MXPA00011295A - Angiostatin receptor - Google Patents

Abstract

The present invention relates, in general, to an angiostatin receptor and, in particular, to an angiostatin receptor present on cellular plasma membranes. More particularly, the present invention relates to the human angiostatin receptor, ATP synthase, or subunit or portion thereof, and to the use thereof in assays designed to screen compounds for their ability to serve as agonists or antagonists of human angiostatin. The invention further relates to nucleic acid sequences encoding ATP synthase, or subunit or portion thereof, and to host cells transformed therewith. The invention also relates to antibodies specific for ATP synthase.

Description

ANGIOSTATIN RECEIVER TECHNICAL FIELD The present invention relates, in general, to an angiostatin receptor and, in particular, to an angiostatin receptor present on the membranes of the cellular plasma. More particularly, the present invention relates to the human angiostatin receptor, ATP synthase, or subunit or portion thereof, and to the use thereof in assays designed to select compounds for their ability to serve as agonists or antagonists of human angiostatin. . The invention further relates to nucleic acid sequences encoding the ATP synthase, or subunit or portion thereof, and to host cells transformed therewith. The invention also relates to antibodies specific for ATP synthase. BACKGROUND The growth of tumors requires the generation of existing blood vessels in the process of angiogenesis. If vascularization is avoided, the growth of the tumor deteriorates dramatically and the size of the tumor is restricted. The modulation of endogenous angiogenic inhibitors in this way plays an important role in the development of the tumor. Angiostatin, a proteolytic fragment of plasminogen, is a potent inhibitor of angiogenesis and the growth of tumor cell metastasis (O'Reilly et al., Cell 79: 315-328 (1994)). Angiostatin can be generated in vitro by limited proteolysis of plasminogen (Sottrup-Jensen et al., Progress in Chemical Fibrinolysis and Thrombolysis 3: 191-209 (1978)) resulting in a 38kDa plasminogen fragment (Val79-Pro353) . Although the enzymatic mechanism by which angiostatin is generated in vivo is unknown, recent studies have shown that the dissociation of plasminogen in angiostatin can be catalyzed by a serine proteinase.

(Gately et al., Cancer Research 36: 4887-4890 (1996)) and a macrophage metalloelastase (Dong et al., Cell 88: 801-810 (1997)). The generation of angiostatin from plasmin reduction has also been demonstrated in vi tro (Gately et al., PNAS 94: 10868-10872 (1997)) and in Chinese hamster ovary and in human fibrosarcoma cells (Stathakis et al. JBC 272 (33): 20641-20645 (1997)). Cellular receptors for plasminogen have previously been demonstrated in human umbilical vein endothelial cells (CEVUH) and are believed to function in the regulation of endothelial cell activities, including angiogenesis (Hajjar et al., J. Biol. Chem. 261 (25): 11656-11662 (1986), Hajjar et al., JBC 269 (3): 21191-21197 (1994)). Plasminogen receptors are also expressed in high numbers on tumor cells, where they have been identified as critical for tumor invasion. Proteins normally found in the cytoplasm have also been shown on cell surface membranes to serve as plasmionogen binding sites (Miles et al., Biochemistry 30: 1682-1691 (1991)). The present invention is a result of the demonstrations that plasminogen and angiostatin are linked to different sites on cell plasma membranes that ATP synthase is the angiostatin binding protein. These findings make possible assays that can be used to select compounds for their ability to modulate angiostatin activities. The compounds identified here have profound utility as a therapeutic substance. SUMMARY OF THE INVENTION The present invention relates to an angiostatin receptor present on cellular plasma membranes. More particularly, the present invention relates to the human angiostatin receptor, ATP synthase, and to the use thereof, or subunits or portions thereof, in assays designed to select compounds for their ability to modulate angiostatin activities. . The invention also relates to nucleic acid sequences encoding ATP synthase, or subunit or portion thereof, and with host cells transformed therewith. The invention also relates to antibodies specific for ATP synthase. BRIEF DESCRIPTION OF THE DRAWINGS Figures IA and IB. Direct binding assay and Scatchard analysis of plasminogen and angiostatin with endothelial cells. CEVUH was plated at a density of 10,000 cells / well and incubated with increasing concentrations of labeled plasminogen, 125I or angiostatin. Figure IA. Plasminogen labeled with 125I was concentration dependent and saturable with an apparent dissociation constant (Kd) of 158 nM and 870,000 sites / cell. Figure IB. In linkage with CEVUH with angiostatin labeled with 125 I was concentration dependent and saturable with a constant Ij of 245 nM and 38,000 sites / cell. Error bars represent the standard deviation. Figure 2. Competition binding assay between plasminogen and angiostatin. CEVUH was plated at a density of 10,000 cells / well and incubated with 1.0 μM of 125 I-labeled plasminogen in the presence of a 100-fold molar excess unlabeled angiostatin for 1 hour at 4 ° C. The cells were washed and the remaining radioactivity was quantified by counting? . (A) Total linkage of 1.0 μM of plasminogen labeled with 125 I was designated as 100 percent. (B) Plasminogen binding was inhibited by approximately 80 percent in the presence of a 25-fold unlabeled plasminogen molar excess. (C) The plasminogen binding was not inhibited in the presence of a 100-fold molar excess of unlabeled angiostatin which suggests distinct binding sites for each on the cells. Similar experiments using angiostatin labeled with 125I (D) showed no inhibition of binding in the presence of a 2-fold molar excess of unlabeled plasminogen (E). Error bars represent the standard deviation. Figures 3A-3D. The affinity purification of plasminogen and angiostatin binding sites. Proteins containing membrane were prepared by SDS-PAGE and then analyzed by Western staining. Membranes were incubated in 10 mM Tris-hcL, 0.15 M NaCl, 0.05 percent NP40, pH 7.5 containing, Figure 3A, streptavidin-alkaline conjugated phosphatase antibody, or, Figure 3B, anti-annexin II antibody and were developed using bromo-4-chloroindol-3-yl-phosphate nitro tetrazolium blue. The membrane stained with Coomassie brilliant blue, Figure 3C, shows affinity purified membrane protein. The membrane incubated with plasminogen labeled with 125I, Figure 3D, showing binding to the membrane purified with plasminogen and not with angiostatin. Lane 1 represents the protein eluted from the sepharose-plasminogen column. Strip 2 represents protein eluted from the sepharose-angiostatin column. The relative molecular weights of ATP of-synthase and ATP β-synthase are approximately 55 and approximately 50 kDa, respectively. Figures 4A-4D. In antibody binding directed against the subunit of ATP synthase on the surface of CEVUH by flow cytometry. The CEVUH was analyzed by FACScan flow cytometry. The histogram graphs are shown for CEVUH (Figure 4A) and A549 (Figure 4B) where (...) represents cells incubated with antibody directed against the subunit of ATP synthase, () preimmune serum and (-) only secondary antibody. The histogram graph of A549 shown in Figure 4C is similar with (...) representing antibodies incubated with a 5-fold molar excess of ATP protein synthase. In Figure 4D, CEVUH demonstrate specific binding, saturated with antibody directed against the ATP synthase subunit. The relative mean fluorescence of CEVUH