DE10148624A1 - Medicaments useful e.g. for preventing thrombosis, inhibiting transplant rejection or treating chronic inflammatory reactions comprises agents inhibiting binding of oxidized proteins to CD36, - Google Patents

Abstract

Medicaments (A and B) for use in humans or animals are new. (A) contains an agent (I) which inhibits the binding of oxidized proteins to CD36 or the functions of CD36 induced by oxidized proteins. (B) contains at least one oxidized protein (II) or oxidized peptide (or a structural analog or mimetic) An Independent claim is included for the preparation of oxidized proteins/peptides, involving reaction with hypochlorous acid or peroxynitrites.

Description

The oxidative modification of proteins is considered a critical step for the Pathogenesis of various diseases caused by atherosclerosis (Arteriosclerosis), neurodegenerative diseases up to Aging process itself, viewed (Holvoet and Collen, 1997 Curr Opin Lipidol, Witztum and Steinberg 1991, J Clin Invest 88, 1785-92, Smith MA et al., 1996, Nature, Oxidative damage in Alzheimers, Stadtmann ER, Protein Oxidation and Aging, 1992, Science 257: 1220-24). High levels of LDL in the blood are considered the main risk factor for the development of arteriosclerotic (atherosclerotic) Vascular disorders (Brown MS, Goldstein IL, 1986, Science 232: 34-37).

However, there is now overwhelming evidence to believe that LDL is not itself, but its oxidized form is the key trigger for that disease-causing changes that lead to atherosclerosis (Steinberg D, Circulation 95: 1062-71, 1997). U.S. Patent No 5,756,067 describes the measurement of cholesterol, triglycerides and Lipoproteins as risk markers for developing atherosclerosis is not yet sufficient, since about half of all heart diseases due to atherosclerosis occurs in patients who are normal Plasma have triglyceride and normal cholesterol levels and because of that angiographic detection of atherosclerosis also in patients with normal lipid levels can be managed. Others not yet published Processes must therefore be the cause of the development of arteriosclerosis be involved.

The oxidation of the lipids in the LDL, either in vitro e.g. B. by Copper-induced oxidation, or in vivo, leads to the formation of reactive Aldehydes (WO 98/59248). Uptake of oxLDL by macrophages leads to the formation of so-called foam cells, a process which is called initiating step in the development of arteriosclerosis is considered (WO 98/59248).

The oxidation of the lipid portion in the LDL is considered to be responsible for this process. For this reason, almost all scientists use oxLDL to investigate the development of arteriosclerosis by adding CuSO ₄ or using malondialdehyde, an end product of lipid peroxidation. The concentration of oxLDL in the plasma of patients with coronary, primary heart disease or transplantation-associated coronary heart disease correlates strongly with the progression of the disease, whereas no elevated oxLDL levels can be measured in healthy control persons (Holvoet et al. Circulation 98: 1487-94, 1998 ). Chronic kidney disease and graft rejection are also associated with high oxLDL levels (Holvoet, Collen Thromb Haemost 76 (5): 663-9, 1996 + ATVB 18 (1) 100-7, 1998).

The oxidation of the protein content in the LDL can also cause physiological / pathophysiological changes. So led delipidated HOCl-oxLDL for induction of the oxidative burst in Macrophages (Nguyen-Khoa et al., BBRC 263: 804-9, 1999) and HOCl-oxLDL caused platelet aggregation (Volf et al. ATVB 20 (8): 2011-18, 2000).

Reactive oxidants are released by the body through phagocytes and play an important role in defense against pathogens tumor monitoring and all inflammatory processes (Bablor, NEJMed 298: 659-663, 1978, Weiss J, NEJMed 320: 365-376, 1989). In addition to "professional" phagocytes, such as granulocytes and monocytes, can also other cells, such as. B. endothelial cells or smooth Muscle cells produce and release reactive oxidants.

