EP1294926A2 - Systeme de test pour determiner l'activite de proteines kinases dependantes des cyclonucleotides et de vasp-phosphatases - Google Patents

Systeme de test pour determiner l'activite de proteines kinases dependantes des cyclonucleotides et de vasp-phosphatases

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Publication number
EP1294926A2
EP1294926A2 EP01938257A EP01938257A EP1294926A2 EP 1294926 A2 EP1294926 A2 EP 1294926A2 EP 01938257 A EP01938257 A EP 01938257A EP 01938257 A EP01938257 A EP 01938257A EP 1294926 A2 EP1294926 A2 EP 1294926A2
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EP
European Patent Office
Prior art keywords
vasp
cnpk
test system
substrate
phosphatase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01938257A
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German (de)
English (en)
Inventor
Peter DRÜCKES
Thomas Jarchau
Ulrich Walter
Benjamin Bader
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Verinos Operations GmbH
Original Assignee
Vasopharm Biotech GmbH
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Publication date
Application filed by Vasopharm Biotech GmbH filed Critical Vasopharm Biotech GmbH
Publication of EP1294926A2 publication Critical patent/EP1294926A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)

Definitions

  • Test system for determining the activity of cyclo-nucleotide-dependent protein kinases and VASP phosphatases
  • the present invention relates to a test system for the detection of the activity of cyclo-nucleotide-dependent protein kinases or of phosphatases which dephosphorylate the vasodilator-stimulated phosphoprotein (“VASP”), process for its production and its use in (high throughput) screening processes ,
  • VASP vasodilator-stimulated phosphoprotein
  • Cyclo-nucleotide-dependent protein kinases (“cNPK”), their substrates (“cNPKS”), and the corresponding protein phosphatases are part of important physiologically, pathophysiologically and pharmacologically relevant cellular signaling pathways (see citations 1, 2 and 3).
  • the cNPK include cAMP-dependent protein kinases (“cAK”) and cGMP-dependent protein kinases (“cGK”).
  • cGK cGMP-dependent protein kinases
  • cGK cGMP-dependent protein kinases
  • cGK In addition to other cGMP effector systems, e.g. cGMP-regulated phosphodiesterases and ion channels, cGK in particular is an important mediator of cGMP-mediated signal transmission.
  • GC-A, GC-B, GC-C membrane-bound guanylyl cyclases
  • GC-A membrane-bound guanylyl cyclases
  • GC-S membrane-bound guanylyl cyclases
  • One of the most important agonists of soluble guanylyl cyclase is the nitrogen monoxide (NO) formed by the NO synthases (NOS III-III).
  • NOS III-III nitrogen monoxide
  • the established effects caused by an increase in cGMP include relaxation of smooth muscle cells (SMCs) and inhibition of platelet activation.
  • cGMP effects are mediated by the cGK (type I) (see citations 1-3). This could be confirmed in corresponding mouse models with cGK-1 deficiency (see citations 4 and 5).
  • cGK are known as homodimers, from which a soluble (monomer 76 kDa; cGK I) and a membrane-based form (monomer 86 kDa; cGK II) can be distinguished.
  • the membrane anchorage of the cGK II is mediated via the N-terminal myristoylation (cf. citations 1-3). There are two isoforms of cGK I and cGK Iß of cGK I.
  • cGK I which is generally referred to as soluble form, is predominantly present in platelets and partially in smooth muscle cells.
  • a particularly high expression of cGK I can be found in human blood platelets and in smooth muscle cells. Activation of cGK-1 in these cells leads to the effects already described above, such as lowering the intracellular Ca (2+) level, inhibiting blood platelets and the contraction of smooth muscle cells.
  • the known substrates of cGK include, among others, e.g. the inositol 1, 4,5-triphosphate receptor (IP3R) and the VASP, as well as p25, cystic fibrosis transmembrane regulator (CFTR) and 6-pyruvoyl tetrahydro-pterine synthase (PTPS).
  • IP3R inositol 1, 4,5-triphosphate receptor
  • VASP p25
  • CFTR cystic fibrosis transmembrane regulator
  • PTPS 6-pyruvoyl tetrahydro-pterine synthase
  • 8-Br-cGMP, 8-pCPT-cGMP have been described as activators of cGK, while certain cGMP analogs (Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS) and isoquinolinesulfonamide derivatives (KT 5823, H8 and H89) which inhibit cGK (cf. quote 12).
