JP2005287503A - Method for identifying risk for thrombogenic disorder by determining tafi-ile347 polymorphism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To identify the risk for thrombogenic disorder, transitory ischemic attack (TIA), atherosclerotic cerebrovascular disease (CVD) and/or coronary heart disease, and also to identify a pharmaceutical for the therapy or prophylaxis of the thrombogenic disorder. <P>SOLUTION: The risk of the thrombogenic disorder is identified by determining the presence of an amino acid exchange from threonine of a thrombin-activable fibrinolysis inhibitor (TAFI) having an amino acid sequence (TAFI-Ile347) to isoleucine. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to thrombogenic disorders such as, but not limited to, atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic stroke (TIA), atherosclerotic cerebrovascular disease (CVD) and / or a method for identifying a risk for coronary heart disease), as well as a method for selecting patients at risk for a thrombogenic disorder, an agent for treating or preventing a thrombogenic disorder As well as methods for producing pharmaceuticals and diagnostic agents by using the TAFI-Ile347 polymorphism.

Thrombin-activated fibrinolysis inhibitor (TAFI) is a known plasma enzyme precursor synthesized in the liver as a prepropeptide and consists of 423 amino acids with a molecular weight of 55 kDa (FIG. 1). The prepropeptide includes a 22 amino acid long signal peptide (amino acid numbers 1-22), a 92 amino acid long activation peptide, and a 309 amino acid long catalytic domain. The nucleic acid sequence contains 1723 nucleotides (Figure 2). The TAFI protein sequence accession number (NCBI protein database) is NP_001863, the nucleotide sequence accession number (NCBI nucleotide database) is NM_001872, and the accession number for TAFI information in OMIM (Online Mendian Inheritance in Man ^™ ) is 603101.

  TAFI is activated by thrombin, plasmin or thrombin / thrombomodulin complex. After processing, TAFI attenuates thrombolysis by removing lysine residues from fibrin clots. Activated and processed TAFI is unstable at 37 ° C. and has a half-life of about 8 minutes. Therefore, TAFI plays a central role in homeostasis, where it functions as an effective fibrinolysis inhibitor. The human TAFI gene has been mapped to chromosome 13q14.11. The human TAFI gene consists of 11 exons and spans a genomic region approximately 48 kb in length (Boffa et al. (1999) Biochemistry, 38, 6547-6558).

  Genetic analysis of the TAFI gene in humans revealed that there are several variable nucleotides (SNPs, single nucleotide polymorphisms) in the promoter and coding regions. With respect to SNPs in the promoter region of the TAFI gene, some of these polymorphisms have been shown to be associated with altered TAFI protein levels in the blood (Franco et al. (2001) Haematologica, 86, 510-517; Henry et al. (2001) Blood, 97, 7, 2053-2058).

  In recent years, two SNPs that cause amino acid substitutions in the corresponding TAFI protein in the coding region of the TAFI gene have been identified. )) And T347I (the threonine at position 347 is I = isoleucine (Ile)). The TAFI-Ile347 mutant appears to exhibit an extended half-life from 8 minutes to 15 minutes under the in vitro conditions tested, and appears to exhibit 60% increased antifibrinolytic ability (Brouwers et al. ( 2001) Blood, 98, 6, 1992-1993; Schneider et al. (2001) J. Biological Chemistry, 277, 2, 1021-1030). A mutation at position 169 in the TAFI protein appears to have little effect on the antifibrinolytic ability of TAFI (Schneider et al. (2001) above). However, there is currently little data available regarding the clinical effects of polymorphisms including TAFI-Ile347 mutations, such as for thrombogenic disorders.

  In accordance with the present invention, it has been found that in particular individuals with the TAFI-Ile347 polymorphism have an increased risk for stroke and transient ischemic attacks (TIA). Therefore, the genetic TAFI-Ile347 polymorphism can be used as a genetic marker, eg, identification of risk for thrombogenic disorders, eg selection of patients at risk for thrombogenic disorders in clinical studies, Identification of drugs for the treatment and / or prevention of thrombogenic disorders, manufacture of pharmaceuticals for the prevention and / or therapeutic treatment of thrombogenic disorders, manufacture of diagnostic agents for identifying thrombogenic disorders, And / or can be used for adaptation of pharmaceutical dosages to treat and / or prevent thrombogenic disorders.

  Therefore, one embodiment of the present invention provides a thrombogenic disorder (eg, but not limited to atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA) And an in vitro or in vivo method for identifying a risk for atherosclerotic cerebrovascular disease (CVD) and / or coronary heart disease), said method being described in SEQ ID NO: 1 in a sample Determining the presence of an amino acid substitution from threonine to isoleucine at position 347 of a thrombin activated fibrinolysis inhibitor (TAFI) having the amino acid sequence of (TAFI-Ile347). Preferably, the sample is from a diabetic patient because diabetes often exhibits reduced fibrinolysis. Generally, the sample is a cell or body fluid, such as blood, or an animal cell or human cell isolated from an animal or human body.