These oxidizing substances include O ₂ , superoxide, hydrogen peroxide, peroxynitrite, OH radicals, hypochlorous acid HOCl, Cl ₂ gas and chloramines. It is still unclear which processes are involved in the creation of these oxidative substances. Ceruloplasmin, 15-lipoxygenase, myeloperoxidase (MPO) and nitric oxide synthase (NO-S) have been found in arteriosclerotic lesions in animals and humans and could contribute to the oxidation of LDL (Carr et al., ATVB 20: 1716-23, 2000 ). A possible oxidation path involves the MPO. MPO, a heme protein enzyme, can halogenate and peroxidize (Carr et al.). The best-described product of myeloperoxidase is hypochlorous acid HOCl ^- , Cl ^- + H ₂ O ₂ + H ⁺ → HOCl + H ₂ O. Hypochlorite-modified proteins, especially HOCl-oxLDL, are found in arteriosclerotic lesions (Hazell et al., J Clin Invest). HOCl modification of proteins also plays a role in other diseases, such as B. Inflammatory joint diseases (Davies et al., Free-Radical-Biol-Med 15 (6): 637-43, 1993), coagulation disorders (oxidation of thrombomodulin) (Glaser et al., J Clin Invest 90 (6): 2565 -73, 1992), tissue destruction by granulocytes in inflammatory reactions in general (Schraufstatter et al., J Clin Invest 85 (2): 554-62, 1290), ischemia, reperfusion damage (Samanta et al., Free Radic Res Commun 7 (2) : 73-82, 1989), glomerulonephritis (Shah et al., J Clin Invest 79 (1): 25-31, 1987), immune regulation (NK-cell apoptosis) (Hansson et al., J Immunol 156 (1) : 42-7, 1996).

CD36, also glycoprotein IIIb (GPIIIb) or glycoprotein IV (GP IV) is a major glycoprotein of platelets, endothelial cells, Monocytes, erythroblasts, epithelial cells and some tumor cell lines, such as Melanoma cells and osteosarcoma cells (Asch et al 1987, Knowles et al. 1984, Kieffer et al 1989). It belongs to the class B family "scavenger" Receptors. Family members are the integral lysosomal Membrane protein LIMP-II (lysosomal integral membrane protein II, Vega et al 1991) the CLA-1 (CD36 and LIMP-II analogous, Calvo and Vega 1993), the FAT protein of the adipocyte membrane (Abumrad et al 1993), PAS IV from breast epithelial cells (Greenwalt et al 1990 and 1992) and the SR-B1 (Acton et al 1994).

The adipocyte's FAT protein is involved in binding and transport long chain fatty acids involved. The PAS IV protein is an integral one Membrane protein of lactating breast epithelial cells and is found in the apical Plasmalemma concentrated. With the secretion of triglycerides it succeeds into the milk and is in the MFGM (milk-fat globule membrane) fraction found. The sequence of PAS IV is almost identical to CD36, it lies but differences in glycosylation.

SR-B1 is a "scavenger receptor" for LDL (Acton et al 1994). CD36 consists of a single, highly glycosylated polypeptide chain with an average molecular weight of 88000 in the reduced and not reduced state and an isoelectric range between 4.4 and 6.3 (McGregor et al 1980, Clemetson 1987). That no clearly defined Isoelectric point can be determined, is due to the variable content Sialic acid (McGregor et al 1981). The carbohydrate content of 26% and strong hydrophobicity gives the CD36 a high resistance to Degradation by proteases as long as the protein is in the membrane (Greenwalt et al 1992). This causes the protein to spread to regions where inflammatory processes take place is protected from "attacks". CD36 has N- and O-glycosidic bonds. The placental cDNA for CD36-derived amino acid sequence (Oquendo et al 1989) shows several hydrophobic areas and probably two transmembrane areas.

Some functions have been postulated for CD36.

It has been described as a receptor for collagen (Tandon et al 1989). Purified CD36 binds to type I, and collagen fibrils Fab fragments of a polyclonal antibody against CD36 inhibit the Collagen-induced aggregation.

However, analyzes of platelets that lack the CD36 show that CD36 is not absolutely necessary for the activation of platelets with collagen. Our joint investigations with colleagues from the working group of JJ Sixma (Utrecht) showed no differences in the adhesion of CD36-deficient platelets and control platelets to bovine or human collagens type I or III in the static system and under the influence of low, medium and high shear rates in the Perfusion chamber when using heparin blood and at physiological Ca ²⁺ concentrations (Saelman et al 1994).

CD36 deficient platelets aggregate normally with hormone collagen, one Mixture of equine type I and III collagen, and with purified bovine and human collagen types I and III (Kehrel et al 1991 and 1993). The Secretion of the α-granules and dense granules induced by type I or III Collagen is no different in CD36 deficient platelets and Control platelets (Kehrel et al 1993). Daniel and co-workers showed that signal transmission after activation with type I collagen CD36 deficient platelets and control platelets run the same way (Daniel et al 1993).