  • cNPK assays are usually carried out by incorporating radioactive phosphorus isotopes ( 32 P or 33 P) in peptide substrates and then separating these reaction products from the starting materials, for example by immobilizing them on a carrier substance (citation 16) or by separating them Reaction products using gel electrophoresis and radio- or immunochemical detection after protein transfer to membranes in the presence of product-specific antibodies (such as in a Western blot, WO 99 / 24,473).
  • a carrier substance citation 16
  • product-specific antibodies such as in a Western blot, WO 99 / 24,473
  • test system described (synonym: assay or diagnostic) is suitable for the effect on a large number of (test) compounds, preferably from chemical or natural substance libraries, in a simple manner to be able to investigate the activity of a cNPK [so-called. High Throughput Screening (short: HTS)].
  • test system can be used to test a large number of (test) compounds from chemical or natural substance libraries in a simple and effective manner for their effect on the activity of a VASP phosphatase [ so-called. High Throughput Screening (short: HTS)].
  • the present invention therefore relates to an HTS-suitable test system for detecting the activity of a cNPK containing a) at least one test compound b) at least one suitable cNPK substrate (cNPKS) c) at least one composition to be incubated containing cNPK and ATP, and d) a suitable detection system for the quantitative determination of the amount of the phosphorylated cNPK substrate.
  • the present invention therefore furthermore relates to an HTS-suitable test system for detecting the activity of the VASP phosphatase containing e) at least one test compound, f) at least one suitable VASP phosphatase substrate, g) at least one composition to be incubated containing VASP phosphatase , and h) a suitable detection system for the quantitative determination of the amount of dephosphorylated VASP phosphatase substrate.
  • test system both objects mentioned are referred to below as the test system according to the invention.
  • test system As a gel-free test system, the test system according to the invention enables a large number of quantitative individual determinations and, by dispensing with gel electrophoretic separation steps, fulfills special requirements with regard to speed, sample size and material consumption. Reproducibility, robustness, solvent tolerance and automation are also guaranteed.
  • test system allows the search for one or more, the same or different test compounds.
  • the cNPK used is preferably a cGK or cAK or a functional variant with the activity of a cNPK.
  • Such protein kinases can be used in purified form or as a crude extract, in particular as a component in protein mixtures from homogenates of biological origin, preferably of human origin.
  • VASP phosphatase used can also be a functional variant with the activity of a VASP phosphatase.
  • VASP phosphatases can be used in purified form or as a crude extract, in particular as a component in protein mixtures from homogenates of biological origin, preferably of human origin.
  • VASP is very particularly preferred as a suitable cNPK substrate according to feature (b) - according to SEQ ID No. 1 (described in C. Haffner et al. In EMBO J., 14 (1), 19-27 (1995)) —containing preferably usable phosphorylation sites, namely: serine-157, serine-239 and threonine-278.
  • cNPK can phosphorylate all three residues, but the cAMP-dependent protein kinase serine 157 is preferred as the main phosphorylation site, while the cGMP-dependent protein kinase Serine-239 preferred.
  • Threonine-278 is phosphorylated by both kinases with a comparable specificity.
  • VASP phosphatase substrate is phosphorylated VASP, corresponding to SEQ ID No. 1, which is phosphorylated at the preferred operable phosphorylation sites, namely: Serin-157, Serin-239 and Threonin-278.
  • such peptide or polypeptide variants or peptoids can also be used as functional variants of a cNPK substrate or a VASP phosphatase substrate, which preferably contain amino acid sequences from VASP, which are present in the vicinity of the phosphorylation or dephosphorylation sites mentioned and although not finally particularly preferably the pentamers with the amino acids 155-159 and / or 237-241 and / or 276-280 according to SEQ ID No. 1 or preferably the decamers with the amino acids 152-161 and / or 234-243 and / or 273-282 from SEQ ID No. 1 or other suitable partial sequences (e.g. see in Fig. 1).