  TIA, PRINT and stroke are neurological disorders with sudden onset due to vascular diseases such as thrombogenic disorders, which differ only in the duration and severity of re-recovery. For example, TIA is a neurological disorder that usually lasts less than 24 hours and does not cause permanent brain damage. PRIND is a neurological disorder that usually lasts for more than 24 hours and does not involve permanent brain damage. Stroke usually causes permanent brain damage.

  In general, the TAFI-Ile347 polymorphism can be determined by methods known to those skilled in the art. One method is to determine amino acid substitutions, which include amino acid sequence analysis, eg, analysis of protein sequence fragments using standard proteolysis or mass spectrometry, enzymatic treatment of proteins, and subsequent degradation. By analysis of the product or by a binding protein or peptide or aptamer specifically directed against TAFI-Ile347, in particular an antibody, an antigen binding site of an antibody, and / or a protein scaffold, preferably Is made by anticalin.

  In the present invention, the term “binding protein” or “binding peptide” means a class of protein or peptide that specifically binds to TAFI or TAFI-Ile347, such as, but not limited to, TAFI or TAFI-Ile347. Monospecific polyclonal or monoclonal antibodies, antibody fragments, and protein scaffolds specifically directed against, such as anticalin, which is specifically directed against TAFI-Ile347 . The term “specifically” means that the binding protein or peptide distinguishes TAFI-Ile347 from other polymorphisms at amino acid position 347 of TAFI (particularly TAFI-Thr347).

  The determination of other polymorphisms at amino acid position 347 of TAFI, particularly TAFI-Thr347, may be used as a reference or control in the methods of the present invention.

  Procedures for producing antibodies or antibody fragments can be achieved by methods well known to those skilled in the art, eg, in mammals with TAFI or TAFI-Ile347, optionally in the presence of eg Freund's adjuvant and / or aluminum hydroxide gel. (E.g., see Diamond, BA, et al. (1981) The New England Journal of Medicine: 1344-1349). Subsequently, polyclonal antibodies formed in animals as a result of an immune response can be isolated from blood using well-known methods and purified, for example, by column chromatography. Monoclonal antibodies can be produced, for example, according to the known methods of Winter and Milstein (Winter, G. and Milstein, C. (1991) Nature, 349, 293-299).

In the present invention, the terms “antibody” or “antibody fragment” are also understood to mean antibodies or their antigen binding sites, which are produced recombinantly and optionally modified, for example , Chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or multispecific antibodies, single chain antibodies and F (ab) or F (ab) ₂ fragments (eg EP-B1-0 368 684, US 4,816,567, US 4,816,397, WO 88/01649, WO 93/06213, or WO 98/24884).

  Typical antibody alternatives include, for example, protein scaffolds for TAFI or TAFI-Ile347, such as lipocalin-based anticalins (Beste et al. (1999) Proc. Natl. Acad. Sci. USA, 96, 1898-1903) can also be used. The natural ligand binding site of lipocalin, eg retinol binding protein or villin binding protein, is modified by a “combinatorial protein design” approach, eg in such a way that they bind to a selected hapten, here TAFI or TAFI-Ile347. (Skerra, 2000, Biochim. Biophys. Acta, 1482, 337-50). Other known protein scaffolds are known as alternatives to antibodies for molecular recognition (Skerra (2000) J. Mol. Recognit, 13, 167-187).

  Aptamers are nucleic acids that bind with high affinity to a polypeptide, here TAFI or TAFI-lLe347. Aptamers can be isolated from large pools of various single-stranded RNA molecules by selection methods such as SELEX (eg, Jayasena (1999) Clin. Chem., 45, 1628-50; Klug And Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; Aptamers can also be synthesized and selected, for example, as L-ribonucleotides in their enantiomers (Nolte et al. (1996) Nat. Biotechnol, 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5). Forms isolated in this way have the advantage of greater stability because they are not degraded by naturally occurring ribonucleases.

Another method of determining the TAFI-Ile347 polymorphism is to analyze the TAFI gene, in particular, to analyze the nucleic acid sequence for detection of a cytidine to thymidine nucleotide substitution at position 1064. In general, the determination of nucleotide substitution can be made by nucleic acid sequence analysis, for example pyrosequencing.
Specifically directed to sequence analysis methods using radiolabeled nucleotides or nucleotides labeled with fluorescent dyes, primer extension analysis, sequence fragment analysis using mass spectrometry, or to mutations in the TAFI gene The antibody, the antigen binding site of the antibody or the protein scaffold, preferably an anticalin, and / or a complementary nucleic acid.

