Antibodies against phosphorylated vasp (vasodilator-stimulated phosphoprotein), hybridoma cells for their preparation, and their use

Abstract

The invention relates to antibodies against VASP (vasodilator-stimulated phosphoprotein) which bind VASP only when it is phosphorylated at amino acid serine 157, i.e. VASP containing phosphoserine 157. Furthermore, the invention relates to hybridoma cells for producing antibodies against VASP containing phosphoserine 157, diagnostic reagent kits containing the antibodies or antibody fragments, and their use for diagnostic and / or therapeutic purposes. The solution according to the invention is suitable for examining the function of the endothelium, ex vivo from biological material (e.g. cell or blood sample), using standard laboratory procedures, whereby damage caused by defective endothelium function can be reversed. à

Description

The subject of the invention are antibodies against VASP (vasodilator-stimulated phosphoprotein), which bind VASP only if it is phosphorylated on serine amino acid 157, i.e. phosphoserine 157 containing VASP. Furthermore, the subject of the invention are hybridoma cells for the production of antibodies against VASP containing phosphoserine 157, diagnostic reagent sets containing antibodies or antibody fragments, and their use for diagnostic and / or therapeutic purposes. The solution according to the invention enables a quick and simple examination of the function of the endothelium, from an ex vivo biological sample (for example, a cell or blood sample), using standard laboratory procedures. Changes in the vascular system are responsible for the development of many chronic and life-threatening diseases, such as myocardial infarction, stroke, arteriosclerosis and countless forms of kidney disease. Endothelial cells - which, among other things, regulate the blood coagulation system, the functioning state of platelets, the migration of inflamed and cancerous cells into the vessel wall, the contraction state and growth of smooth muscle cells, and accordingly blood pressure and the structure of the vessel wall, as well as the formation of blood vessels - play a particularly important role in regulating the functioning of the vascular system. During serious vascular diseases, these functions of the vessel wall are damaged to such an extent that it can eventually lead to a heart attack, a heart attack ("grave stroke"), or the development of one of the countless kidney diseases. The endothelium produces prostacyclin (PG12), which plays a very important role, and nitric oxide (NO), the latter of which is classified as endothelium-derived relaxation factors (EDRF). PGL2 and NO inhibit both platelets and vascular wall muscles. The functioning of the endothelium is regulated by the mentioned factors of endothelial origin - such as prostacyclin (PG12) or EDRF, for example NO. Thus, in order to treat diseases related to endothelium malfunction in a specific way, it is necessary to determine biological characteristics that allow the diagnosis of endothelium malfunction and the necessary correction. It is desirable to recognize the malfunctioning of the endothelium as early as possible, i.e. at the stage when the irreversible damage caused by the malfunctioning of the endothelium - such as atherosclerosis - has not yet developed. Early detection of endothelium malfunction allows the development of new medical procedures that can reverse the damage caused by endothelium malfunction. Known methods for determining the function of the endothelium can be invasive methods, such as quantitative angiography, or non-invasive methods, such as image-providing methods. The disadvantages of these procedures are that the tests are performed directly on the person to be tested, and their quantitative determination is difficult and very expensive. Accordingly, it is necessary to develop biochemical / immunological procedures that enable a quick and simple examination of the functioning of the endothelium, from an ex vivo biological sample (for example, a cell or blood sample), using standard laboratory procedures. The functioning of the endothelium consists of processes that can be regulated by endothelium-derived factors such as prostacyclin (PGI2) or EDRF, such as NO. The purpose of the invention is to provide reagents for the evaluation and regulation of endothelium function. According to this and in accordance with the other objectives of the invention, the subject of the invention is antibodies against VASP ("vasodilator-stimulated phosphoprotein"), which bind VASP only if it is present in phosphorylated form. According to one aspect of the solution according to the invention, an antibody is disclosed that binds VASP only if VASP is phosphorylated on serine amino acid 157 (VASP containing phosphoserine 157, abbreviated: "phosphoserine 157 VASP). Various embodiments of the invention include polyclonal antibodies, monoclonal antibodies, and antibody fragments. Other embodiments include a monoclonal antibody produced by the hybridoma cell line 16C2, and in particular Mab 16C2. A further aim of the invention is the production of hybridoma cells suitable for the production of the antibody according to the invention. In accordance with this aim, a further object of the invention is a hybridoma cell line which produces the antibody which binds VASP only in the event that VASP is phosphorylated. According to one embodiment of the invention, the subject of the invention is the 16C2 hybridoma cell line (ACC2330). During the development of the solution according to the invention, our further goal was to develop a procedure suitable for evaluating the functioning of the endothelium. Accordingly, the invention provides methods for determining the phosphorylation status of VASP. According to one aspect of the solution according to the invention, a biological substance is brought into contact with an antibody against VASP (vasodilator-stimulated phosphoprotein), which binds VASP only if VASP is phosphorylated. According to another aspect of the solution according to the invention, the subject of the invention is a quantitative method, which also includes the quantitative determination of the VASP antibody bound to the biological material. A special embodiment of the