incubated with preimmune rabbit serum subtracted from the relative mean fluorescence of CEVUH incubated with the same volume of anti-ATP synthase gave the relative mean fluorescence resulting from specific binding of antibodies directed against the synthase subunit. ATP on the surface of CEVUH. Figures 5A-5F. The immunofluorescence microscope of ATP-synthase on the surface of CEVUH. CEVUH were incubated with rabbit polyclonal antiserum grown against the ATP synthase subunit of E. coli. Figure 5A, CEVUH under epi-illumination showing the immunofluorescent surface spotting for the ATP synthase subunit. Figure 5B, the same CEVUH field under visible light. Figure 5C, human dermal microvascular endothelial cells also showed immunofluorescent surface staining for the ATP synthase subunit. Control experiments were performed with Figure 5D preimmune serum and Figure 5E secondary antibody alone. Figure 5F, CEVUH was permeabilized by ketone binding before adding antibody to the ATP synthase subunit. Figure 6. Competition binding assay between angiostatin and the antibody against the ATP synthase subunit of E. Coli. CEVUH was plated at a constant density 10,000 cells / well and incubated with 0.5 μM angiostatin labeled with 125 I in the presence of a 1:10 dilution of antibody against the ATP synthase subunit of E. coli for 1 hour at 4 hours. ° C. The cells were washed and remained bound, the radioactivity was quantified by counting and the nonspecific binding was measured in the presence of excess unlabeled angiostatin and subtracted from the total binding. (A) total linkage of 0.5 μM angiostatin labeled with 125 I was designated as 100 percent. (B) Angiostatin binding was inhibited by 59 percent in the presence of a 1:10 dilution of anti-ATP synthase subunit antibody. Competition studies were also performed simultaneously using rabbit pre-immune serum to determine non-specific inhibition. Error bars represent the standard deviation. A homoscedastic test with 1 tail was used for the statistical analysis; p < 0.10. • Figures 7A-7E. In angiostatin binding with the recombinant subunit to human ATP synthase. The a subunit of human ATP synthase was cloned and expressed in E. coli and purified using Qiagen sepharose-nickel protein purification system before dialyzing into SRF, pH 7.0. The recombinant protein was electrophoresed at 5-15 percent jk 10 of SDS-PAGE, electro-anchored on ImmobilonM membrane and incubated 18 hours in 10 mM Tris-HCl, 0.15 M NaCl, 0.05 percent NP40, pH 7.5 (TSN) that contained angiostatin - 125I. For competition studies, unlabeled ligand was added 4 hours before the radiolabelled ligand. The spots were washed in TSN regulator containing 0.05 percent Tween 80 and bound radioactivity was quantified in a Molecular Dynamics PhosphorImagerMR. Figure 7A. Stained membrane Commassie Immobilon containing the α subunit of human ATP synthase. Figure 7B. Linkage of 0.5 μM angiostatin labeled with 125I. Figure 7C. Linkage of 0.5 μM angiostatin labeled with 125 I in the presence of a 250-fold molar excess of unlabeled angiostatin. Angiostatin binding is inhibited by approximately 56 percent. Figure 7D. Linkage of 0.5 μM angiostatin labeled 125I in the presence of a molar surplus of 2500 times of unlabeled plasminogen.

The angiostatin binding was not inhibited. Figure 7E. Linkage of 0.5 μM of plasminogen tagged with 125 I with the subunit a of the human ATP synthase. Plasminogen was not linked to the recombinant subunit of ATP synthase, however, if it was linked to control annexin II (as shown in Figure 3). Figure 8. Antibody binding directed against the β subunit of ATP synthase on the surface of CEVUH by flow cytometry. CEVUH was analyzed by FACScan flow cytometry as described above and in the examples. Histogram plots are shown for CEVUH cells incubated with antibody directed against the β-subunit of ATP synthase. DETAILED DESCRIPTION OF THE INVENTION The present invention is a result of the demonstration that plasminogen and angiostatin are linked at different sites on the surface of endothelial cells, Annexin II and ATP synthase, respectively. The invention provides methods for identifying compounds that can be used to model the effects of angiostatin in vivo, and including its inhibitory effects on angiogenesis and the migration and proliferation of endothelial cells. In one embodiment, the present invention relates to methods of compound selection for their ability to bind ATP synthase and thereby potentially function as angiostatin agonists or antagonists. The ATP synthase includes two major domains, a F0 portion extending the asymmetric membrane containing a proton channel and a F? soluble that contains three catalytic sites that cooperate in synthetic reactions. The F- region contains the subunits a, β, α, d and C. (See Elston et al., Nature 391: 510 (1998)). The entire ATP synthase molecule can be used in the present assays or a subunit a of the same can be used, for example, the subunit a and / or the β subunit, the angiostatin binding domain of ATP synthase can also be used. use, as well as a fusion protein comprising the synthase, the subunit thereof or the angiostatin binding domain thereof. The following examples indicate that the ATP synthase a and β subunits are present in the plasma membrane of endothelial cells. In addition, the examples indicate that angiostatin binds to subunit a. The α and β subunits present in cell plasma membranes may be identical to those present in mitochondria membranes or may be represented by a truncated form thereof (eg, terminal C or truncated N). The binding assays of the invention include the use of any of truncated forms. The binding assays of this embodiment of the invention include cell-free assays in which the ATP synthase, or a subunit thereof or the angiostatin binding domain thereof (or the fusion protein containing the same) , incubated with a test compound (proteinaceous or non-proteinaceous) which, advantageously, has a detectable label (e.g., a radioactive or fluorescent label). After incubation, the ATP synthase, or the subunit thereof or the angiostatin binding domain thereof (or the fusion protein), free or bound to the test compound, can be separated from the test compound without bind using any of a variety of techniques (for example, the ATP synthase (or fusion protein binding domain or subunit) can be bound to a solid support (e.g., a plate or column) and washed free of unbound test compound). The amount of test compound bound to the ATP synthase or the subunit thereof or the angiostatin binding domain thereof (or fusion protein), is then determined, for example, using a suitable technique to detect the label used ( for example, liquid scintillation counting and labeled gamma counting or by fluorometric analysis). The binding assays of this modality may also take the form of competition binding assays without a cell. In this type of assays, the ATP synthase, or subunit thereof or angiostatin binding domain thereof, or fusion protein containing the same, is incubated with a known compound to interact with ATP synthase ( example, angiostatin or ATP synthase binding portion thereof), this compound, advantageously, bears a detectable label (eg, a radioactive or fluorescent label) a test compound (proteinaceous or non-proteinaceous) is added to the reaction and it is tested to determine its ability to compete with the known compound (tagged) to bind to the ATP synthase, or subunit thereof or the angiostatin binding domain thereof (or fusion protein). The free known compound (labeling) can be separated from the known compound bound, and the amount of bound known compound is determined to assess the ability of the test compound to compete.

