace, M.E., and MacSwiney, F.J. (1976). A major gene controlling warfarin-resistance in the house mouse, J Hyg (Lond) 76, 173-181. Wallin, R., and Martin, L.F. (1985). Vitamin K-dependent carboxylation and vitamin K metabolism in liver. Effects of warfarin, J Clin Invest 76, 1879-1884. Winter and Milstein, 1991, Nature 349:293-299. Wood, Retrovirus-Mediated Gene Transfer, page 147 to 176 in Pinkert, 1994, supra. Zheng and Kemeny, 1995, Clin. Exp. Immunol. 100: 380-2.

51 Other embodiments of the invention as described herein are defined in the following paragraphs: 1. A vitamin K epoxide recycling polypeptide (VKORC 1) as a target for coumarin and its derivatives in mammals comprising a polypeptide sequence selected from the group 5 consisting of: (a) a polypeptide sequence selected from the group consisting of a sequence according to SEQ ID No. 1, 12, and 17; (b) a polypeptide sequence of an allele of the polypeptide sequence defined in (a); (c) a polypeptide sequence having at least 80% homology with the polypeptide 0 sequence defined in (a) or (b), wherein the polypeptide sequence has VKORCI activity; and (d) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a), (b) or (c) having VKORCI activity. 2. A VKORCl nucleic acid comprising a nucleic acid sequence selected from the group 5 consisting of: (a) a nucleic acid sequence coding for the VKORCI polypeptide of claim 1; (b) a nucleic acid sequence selected from the group consisting of a sequence according to SEQ ID No. 2, 13, and 18; (c) a nucleic acid sequence which hybridizes under stringent conditions to the 0O nucleic acid sequence defined in (a) or (b), wherein the nucleic acid sequence codes for a polypeptide having VKORC 1 activity; (d) a nucleic acid sequence which, but for the degeneracy of the genetic code, would hybridize to the nucleic acid defined in (a), (b) or (c), and wherein the nucleic acid sequence codes for a polypeptide having VKORC 1 activity; and 25 (e) a fragment of the nucleic acid sequence defined in (a), (b), (c) or (d), wherein the fragment codes for a polypeptide having VKORCI activity. 3. A fusion protein comprising (a) the VKORCI polypeptide of claim I or a polypeptide encoded by the VKORCI nucleic acid of claim 2, and 30 (b) a heterologeous part. 4. A vector comprising the VKORC 1 nucleic acid of claim 2. 5. The vector of claim 4, wherein the vector is an expression vector. 6. The vector of claim 4 or 5, wherein the vector is a knock-out gene construct.

52 7. A host cell containing the VKORCl nucleic acid of claim 2 or the vector of any of claims 4 to 6. 8. The host cell of claim 7, wherein the host cell is a non-human embryonic stem cell. 9. A transgenic non-human mammal, wherein the transgenic mammal contains the host cell 5 of claim 8. 10. A DNA or a RNA probe directed against the VKORCI nucleic acid of claim 2. 11. A PCR primer directed against the VKORC 1 nucleic acid of claim 2, preferably a PCR primer selected from the group consisting of a PCR primer according to SEQ ID No. 53 to 69, and 70. 0 12. A small interfering RNA molecule (siRNA) or a short hairpin RNA (shRNA) directed against the VKORCl nucleic acid of claim 2, preferably a siRNA selected from the group consisting of SEQ ID No. 29, 30, 33, 34, 37, 38, 41, 42, 45, 46, 49, and 50. 13. An antisense RNA or DNA directed against the VKORCI nucleic acid of claim 2. 14. An RNA-aptamere directed against the VKORCl polypeptide of claim 1, wherein the 5 RNA-aptamere exerts an effect on the activity of the VKORC 1 polypeptide. 15. An antibody or a fragment thereof, which specifically recognizes and binds the VKORCl polypeptide of claim 1. 16. A method of producing a VKORCl polypeptide comprising the steps of: (I) providing a host cell having been introduced the VKORC I nucleic acid of claim 0 2 or a vector containing the VKORCl nucleic acid of any of claims 4 to 6; (II) expressing the VKORCI polypeptide in the host cell; and (III) isolating the VKORCI polypeptide from the host cell. 17. A method of identifying a coumarin derivative which exerts an effect onto the activity of VKORCI polypeptide of claim 1 comprising the steps of: 25 (I) providing a host cell having been introduced the VKORCl nucleic acid or a vector containing the VKORCI nucleic acid; (II) expressing the VKORCl polypeptide in the host cell; (III) administering a candidate coumarin derivative; (IV) determining the activity of VKORC 1 polypeptide (candidate activity value); 30 (V) comparing the candidate activity value with a control activity value; and (VI) identifying the candidate cournarin derivative as a coumarin derivative exerting an effect onto the activity of the VKORC1 polypeptide, provided the candidate activity value is significantly different from the control activity value.