  In this form, the term “specifically” refers to an antibody, an antigen binding site of an antibody or a protein scaffold, preferably an anticalin, and / or a complementary nucleic acid at the amino acid position 347 of the TAFI-Ile347 gene and TAFI. It is meant to distinguish from other polymorphisms of nucleotide codons (especially TAFI-Thr347). A preferred nucleotide substitution for the amino acid polymorphism at position 347 is the nucleotide at position 1064.

Complementary nucleic acids are preferably single-stranded DNA molecules, which can be chemically synthesized according to, for example, the phosphotriester method (eg, Uhlmann, E. and Peyman, A. (1990) Chemical Reviews, 90 , 543-584). These complementary nucleic acid, for example as hybridization probes on a DNA microarray, or can be used as amplification probes, for example TaqMan fluorogenic 5 'nuclease analysis ^(R) analysis (Taqman ^(R) Laboratory).

  An antibody, an antigen-binding site of an antibody, or a protein scaffold can be appropriately produced as described above.

  In any case, the invention preferably further determines other possible polymorphisms at amino acid position 347 of TAFI, for example TAFI-Thr347, as a reference or control, preferably using the method as described above. This method is advantageous.

  In general, the methods of the invention are used to increase vascular disease relative to a reference or control (eg, but not limited to atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient Risk for traumatic ischemic attack (TIA), atherosclerotic cerebrovascular disease (CVD), and / or coronary heart disease) can be identified, thereby preventing and / or preventing an individual (eg, a diabetic patient) It is possible to obtain therapeutic treatment or adaptation of the dose of the drug administered (further described below).

  Therefore, other embodiments of the present invention provide thrombogenic disorders such as, but not limited to, atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA) ), Atherosclerotic cerebrovascular disease (CVD), and / or coronary heart disease), an in vitro or in vivo method, said method comprising a sample (preferably a diabetic patient) In), determining the presence of an amino acid substitution from threonine to isoleucine at position 347 of the thrombin activated fibrinolysis inhibitor (TAFI) having the amino acid sequence set forth in SEQ ID NO: 1

  The method for selecting a patient at risk for a vascular disease of the present invention can be characterized and implemented in a manner similar to the method for identifying a risk for a vascular disease described above.

Other embodiments of the present invention include thrombogenic disorders such as, but not limited to, atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA), and A method for identifying an agent, preferably an inhibitor of TAFI-Ile347, for the treatment and / or prevention of atherosclerotic cerebrovascular disease (CVD) and / or coronary heart disease), said method comprising: Including steps:
(A) providing a TAFI-Ile347 or TAFI-Ile347 gene;
(B) providing a test compound; and
(C) A step of measuring or detecting the effect of the test compound on TAFI-Ile347 or TAFI-Ile347 gene.

  In the present invention, the term “inhibitor” refers to a TAFI-Ile347 enzyme precursor, in particular a biochemical or chemical compound that inhibits or reduces the removal of lysine residues from fibrin clots, and / or TAFI-Ile347. Means a biochemical or chemical compound that reduces the half-life of at least about 20% to about 50%, preferably at least about 30% to about 45%, especially about 45%. The term “about” generally means that the error range is +/− 20%, in particular +/− 10%, preferably +/− 5%.

  Generally, the TAFI-Ile347 or TAFI-Ile347 gene is provided, for example, in an analytical system, for example directly or indirectly with a test compound, in particular a biochemical or chemical test compound, for example in the form of a chemical compound library. Make contact. Next, the effect of the test compound on the TAFI-Ile347 or TAFI-Ile347 gene is measured or detected. The appropriate inhibitor can then be analyzed and / or isolated. For screening chemical compound libraries, the use of high-throughput analysis known to those skilled in the art or commercially available is preferred.

  In the present invention, the term “chemical compound library” refers to a large number of chemical compounds collected from all diverse sources such as chemically synthesized molecules and natural products, or a large number of chemical compounds produced by combinatorial chemistry techniques. means.

  In general, the effect of the test compound on TAFI-Ile347 or TAFI-Ile347 gene is measured or detected by a heterogeneous assay or a homogeneous assay. The heterogeneous assay used in the present invention is an analysis that includes one or more washing steps, whereas the homogeneous assay does not require such washing steps. Reagents and compounds are simply mixed and measured.