CD36 is a receptor for thrombospondin-1 (TSP-1) (Asch et al 1987, McGregor et al 1989). CD36 threonine (92) is phosphorylated on resting platelets. Dephosphorylation enables the binding of thrombospondin (Asch, Science 1993). Purified CD36 specifically binds to thrombospondin. This binding is dependent on Ca ²⁺ and cannot be inhibited by RGD peptides. The monoclonal antibody OKM5, which is directed against CD36, inhibits the binding of thrombospondin to platelets activated with thrombin (Asch et al 1989). Leung and co-workers described that two peptide regions on the CD36 influence the binding of thrombospondin. Peptide 139-155 enhances platelet-rich platelet aggregation induced by ADP or collagen. Peptide 93-110, on the other hand, partially inhibits collagen-induced aggregation and also blocks the binding of CD36 to immobilized thrombospondin. This peptide cannot bind thrombospondin alone, but in the presence of peptide 139-155 (Leung et al 1992, Pearce et al 1993). The SVTCG sequence of thrombospondin binds to CD36 with high affinity (Li et al 1993). Silverstein et al (1992) demonstrated by experiments with "sense" and "antisense" transfected melanoma cells the importance of CD36 for thrombospondin binding. The binding site on CD36 for thrombospondin is between amino acid 93-120 (Frieda et al 1995).

CD36 is also described as a binding mediator between platelets, endothelial cells, monocytes or C32 melanoma cells on the one hand and erythrocytes infected with the malaria parasite Plasmodium falciparum on the other (Barnwell et al 1989). The binding of infected erythrocytes to capillary endothelial cells, called sequestration, is of crucial importance for the often fatal outcome of malaria tropica when sequestration takes place in the brain (celebrated malaria). Infected erythrocytes bind to immobilized, purified CD36 (Ockenhouse et al 1989). COS cells in which the cDNA for CD36 was expressed acquire the ability to bind malaria-infected erythrocytes (Oquendo et al 1989). With the help of anti-idiotype antibodies against the monoclonal anti CD36 antibody OKM5, a binding partner on infected erythrocytes, the sequestrin, was found (Ockenhouse et al 1991). Contact with infected erythrocytes activates platelets and monocytes (Ockenhouse et al 1989). In the hands of Tandon et al (1991), CD36-deficient platelets show no binding ability for infected erythrocytes. In contrast, we observed that the binding is only disturbed in the presence of EDTA. In the presence of Ca ²⁺ and Mg ²⁺ , we clearly observed rosetting of Plasmodium falciparum-infected erythrocytes and CD36-deficient platelets (Kronenberg et al 1992). In addition to CD36, other binding mediators for malaria-infected erythrocytes have been described, including thrombospondin, which requires divalent cations for this function (Berendt et al 1989, Roberts et al 1985). Thrombospondin could mediate the observed binding of CD36 deficient platelets to infected erythrocytes. The binding of Plasmodium falciparum-infected erythrocytes to CD36 is inhibited by the CD36 peptides 145-171 and 156-184 (Baruch et al 1999). The role of the CD36 in signal transmission is also frequently discussed (Shattil and Brugge 19919). In the immunoprecipitation of CD36 from resting platelets, the tyrosine kinases of the src gene family pp60 ^fyn , pp62yes and pp54 / 58 ^{lyn are also} precipitated, which suggests a close association with the CD36 (Huang et al 1991). The importance of this discovery is still unclear. Some antibodies against CD36 activate platelets and monocytes (Aiken et al 1990, Schüepp et al 19919). The IgM antibody NLO7 activates platelets using the complement system (Alessio et al 1993). As a further function of CD36, a role in thrombotic thrombocytopenic purpura (TTP) is described (Lian et al 1991). A protein (p37), which occurs in the plasma of TTP patients, agglutinates platelets, mediated via CD36. The meaning of this finding is still unknown. The peptide VTCG from thrombospondin inhibits phosphatidylinositol (3.4) biphosphate synthesis in platelets activated with thrombin. CD36 is one of the thrombospondin receptors that mediate late activation of PI-3 kinase and phospholipase C (Trumel, Payrastre, Mazarguil, Chap, Plantavid, personal communication).

CD36 is involved in the transport of long chain fatty acids in Muscle tissue cells.

Overexpression of CD36 in muscle cells from transgenic mice led to increased cellular intake of fatty acids, increased the Fatty acid oxidation by contractile muscles and reduced the Concentration of triglycerides and free fatty acids in plasma. The Mice had a lower body weight, especially a lower one Body fat than her control relatives.