53 18. The method of claim 17, wherein the control activity value is determined by a method comprising the steps of: (A) providing a host cell according to step (I); (B) expressing the VKORCI polypeptide in the host cell; and 5 (C) determining the activity of VKORCI polypeptide (control activity value). 19. The method of claim 17, wherein the determined activity of VKORCI polypeptide is dithiothreitol-dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the significantly different activity value is a candidate activity value which is significantly higher than the control activity value. 0 20. The method of claim 17, wherein at least one additional compound is introduced into the host cell, which compound is selected from the group consisting of vitamin K, cytochrome B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase. 21. A method of determining a VKORCl polypeptide sequence which conveys a coumarin 5 effect exerted onto VKORC 1 activity, comprising the steps of: (I) providing a cell expressing the VKORC1 polypeptide of claim 1, which VKORC1 polypeptide has at least one sequence abnormality; (II) administering coumarin or a derivative thereof to the cell; (III) determining the activity of the VKORC1 polypeptide (sequence abnormality 0 activity value); and (IV) comparing the sequence abnormality activity value with the control sequence activity value, wherein a significant deviation of the sequence abnormality activity value from the control sequence activity value is indicative that the sequence abnormality of the 25 VKORC 1 polypeptide conveys the coumarin effect exerted onto VKORC 1 polypeptide. 22. The method of claim 21, wherein the control sequence activity value is determined by a method comprising the steps of: (I) providing a cell expressing the VKORC 1 polypeptide of claim 1; (II) administering coumarin or a derivative thereof to the cell; 30 (III) determining the activity of the VKORCl polypeptide (control sequence activity value). 23. The method of determining of claim 21, wherein the determined activity is dithiothreitol dependent conversion of vitamin K 2,3-epoxide to vitamin K quinone and wherein the 54 significantly different value is a sequence abnormality activity value which is significantly higher than the control sequence activity value. 24. The method of claim 21, wherein at least one additional compound is introduced into the cell which compound is selected from the group consisting of vitamin K, cytochrome 5 B5, and a nucleic acid coding for gamma-glutamyl-carboxylase, for microsomal epoxidehydrolase, for calumenin, or for glutathion-S-transferase. 25. The VKORCI polypeptide of claim 1, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which exerts an effect on the activity of the VKORC1 polypeptide. 0 26. The VKORCI polypeptide of claim 25, wherein the VKORC1 polypeptide is the polypeptide according to SEQ ID No. I or 12 and the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C. 27. A VKORCI nucleic acid selected from the group consisting of: (a) a nucleic acid coding for the VKORCI polypeptide of claim 25 or 26, 5 (b) a nucleic acid sequence selected from the group consisting of a sequence according to SEQ ID No. 3, 4, 5, 6, 7, 14,and 94, (c) a nucleic acid sequence which, but for the degeneracy of the genetic code, would hybridize to the nucleic acid defined in (a) or (b), and wherein the nucleic acid sequence codes for the polypeptide of claim 25 or 26. 0 28. A vector containing the VKORCl nucleic acid of claim 27. 29. A DNA or a RNA probe directed against the VKORC1 nucleic acid of claim 27. 30. A PCR primer directed against the VKORCI nucleic acid of claim 27. 31. An antibody or a fragment thereof, which specifically recognizes and binds the VKORCI polypeptide of claim 25 or 26. 25 32. A transgenic non-human mammal wherein the mammal contains a stem cell containing the VKORCI nucleic acid of claim 27 or the vector according to claim 28. 33. A diagnostic comprising a compound selected from the group consisting of the VKORC1 nucleic acid of claim 27, the DNA or the RNA probe of claim 29, the PCR primer of claim 30, and the antibody of claim 31. 