  • Such peptides phosphorylated according to the invention can be detected particularly advantageously with suitable antibodies in terms of features (d) or (h), preferably monoclonal antibodies.
  • the monoclonal antibody 16C2 which is selected and obtainable from the hybridoma cell line DSM ACC2330, is very particularly preferably used (WO 99/24473).
  • This antibody or a functional variant thereof specifically detects a phosphorylation event, very particularly preferably it recognizes an epitope which contains the phosphorylated serine 239 of the VASP sequence.
  • a functional variant relates in particular to those antibodies with an agreement of preferably 90-99% or 70-90% in the amino acid sequence of 16C2 with the homologous function of quantitatively determining a reaction product in the sense of feature (d) or (h).
  • the test system according to the invention can be run both heterogeneously (for example after immobilization of the reaction products) but preferably homogeneously.
  • the immunological detection in the sense of features (d) or (h) can be carried out according to the methods known to the person skilled in the art, such as, for example, coupled enzyme reaction (alkaline phosphatase or luciferase) or general sandwich technologies (for example ELISA), and fluorimetric methods (fluorescence , time-resolved fluorescence, fluorescence resonance energy transfer (FRET)).
  • coupled enzyme reaction alkaline phosphatase or luciferase
  • sandwich technologies for example ELISA
  • fluorimetric methods fluorescence , time-resolved fluorescence, fluorescence resonance energy transfer (FRET)
  • test system according to the invention can preferably be automated as a homogeneous HTS and is therefore suitable for 96, 384, 1536-well plates and more.
  • the present invention also relates to a method for producing a test system, in which at least one compound to be examined and at least one composition containing cNPK, ATP and cNPK substrate, optionally a phosphorylation reaction stopper, at least one suitable detection system, such as an antibody, including an immunological detection agent and if necessary, other aids are put together
  • the present invention also relates to a method for producing a test system, in which at least one compound to be examined, at least a composition containing VASP phosphatase and at least one suitable detection system for quantifying the dephosphorylation of a VASP phosphatase substrate are put together.
  • the present invention further provides an HTS-suitable method for finding one or more active compounds which modulates the activity of the cNPK, comprising the steps: i) bringing the compound to be investigated or a multiplicity of compounds to be investigated into contact with cNPK, ATP and a suitable cNPK substrate, and j) quantification of the phosphorylated cNPK substrate with a suitable detection system, if necessary after termination of the phosphorylation reaction with a suitable phosphorylation reaction stopper.
  • the present invention furthermore relates to an HTS-suitable method for finding one or more active compounds which modulate the activity of VASP phosphatase, comprising the steps: k) bringing the compound to be investigated or a multiplicity of compounds to be investigated into contact VASP phosphatase and a suitable VASP phosphatase substrate, and I) quantification of the dephosphorylated VASP phosphatase substrate with a suitable detection system, if necessary after termination of the dephosphorylation reaction with a suitable dephosphorylation reaction stopper
  • the method for finding a chemical compound as described above takes place in a microtiter plate.
  • the microtiter plate can contain different numbers of vessels. For example, it may contain 96, 384, 768, 1536, 3072 or more vessels. Preferred individual components of the method according to the invention have already been described in more detail above.
  • the active (test) compound can be a pharmaceutically active compound in the function of a modulator - such as (hyper) activator / or (total) inhibitor - for the activity of a cNPK or a natural product in the broadest sense, in particular a raw extract, or a component contained therein.
  • the substance to be examined is generally a naturally occurring, a naturally occurring and chemically modified and / or a synthetic substance. In particular, so-called combinatorial substance libraries can be searched particularly easily and quickly with the methods according to the invention.