This assay can be formatted so as to facilitate the selection of large numbers of test compounds by binding the ATP synthase, or subunit thereof or the angiostatin binding domain thereof (or the fusion protein), to a solid support so that it can be easily washed free of unbound reagents. A plastic support, for example, a plastic plate (e.g., a 96-well plate), is preferred. The ATP synthase, or subunit thereof or angiostatin binding domain thereof (or fusion protein), suitable for use in the cell-free assays described above can be isolated from natural sources (e.g. membrane preparations) or prepare recombinantly or chemically. The ATP synthase, or subunit thereof, or angiostatin binding domain thereof, can be prepared as a fusion protein using, for example, known recombinant techniques. Preferred fusion proteins include a GST (glutathione-S-transferase) fraction, a PFV (green fluorescent protein) fraction (useful for cell localization studies) or a His tag (useful for affinity purification). The fraction that is not ATP synthase can be present in the N-terminal or C-terminal fusion protein of the ATP synthase, subunit or binding domain. As indicated above, the ATP synthase, or subunit thereof or angiostatin binding domain thereof, or fusion protein, may be present bound to a solid support, including a plastic or glass plate or bed, a chromatographic resin (for example, sepharose), a filter or a membrane. Methods of binding proteins with these supports are well known in the art and include direct chemical binding and binding via a binding pair (eg, biotin and avidin or biotin and streptavidin). It will also be appreciated that, whether free or bound to a solid support, the ATP synthase, or subunit thereof or angiostatin binding domain thereof, or fusion protein, may be unlabelled or may carry a detectable label. (for example, a radioactive fluorescent label). The binding assays of the invention also include cell-based assays in which the ATP synthase, or subunit thereof or angiostatin binding domain thereof or fusion protein, is presented on a cell surface. Suitable cells for use in these assays include cells that are naturally expressing ATP synthase and cells that have been technically designed to express ATP synthase (or subunit thereof or angiostatin binding domain thereof or protein). fusion comprising the same). The cells may be normal or tumorigenic. Advantageously, cells expressing ATP synthase are used. Examples of suitable cells include prokaryotic cells (e.g., bacterial cells (e.g., E. coli)), lower eukaryotic cells (e.g., yeast cells (e.g., Promega hybrid set (CG 1945 and Y190), and strains YPH500 and BJ5457)) and higher eukaryotic cells (e.g., insect cells and mammalian cells (e.g., endothelial cells, including bovine aortic endothelial cells (CEAB), bovine adrenal medullary endothelial cells (CEMAB), cells murine CP-4-l endothelial cells, CEVUH or any human endothelial cell line, or cells such as human lung carcinoma cells (e.g., A549 cells)).

The cells can be engineered to express the ATP synthase (advantageously, human ATP synthase, or a subunit thereof or an angiostatin binding domain thereof, or fusion protein including it) by introducing it into a host. selected an expression construct comprising a sequence encoding ATP synthase, or subunit thereof or an angiostatin binding domain thereof or fusion protein, operably linked to a promoter. A variety of vectors and promoters can be used. For example, pET-24a (+) (Novagen) containing a T7 promoter is suitable for use in bacteria, likewise, pGEX-5X-1. Suitable yeast expression vectors include pYES2 (Invitron). Suitable baculovirus expression vectors include p2Bac (Invitron). Suitable mammalian expression vectors include pBK / CMV (Stratagene). The introduction of the construct into the host can be performed using any of a variety of standard transfection / transformation protocols (see Molecular Biology, A Laboratory Manual, Second Edition, J. Sambrook, EF Fritsch and T. Maniatis, Cold. Press, 1989). The cells thus produced can be cultured using established culture techniques convenient for the participating host. Culture conditions can be optimized to ensure expression of the ATP synthase (or subunit, or binding domain or fusion protein) coding sequence. Although for cell-based binding assays the ATP synthase (or subunit, or binding domain or fusion protein) can be expressed on a membrane of the host cell although for cell-based binding assays the ATP synthase (FIG. or subunit, or binding domain or fusion protein) can be expressed on a membrane of the host cell (e.g., on the surface of the host cell), for other purposes the coding sequence can be selected so as to ensure that the product of the expression is secreted in the culture medium. The cell-based binding assays of the invention can be carried out by adding test compound (advantageously, having a detectable label (eg, radioactive or fluorescent)), with a medium in which the cells expressing the ATP synthase (or subunit thereof, or angiostatin binding domain thereof or fusion proteins containing the same), incubating test compound with the cells under favorable conditions to bind them and then removing the unbound test compound and determining the amount of test compound associated with the cells. The identification of ATP synthase on a cell membrane (eg, on the surface of the cell) can be done using techniques such as those in the examples that follow (eg the cell surface can be labeled with biotin and the protein followed). by a fluorescent tag). The associated membrane proteins • (for example, cell surface proteins) can also be analyzed on a Western blot and the bands subjected to mass spectroscopy analysis. For example, a fluorescently labeled antibody can be used with permeabilized cells where these cells can be tested with another fluorescently labeled protein. Each F 10 label can be monitored at a different wavelength, for example, using a confocal microscope to demonstrate colocalization. As in the case of cell-free assays, cell-based assays can also take the form of competitive assays wherein a compound known to bind to ATP synthase (and preferably labeled with a detectable label) is incubated with cells expressing ATP synthase (or subunit thereof, or angiostatin binding domain thereof) or fusion protein comprising the same) in the presence and absence of test compound. The affinity of a test compound for ATP synthase can be assessed by determining the amount of known compound associated with the cells incubated in the presence of test compound, as compared to the amount associated with the cells in the absence of the test compound.