  Appropriate functional analysis can be based on gene expression of TAFI-Ile347. When the biochemical or chemical compound to be tested is present as an inhibitor of TAFI-Ile347 gene expression, direct inhibition is generally measured by means known to those skilled in the art or as described above. be able to. For example, to measure TAFI (eg, as a reference or control) and / or TAFI-Ile347 enzymatic activity, thrombolysis and / or removal of lysine residues from fibrin, or, in general, For example, its carboxypeptidase activity using the synthetic carboxypeptidase substrate anisylazoformyl lysine can be measured by a suitable assay known to those skilled in the art (see, eg, Schneider, M. et al. (2002) above). .

  Heterogeneous assays are, for example, ELISA (enzyme-linked immunosorbent assay), DELFIA, SPA and flash plate analysis.

  ELISA is generally a known assay that uses an enzyme as a marker molecule, eg, a peroxidase staining reaction is initiated by the addition of a peroxidase substrate and the optical density is measured with a suitable densitometer.

The analysis based on DELFIA (dissociation enhanced lanthanide fluorescence immunoassay) is a solid phase analysis. The antibody is usually labeled with europium or other lanthanide, and europium fluorescence is detected after washing away unbound europium labeled antibody.

  SPA (Scintillation Proximity Analysis) and flashplate analysis typically utilize biotin / avidin interaction to capture radiolabeled substrate. Generally, the reaction mixture contains a biotinylated peptide substrate. After the reaction, the biotinylated peptide is captured by streptavidin. In SPA detection, streptavidin binds on beads containing scintillant, whereas in flash plate detection, streptavidin binds inside the wells of microplates containing scintillant. Once immobilized, the radiolabeled substrate is close enough to the scintillant to stimulate light emission.

  Examples of other homogeneous assays include TR-FRET, FP, ALPHA, EFC, and gene analysis.

  Analyzes based on TR-FRET (time-resolved fluorescence resonance energy transfer) are usually europium and APC, modified allophycocyanin, or other dyes with overlapping spectra (eg, Cy3 / Cy5 or Cy5 / Cy7) (Schobel, U., et al. (1999) Bioconjugate Chem. 10, 1107 to 1114). For example, after exciting europium with 337 nm light, the molecule fluoresces at 620 nm. However, if this fluorophore is sufficiently close to APC, europium transfers its excitation energy to APC and fluoresces at 665 nm. After the reaction, an antibody labeled with europium is added together with streptavidin-APC. Due to the proximity of the APC to the europium fluorophore, quenching of the europium fluorescence occurs when APC fluorescence is imparted (FRET).

  Analysis based on fluorescence polarization (FP) is an analysis that uses polarized light to excite a fluorescent substrate peptide in solution. These fluorescent peptides are free in solution, disintegrate, and depolarize the emitted light. However, when the substrate peptide binds to a larger molecule, its decay rate becomes very low and the emitted light remains strongly polarized.

  Analysis based on ALPHA (Amplified Luminescence Proximity Homogeneous) is based on the movement of singlet oxygen between an adjacent donor and acceptor beads. Upon excitation at 680 nm, the photosensitizer in the donor bead converts ambient oxygen to singlet state oxygen, which diffuses to a distance of 200 nm. The chemiluminescent group in the acceptor bead transfers energy to the fluorescent acceptor in the bead and subsequently emits light at about 600 nm.

  Analysis based on EFC (enzyme fragment complementation) or equivalent analysis can be used in particular for high-throughput screening of compounds. EFC analysis is based on a processed β-galactosidase enzyme consisting of two fragments: an enzyme acceptor (EA) and an enzyme donor (ED). When the fragments are separated, β-galactosidase activity is lost, but when the fragments are combined, they work together (complement) to form an active enzyme. EFC analysis utilizes an ED-analyte conjugate, where the analyte can be recognized by a specific binding protein such as an antibody or receptor. In the absence of a specific binding protein, the ED-analyte conjugate can supplement EA and form active β-galactosidase, producing a positive luminescent signal. When the ED-analyte conjugate and the specific binding protein bind, complementation with EA is inhibited and no signal is generated. If free analyte is provided (in the sample), it will compete with the ED-analyte conjugate for binding to the specific binding protein. Free analyte releases the ED-analyte conjugate for complementation with EA and produces a signal depending on the amount of free analyte present in the sample.