  • connection is very particularly preferably suitable which, bypassing the NO / cGMP signal path, directly modulates - activates or inhibits a cNPK; optionally modulated selectively - activated or inhibited.
  • the invention therefore also relates to a method in which the pharmaceutically active compound modulates a cNPK signaling pathway.
  • cNPK cyclo-nucleotide-dependent protein kinases
  • Another object of the present invention is therefore a method for diagnosis by the direct and gel electrophoresis-independent determination of the cNPK activity from samples, comprising the steps: m) incubating a sample in the presence of at least one composition containing suitable cNPKS and possibly ATP, n ) Adding at least one suitable detection system to the composition to quantify the phosphorylation of the cNPKS; and o) determining the activity of the cNPK, in particular in blood / cell or tissue extracts or in the form of permeabilized and / or intact cells.
  • Another object of the invention is a method for diagnosis by the direct and gel electrophoresis-independent determination of the VASP phosphatase activity from samples, comprising the steps: p) incubating a sample in the presence of at least one composition containing a suitable VASP phosphatase substrate q) adding at least one suitable detection system to the composition to quantify the dephosphorylation of the VASP phosphatase substrate; and r) determination of the activity of VASP phosphatase, in particular in blood / cell or tissue extracts or in the form of permeabilized and / or intact cells.
  • the diseases to be diagnosed are preferably cardiovascular diseases and diseases with associated vascular damage, e.g. Hypertension, thrombosis and endothelial dysfunction syndrome, e.g. in arteriosclerosis, diabetes, vasculitis and diseases of hematopoietic cells such as acute leukaemias, myeloproliferative diseases, myelodysplasias (see quote 17).
  • cardiovascular diseases and diseases with associated vascular damage e.g. Hypertension, thrombosis and endothelial dysfunction syndrome, e.g. in arteriosclerosis, diabetes, vasculitis and diseases of hematopoietic cells such as acute leukaemias, myeloproliferative diseases, myelodysplasias (see quote 17).
  • FIG. 1 shows a preferred embodiment of the test system according to the invention.
  • the cGK activity assay is based on the observation of the in vitro phosphorylation of a biotinylated substrate peptide by activated cGK (cf. FIG. 1A).
  • the VASP substrate peptide consists of a partial sequence of the amino acid sequence of human VASP (vasodilator stimulated phosphoprotein), a natural cGK substrate protein.
  • the phosphorylated VASP substrate peptide was surprisingly detectable with the aid of a detection system based on time-resolved fluorescence resonance energy transfer (FRET) (compare FIG. 1B).
  • FRET fluorescence resonance energy transfer
  • the anti-pSer239-VASP (mAb 16C2) monoclonal antibody which surprisingly specifically recognized the phosphorylated VASP substrate peptide (and left non-phosphorylated VASP substrate peptide undetected).
  • mAb 16C2 anti-pSer239-VASP
  • SA-APC Fluorescence-labeled streptavidin
  • FIG. 2 shows the kinetics of the phosphorylation of the biotinylated substrate peptide by the cGK.
  • the ordinate of FIG. 2 shows the measured FRET signal in cps.
  • the VASP substrate peptide is only phosphorylated in the presence of the cGK and its activator cGMP (tube A). No signal change was observed without cGMP and with cGK (tube B) and without cGK and with cGMP (tube C).
  • FIG. 3 shows the activation of the cGK with the natural activator cGMP and a cGMP-analogous molecule (8-pCPT-cGMP), measured with the test system described.
  • the enzyme activity (in cps / min) is plotted against the activator concentration (in mol / l).
  • FIG. 4 shows the surprising finding that with the test system according to the invention, the enzyme activity of the cGK contained in platelet extracts in this complex biological protein mixture can be observed and quantitatively measured.
  • FIG. 4A shows no difference between the phosphorylation kinetics without (D) and with the specific cAK inhibitor PKI (x).
  • FIG. 4B shows the influence of 100 nmol / l PKI on the phosphorylation of the VASP substrate peptide by purified cGK (0.3 nmol / l) without (D) and with PKI (x) and the influence of PKI on purified cAK (catalytic subunit, 3 nmol / l) without (O) and with PKI (•).