invention is, for example, a Westem-blot method, which consists of the electrophoretic separation of VASP and the reaction of VASP with an anti-VASP antibody, which antibody binds VASP only if VASP is phosphorylated. . Another embodiment of the invention is a flow cytometry method in which the sample is treated with an antibody against VASP HU 222 411 BI contact, which binds VASP only if VASP is phosphorylated. According to another aspect of the solution according to the invention, the subject of the invention is a diagnostic method. In a typical procedure, the sample is contacted with an anti-VASP antibody that binds VASP only when VASP is phosphorylated, and the phosphorylation status of VASP is evaluated. According to one embodiment of the invention, this method includes determining the amount of phosphorylation of VASP in the sample. Another embodiment of the invention provides methods in which the antibody binds to phosphoserine 157 VASP. According to another embodiment of the invention, the sample is a human platelet or a human whole blood sample. Another goal of the solution according to the invention is to provide methods for identifying markers that characterize the functioning of the endothelium. According to one aspect, the subject of the invention is a method for identifying substances that affect cGMP and / or cAMP levels. According to the procedure, samples from patients treated with substances affecting cGMP and / or cAMP levels are used. The samples are contacted with an anti-VASP antibody that binds VASP only when VASP is phosphorylated, and the VASP phosphorylation status is evaluated in comparison to a control sample. We also describe in detail a procedure for detecting endothelium malfunction, during which a sample from a patient is brought into contact with an antibody against VASP, which binds VASP only if VASP is phosphorylated, and then the healthy Compared with a (normal) sample, the phosphorylation state of VASP is evaluated. Another goal of the solution according to the invention is to produce a set of reagents suitable for implementing the diagnostic procedure described here. Furthermore, the invention provides a reagent kit comprising an antibody that binds VASP only when VASP is phosphorylated. Another goal of the solution according to the invention is to create procedures for the treatment of patients suffering from malfunctioning of the endothelium. Accordingly, and in accordance with other purposes of the present invention, we provide a method of treatment in which a patient is treated with a therapeutically effective amount of an antibody that binds VASP only when VASP is phosphorylated. Human VASP ("vasodilator-stimulated phosphoprotein"), which is phosphorylated in platelets and vessel wall cells under the influence of hormones and active substances, was recently identified, isolated and characterized from a molecular genetic point of view [Haffner et al., EMBO J. 14, 19-27 (1995)] . VASP is an important component of the ANF / NO / cGMP / cGMP protein kinase signaling pathway, which plays an extremely important role in the physiological, pathophysiological and medical aspects, as well as in the cAMP / cAMP protein kinase signaling pathway [U. Walter, Blick 1 / 97 Würzburg University, 79-81 (1997)]. VASP is expressed by almost all human and animal cells, but is found in particularly high concentrations in platelets, vascular smooth muscle and fibroblasts. In cell cultures, VASP is associated with surface junctions (cell / matrix interfaces), cell / cell contacts, microfilaments, and dynamic membrane regions (e.g. leading edges) [Walter et al., Agents and Actions 45S, 255-268 (1995 )]. In the vasculature, VASP phosphorylation is associated with inhibition of platelet adhesion / aggregation, inhibition of smooth muscle contraction / migration, and inhibition of paracellular endothelium permeability. VASP is phosphorylated and dephosphorylated at three different sites [serine 157, serine 239 and threonine 278, [cf. Horstrup et al., Eur. J. Biochem. 225, 21-27 (1994)]}. Serine 239 is sometimes referred to as 238-like, especially when the first methionine is not taken into account when numbering the amino acid sequence of VASP. According to him, VASP 239 is phosphorylated in the cells of the healthy human circulatory system (platelets, endothelial cells and smooth muscle cells) under the influence of physiological and medicinal NO donors, platelet inhibitors and vasodilators. VASP serine 239 is phosphorylated by cGMP-dependent protein kinases. The cGMP-dependent protein kinases are activated - via cGMP-η - by important hormones, such as natriuretic peptides that stimulate the excretion of sodium in the urine, or substances and active substances that release NO. In addition, VASP serine 239 is also phosphorylated by cAMP-dependent protein kinases activated by hormones and active substances that increase cAMP levels. Although cAMP-dependent protein kinases mainly phosphorylate serine at position 157, they are also phosphorylated at position 239 of the VASP amino acid sequence. According to some observations, the phosphorylation of VASP 157-like in human platelets is closely related to the inhibition of the connection between fibrinogen and Ilb-IIIa glycoprotein [Horstrup et al., Eur. J. Biochem. 225, 21-27 (1994)]. According to him, the determination of the degree of phosphorylation of VASP, especially VASP 239 and / or 157, from biological samples, such as extracts of tissues and cells, can be a very significant biochemical parameter. This biochemical parameter enables the development of a diagnostic system that can be used to determine those hormones or active substances that increase cGMP and / or cAMP levels [for example, atrial sodium-releasing factor (ANF), guanylin, NO- ot-releasing substances and active substances], and with the help of which we can draw conclusions about the functions of the endothelium in vivo. Antibodies that reversibly bind phosphorylated proteins have been previously described in US Patent No. 5,599,681, WO 93 / 21230 HU 222 411 Β1 in international publication document and described by U. Walter [Blick 1 / 97 Würzburg University, 79-81 (1997)]. During the development of the solution according to the invention, our goal was to develop a