It will be appreciated from reading this disclosure that the selectivity of a test compound for ATP synthase on the surface of cells, compared to mitochondrial ATP synthase, can be easily assessed. Compounds which, by virtue of their physico-chemical properties, can not be diffused across cell membranes (and which are not natural or artificial ligands for cellular transporters) can be considered selective for ATP synthase on the surface of the cell. For example, compounds that bind ATP synthase to the cell surface but are positively charged by this can prevent them from diffusing through the membranes. A test compound identified in one or more of the assays described above as being capable of binding to ATP synthase can, potentially, mimic or enhance the effects of angiostatin on angiogenesis, cell migration, proliferation and pericellular proteolysis or, potentially, , antagonize the effects of angiostatin, for example, by preventing angiostatin from binding to its receptor. To determine the specific effect of any particular test compound selected based on its ability to bind ATP synthase (or inhibit (competitively or non-competitively) the angiostatin that binds to ATP synthase), assays can be carried out to determine, for example, the effect of various concentrations of the selected test compound on the activity, for example, of cell proliferation (eg, endothelial cell), metabolism or cytosolic / cytoplasmic pH. (Tests can be carried out to determine the effect of test compounds on the ATP (and ATPase) synthase activity using standard enzyme assay protocols). Cell proliferation can be monitored by measuring the assimilation of labeled bases (e.g., radioactively (e.g., 3H, 51C, 14C), e.g., fluorescently (e.g., CYQUANT (Molecular Probes)) or colorimetrically (e.g., ßrdU ( Boehringer Manheim or MTS (Promega)), in cellular nucleic acids Cytosolic / cytoplasmic pH determinations can be made with a digital imaging microscope using substrates such as BCECF (bis (carboxyethyl) -carbonyl fluorescein) (Molecular Probes, Inc.) A test compound that reduces or replaces the concentration of angiostatin required to inhibit cell proliferation or lower intracellular pH can be expected to do so by acting as an angiostatin agonist.A test compound that increases proliferation in the presence of angiostatin (or functional portion thereof or functional equivalent thereof) can be expected to do so by acting as an antagonist angiostatin nist. A test compound that raises the intracellular pH in the presence of angiostatin (or functional portion thereof or functional equivalent thereof) can do so by acting as an angiostatin antagonist. These functional tests can also be carried out in the absence of angiostatin (ie, the test compound alone), with angiostatin (or functional portion thereof or functional equivalent thereof) running as a separate control. The test compound which, for example, modulates the intracellular pH in the absence of angiostatin may be an angiostatin agonist or antagonist. Other types of assays that can be carried out to determine the effect of a test compound on angiostatin binding with ATP synthase include the Lewis lung carcinoma assay (O'Reilly et al., Cell 79: 315 (1994)). ) and extracellular migration assays (Boyden Chamber assay: Kleinman et al., Biochemistry 25: 312 (1986) and Albini et al., Can. Res. 47: 3239 (1987)). Das et al. (J. Exp. Med. 180: 273 (1994)) have reported the presence of the β-subunit of ATP synthase that transports H + on the plasma membrane of human tumor cell lines. The present demonstration of the α-subunit of ATP synthase on plasma membranes, and the linkage thereto of angiostatin, indicates that angiostatin can be directly involved in effecting cytolysis, for example, of tumor cells. The binding of angiostatin with its receptor can result in the transport of protons through the membranes of the plasma and into the cells, resulting in cytolysis by osmotic shock. In accordance with the foregoing, the present invention includes within its scope methods of selection composed of its ability to modulate the effect of angiostatin or proton pump which is the result of the binding of angiostatin with its receptor. In one of these assays, cells expressing ATP synthase (or subunit (eg, a_ or β) or portion thereof) are incubated with the test compound in the presence of angiostatin (or functional portion thereof or functional equivalent). of the same) and the proton influx of the cells is determined and compared with the proton influx observed in the absence of the test compound. Compounds that reduce the angiostatin concentration (or functional portion thereof or functional equivalent thereof) necessary to effect a particular level of proton influx can be expected to do so by acting as an angiostatin agonist. Compounds that reduce the amount of proton pump induced by angiostatin observed can be expected to do so by acting as an angiostatin antagonist. The amount of proton pumping can be determined using any of a variety of approaches, including using cells previously loaded with a pH-sensitive reporter (for example, BCECF can be used to measure pH (Misra et al., Biochem J. 309 : 151 (1995)) and monitor the effect of the test compound on the reporter Alternatively, the effect of a test compound on proton pumping can be determined by monitoring the lysis of the cells using, for example, a test of chromium 51 release (McManus et al., Exper. Lung Res. 15: 849 (1989); Zucker et al., Res. Comm. Chem. Path. Pharm. 39: 321 (1983)). In addition to the various approaches described above, assays can also be designed to be colorometrically monitorable or using time resolved fluorescence. In another embodiment, the invention relates to compounds identified using the aforementioned assays for being capable of binding to ATP synthase (and / or inhibiting angiostatin from binding to ATP synthase). (competitively or non-competitively) and / or modulate the effects of angiostatin on cellular bioactivities and / or modulate the ATP synthase activity. These compounds may include novel small molecules (e.g., organic compounds (e.g., organic compounds less than 500 Daltons), and novel polypeptides, oligonucleotides, as well as novel natural products (preferably in isolated form) (including alkyloids, tannins, glycosides, lipids, carbohydrates and the like) Compounds that mimic or enhance the activities of angiostatin can be used to inhibit angiogenesis, for example, to patients who have tumors and in patients suffering from retinopathies related to vascular tissue (including diabetics) and Terigio Other diseases in which angiogenesis is a significant component of tissue pathology include rheumatoid arthritis and keloid formation. Compounds that inhibit angiostatin activities can be used to promote angiogenesis in conditions of vascular insufficiency, ^ f 10 including ischemic heart disease, peripheral vascular disease, thromboembolic disease, stroke and vasculitis (Buerger's disease, Wegener's granulomatosis, and giant cell arteritis). These compounds can also be used at wound sites to promote healing, and at sites of transplant and grafts (for example, skin grafts). Other diseases / disorders amenable to treatment using compounds selected in accordance with the assays described above include obesity, osteoarthritis, diseases / vascular disorders of the eye, including diabetic retinopathy, macular degeneration, retinopathy of prematurity, corneal inflammation, and viral infections, as well as psoriasis, spinal cord damage, and other diseases and disorders that can be expected to benefit from the intervention of vascularization. The compounds identified according to the above tests can be formulated as pharmaceutical compositions. These compositions comprise the pharmaceutically acceptable carrier compound and diluent. The compound