  • Example 1 FRET detection system and titration of the measuring range with phosphopeptide
  • a synthetic phosphopeptide (phosphorylated version of the VASP substrate peptide) was used as a "reaction product" instead of a kinase reaction and quantified with a detection mix.
  • reaction product phosphorylated version of the VASP substrate peptide
  • different mixtures of non-phosphorylated peptide and phosphopeptide were produced.
  • the total concentration of peptide was kept constant at 500 nmol / l. 50 ⁇ l of these mixtures were placed in the wells of a “96 well” microtest plate and 200 ⁇ l each of a detection mix containing the following reagents, diluted in PBS:
  • anti-pSer239-VASP mAb 16C2
  • anti-mouse-Eu W1024, PE Wallac
  • Bovine serum albumin 0J g / l
  • the signal-to-background ratio defined by the quotient of the maximum signal and the signal without phospho-peptide, was about 5.
  • the linearity of the measuring range in titration with the help of synthetically produced phosphopeptide found in Example 1, Table 1 enables the standardization and quantification of the enzymatic kinase reactions based on the test system according to the invention.
  • Vessel A also contained 5 ⁇ mol / l cGMP and 25 ng / ml purified cGK.
  • Vessel B also contained 25 ng / ml purified cGK.
  • Vessel C also contained 5 ⁇ mol / l cGMP.
  • the kinase reaction was started with 50 // mol / l ATP with further incubation at 30 ° C. All concentrations given above are to be understood as final concentrations in a volume of 500 ⁇ ⁇ . At the times indicated, 50 ⁇ ⁇ were removed from the vessels at the end of the reaction and mixed with 50 ⁇ ⁇ of a 30 mmol / l EDTA solution. The phosphorylated substrate peptide was detected as described in Example 1 in the wells of a "384 well" microtest plate.
  • VASP substrate peptide is phosphorylated only in the presence of the cGK and its activator cGMP (vessel A). Without cGMP and with cGK (tube B), without cGK and with cGMP (tube C), no signal change was observed.
  • test system is suitable in the form shown for high-throughput screening.
  • pipetting robots e.g. the Biomek systems from Beckman or the Genesis Workstation from Tecan
  • microtest plates in 96, 384 and even 1536 well format can be processed.
  • the cGK assay could be implemented for such a robot system in 384 well format as follows for screening for activators of the cGK. The following are pipetted sequentially:
  • the dose-dependent activation of the cGK by the natural activator cGMP and a cGMP-analogous molecule (8-pCPT-cGMP) can be analyzed with the test system according to the invention.
  • 8 kinase reactions (as described in Example 3, total volume 500 ⁇ ) were carried out, which contained the following components:
  • biotinylated VASP substrate peptide biotin-VASP 231 -245
  • test system is suitable for specifically measuring the activity of the cGK in tissue extracts or platelet lysates.
  • Platelets were prepared using a known method (cf. quote 15). After producing a soluble platelet extract, this was tested without activator and with 8-pCPT-cGMP (0.5 ⁇ mol / l) according to Example 3. In addition to 8-pCPT-cGMP, a third reaction vessel also contained 100 nmol / l PKI. This inhibitor specifically inactivates the cAMP-dependent protein kinase (cAK), which is also present in platelets.
  • FIG. 4A shows no difference between the phosphorylation kinetics without and with PKI.
  • FIG. 4A shows no difference between the phosphorylation kinetics without and with PKI.
  • 4B shows the influence of 100 nmol / l PKI on the phosphorylation of the VASP substrate peptide by purified cGK (0.3 nmol / l) and purified cAK (catalytic subunit, 3 nmol / I).
  • concentration of PKI used is sufficient to completely inhibit cAK.
  • the activity of the cGK is not affected.
  • ATP adenosine 5'-triphosphate
  • BSA bovine serum albumin
  • cAK cAMP-dependent protein kinase
  • cGK cGMP-dependent protein kinases
  • cGMP cyclo-guanosine-3 ', 5'-monophosphate
  • cNPKS substrate for cyclo-nuceotide-dependent protein kinases
  • cNPK cyclo-nucleotide-dependent protein kinases
  • HTS high-throughput screening
  • PKI protein kinase A specific inhibitor
  • TR-FRET Time-Resolved-Fluorescence-Resonance-Energy-Transfer
  • VASP Vasodilator-stimulated phosphoprotein
  • Vasodilator dysfunction in aged spontanously hypertensive rat changes in NO synthases III and soluble guanylyl cyclase expression, and in Superoxide anion production. Cardiovascular Res 37, 772-779