biochemical / immunological procedure that enables a quick and simple qualitative and / or quantitative determination of the phosphorylation state of VASP in a biological sample. This goal was achieved by producing an antibody that binds VASP only when VASP is phosphorylated. Thus, the subject of the invention - in a completely generally accepted way - is an antibody, which antibody binds VASP only if VASP is phosphorylated. By the antibody according to the invention, we mean polyclonal, monoclonal antibodies (Mabs) and their fragments, as well as SCFV fragments or other synthetic or recombinant protein domains, which specifically recognize the phosphorylated sections of VASP. Antibody fragments include any part of an antibody capable of binding the target antigen - in this case VASP or a specific part thereof. Antibody fragments are in particular F(ab')2, Fab, Fab' and Fv fragments. These fragments can be produced from any antibody class, but are typically made from IgG or IgM. These fragments can be produced using conventional recombinant DNA techniques or by proteolytic digestion with papain or pepsin in the classical way. [See "Current protocols in immunology", II. chapter, ed.: Coligan et al., John Wiley and Sons (1991-1992)]. F(ab')2 fragments are typically around 110 kDa (IgG) and 150 kDa (IgM) in size and contain two antigen-binding parts. The antigen-binding regions are connected to each other by disulfide bridge(s). These fragments lack virtually all, if not all, Fc. Fab' fragments are typically around 55 kDa (IgG) and 75 kDa (IgM). Fab' fragments can be produced from an F(ab')2 fragment by reducing the disulfide bond(s). By using the free sulfhydryl group(s) formed after the reduction, the Fab' fragments can be easily conjugated with other molecules, such as reagents suitable for identification (e.g. enzymes). Monovalent Fab fragments are generally about 50 kDa, regardless of origin. Fab fragments include the light and heavy chains, as well as the variable (VL and VH) and constant (CL and Ch,) regions of the antigen-binding part of the antibody. The H and L parts are connected by an intramolecular disulfide bridge. Fv fragments - regardless of origin, are typically about 25 kDa in size and contain both light (VL) and heavy (VH) chains. Generally, VL and VH chains are held together by non-covalent bonds, so they dissociate easily. They have the same binding properties as larger Fab fragments, but their size is advantageously small. Accordingly, different methods have been developed for cross-linking the VL and VH chains, such as the use of glutaraldehyde (or other chemical cross-linking compounds), the construction of an intramolecular disulfide bridge (incorporation of cysteines), or the use of peptide linkers. The resulting Fv consists of one chain (ie, SCFv; "single chain Fv"). A preferred method involves the production of SCFvs using recombinant methods. The process allows us to mix and match variable chains from different antibodies to produce Fv fragments with new properties. In a typical procedure, a recombinant vector can be prepared that contains an expression cassette driven by suitable regulatory elements. The cassette region may contain a DNA segment encoding a peptide linker sequence suitable for the creation of fusion proteins, with convenient cloning sites at the 5' and 3' ends. In order to make a fusion protein with a linker, an SCFv can be produced by cloning the DNA segment encoding the desired variable region(s) into the vector. In an exemplary alternative approach, two Fv-encoding DNAs are ligated to the DNA containing the linker, and the resulting triple fusion construct is ligated directly into a conventional expression vector. Depending on the chosen vector, DNA produced according to any of these methods can be expressed in eukaryotic or prokaryotic cells. According to a preferred embodiment of the invention, monoclonal antibodies are used which bind VASP only if VASP is phosphorylated. In the sense used in the description of the invention, the term VASP also includes VASP derivatives. Examples include functionally identical derivatives, mutants, VASP fragments and variants, such as phosphoserine 239 VASP, which is additionally phosphorylated at serine 157 and / or threonine 278, as well as glycosylation mutants and other covalent changes, and the structural elements that are significant from the point of view of the protein / protein interaction. In the description, the term VASP is understood to mean both human and VASP from other species, specifically from mammals (for example, rat, mouse, rabbit, dog, pig and monkey). Antibodies can be produced by any conventional method. An example of this is the procedure described by Köhler and Milstein [Köhler and Milstein, Naturé 256, 495 (1975)]. If the selectivity of the antibody must depend on the phosphorylation state, the antibody is produced against phosphorylated VASP. The isolated antibodies are selected in a traditional way, for a specific selectivity. Antibodies are preferably produced against the fragment containing threonine 157, 239 and / or 278 of VASP. The length of the VASP antigen is unimportant as long as it is capable of eliciting a humoral response. Typical peptides are shorter than 50 amino acids. Some are beneficial HU 222 411 Β1 peptide is less than about 20 amino acids. Most preferred peptides are between about 6 and about 12 amino acids in length. Preferred peptides include the KLRKVS239KQ and RKVS239KQE segments. The peptide is preferably phosphorylated at serine 15, serine 239 and / or threonine 278. The peptides may be of recombinant origin. Peptides are typically produced by proteolytic degradation of VASP or by in vitro synthesis and phosphorylation. Monoclonal antibodies which show the above-mentioned properties and which can be used in flow cytometry are also preferred. For this, the new antibody must retain its specificity even when the antibody is exposed to conditions that may cause the antibody to change its conformation. Such a situation can be, for example, in the recording step used in flow cytometry. Therefore, the new antibody can be tested, for example, in terms of suitability for flow cytometry. The monoclonal antibody designated 16C2 is particularly preferred. The new antibodies