can be presented as a dosage unit (e.g., as a tablet or capsule) or as a solution, preferably sterile, particularly when its administration by injection is anticipated. The compound may also be present as a cream, gel or ointment, for example, when topical administration is preferred. The dosage and the dosage regimen will vary, for example, with the patient, the compound and the effect sought. The optimal dose and regimens can be easily determined by one skilled in the art. In a specific embodiment, the invention relates to a method of antagonizing the effect of angiostatin in a patient by administering ATP synthase, or a soluble angiostatin binding portion thereof. The ATP synthase, or portion thereof, suitable for use in this method can be prepared recombinantly or chemically and can be formulated with an acceptable carrier (including a liposome) as a pharmaceutical composition. The ATP synthase, or portion thereof, may be present as a fusion protein, for example, fused to the heavy chain of IgG. The ATP synthase, or portion thereof, can be derived (eg, with polyethylene glycol) to modify its half-life in vivo. The method of this modality finds application, for example, in wound healing. In another embodiment, the invention relates to antibodies specific for ATP synthase, and antigen binding fragments thereof, including F (ab) 2 'or F (ab) fragments. The antibodies can be monoclonal or polyclonal and can be prepared using standard techniques (Harlow and Lane Antibodies, A Laboratory Manual, (1988) Cold Spring Harbor, F 10 Laboratories). The antibodies can be used in ATP purification protocols or the antibodies can be formulated as pharmaceutical compositions and used therapeutically to mimic or enhance the effects of angiostatin on endothelial cells or to antagonize these effects. 15 In yet another modality, the invention relates to games, for example, convenient games for carrying out the assays described herein. These assays may include ATP synthase, or subunit thereof or angiostatin binding domain thereof, or fusion proteins that comprise the same, and / or angiostatin. These components can carry a detectable label. The kit may include an antibody specific for ATP synthase or specific for angiostatin. Plasminogen may also be present. The game can include any of the above components arranged within one or more container elements. The game may also include auxiliary reagents (eg, regulators) for use in trials. In another embodiment, the present invention relates to diagnostic methods that are based, for example, on 5 assays for the binding of angiostatin with ATP synthase. These methods make it possible to identify patients suffering from diseases, disorders or conditions associated with abnormal angiogenesis. The demonstration of ATP synthase is the angiostatin linker αFk 10 protein, and the resulting availability of methods of identifying substances that can be used to modulate the effects of angiostatin, makes it possible to determine which individuals will likely respond to particular therapeutic strategies. Treatment strategies for individuals suffering from a disease, disorder or condition associated with abnormal angiogenesis can be designed more effectively and with greater ability to predict a successful outcome. Thus, for a given clinical disease that is associated with both abnormal angiogenesis and that is of origin polygenic (not Mendelian), you can select the genotype that is involved not only in the disease, but also in which variant of the disease is associated with abnormal angiogenesis and proceeds to select, via a diagnostic procedure, all patients futures that have the same genotype in order to choose the therapeutic strategy most associated with a successful outcome or at least associated with a toxic side effect, for that genotype. Certain aspects of the present invention are described in greater detail in the non-limiting examples that follow. EXAMPLES The following experimental details are described in the specific examples that follow. Purification of proteins? Plasminogen was purified from human plasma by affinity chromatography and separated into isoforms 1 and 2 as previously described (Deutsch et al., Science 170: 1095 (1970), Gonzalez-Gronow et al., Biochemistry 23: 190- 194 (1984)). Based on kinetics and electrophoretic analysis, all plasminogen preparations proved to be plasmin-free. The concentration of plasminogen was determined spectrophotometrically at 280 nm using an A1% / lcm value of 1.67 and a molecular mass of 92 kDa for plasminogen Glu1 20 (Castellino et al., Chem. Rev. 81: 431 (1981)). The human plasminogen kringles 1-3 (angiostatin) was purified as previously described (Sottrup-Jensen et al., Progress in Chemical Fibrinolysis and Thrombolysis 3: 191-209 (1978)). The angiostatin concentration was determined spectrophotometrically at 280 nm using an A1% / lcm value of 0.8 and a molecular mass of 38 kDa (Sottrup-Jensen et al., Progress in Chemical Fibrinolysis and Thrombolysis 3: 191-209 (1978)). The protein endotoxin levels were < 50.0 pg endotoxin / milliliter valued by coagulation times of amoebic lysate Limulus of Pyrotell (Associates of Cape Cod, Woods Hole, MA). Cell culture Primary human umbilical vein endothelial cells (CEVUH) were cultured as previously described (Morales et al., Circulation 91: 755-763 (1995)) in 150 millimeter petri dishes and retained for up to 6 passages. Human thermal microvascular endothelial cells (CEMH) were obtained at Clonetics (San Diego, California), were cultured according to specifications and retained for up to 6 passages. Human lung carcinoma cells A549 were obtained from ATCC (Rockville, MD) and were grown according to specifications. For all experiments the cells were discarded by incubation with SRF containing 2 mM EDTA, pH 7.4. Antibody Purification The antibody to ATP synthase synthase subunit alpha-tagged His was generated in rabbits by intranodal injection (Covance Laboratories, Vienna, VA). The production bleeds were centrifuged and the serum obtained was precipitated with ammonium sulfate. The precipitate was resuspended in SRF / 0.5M NaCl, pH 7.5 and passed on sepharose protein A (Sigma, St. Louis, MO), sepharose-plasminogen and ATP synthase columns subunit A-sepharose (CNBr coupling, Pharmacia Amershamn, Piscataway, NJ). Each column was eluted with 20 mM glycine, pH 2.5. Neutralized IgG fractions were tested by immunodiffusion, enzyme linked immunosorbent assay and Western blotting. Antibodies to the alpha subunit of ATP synthase showed no cross-reactivity in plasminogen or other proteins by Western blot analysis. The polyclonal antibody obtained from Dr. A.E. Senior (Rochester Medical Center, Rochester, NY) directed against the ATP synthase alpha subunit of E. coli was characterized by enzyme-linked immunosorbent assay and Western blot analysis and showed no cross-reactivity with other proteins in the Fl portion or in the membranes of E. coli (Perlin et al., Archives of Biochem, and Biphys 236 (2): 603-611 (1985), Rao et al., Archives of Biochem, and Biophys. 