  Examples of genetic analysis include two-hybrid system analysis (Fields and Sternglanz (1994) Trends in Genetics, 10, 286-292; Colas and Brent (1998) TIBTECH, 16, 355-363). In this test, cells are transformed with an expression vector that expresses a fusion protein consisting of a polypeptide according to the invention and a DNA binding domain of a transcription factor (eg, Gal4 or LexA). The transformed cell further comprises a reporter gene, whose promoter contains a binding site for the corresponding DNA binding domain. By transforming the second fusion protein with this polyprotein by transformation with another expression vector that expresses a second fusion protein consisting of a known or unknown polypeptide and an activation domain (eg, from Gal4 or herpes simplex virus VP16). When interacting with a peptide, the expression of the reporter gene can be greatly increased. As a result, this test system can be used to screen for biochemical or chemical compounds that inhibit the interaction of TAFI-Ile347 and its substrate (eg, fibrin). In this way it is possible to rapidly identify new active compounds.

  Other analyzes are based on polypeptides bound to a solid phase (eg TAFI-Ile347). Thus, a test compound includes, for example, a detectable marker, for example, such a compound can be a radioactive label, a fluorescent label, or a luminescent label. In addition, the compound may be coupled to a protein, such as by enzymatic catalysis by peroxidase analysis using a chromogenic substrate or indirectly by coupling a detectable antibody. Detection is possible. Another possibility is to investigate protein complexes bound to the solid phase by mass spectrometry (SELDI). For example, a conformational change of TAFI-Ile347 in an activated state as a result of interaction with the test substance can be detected, for example, by a fluorescent change of an endogenous tryptophan residue in the polypeptide.

  Polypeptides bound to a solid phase can also form part of an array. Methods for producing such arrays using solid phase chemistry and photolabile protecting groups are disclosed, for example, in US Pat. No. 5,744,305. These arrays can also be contacted with a test compound or compound library and tested for interactions, eg, binding, or conformational changes.

  Advantageously, the method of the present invention is performed in a robotic system, including, for example, robotic plating and robotic liquid transfer systems using, for example, microfluidics (ie, with a grooved structure).

  In other embodiments of the invention, the method is performed in the form of a high-throughput screening system. Advantageously in such a system, the screening method is automated and miniaturized, in particular using robotic controlled miniaturized wells and microfluidics.

Other embodiments of the present invention include thrombogenic disorders such as, but not limited to, atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA), and , Atherosclerotic cerebrovascular disease (CVD) and / or coronary heart disease), a method for producing a medicament for the prophylactic and / or therapeutic treatment comprises the following steps:
(A) identifying an agent for the treatment and / or prevention of a thrombogenic disorder according to the method described above,
(B) providing a sufficient amount of the agent identified according to step (a); and
(C) formulating said drug using one or more pharmaceutically acceptable carriers or adjuvants.

  In order to produce the medicaments of the present invention, the identified agents are usually formulated with one or more pharmaceutically acceptable carriers or adjuvants, such carriers depending on the type of administration, for example Physiological buffer solutions such as sodium chloride solution, demineralized water, stabilizers such as protease or nuclease inhibitors, preferably aprotinin, ε-aminocaproic acid or pepstatin A, or sequestering agents such as EDTA, gelling agents such as Examples include white petrolatum, low viscosity paraffin, and / or yellow wax.

  Suitable further additives include, for example, surfactants such as Triton X-100, or sodium deoxycholate, as well as polyols such as polyethylene glycol or glycerol, sugars such as sucrose or glucose, zwitterionic Compounds such as amino acids such as glycine, or in particular taurine or betaine, and / or proteins such as bovine or human serum albumin. Surfactants, polyols, and / or zwitterionic compounds are preferred.

  The physiological buffer solution preferably has a pH of about 6.0 to 8.0, in particular a pH of about 6.8 to 7.8, in particular a pH of about 7.4, and / or an osmolarity of about 200 to 400 milliosmol / liter, preferably about 290 to 310 milliosmol / liter. The pH of the medicament is generally determined using a suitable organic or inorganic buffer, for example, preferably phosphate buffer, Tris buffer (Tris (hydroxymethyl) aminomethane), HEPES buffer ([4 -(2-hydroxyethyl) piperazino] -ethanesulfonic acid) or MOPS buffer (3-morpholino-1-propanesulfonic acid). The choice of each buffer generally depends on the desired buffer molarity. For example, phosphate buffers are suitable for injection solutions and infusion solutions.

  The medicament can be administered in a conventional manner, for example, an injection comprising a medicament according to the invention in an oral dosage form, for example a tablet or capsule, in a processed form implanted subcutaneously via the mucosa, for example via the nose or mouth. Can be administered by infusion or gel. Furthermore, in order to treat the specific joint diseases as described above, it is possible to administer the pharmaceutical locally and locally in the form of a liposome complex as required. Furthermore, treatment can be performed by a transdermal therapeutic system (TTS) that allows for temporary release control of the drug. TTS is known for example from EP0 944 398 (A1), EP0 916 336 (A1), EP0 889 723 (A1) or EP0 852 493 (A1).