can be prepared by methods known to the expert. For the production of monoclonal antibodies, the technique described by Köhler and Milstein [Köhler and Milstein, Naturé 256, 495 (1975)] can advantageously be used for the production of hybridoma cells. The specificity of the purified antibodies can be tested with the following test procedures: a) Westem-blot procedure using recombinant VASP phosphorylated to different degrees with cAMP- or cGMP-dependent protein kinases, with the association that a positive signal should only be obtained in the case of phosphorylated VASP. b) Westem-blot procedure using cell extracts, for example extracts of Pkt2 cells, which were transfected with human cGMP protein kinase, human VASP or VASP mutants created in different ways. In each of the mutants, the mutation occurred in one of the three phosphorylation sites, and the mutation—(S157A)VASP, (S239A)VASP, and (T278A)VASP—resulted in deletion of the phosphorylation site. Antibodies whose positive signal was canceled by the S239A mutation, but not by the other two mutations, are suitable. c) Westem-blot method using human platelets treated with various vasodilators (for example, prostacyclin or NO donors) or with cAMP and cGMP protein kinase activators. The antibody that can be used to identify a specific band in the extract only for phosphorylated VASP is suitable. In a similar way, antibodies can be screened using human fibroblasts and rat and mouse platelets. d) Immunofluorescence test using fixed human platelets and endothelial cells. A positive signal is obtained if VASP is present in phosphoprotein form. The subject of the invention is also a hybridoma cell line that produces the new monoclonal antibody. The 16C2 hybridoma cell line producing the 16C2 monoclonal antibody is particularly preferred. The 16C2 monoclonal antibody-producing hybridoma cell line 16C2 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures), Mascheroder, on November 6, 1997, according to the rules of the Budapest Convention, under identification number DSM ACC2330. Weg lb, D-38124 Braunschweig, Germany. The new antibody is suitable for the qualitative and / or quantitative - preferably quantitative - determination of the phosphorylation of VASP in vivo and in vitro, preferably for the determination of phosphorylation on serine 239 and / or serine 157 of VASP, particularly preferably serine 239 of VASP to determine its phosphorylation. Quantitative methods based on the use of antibodies are well known in the art. Such procedures can be found, for example, in "Current protocols in immunology [II. chapter, ed.: Coligan et al., John Wiley and Sons (1991-1992)] c. in a publication. These methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and the like. The new antibodies are also suitable for the qualitative and / or quantitative, preferably quantitative, determination of VASP phosphorylation in biological material, preferably for determining phosphorylation on serine 239 and / or serine 157 of VASP, particularly preferably for determining phosphorylation on serine 239 of VASP. Biological material includes, for example, cell extracts, tissue extracts, cell sections, cell tissue, and cells such as platelets, leukocytes, endothelial cells, smooth muscle cells, and fibroblasts. The biological material may be of human or other mammalian origin, such as rat, mouse, rabbit, dog, pig, and monkey. Biological material of human origin is preferred. The new antibodies are suitable for the quantification of phosphorylated VASP, especially phosphoserine 157 VASP and / or phosphoserine 239 VASP, in biological material by using Westem-blot autoradiograms using procedures known to the expert - for example, NIH gel blotting image 1.6. software, or other quantitative evaluation based on immunofluorescence. Because of the correlation between VASP phosphorylation at serine 239 and 157 and the activity of cGMP- and / or cAMP-dependent protein kinases, the new antibodies can be used as reagents to diagnose cGMP signaling pathways and cAMP signaling pathways, especially cGMP signaling pathways. The use of new antibodies for the determination of phosphoserine 239 VASP in biological materials by Western blotting and immunofluorescence allows the development of diagnostic procedures for the identification of cGMP-increasing substances, hormones and drugs. Examples of such substances may be: ANF, guanylin, NO-releasing substances or drugs. For example, NO-dono5 HU 222 41 It is possible to monitor the regulation of IBI disease therapy and its course, which - among other things - provides data regarding the symptoms characteristic of nitrate tolerance that may develop. From the correlation between VASP serine 157 phosphorylation and the activity of cAMP-dependent protein kinases, it follows that the new antibodies may be suitable as reagents for the diagnosis of substances, hormones and drugs that increase the cAMP level. Consequently, the invention relates to diagnosis or therapy utilizing the novel antibodies or fragments thereof. New procedures are procedures that can be performed in a laboratory, outside the human body (ex-carrier). The new antibodies can be used to determine the degree of phosphorylation of VASP in biological materials from different species. Examples of such species include: human, rat, mouse, rabbit, dog, pig or monkey. It is preferred to use the new antibody to detect phosphorylated VASP in human, rat, mouse or dog biological material. The use of a new antibody for the determination of phosphorylated VASP in biological materials of human origin is particularly advantageous. The new antibodies or their fragments can also be used as biosensors. Biosensors are known to those skilled in the art. The method that uses a specific secondary binding partner - an antibody, a lectin or a receptor - is particularly advantageous. In this case, the identifiable signal required for identification and quantification is carried by the specific binding partner. The indications are known by themselves to the specialist. A signal can be, for example, a chromophore, a luminophore, a fluorophore group, an enzyme, a radioactive isotope, or a colorless or colored particle. A method in which the unlabeled specific binding partner is attached to a solid phase