255 (2): 309-315 ( 1987)). Linkage assays Ligands were radioiodinated using Iodobeads® (Pierce), repurified in L-lysine-sepharose, eluted with 100 mM 6-aminocaproic acid (EACA) and dialyzed in PBS, pH 7.0, before its use in binding assays. CEVUH were plated at a density of 5000 or 10,000 cells per well and incubated with increasing concentrations of 125I-labeled ligand in medium containing 1 percent bovine serum albumin (BSA) for one hour at 4 ° C in 96-well plates. wells The wells were washed and remained bound, the radioactivity was quantified using the LKB 1272 gamma radiation counter. The non-specific binding was measured in the presence of excess unlabeled ligand. Membrane purification The plasma membrane extracts of CEVUH labeled with NHS-biotin were prepared by cavitation and ultracentrifugation of Parr nitrogen pump 21 kilograms / square centimeter (Young et al., J. Biol. Chem. 270 (3): 999- 1002 (1995)). The membrane extracts were incubated with plasminogen-sepharose or angiostatin-sepharose columns in an inhibitor cocktail regulator (Young et al., J. Biol. Chem. 270 (3): 999-1002 (1995)). Each sepharose column was eluted with 50 mM Tris / 100 M EACA, pH 7.5, 50 mM Tris / 1 M Nací, pH 7.5, 50 mM Tris / 7 percent DMSO and 20 mM glycine, pH 2.5 to perform all types of link. The glycine eluates were dialyzed, lyophilized, electrophoresed in 5-15 percent gradient SDS-PAGE (Laemmli, Nature (London) 227: 680-685 (1979)) and electro-spiked on Immobilon® membrane (Matsudaira, J. Biol. Chem. 262: 10035-10038 (1987)) before experiments to identify plasminogen and angiostatin binding proteins. Mass spectrometer analysis The plasma membrane proteins were separated on SDS-PAGE gels and the bands of interest were separated from the gels and digested at the site with trypsin. A portion (1/20) of each sample was analyzed by MALDI-MS and the mass spectrometric peptide maps obtained were used to identify the protein in the OWL Protein version 29.6 database (Mann et al., Biol. Mass Spectrom, 22: 338-345 (1993), Pappin et al., Curr. Biol. 3: 327-332 (1993)). Flow cytometry CEVUH and A549 cells were resuspended in ice-cold staining buffer (HBSS, 1 percent BSA, 0.1 percent sodium azide) and incubated on ice for 30 minutes with either rabbit polyclone antiserum grown against the ATP synthase alpha subunit derived from E. coli or with preimmune rabbit serum. Cells were washed with ice-cold spotting buffer and agglomerated in a microfuge at 4 ° C. This wash was repeated twice and the cells were resuspended in cold stain regulator on ice before incubation on ice for 30 minutes in the dark with fluorescein isothiocyanate-conjugated charge anti-rabbit IgG (FITC). After the final wash (as above), the cells were agglomerated and fixed in 10 percent neutral regulated formalin at a density of 10 x 10 cells / milliliter. Control experiments were performed using antibody directed against the alpha subunit of ATP synthase which was preincubated with a 5-fold molar excess of ATP synthase protein from recombinant alpha subunit. The relative mean fluorescence after excitation at a wavelength of 488 nm was determined for each sample on a FACScan flow cytometer (Becton-Dickenson) and analyzed with CellQuest software (Becton-Dickenson). Immunofluorescence microscopy CEVUH and HMVEC were plated at 5xl05 cells / milliliter on glass-covered slides and allowed to adhere during the night. Cells were incubated at 4 ° C for one hour in PBS, pH 7.0 containing 1 percent BSA either with rabbit polyclonal antiserum grown against the ATP synthase alpha subunit derived from E. coli, preimmune rabbit serum, IgG preimmune, or anti-rabbit IgG. The cells were washed and incubated at 4 ° C for one hour in the dark with goat anti-rabbit IgG conjugated with indocarbocyanin (Cy3) before washing and fixed at 4 percent paraformaldehyde. Immunofluorescence microscopy was performed using a microscopic Olympus BX-60 (Olympus Cor., Lake Success, NY). Cloning of the alpha subunit of ATP synthase Polyva A + mRNA from CEVUH was isolated using Oligotex resin (Qiagen). The RNA was reverse transcribed into single-stranded cDNA using AMV reverse transcriptase (Boeh, Mann). The alpha subunit of ATP synthase was amplified by polymerase chain reaction using extended high-fidelity polymerase chain reaction system (Boeh, Mann). The product of the 1.7 kb polymerase chain reaction was purified from an agarose gel of 0.8 percent TAE (Tris-acetate / EDTA) using a gel extraction set (QIAEX II.) Restriction enzyme digestions of the polymerase chain reaction fragment and the pLEl vector were carried out at 37 ° C for one hour, both digests were passed over Qiaquick purification columns, then ligated overnight at 16 ° C using T4 DNA ligase. DH5alpha competent from E. coli (Gibco BRL) were transformed with the ligation mixture, plated on 2xYT agarose plates and cultured overnight at 37 ° C. Colonies were selected for the insert via enzyme digestion Restriction and sequencing of DNA Purification of the ATP BL21DE3 ATP synthase alpha subunit competent from E. coli were transformed with the pLEl vector containing the alpha subunit, plated on 2xYT agarose and cultured overnight at 37 ° C. 20 milliliters of 2xYT (YT = bacto-yeast tryptone) containing 50 micrograms / milliliter of kanamycin were inoculated with a colony and cultured overnight at 37 ° C (200 rpm). One liter of culture (2xYT, 50 micrograms / milliliter of kanamycin) was inoculated with 20 milliliters of the non-induced culture overnight and cultured at 37 ° C at an A of 0.6 at a wavelength of 600 nm. Isopropylthio-b-D-galactosidase (IPTG) was added to the final concentration of 1 mM and cultured for an additional 3 hours. The cells were harvested by centrifugation at 8000 rpm for 10 minutes and stored overnight at -20 ° C. Lysates were prepared under denaturing conditions and the batch was purified using Qiagen Ni-NTA agarose (Qiagen). The resulting protein was dialyzed against PBS, pH 7.0 for use in all experiments. Proliferation assay CEVUH were plated at a density of 5000 cells / well in medium devoid of fetal calf serum overnight to allow the cells to remain still. Fresh medium containing fetal calf serum was added to the wells together with angiostatin at a final concentration of 0.5, 0.75 and 1.0 μm. In some experiments, antibody directed against the ATP synthase alpha subunit derived from E. coli was also added at a 1:10 dilution. A solution of MTS / PMS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl-2H-tetrazolium, internal salt / phenazine methosulfate)) was added. after 24 hours and the absorbance of formazan was quantified in a Thermomax® plate reader at a wavelength of 490 nm according to the manufacturer's specifications (Promega, Madison, Wl). The absorbance values used to calculate the percentage proliferation of the cells ranged from 0.81 for the untreated, 0.60 for the treated ones and 0.47 for the still cells of the baseline. EXAMPLE 1 Competition binding assay To determine whether angiostatin blocks angiogenesis by competitive interaction with the plasminogen receptors of endothelial cells, the effects of angiostatin on the binding of plasminogen to endothelial cells were analyzed. In control experiments, the plasminogen bound to CEVUH in a saturable manner dependent on concentration with an apparent dissociation constant (K ^) of 158 nM and approximately 80, 700 sites per cell (Figure IA), comparable with values previously reported (Hajjar et al., JBC 269 (3): 21191-21197 (1994)). Angiostatin was also linked to CEVUH in a saturable manner dependent on concentration with a similar affinity (Ij of 245 nM), with approximately 38,000 sites per cell (Figure IB). Linkage studies using 125 I-labeled plasminogen and a 100-fold molar excess of unlabeled angiostatin demonstrated no inhibition of plasminogen binding (Figure 2). Similar studies were performed using angiostatin labeled with 125I. The excess of unlabelled plasminogen had little or no effect on the angiostatin binding (Figure 2). In contrast to plasminogen, the angiostatin binding labeled with 125 I with CEVUH in the presence of 100 mM 6-aminocaproic acid (EACA) was only slightly inhibited, suggesting that the binding of angiostatin with endothelial cells is not a process depending on the site of lysine binding. Together, these data indicate the presence of a distinct angiostatin binding site on CEVUH. EXAMPLE 2 Purification of angiostatin binding site from endothelial cells Cell surface proteins participating in plasminogen or angiostatin binding with CEVUH were identified by submitting CEVUH plasma membranes labeled with NHS-biotin with