Injectable solutions are generally used when relatively small amounts of solutions or suspensions (eg, about 1 to about 20 ml) may be administered to the body. In general, infusion solutions are used when larger amounts of solutions or suspensions (eg, 1 liter or more) are administered. Unlike infusion solutions, only a few milliliters are administered in the case of injection solutions, so slight differences in blood and tissue fluid pH and osmotic pressure in injection solutions are not per se important with respect to pain sensations, or It is only as important as it does not matter. Therefore, it is usually not necessary to dilute the preparation according to the invention before use. However, for relatively large doses, the formulation according to the invention should be diluted easily to such an extent that at least a nearly isotonic solution is obtained prior to administration. An example of an isotonic solution is a 0.9% strength sodium chloride solution. In the case of infusion, dilution can be performed, for example, using sterile water, while administration can be performed, for example, via a so-called bypass.

  In other embodiments, the TAFI-Ile347 or TAFI-Ile347 gene is a particularly diabetic vascular disease (eg, but not limited to atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient It can be used to produce a diagnostic agent for identification of ischemic stroke (TIA), atherosclerotic cerebrovascular disease (CVD), and / or coronary heart disease).

  For example, TAFI-Ile347 can be used to produce a binding protein or binding peptide or aptamer as described above, which can be used as a diagnostic agent tool. The TAFI-Ile347 gene itself can be used in particular in its single-stranded form, or for the production of complementary nucleic acids, preferably complementary single-stranded DNA can be used. This double-stranded DNA can generally be used as a hybridization and / or amplification probe known to those skilled in the art.

Other embodiments of the invention include thrombogenic disorders such as, but not limited to, atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA), atheroma With respect to a method of adapting the dosage of a medicament for treating and / or preventing sclerosing cerebrovascular disease (CVD) and / or coronary heart disease), said method comprises:
(A) Presence of an amino acid substitution from threonine to isoleucine at position 347 of thrombin-activated fibrinolysis inhibitor (TAFI) having the amino acid sequence of SEQ ID NO: 1 (TAFI-Ile347) in a sample derived from diabetes, for example Determining, and
(B) adapting the dosage of the medicament according to the result of step (a),
including.

  As explained above, TAFI-Ile347 exhibits a long half-life and high antifibrinolytic activity in vitro, and preferably high risk for stroke and TIA. Therefore, individuals with the TAFI-Ile347 polymorphism need larger amounts of pharmaceuticals, for example, to treat and / or prevent thrombogenic disorders. In general, the dosage of the pharmaceutical should advantageously be matched to the genetic TAFI profile of the individual or patient, in particular the individual or patient homozygous for isoleucine at position 347. The determination step (a) of the method of the present invention can be performed as described above.

The following examples, tables, and drawings illustrate the invention without limiting the scope of the invention.
FIG. 1 shows the protein sequence of TAFI.
FIG. 2 shows the nucleotide sequence of TAFI.

As a result of polymorphisms at corresponding positions on both alleles of the abbreviation TAFI gene, the TAFI variant with threonine at position 347 of the protein is called TAFI-347-TT.

  As a result of polymorphism at the corresponding position on one of both alleles of the TAFI gene, the TAFI variant with threonine and isoleucine at position 347 of the protein is called TAFI-347-TI.

As a result of polymorphisms at corresponding positions on both alleles of the TAFI gene, the TAFI variant with an isoleucine at position 347 of the protein is called TAFI-347-II.

  Analyzes TAFI polymorphism at position 347 of TAFI protein (NCBI accession number of protein sequence: NP — 001863; FIG. 1) in a group of patients with or without cardiovascular events or endpoints did.

1. SNP (single nucleotide polymorphism) detection by sequence analysis and analysis
1.1 Amplification of genomic DNA region with polymorphism of interest For detection of a nucleotide substitution from cytidine to thymidine at position 1064 of the TAFI sequence of NCBI accession number NM_001872 (Table 2) resulting in a TAFI-Ile347 protein variant The following amplification primers were used:
Forward primer: 5′-CACACCAGCTTTGCTCACC-3 ′,
Reverse primer: 5′-CATTTTCACTGTTTTAGCTCC-3 ′.

For amplification, the following PCR protocol was used and all of the following reagents for amplification were obtained from Applied Biosystems (Foster City, USA): genomic DNA (20 ng); Taq Gold polymerase (1 unit) ); 1 × Taq polymerase buffer; 500 μM dNTP; 2.5 mM MgCl ₂ ; 200 nM each amplification primer pair (for sequence see amplification primer pair 1 above); add H ₂ O to 5 μl To do.