directly or indirectly - for example with the help of another antibody or avidin / biotin bridge - by methods known per se to the expert is preferred. The new antibody or its fragments can be radioactively labeled using methods known to those skilled in the art. The indicated new antibody or its fragments can be used in immunoscintigraphy or for immunotherapeutic purposes. In addition, these monoclonal antibodies can also be used as drug carriers in the treatment of diseases caused by the malfunctioning of the endothelium. After determining the complete nucleotide sequence of the Mab 16C2 "V" gene, it is also technically possible to produce antibody constructs in which hypervariable regions are inserted into the human Mab framework [Jones et al., Naturé 321, 522-525 (1986); Verhoyen et al., Science 239, 1534-1536 (1988)]. It is preferable to quantify the antibody bound by the antigen in platelets by flow cytometry, for example after recording a suitable part of the whole blood sample by methods known to the person skilled in the art. Flow cytometry procedures known to those skilled in the art are described, for example, by G. Otten and W. M. Yokohama [G. Otten and W.M. Yokohama, “Flow cytometry analysis using the Becton Dickinson FACScan. Current protocols in immunology, 5.4.1-5.4.19 (1992)] are used. Flow cytometric analysis of VASP phosphorylation, especially VASP serine 239 phosphorylation, using the new antibodies is not limited to human platelets. The assay can also be performed on other cell types such as lymphocytes, monocytes, leukocytes, endothelial cells and smooth muscle cells. With the help of the new antibodies, the flow cytometric examination of VASP phosphorylation, especially VASP phosphorylation of phosphoserine 239, in freshly collected human platelets allows us to determine the activity of endothelium-derived factors - for example NO and prostacyclin - and, consequently, we can determine the functioning and malfunctioning of the endothelium. its role in, for example, arteriosclerosis, high blood pressure, diabetes, heart failure and kidney failure in cardiovascular disorders. With the help of the new antibodies, determining the phosphorylation of VASP, especially the phosphorylation of serine 239 of VASP, makes it possible to regulate the treatment of the above-mentioned diseases. Resistance that develops during therapy - such as nitrate tolerance - can also be determined. The new antibodies can also be used as pharmaceutical agents. As an active ingredient in biological materials, they influence the phosphorylation state of VASP and / or its relationship with proteins. Consequently, the subject of the invention are those pharmaceutical preparations which contain at least one new antibody. The new formulations can be used internally (orally), parenterally (intravenously), through the anus, or topically (topically). These preparations can be administered in the form of solution, powder (tablets or capsules, including microcapsules), liposome preparations, lipid complexes, colloidal dispersions, injection solutions or suppositories. Suitable materials for formulations of this nature are conventional pharmaceutical solutions or solid additives and additives, solutions, emulsifiers, "glidants", flavor additives, colorants and / or buffers. It is advisable to dose 0.1-100 mg per kilogram of body weight. It is advisable to administer these in dosage units that contain at least the effective daily amount of the new antibodies - for example 30-3000 mg. The daily dose depends on the body weight, age, gender and condition of the mammal. However, higher or lower daily doses may be used. The daily dose can be administered once in one dosage unit or in several smaller dosage units, or in divided doses several times - at predetermined intervals. HU 222 411 Bl In the case of pharmaceutical preparations, the new antibodies are processed with pharmaceutically inactive organic or inorganic additives. Additives suitable for the preparation of tablets, coated tablets and hard gelatin capsules are, for example, lactose, corn starch or its derivatives, tallow and stearic acid (and palmitic acid) or their salts. Additives suitable for the preparation of solutions are water, polyhydric alcohols, sucrose, inverse sugar and glucose. Additives suitable for the preparation of injections are water, polyhydric alcohols, glycerin and vegetable oils. Additives suitable for making suppositories are vegetable oils and hardened oils, waxes, fats and semi-solid polyhydric alcohols. Pharmaceutical preparations may contain preservatives, solutions, stabilizers, wetting agents, emulsifiers, sweeteners, dyes, flavorings, salts suitable for changing the osmotic pressure, buffers, coating materials, antioxidants and, where necessary, other medically active compounds. The subject of the invention is the production process of the new drug, during which at least one new antibody is brought into a formulation suitable for administration with medically suitable and physiologically tolerable additives and, where necessary, other suitable active compounds, additives or supplementary substances. The subject of the invention is, quite generally, a diagnostic method which is suitable for determining the phosphorylation of VASP, preferably the phosphorylation of VASP serb 239, other specific probes, for example monoclonal antibodies, polyclonal antibodies, SCFV fragments or other synthetic or recombinant protein domains, which are specifically identify phosphorylated sequences of VASP, particularly phosphoserine 239 VASP. The subject of the invention is also a diagnostic method suitable for determining the phosphorylation of serine 157 and / or threonine 278 of VASP, other specific probes, such as monoclonal antibodies, polyclonal antibodies, SCFv fragments or other synthetic or recombinant protein domains, which specifically identify the sequences of VASP that are phosphorylated according to 157 and / or contain -threonine. By binding to proline-rich proteins such as zyxin and vinculin, and simultaneously by binding its own proline-rich domain to profilin, VASP enables the formation of actin filaments, in which VASP participates as an adapter molecule. Disruption of this protein / protein relationship often leads to defective platelet