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Abstract

L'invention concerne un système de test, approprié pour un criblage à haut débit (HTS), servant à détecter l'activité de protéines kinases dépendantes des cyclonucléotides (cNPK). Ce système comprend a) au moins un composé de test, b) au moins un substrat cNPK approprié, c) au moins une composition à incuber contenant des cNPK et ATP, éventuellement un moyen pour arrêter la réaction de phosphorylation, d) un système de détection approprié pour quantifier la phosphorylation du substrat cNPK. L'invention concerne en outre un système de test approprié HTS, servant à détecter l'activité de VASP-phosphatases. Ce système comprend e) au moins un composé de test, f) au moins un substrat VASP-phosphatase approprié, g) au moins une composition à incuber contenant des VASP-phosphatases et h) un système de détection approprié pour quantifier la déphosphorylation du substrat VASP-phosphatase. Ces systèmes de test s'utilisent pour détecter, dans une bibliothèque de substances chimiques ou naturelles, des composés qui modulent l'activité d'une cNPK ou d'une VASP-phosphatase.
EP01938257A 2000-06-14 2001-06-12 Systeme de test pour determiner l'activite de proteines kinases dependantes des cyclonucleotides et de vasp-phosphatases Withdrawn EP1294926A2 (fr)

Applications Claiming Priority (3)

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DE10029210 2000-06-14
DE10029210A DE10029210A1 (de) 2000-06-14 2000-06-14 Testsystem sowie dessen Verwendung
PCT/EP2001/006621 WO2001096594A2 (fr) 2000-06-14 2001-06-12 Systeme de test pour determiner l'activite de proteines kinases dependantes des cyclonucleotides et de vasp-phosphatases

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EP1294926A2 true EP1294926A2 (fr) 2003-03-26

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EP (1) EP1294926A2 (fr)
AU (1) AU2001263956A1 (fr)
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WO (1) WO2001096594A2 (fr)

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DE10058596A1 (de) * 2000-11-25 2002-06-06 Aventis Pharma Gmbh Verfahren zum Screening von chemischen Verbindungen zur Modulierung der Wechselwirkung einer EVH1-Domäne oder eines Proteins mit einer EVH1-Domäne mit einer EVH1-Bindedomäne oder einem Protein mit einer EVH1-Bindedomäne sowie ein Verfahren zum Nachweis besagter Wechselwirkung
US7727752B2 (en) 2003-07-29 2010-06-01 Life Technologies Corporation Kinase and phosphatase assays
CA2445420A1 (fr) * 2003-07-29 2005-01-29 Invitrogen Corporation Tests de kinase et de phosphatase
WO2007051207A2 (fr) 2005-10-28 2007-05-03 Invitrogen Corporation Dosages de l'activite kinase et d'ubiquitination
EP3091359A1 (fr) 2015-05-08 2016-11-09 Leibniz - Institut für Analytische Wissenschaften - ISAS - E.V. Procédé d'identification de protéines marqueurs destinées au diagnostic et stratification de risques des troubles de la coagulation sanguine

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US6346406B1 (en) * 1997-08-20 2002-02-12 University Of Medicine & Dentistry Of New Jersey Elongation factor-2 kinase (EF-2 kinase), and methods of use therefor
RU2218178C2 (ru) * 1997-11-07 2003-12-10 Вазофарм Биотех Гмбх Антитела против фосфорилированного vasp (стимулированного вазодилататором фосфопротеина), гибридомные клетки для их получения и их применение

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DE10029210A1 (de) 2002-01-31
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US20030166005A1 (en) 2003-09-04
WO2001096594A3 (fr) 2002-05-16

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