Amplification program for PCR / genotype analysis :
95 ° C. × 10 minutes × 1 cycle 95 ° C. × 30 seconds 70 ° C. × 30 seconds × 2 cycles;
95 ° C x 30 seconds 65 ° C x 30 seconds x 2 cycles;
95 ° C x 30 seconds 60 ° C x 30 seconds x 2 cycles;
95 ° C. × 30 seconds 56 ° C. × 30 seconds 72 ° C. × 30 seconds × 40 cycles;
72 ° C. for 10 minutes 4 ° C. for 30 seconds × 1 cycle.

1.2 Identification of polymorphisms of interest The following protocol was used for minisequence and detection of polymorphisms, while all of the following reagents were obtained from Applied Biosystems (Foster City, USA) . Purified PCR product (2 μl); BigDye terminator kit (1.5 μl); 200 nM one sequence analysis primer (for sequence see forward or reverse amplification primer 1 above); add H ₂ O To 10 μl.

Amplification program for sequence analysis:
96 ° C. × 2 minutes × 1 cycle;
96 ° C for 10 seconds 55 ° C for 10 seconds 65 ° C for 4 minutes x 30 cycles;
72 ° C x 7 minutes 4 ° C x 30 seconds x 1 cycle.

In order to extract raw data, first, the sequence was analyzed using a sequence analysis tool (Applied Biosystems, Foster City, USA), and then Phred (base caller), Phrap (assembler), polyphred (SNP caller) And Consed (result viewer). Phred, Phrap, Polyphred, and Consed are software designed by Phil Green at the University of Washington ( http://www.genome.washington.edu).

1.3 Statistical approach to genotype / phenotype association All SAS statistical packages (version 6.12 or higher, SAS Institute GmbH, Heidelberg / Germany) Analysis was performed. To detect associations between genetic polymorphisms and a number of clinically relevant parameters, descriptive statistics were calculated (median, quartile) and Wilcoxon rank-sum tests were performed. Wilcoxon rank sum test was used to compare two independent samples. Calculation of test statistics is based on rank in the pooled sample.

  To search for associations between SNPs, risk factors, and illnesses, a chi-square test is performed to calculate numbers and percentages and explain the data. Chi-square test is a statistical test to calculate the dependence of two variables. Variable values are contained in two or more classes. A contingency table was used to analyze the relationship between these variables. The table includes a number of rows as the number of recognized first variables and a number of columns as the number of recognized second variables. All cells contain specific patient indicators. To build the study statistics, the difference between the calculated frequency and the observed frequency was calculated.

  After examining the results, relevant variables were selected. Logistic regression was used to confirm the results, taking into account the confounding covariates. Logistic regression was used to analyze the effects of several explanatory variables on specific response variables. P values were obtained by related statistical tests. From this p-value, it is interpreted that there is a significant influence of the relevant explanatory variables.

  Odds ratios were calculated for binary variables. The odds ratio is the ratio of the odds at which an event occurs in one group to the odds at which the event occurs in another group.

2. Results Results were obtained by analyzing a subgroup of patient groups, and the subgroups had the following characteristics: all diabetics, no treatment with the ACE inhibitor ramipril, no alcohol.

  As can be seen from the table, individuals with all TAFI-I347I polymorphisms have a risk for stroke (7.35% vs. 3.35%) compared to individuals with TAFI-I347T and TAFI-T347T polymorphisms, And the risk for TIA (4.41% vs. 0.7%) was great.

The protein sequence of TAFI is shown. The nucleotide sequence of TAFI is shown.

Claims (33)