aggregation or muscle contraction. for example, the creation of actin / myosin bridges is a prerequisite for the contraction of smooth muscles. A correlation was found between VASP serine 157 phosphorylation and fibrinogen receptor glycoprotein Ilb / IIIa inhibition [Horstrup et al., Eur. J. Biochem., 225, 21-27 (1994)]. Determining the phosphorylation of serine 157 of VASP thus enables a systematic search for substances or drugs that affect the relationship between VASP and its intracellular binding partner, and - for example - they are suitable for the treatment of cardiovascular diseases related to impaired circulation. Advantageous embodiments of the invention are described in the following examples that supplement the description. The following examples - without limiting it in any way - serve to provide a clearer understanding of the solution according to the invention. Examples Example 1 Isolation of a monoclonal antibody against phosphoserine 239 VASP A phosphorylated and a non-phosphorylated peptide containing KLRKVS239KQ located in the vicinity of serine 239 and the RKVS239KQE peptide sequence segment were synthesized using an Applied Biosystems peptide synthesizer according to the Fmoc reaction known to those skilled in the art. Phosphoserine was incorporated into the phosphorylated peptide during peptide synthesis using Fmoc-serine [PO(Obzl)OH-OH] (Calbiochem). Peptides confirmed by mass spectrometry were purified by reversed-phase column chromatography (RPC) on a Vydac 218TP column to >98% purity. Peptides activated with bromoacetic acid or bromoacetic acid-N-hydroxysuccimid ester (Sigma) were linked with thiolated KLH (Keyhole limpet hemocyanin, nanoTools). Female Balb / c mice (6 weeks old) were immunized subcutaneously with KLH-phosphopeptide (10 pg / individual) 4 times, every 14 days. In the case of the first injection, the phosphopeptide was mixed with complete Freund's adjuvant, and for the other three injections with incomplete Freund's adjuvant. After the second week after the last injection, the mice received a "booster" injection containing 10 pg of immunogen dissolved in PBS ("Phosphate Buffered Saline") per day for three days. The day after the last "booster" injection, the mice were sacrificed and the spleen was removed. Spleen cells were isolated and fused with non-producing myeloma cells [eg, PAI-Zellen, J.W. Stocker et al. (1982) Res. Disclosure, 21713] according to the methodology established by Köhler / Milstein. Phosphorylated / non-phosphorylated peptides and phosphorylated / non-phosphorylated recombinant human VASP were also used in the screening procedures for distinguishing hybridoma cells capable of producing antibodies against phosphoserine 239 VASP. In tests with phosphorylated / non-phosphorylated peptides, peptides were covalently bound to DNA-BIND-ELISA plates (Costar) and hybridoma supernatants were screened by ELISA. The supernatants that were able to identify the phosphopeptide were examined to see if they could differentiate between fully phosphorylated recombinant VASP produced in the E. coli system and the corresponding dephosphorylated VASP. Monoclonal an7 identified from the supernatant of hybridoma cells found positive in the above-mentioned procedure HU 222 41 IBI antibodies can be purified from serum-free hybridoma supernatant, preferably by thiophylla adsorption chromatography (POROS 50-OH, nanoTools). Using different test procedures, it was possible to identify and characterize one of the isolated antibodies (clone 16C2) as a monoclonal antibody belonging to the IgGlK class, which recognizes VASP only when this protein is phosphorylated at serine 239. Under the conditions used, the 16C2 antibody does not recognize other proteins and other VASP phosphorylation sites. Monoclonal antibodies that specifically recognize phosphoserine 157 VASP or phosphothreonine 278 VASP can be developed analogously to the above-mentioned procedure. In this case, phosphorylated peptides containing a known peptide segment located around serine 157 or threonine 278 of VASP are used as antigens. The production of monoclonal antibodies recognizing VASP phosphorylated at position 239 was described by Smolenski et al. in their communication [J. Bio. Chem. 237, 20029-20035 (1998)] was also described. Example 2 A flow cytometric study of the phosphorylation of VASP site 239 Investigation of serine 239 phosphorylation of VASP in washed human platelets Those described by Eigenthaler et al. [Eur. J. Biochem., 205, 471-481 (1992)], human platelets were prepared, and VASP phosphorylation was stimulated with substances affecting the circulatory system. The reaction was stopped by fixation with formaldehyde (final concentration approximately 3.5%) (10 min at room temperature). After the optional washing, the cells were permeabilized with Triton X-100 and then washed. Staining was performed by incubating with the appropriate antibody (which was either directly fluorescently labeled or a secondary antibody was fluorescently labeled). For this purpose, the concentration of the primary antibody is preferably 0.5-5 pg / ml, preferably 1-2 pg / ml. For example, the primary antibody Mab 16C2 is used at a concentration of 1.7 pg / ml. The binding of the 16C2 antibody was then confirmed by, for example, G. Otten and W. M. Yokohama [Flow cytometry analysis using the Becton Dickinson FACScan. Current protocols in immunology, 5.4.1-5.4.19 (1992)], determined and studied in a flow cytometer. Example 3 The use of Western blot to determine the phosphorylation of serine 239 of VASP in cell extracts For the determination of VASP phosphorylation in cell extracts in parallel with flow cytometric measurements (example 2), it is known to the expert, for example Eigenthaler et al. [Eur. J. Biochem., 205, 471-481 (1992)] using the Western blot method. The antibody concentration used, depending on the VASP content to be determined in the sample, is ideally between 0.1 and 5 pg / ml, preferably between 0.5 and 1.0 pg / ml. For example, while a concentration of 0.5 pg / ml is preferred for testing human or rat platelets, an antibody concentration of 1.0 pg / ml is preferred for testing endothelial cells from human umbilical cords. In principle, when determining the phosphorylation of serine 239 of