affinity chromatography on plasminogen-sepharose or angiostatin-sepharose. Two distinct bands were identified in Western blot analysis using streptavidin-alkaline phosphatase conjugate (Figure 3A) or by staining with Coomassie bright blue (Figure 3C). A companion stain, tested with an antibody to the known plasminogen receptor, annexin II, demonstrated immunological cross-reactivity with the 44 kDa membrane protein isolated from the plasminogen-sepharose column (Figure 3B, lane 1), but not with the 55 kDa protein isolated from the angiostatin-sepharose column (Figure 3B, lane 2) analysis of ligand staining of affinity-purified plasma membranes using 125 I-labeled plasminogen (Figure 3D, stripes 1 and 2) demonstrated linkage of plasminogen only to the 44 kDa protein and not to the 55 kDa species. These data give additional evidence of the fact that CEVUH contains an angiostatin binding site other than the plasminogen binding protein, annexin II. EXAMPLE 3 Peptide mass fingerprint of the angiostatin binding site To identify the component of the exclusive angiostatin binding site, affinity purified proteins were analyzed by amino terminal sequencing, mass spectrometer analysis and peptide mass fingerprint. Both 44 and 55 kDa proteins were analyzed by reduced SDS-PAGE and digested with trypsin on site (Matsui et al., Electrophoresis 18: 409-417 (1997)). The resulting peptides were extracted and the mass of approximately 30 peptides was determined using a Bruker Reflex MALDI-TOF mass spectrometer, providing a unique signature by which to identify the protein by peptide mass searches (Mann et al., Biol. Mass Spectrom .22: 338-345 (1993), Pappin et al., J. Exp. Med. 180: 273-281 (1994)). The 55 kDa angiostatin binding membrane protein was identified as the alpha / beta subunit of ATP synthase (Table I), whereas the plasminogen binding protein was confirmed as annexin II. Although expression of the beta subunit of ATP synthase has been reported on the surface of several tumor cell lines (Das et al., J. Exp. Med. 180: 273 (1994)), this is the first evidence of the expression on the surface of the ATP synthase subunits on CEVUH. Table I Bruker Reflex mass spectrometer MALDI-TOF analysis of 55 kDa peptides Sequence Peptide mass (monoisotopic) Measure (Da) Calculated (Da) QMSLLLR 859.48 859495 AVDSLVPIGR 1025.58 1025,587 VGLKAPGIIPR 1119.68 1119,713 TIAMDGTEGLVR 1261.40 1261,634 ISVREPMQTGIK 1357.70 1357,739 IMNVIGEPIDER 1384.68 1384,702 AHGGYSVFAGVGER 1405.66 1405,674 FTQAGSEVSALLGR 1434.73 1434,747 TSIADTIINQKR 1471.81 1471,836 EAYPGDVFYLHSR 1552.71 1552,731 VALVYGQMNEPPGAR 1600.79 1600,803 TGAIVDVPVGEELLGR 1623.87 1623,883 LVLEVAQHLGESTVR 1649.88 1649,910 IMDPNIVGSEHYDVAR 1814.85 1814,862 VLDSGAPIKIPVGPETLGR 1918.08 1918,089 AIAELGIYPAVDPLDSTSR 1986.99 1987,026 IMNVIGEPIDERGPIKTK 2009.10 2009.098 IPSAVGYQPTLATDMGTMQER 2265.06 2265.077 EVAAFAQFGSDLDAATQQLLSR 2337.15 2337.160 EXAMPLE 4 Linking of ATP synthase antibody subunit alpha to the surface of CEVU? by flow cytometry and immunofluorescence microscopy To further confirm the surface location of ATP synthase, the CEVUH were analyzed by flow cytometry and immunofluorescence microscopy. A rabbit polyclonal antiserum cultured against the ATP synthase alpha subunit of E. coli reacted with the CEVUH cell membranes as determined by fluorescence activated flow cytometry (Figure 4). Control flow cytometry studies were performed using A549 cells which are known to express the alpha / beta subunits of ATP synthase (Das et al., J. Exp. Med. 180: 273-281 (1994)) (FIG. 4B). A549 cells were also analyzed with anti-alpha subunit ATP antibody synthase preincubated with a 5-fold molar excess of recombinant alpha subunit of ATP protein synthase and showed a decreased binding affinity (Figure 4C). CEVUH were incubated with increasing concentrations of antibody to determine saturation. Figure 4D demonstrates specific, saturable binding of antibody directed against the alpha subunit of ATP synthase on CEVUH membranes. Immunofluorescence microscopy of CEVUH confirmed the immunoreactivity associated on the surface of the antibody of the alpha subunit of ATP synthase on the cell membranes of CEVUH (Figure 5A). Control experiments were performed with secondary antibodies only (Figure 5D), preimmune serum (Figure 5E) and CEVUH permeabilized in the presence of anti-ATP alpha synthase subunit antibody (Figure 5F). Human dermal microvascular endothelial cells were also reacted with antiserum grown against the alpha subunit of ATP synthase (Figure 5C). EXAMPLE 5 Inhibition of angiostatin binding in the presence of the antibody with the ATP synthase alpha subunit polyclonal rabbit antibody cultured against the alpha synthase subunit of ATP blocked the angiostatin binding for CEVUH by 59 percent, demonstrating that this protein functions in a binding of angiostatin (Figure 6). In addition, angiostatin labeled with 125 I linked to purified recombinant alpha subunit of human ATP synthase (Figure 7B) and binding was inhibited by about 56 percent in the presence of a 250-fold molar excess of unlabeled angiostatin (Figure 7C). Complete inhibition of the binding was not obtained and may be due in part to unspecific binding, inadequate folding of the recombinant protein or binding epitopes only found in the presence of the alpha / beta heterodimer. In addition, the binding to the ATP synthase alpha subunit was not inhibited by a 2500-fold molar excess of unlabeled plasminogen (Figure 7D). In addition, the 125 I -labeled plasminogen was not bound to the recombinant alpha subunit of ATP synthase (Figure 7E), but was bound to annexin II (Figure 3D).

EXAMPLE 6 Inhibition of proliferation in the presence of antibody to the alpha subunit of human ATP synthase To determine whether the antiproliferative effects of angiostatin were mediated by ATP synthase binding, proliferation assays were performed in the presence of antiserum grown against the ATP alpha synthase subunit of E. coli. The inhibitory effects of angiostatin on the proliferation of CEVUH were abrogated at approximately 81 percent in the presence of the antibody to the ATP synthase alpha subunit (Table II), providing direct evidence that the angiostatin binding to the ATP synthase alpha subunit functions as a mechanism for the inhibition of endothelial cell growth. These data indicated that this binding site serves as a receptor for angiostatin. • twenty Table II The anti-proliferative effect of angiostatin is reversed by anti-ATP synthase alpha subunit antibody Percentage proliferation inhibited, +/- SEM Concentration of S i n C or n% angiostatin added, μm antibody antibody recovery 0 0 0 0 0.5 10 ± 1.4 1 ± 0.2 90 0.75 25 ± 4.2 5 ± 4.1 80 1.0 23 ± 9.0 6 ± 0.8 74 CEVUH was plated at a density of 5000 cells / well in medium containing angiostatin at a final concentration of 0.5, 0.75 and 1.0 μM. Anti-ATP synthase alpha subunit antibody derived from E. coli was added concomitantly at a 1:10 dilution. MTS / PMS solution was added and the absorbance of the formazan was quantified according to the manufacturer's specifications (Promega, Madison, Wl). The average proliferative effect of pre-immune serum and anti-alpha subunit antibody alone increased 4.6 percent over the control of the regulator. The results represent three separate experiments performed in duplicate with S.E.M. Percent recovery represents the ability of the anti-ATP synthase alpha subunit antibody to block the antiproliferative effect of angiostatin, and thereby restore proliferation to an average of 81 percent of that obtained with the control cells. The ATP synthase is composed of two functional domains called F1 and F0. The F- portion contains multiple subunits (a3ß3? Dc) and acts as the catalytic site for the synthesis of ATP, while the F0 portion immersed in the membrane is a proton channel (Penefsky et al.