30 34. A method of diagnosing a VKORC 1 associated deficiency in a patient comprising the steps of: (I) amplifying a DNA sample obtained from the patient or reverse transcribing a RNA sample obtained from the patient into a DNA and amplifying the DNA; and 55 (II) analyzing the amplified DNA of step (I) to determine at least one sequence abnormality in a nucleic acid sequence coding for the VKORCl polypeptide of claim I or in an amino acid sequence of the VKORCI polypeptide; wherein the determined sequence abnormality is indicative of the patient suffering from 5 a VKORC I associated deficiency; preferably warfarin resistance. 35. The method of claim 34, wherein the amplified DNA encodes at least a partial sequence of the VKORCl polypeptide according to SEQ ID No. I and wherein the sequence abnormality is selected from the group consisting of V29L, V45A, R58G, R98W, L128R, and Y139C. 0 36. The method of claim 34, wherein the amplified DNA is analyzed by a technique selected from the group consisting of PCR- based analysis, restriction digestion analysis, and DNA sequencing analysis. 37. A method of diagnosing a VKORC1 associated deficiency in a patient comprising the steps of: 5 (I) providing a sample obtained from the patient; and (II) detecting a VKORCI polypeptide having a sequence abnormality in the sample using the antibody of claim 31, wherein the determined sequence abnormality is indicative of the patient suffering from a VKORCI associated deficiency. .0 38. The method of claim 37, wherein the sample is analyzed by a technique selected from the group consisting of immunohistochemical detection, immunoblotting, preferably Western blotting, and ELISA. 39. A method of identifying a coumarin derivative which exerts an inhibitory effect onto the activity of a VKORC1 polypeptide having at least one sequence abnormality, 25 comprising the steps of: (I) providing a cell expressing the VKORCI polypeptide according to claim 25 or 26, preferably a polypeptide encoded by a sequence selected from the group consisting of SEQ ID No. 3, 4, 5, 6, 7, 14, and 94; (II) administering a candidate coumarin derivative to the cell; 30 (III) determining the activity of the VKORC1 polypeptide (sequence abnormality activity value); and (IV) comparing the sequence abnormality activity value with the control sequence activity value, 56 (V) identifying the candidate coumarin derivative as the coumarin derivative exerting an inhibitory effect onto the activity of a VKORCI polypeptide, if the administration of the candidate coumarin derivative results in a sequence abnormality activity value which is significantly lower than the control sequence 5 activity value. 40. A method of identifying a coumarin derivative which is toxicologically effective in warfarin-resistant rodents comprising the steps of: (I) providing a warfarin-resistant rodent; (II) administering a candidate coumarin derivative to the rodent; 0 (III) determining the toxicity of the candidate coumarin derivative onto the rodent (candidate coumarin derivative toxicity value); (IV) comparing the candidate coumarin derivative toxicity value with a control coumarin toxicity value; (V) identifying the candidate coumarin derivative as a toxicologically effective 5 coumarin derivative, provided that the candidate coumarin derivative toxicity value is significantly larger than the control coumarin toxicity value. 41. The method of claim 40, wherein warfarin-resistant rodent is a rodent transgenic for VKORC1 polypeptide of claim 1, wherein the VKORC1 polypeptide contains at least one sequence abnormality, which causes warfarin resistance, preferably a polypeptide .0 encoded by the sequence according to SEQ ID No. 14. 42. The method of claim 40, wherein VKORC1 polypeptide is the polypeptide according to SEQ ID No. 12 and the sequence abnormality is selected from the group consisting of V29L (85 G>T), V45A(134 T>C), R58G (172 A>G), R98W (292 C>T), and L128R (383 T>G), and Y139C (416 A>G).). 25 43. A composition for killing rodents, comprising a rodenticidally effective amount of the coumarin derivatives identified by the method according to any of claims 39 to 41. 44. Use of the PCR primers according to SEQ ID No. 88 to 91 for determining whether or not a rat has a warfarin resistance genotype in a sample obtained from a rat. Still further embodiments are within the scope of the following claims. 30