VASP, the Westem-blot test with cell extracts and the flow cytometric analysis with fixed cells ended with the same result. Example 4 Determination of serine 239 phosphorylation of VASP in whole blood or platelet-rich plasma (PRP) Whole blood samples and PRP were incubated with substances affecting the circulatory system. The reaction was stopped by fixation with formaldehyde (final concentration approximately 3.5%) (10 min at room temperature). PRP from whole blood, Eigenthaler et al. [Eur. J. Biochem., 205, 471-481 (1992)] was prepared as described. Platelets from PRP were sedimented by centrifugation and then suspended in physiological buffer [Eigenthaler et al., (1992) see above]. Platelets were permeabilized and then stained as described for washed platelets. Example 5 Time dependence of VASP serine 239 phosphorylation in washed human platelets after stimulation with sodium nitroprusside (SNP) Human platelets were treated with 100 μΜ SNP. The cells were sampled at 0, 1, 3 and 5 minutes. The 16C2 antibody was used to determine VASP serine 239 phosphorylation. The measurements were carried out on the one hand with Westem-blot analysis of the collected cell extracts, and on the other hand, in parallel with this, on human platelets fixed and permeabilized as mentioned above, by flow cytometry. The autoradiograms obtained during the Westem-blot procedure were quantitatively analyzed with NIH gel-blotting image 1.6 software. The increase in VASP binding of 16C2 antibody and phosphoserine 239 [% increase (5 min value=maximum effect=100%)] was Table 1 contains. Table 1 t(min) % increase in platelets on cell extract cytometry Westem-blot 0. 0 0 1. 62.5 83.5 3. 89.3 95.2 5. 100.0 100.0 HU 222 411 Bl Example 6 Use of the 16C2 antibody to analyze the phosphorylation of recombinant VASP by c-AMP-dependent protein kinases and c-GMP-dependent protein kinases pg / ml purified recombinant hexahistidine-tagged VASP with 11 pg / ml cAMP protein kinase purified catalytic subunit (cAPK) and 24 pg / ml was phosphorylated with purified cGMP protein kinase (cGPK). Samples were taken from the reaction mixture at specified times, immediately mixed with a stop solution containing SDS, boiled and fractionated by SDS-PAGE. The phosphorylation of VASP was analyzed by staining with Coomassie Blue and Westem-blotting. For the Western blot, M4 polyclonal VASP antibody [Halbrüge M. et al., J. Bioi. Chem. 265, 3088-3093 (1990)] (available from Alexis Corporation, Alté Hauensteinstrasse 4, CH-4448 Laufelfingen, Switzerland) and 16C2 monoclonal VASP antibody were used. In the gel stained with Coomassie Blue, the band below VASP is cAPK, while the band above VASP is cGMP-PK. SDS-PAGE analysis showed that a change (from 46 kDA to 50 kDA) occurred in the migration of VASP after cAPK-preferred phosphorylation of serine 157. During phosphorylation, cGMP-PK prefers serine amino acid 239 (which can be detected with the 16C2 antibody). Example 7 Analysis of phosphorylation in Ptk2 cells transfected with VASP containing different phosphorylation mutations Phosphorylation of transfected human VASP and VASP-mutants was investigated in Ptk2 cells. Ptk2 cells were transfected together with VASP containing wild-type and mutant (inactivated) phosphorylation sites (modified serine 157, serine 238, and threonine 277) and human cGMP protein kinase Ιβ expressed under the control of the CMV promoter. Ptk2 cells contain very little intrinsic bound VASP and cGMP protein kinase. 2 days after transfection, cells were incubated with 30 μΜ 8pCPT-cGMP for 30 minutes and cell extracts were isolated. Then, the phosphorylation of VASP in the cell extracts was examined by Western blotting. M4 polyclonal and 16C2 monoclonal antibodies were used for Western blotting. In the case of serine 157 inactivation (SÍ 57A mutation), the phosphorylation-dependent change from 46 kDA to 50 kDA was not present. The signal recognized by the 16C2 antibody was not present after inactivation of serine 239 (S239A). In these studies, threonine 277 mutants behaved like wild-type VASP. Example 8 Analysis of VASP phosphorylation after stimulation with various vasodilators and cAMP / cGMP analogs in human platelets Washed human platelets (0.7 χ 109 cells / ml) were treated with 1 μΜ Pg-I2 (prostacyclin), 0.5 mM 5,6-DCIcBIMPS (a cAMP analog capable of crossing the cell membrane), 10 μΜ SNP (nitroprusside -sodium) or 1 mM 8pCPTcGMP (a cGMP analogue that can pass through the cell membrane) was incubated. Samples (2.8 x 107 cells) were taken at given times. In the samples mixed with SDS stop solution and boiled, the phosphorylation of VASP was investigated using the Western blot method. M4 polyclonal and 16C2 monoclonal antibodies were used in the study. Phosphorylation of serine 157 was quantified by shifting the band containing VASP from 46 kDA to 50 kDA, while phosphorylation of serine 239 was quantified using the signal provided by the 16C2 antibody. Example 9 Phosphorylation of VASP in rat platelets Rat platelets (0.7 xlO8 9 cells / ml) were incubated with or without 100 μΜ SNP (nitroprusside sodium) or 10 μΜ prostaglandin El (PGE1) for 5 minutes. Extracts of these platelets were analyzed by Western blotting. The biot showed that a phosphorylation-dependent (phosphorylation of serine 157) shift of the VASP band from 46kDA to 50kDA and the signal given by the 16C2 antibody are also present in rat platelets. A similar result was obtained when mouse platelets were used. Example 10 VASP and phospho-VASP immunofluorescence studies on human platelets Human platelets (platelet-rich plasma, PRP) were placed on a glass microscope slide and allowed to bind and spread for 45 minutes. The platelets on the glass slide were incubated for 15 minutes in the absence (A and C) and presence (B and D) of 100 μΜ 8pCPTcGMP. Cells were then fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton Χ-100. The slides were first incubated with M4 polyclonal antibody (1:1000) or 16C2 monoclonal antibody and then with the usual second antibody. The photos show the appearance of the phosphorylation-dependent signal when using the 16C2 antibody. The signal obtained with the M4 antibody (in immunofluorescence studies, since there is no separation, 46 kDA and 50 kDA are summed) is independent of phosphorylation.