Advances in Enzymology and Related Areas of Molecular Biology 64: 173-214 (1991)). Isolated alpha and beta subunits bind to ATP and have weak ATPase activity; without ^ fc 10 However, the synthesis of ATP requires both subunits F1 and F0 (Boyer, Ann. Rev. Biochem, 66: 717-749 (1997)). Endothelial cells represent a strategic role within the vasculature, serving as a barrier between the intravascular compartment and tissues and are frequently exposed to hypoxic tension. In relation to other cell types, endothelial cells are more resistant to hypoxic threat because of their ability to maintain a high level of intracellular ATP (Graven et al., Kidney International 51: 426-437 (1997)). A The ATP synthase associated with the plasma membrane can produce extracellular ATP that can diffuse back into the cell providing an additional, although limited source of ATP (Unno et al., Am. J. Physiol. 270: G1010 (1996), Unno et al., Surgery 121: 668 (1997)). The Angiostatin, by binding to the alpha / beta subunits of the ATP synthase located in the plasma membrane, can interrupt this production of ATP, rendering the endothelial cells more vulnerable to hypoxic threat and eventual irreversible cell damage. In the microenvironment of a growing tumor, tissue hypoxia provides a powerful stimulus for the production of angiogenic growth factors such as VEGF, bFGF, and angiopoietin. The ability of host endothelial cells to respond to these growth factors by increased proliferation probably depends on the ability to survive the hypoxic threat. Abolishing the ability to withstand low oxygen tension, angiostatin can decrease the survival of endothelial cells in the tumor microenvironment. It has recently been reported that angiostatin It can also function by inducing endothelial cell apoptosis, providing an additional independent mechanism for the anti-angiogenic action of this polypeptide (Claesson-Welsh et al., Proc. Natl. Acad. Sci., 95: 5579-5583 (1998)). ). EXAMPLE 7 Antibody link directed against the beta subunit of ATP synthase on the surface of CEVUH by flow cytometry The CEVUH cells were resuspended in the regulator ice-cold spotting (HBSS, 1 percent BSA, 0.1 percent sodium azide) and incubated on ice for 30 minutes with either rabbit polyclonal antiserum raised against beta subunit of ATP synthase derived from E. coli or • pre-immune rabbit serum. The cells were washed with ice-cold spotting buffer and agglomerated in a microfuge at 4 ° C. This wash was repeated twice and the cells were resuspended in cold ice staining buffer before incubation on ice for 30 minutes in the dark with goat anti-rabbit IgG conjugate with fluorescein isothiocyanate (FITC). After the final wash (as above), the cells were agglomerated and fixed in 10 percent neutral regulated formalin at a density of 10 x 10 cell / milliliter. Control experiments were carried out using antibody directed against the beta subunit of synthase of ATP which was preincubated with a 5-fold molar excess of recombinant beta subunit of ATP protein synthase. The relative mean fluorescence after excitation at a wavelength of 488 nm was determined for each sample on a FACScan flow cytometer (Becton-Dickenson and analyzed with CellQuest software (Becton-Dickenson). All the documents cited above are hereby incorporated by reference in their entirety. A person skilled in the art will appreciate from reading this description that various changes in form and detail can be made. do without departing from the true scope of the invention.

Claims (24)

1. NOVELTY OF THE INVENTION Having described the foregoing invention, it is considered a novelty and therefore the content of the following is declared as property: CLAIMS 1. A method for selecting a test compound for its ability to inhibit or increase the binding of angiostatin with ATP synthase comprising: (i) contacting the test compound and angiostatin with ATP synthase, or the angiostatin binding portion thereof, under conditions such that angiostatin can bind to the synthase of ATP, or the angiostatin binding portion thereof, in the absence of the test compound, and (ii) determining the amount of angiostatin bound to the ATP synthase, or the angiostatin binding portion thereof, and comparing that amount with an amount of angiostatin bound to the ATP synthase, or angiostatin binding portion thereof, in the absence of the test compound, characterized in that a reduction in the The angiostatin bound to the ATP synthase, or angiostatin binding portion thereof, in the presence of the test compound indicates that the test compound inhibits the binding of angiostatin with ATP synthase, or the binding portion. of angiostatin thereof, and wherein an increase in the amount of angiostatin bound to the ATP synthase, or angiostatin binding portion thereof, in the presence of the test compound indicates that the test compound increases the binding of the angiostatin with the ATP synthase, or the angiostatin binding portion thereof.
2. 2. The method according to claim 1, characterized in that angiostatin has a detectable label.
3. 3. The method according to claim 1, characterized in that the ATP synthase, or the angiostatin binding portion thereof, is attached to a solid support.
4. 4. The method according to claim 1 wherein the ATP synthase or the angiostatin binding portion thereof is associated with a lipid membrane.
5. 5. The method according to claim 4, characterized in that the membrane is a membrane of an intact cell.
6. 6. The method according to claim 5, characterized in that the cell naturally expresses ATP synthase.
7. 7. The method according to claim 1 wherein the cell has been transformed with a nucleic acid sequence encoding the ATP synthase, or the angiostatin binding portion thereof.
8. 8. A compound identified in the method according to claim 1 of claim 1 • characterized by inhibiting angiostatin binding with the 5 ATP synthase or the angiostatin binding portion thereof.
9. 9. A compound identified in the method according to claim 1, characterized by increasing the angiostatin binding to the ATP synthase or the angiostatin binding portion thereof.
10. 10. A method for selecting a test compound for its ability to modulate a bioactivity resulting from the binding of angiostatin with ATP synthase that 15 comprises: (i) contacting the test compound and angiostatin with a cell expressing ATP synthase, or the angiostatin binding portion thereof, under conditions such that angiostatin can be linked to ATP synthase, or the angiostatin binding portion thereof, in the absence 20 of the test compound, and (ii) determine the amount of angiostatin required to achieve the same bioactivity in the presence of the test compound as in the absence of the test compound, wherein a reduction in the amount of angiostatin required to achieve the same bioactivity in The presence of the test compound indicates that the test compound is an angiostatin agonist., and wherein an increase in the amount of angiostatin required to achieve the same bioactivity in the presence of the test compound indicates that the test compound is an angiostatin antagonist.
11. 11. An angiostatin agonist identified according to the method claimed in claim 10.
12. 12. An angiostatin antagonist identified in accordance with the method claimed in claim 10.
13. 13. The method in accordance with that claimed in dF 10. claim 10 characterized in that the bioactivity is the inhibition of cell proliferation.
14. 14. The method according to claim 10, characterized in that the bioactivity increases the pumping of protons.
15. 15. A method for inhibiting the inhibitory effect of angiostatin angiogenesis in a patient comprising administering to the patient an amount of an angiostatin antagonist that binds to the angiostatin binding portion of the ATP synthase sufficient to effect 20 said inhibition.
16. 16. A method for inhibiting the inhibitory effect of angiostatin angiogenesis in a patient comprising administering to the patient an amount of a soluble angiostatin binding portion of ATP synthase sufficient to 25 perform said inhibition.
17. 17. A method for increasing the inhibitory effect of angiogenesis of angiostatin in a patient comprising administering to the patient an amount of an angiostatin agonist that binds to a binding portion of 5 angiostatin of sufficient ATP synthase to effect said increase.
18. 18. An expression construct comprising a vector and a nucleic acid sequence encoding the ATP synthase subunit a, or a binding portion of ^ fc 10 angiostatin of the same operably linked to a promoter.
19. 19. A host cell comprising the construct of claim 18.
20. 20. A method for producing the a subunit of ATP synthase, or the angiostatin binding portion of the Same, which comprises culturing the host cell according to claim 19 in conditions such that the nucleic acid is expressed and the a subunit of the ATP synthase is produced, or the angiostatin binding portion thereof. , through the above.
21. 21. An antibody specific for the α-subunit of ATP synthase, or an angiostatin-binding portion thereof, or the antigen-binding portion thereof.
22. 22. A kit comprising ATP synthase, or the angiostatin binding portion thereof, and angiostatin, 25 or the truncated form thereof.
23. 23. An isolated complex comprising angiostatin and siritasa ATP, or the angiostatin binding portion thereof. • The complex according to claim 23, characterized in that the complex is attached to a solid support. •