  Determining the presence of an amino acid substitution from threonine to isoleucine at position 347 of a thrombin activated fibrinolysis inhibitor (TAFI) having the amino acid sequence set forth in SEQ ID NO: 1 (TAFI-Ile347) in the sample, A method for identifying a risk associated with a thrombogenic disorder.   Risks related to thrombogenic disorders are atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA), atherosclerotic cerebrovascular disease (CVD), and / or The method of claim 1, comprising a risk for coronary heart disease.   The method according to claim 1 or 2, wherein the sample is derived from a diabetic patient.   4. A method according to any one of claims 1 to 3, wherein the amino acid substitution is determined in the TAFI protein.   5. A method according to claim 4, wherein the amino acid substitution is determined by amino acid sequence analysis or by a binding protein or peptide or aptamer specifically directed against TAFI-Ile347.   6. The method according to claim 5, wherein the binding protein or binding peptide is an antibody, an antigen binding site of an antibody, and / or a protein scaffold, preferably an anticalin.   4. A method according to any one of claims 1 to 3, wherein the amino acid substitution is determined in the TAFI gene, preferably on both alleles of the TAFI gene.   The determination in the TAFI gene is determined by sequencing the nucleic acid or by an antibody specifically directed against a mutation in the TAFI gene, an antigen binding site of the antibody or a protein scaffold, preferably an anticalin, and / or Alternatively, the method according to claim 7, wherein the method is performed with complementary nucleic acids.   Relating to a thrombogenic disorder comprising determining the presence of an amino acid substitution from threonine to isoleucine at position 347 of a thrombin-activated fibrinolysis inhibitor (TAFI) having the amino acid sequence set forth in SEQ ID NO: 1 in a sample How to select patients at risk.   Risks related to thrombogenic disorders are atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA), atherosclerotic cerebrovascular disease (CVD), and / or The method of claim 9, comprising a risk for coronary heart disease.   The method according to claim 9 or 10, wherein the sample is derived from a diabetic patient.   12. A method according to any one of claims 9-11, wherein the amino acid substitution is determined in the TAFI protein.   13. The method of claim 12, wherein the amino acid substitution is determined by a binding protein or binding peptide or aptamer specifically directed against TAFI-Ile347.   14. The method of claim 13, wherein the binding protein or binding peptide is an antibody, an antigen binding site of an antibody, and / or a protein scaffold, preferably an anticalin.   12. A method according to any one of claims 9 to 11, wherein the amino acid substitution is determined in the TAFI gene, preferably on both alleles of the TAFI gene.   The determination in the TAFI gene is made by an antibody specifically directed against a mutation in the TAFI gene, an antibody antigen binding site or protein scaffold, preferably an anticalin, and / or a complementary nucleic acid. The method of claim 15. (A) providing a TAFI-Ile347 or TAFI-Ile347 gene;
(B) providing a test compound; and
(C) measuring or detecting the effect of the test compound on TAFI-Ile347 or TAFI-Ile347 gene;
A method of identifying a TAFI-Ile347 agent, preferably an inhibitor, for the treatment and / or prevention of a thrombogenic disorder.   The method of claim 17, wherein the test compound is provided in the form of a chemical compound library.   The method according to claim 17 or 18, wherein the effect of the test compound on TAFI-Ile347 or TAFI-Ile347 gene is measured or detected by a heterogeneous assay or a homogeneous assay.   20. The method of claim 19, wherein the heterogeneous assay is ELISA (enzyme-linked immunosorbent assay), DELFIA (dissociation-promoting lanthanide fluorescence immunoassay), or SPA (scintillation proximity analysis).   The homogeneous assay is TR-FRET (time-resolved fluorescence resonance energy transfer) analysis, FP (fluorescence polarization) analysis, ALPHA (amplified luminescence proximity homogeneous assay), EFC (enzyme fragment complementation) analysis, or genetic analysis The method of claim 19.   The method according to any one of claims 17 to 21, wherein the method is performed on an array.   The method according to any one of claims 17 to 22, wherein the method is performed in a robotic system.   24. The method according to any one of claims 17 to 23, wherein the method is performed using microfluidics.   The method according to any one of claims 17 to 24, wherein the method is a high-throughput screening method. (A) identifying an agent for the treatment and / or prevention of a thrombogenic disorder according to the method of any one of claims 17 to 25;
(B) providing a sufficient amount of the agent identified according to step (a); and
(C) formulating the drug with one or more pharmaceutically acceptable carriers or adjuvants;
A method for producing a medicament for the prophylactic and / or therapeutic treatment of a thrombogenic disorder. The thrombogenic disorder comprises atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA), and / or atherosclerotic cerebrovascular disease (CVD) Item 27. The method according to Item 26.   Use of the TAFI-Ile347 or TAFI-Ile347 gene for the manufacture of a diagnostic agent for identifying thrombogenic diseases.   Thrombogenic disorders include atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA), atherosclerotic cerebrovascular disease (CVD), and / or coronary heart 29. Use according to claim 28, comprising a risk for the disease.   30. Use according to claim 28 or 29 for identifying a thrombogenic disease in a diabetic patient. (A) determining the presence of an amino acid substitution from threonine to isoleucine at position 347 of thrombin-activated fibrinolysis inhibitor (TAFI) having the amino acid sequence described in SEQ ID NO: 1 (TAFI-Ile347) in the sample ,and,
(B) adapting the pharmaceutical dosage according to the result of step (a),
A method of adapting the dosage of a medicament for the prophylactic and / or therapeutic treatment of thrombogenic disorders.   Thrombogenic disorders may include atrial fibrillation, stroke, long-term intermittent neuropathy (PRIND), transient ischemic attack (TIA), atherosclerotic cerebrovascular disease (CVD), and / or coronary heart disease. 32. The method of claim 31 comprising:   33. The method of claim 31 or 32, wherein the sample is from a diabetic patient.

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