Claims

CLAIMS 1. An antibody against VASP or a fragment thereof that binds VASP as an antigen only when VASP is phosphorylated at serine amino acid 157, i.e., VASP containing phosphoserine 157.

2. In vitro use of an antibody or antibody fragment as defined in claim 1 as a diagnostic agent.

3. Use according to claim 2 for the qualitative or quantitative determination of VASP containing phosphoserine 157 in a biological sample.

4. The use according to claim 3, wherein the biological sample comprises human platelets or whole blood.

5. Use according to claim 3 or 4, wherein the determination of phosphoserine 157 is carried out by Western blotting. HU 222 41 IBI 6. The use according to claim 3 or 4, wherein the determination of phosphoserine 157 is performed by flow cytometry.

7. Use according to any one of claims 2-6 for detecting substances affecting cGMP and / or cAMP levels in a biological sample.

8. Use according to any one of claims 2-6 for determining the in vivo activity of endothelial factors.

9. Use according to any one of claims 2-8 for detecting endothelial dysfunction.

10. A hybridoma cell line which produces the specific antibody of claim 1.

11. A diagnostic reagent kit comprising an antibody or antibody fragment as defined in claim 1.

12. In vitro diagnostic method, characterized in that an antibody or antibody fragment defined according to claim 1 is used to determine or influence the phosphorylation state of phosphoserine 157 of VASP and / or the protein interactions of VASP in a biological material.