Claims

1. Use of a VKORC 1 nucleic acid in a method for gamma-carboxylating a vitamin-K dependent polypeptide in a host cell. 5

2. The use of claim 1, comprising: a) introducing said VKORCI nucleic acid into the host cell; and b) expressing a VKORCI polypeptide in the host cell.

3. The use of a claim I or claim 2, wherein the VKORCI polypeptide is selected from the 0 group consisiting of: (a) a polypeptide sequence consiting of SEQ ID NO: 1; (b) a polypeptide sequence having at least 90% identity with the polypeptide sequence defined in (a) and having VKORC I activity; and (c) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a) 5 or (b) having VKORCl activity.

4. The use of any one of claims 1 to 3, wherein said host cell is a eukaryotic cell.

5. The use of claim 4, wherein said eukaryotic cell is selected from the group consisiting of 20 HEK293-EBNA cells, primary hepatocytes, yeast cells, insect cells, COS cells, hepatocyte cell lines, and non-human embryonic stem cells.

6. The use of any one of claims I to 5, wherein said vitamin K dependent polypeptide is selected from the group consisiting of blood coagulation factor II, VII, IX, X, protein C, protein 25 S, protein Z, matrix gla protein and osteocalcin.

7. The use of any one of claims 2 to 6, wherein said VKORCI polypeptide facilitiates gamma-carboxylation of said vitamin-K dependent polypeptide in said host cell. 58

8. The use of any one of claims I to 7, wherein said VKORCI nucleic acid is used in combination with at least one additional compound selected from the group consisting of vitamin K, cytochrome B5, a nucleic acid coding for gamma-glutamyl-carboxylase, a nucleic acid coding for microsomal epoxidehydrolase, a nucleic acid coding for calumenin, and a 5 nucleic acid coding for glutathion-S-transferase.

9. Use of a VKORCI nucleic acid for facilitating gamma-carboxylation of a vitamin-K dependent protein in a host cell, comprising: a) introducing said VKORCI nucleic acid into the host cell; and 0 b) expressing a VKORCI polypeptide in the host cell.

10. The use of claim 9, wherein said VKORCI polypeptide is selected from the group consisting of: a) a polypeptide sequence consisting of SEQ ID NO: 1; 5 b) a polypeptide sequence having at least 90% identity with the polypeptide sequence defined in (a) and having VKORCl activity; and c) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a) or (b) having VKORC 1 activity. 0

11. The use of claim 9 or claim 10, wherein said host cell is a eukaryotic cell.

12. The use of claim 11, wherein said eukaryotic cell is selected from the group consisting of HEK293-EBNA cells, primary hepatocytes, yeast cells, insect cells, COS cells, hepatocyte cell lines, and non-human embryonic stem cells. 25

13. The use of any one of claims 9 to 12, wherein said VKORC1 nucleic acid is used in combination with at least one additional compound selected from the group consisting of vitamin K, cytochrome B5, a nucleic acid coding for gamma-glutamyl-carboxylase, a nucleic acid coding for microsomal epoxidehydrolase, a nucleic acid coding for calumenin, and a 30 nucleic acid coding for glutathion-S-transferase. 59

14. Use of a VKORCI nucleic acid in a method to enzymatically convert or support the enzymatic conversion of vitamin K2, 3-epoxide to vitamin K-quinone and/or the conversion of vitamin K quinone to vitamin K hydroquinone, comprising: a) introducing said VKORCl nucleic acid into a host cell; and 5 b) expressing a VKORCI polypeptide in said host cell, wherein said VKORC 1 polypeptide enzymatically converts or supports the enzymatic conversion of vitamin K2, 3-epoxide to vitamin K-quinone and/or the conversion of vitamin K quinone to vitamin K hydroquinone. 0

15. The use of claim 14, wherein the VKORCI polypeptide is selected from the group consisting of: a) a polypeptide sequence consisting of SEQ ID NO: 1; b) a polypeptide sequence having at least 90% identity with the polypeptide sequence defined in (a) and having VKORCI activity; and 5 c) a polypeptide sequence of a fragment of the polypeptide sequence defined in (a) or (b) having VKORCI activity.

16. The use of claim 14 or claim 15, wherein said host cell is a eukaryotic cell. 0

17. The use of claim 16, wherein said eukaryotic cell is selected from the group consisting of HEK293-EBNA cells, primary hepatocytes, yeast cells, insect cells, COS cells, hepatocyte cell lines, and non-human embryonic stem cells. Date: 16 November 2012 25