Absence of CD36 in humans leads to loss of intake of long chain fatty acids, the main energy source for the heart muscle, in the Cardiac muscle cells and consequently increased occurrence of congenital hypertrophic cardiomyopathies (Fuse et al 1998). Even CD36 deficient Mice show a defective transport of fatty acids into the cells and an impaired lipoprotein metabolism (Febbraio et al 1999).

CD36 mediates arachidonic acid mediated platelet aggregation (Dutta-Roy et al 1996) and binds to negatively charged phospholipids in Cell membranes (Ryeom et al 1996). In particular phosphatidylserines (PS) and phosphatidylinositol (PI) are specifically high with CD36 Affinity bound (Rigotti et al 1995). Because apoptotic cells Exposing phosphatidylserines on their surface can be via CD36 Contact with phagocytotic cells (Fadok et al 1998, Alberts et al 1998). Phosphatidylserine probably binds to the CD36 sequence 162-183 (Yamaguchi et al 2000). CD36 connects Cholesteryl hemisuccinate and can be easily cleaned through this reaction become (Kronenberg et al 1998).

The main function of CD36 is probably its role as a receptor for oxidized LDL. This role was first described by Endemann et al in 1993. Transfection experiments with a cDNA clone, which codes for CD36, in the human macrophage-like THP cell line led to a newly acquired binding ability of the cell for Cu ²⁺ -oxidized LDL. The monoclonal CD36-specific antibody OKM5 inhibits the binding of Cu ²⁺ -oxLDL to platelets by 54%. The binding site for Cu ²⁺ -oxLDL is in the region of CD36 sequence 155-183 (Puente-Navazo et al 1996).

Nicholson et al described that Cu ²⁺ -oxLDL presumably binds to CD36 via its lipid portion (Nicholson et al 1995). Macrophages from blood donors who lack CD36 on the monocytes have a significantly reduced (-40%) uptake of oxLDL compared to controls (Nozaki et al 1995).

Vitamin E (alpha-tocopherol) inhibits the absorption of Cu ²⁺ -oxLDL in smooth muscle cells of the aorta by inhibiting CD36 (Ricciarelli et al 2000). The binding of oxLDL to mouse CD36 is partially carried out by oxidized phospholipids, which are associated with lipid and protein content (Boullier et al 2000). Recently it was found that CD36 is not only the receptor for Cu ²⁺ -oxLDL, but also for NO ₂ -LDL and that CD36 is responsible for the formation of NOz-LDL mediated foam cells (Podrez et al 2000).

This bond is caused by lipid oxidation products from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholinekompetitivinhibiert. The authors therefore speculate that the myeloperoxidase catalyzed Peroxidation of lipids serves to target targets containing phospholipid to mark the phagocytosis by cells containing CD36. In harmony with its role as a receptor for oxLDL is CD36 loaded with lipids Macrophages found in arteriosclerotic plaques (Nakata et al 1999) and smooth muscle cells can be expressed in vivo by expression of CD36 Foam cells (Ohya et al 2000).

The lack of CD36 appears to protect against arteriosclerosis. So develop mice that lack the ApoE protein on an appropriate diet arteriosclerotic plaques. If these mice are also given the CD36 gene knocked out, the mice develop under the same conditions as theirs Control relatives containing CD36 were 76% less arteriosclerotic Aortic tree lesions on a high-fat diet and 45% fewer plaques in the aortic sinus with a normal diet.

Macrophages from CD36 and ApoE double knockout mice internalized more than 60% less copper-oxidized LDL and NO ₂ -LDL (Febbraio et al 2000).

A blockade of thrombotic and arteriosclerotic functions of CD36 while not influencing the important CD36 mediated uptake of long chain fatty acids into the cell would be a important step forward to fight vascular diseases.

This object has been achieved by the present invention. In the context of the present invention it was found that Cell functions that oxLDL triggers via CD36, also from others Substances can be triggered. Surprisingly, it is these other substances are oxidized proteins that do not Must have lipid content. Proteins are used in the body to fight infection, or in arteriosclerotic plaques, or in acute and chronic Inflammation oxidized.

• 1. Aktivierung von Thrombozyten
  • - einerseits Thrombosen, Herzinfarkt, Schlaganfall, aber andererseits auch Blutstillung
• 2. Schädigung von Endothelzellen
  • - Ischämie, Entzündungsreaktionen, Ödembildung, Störung der Prostacyclin-Freisetzung
• 3. Aktivierung von Leukozyten, z. B.
  • a) Erhöhte Adhäsion von Leukozyten an Endothelzellen
  • b) Förderung der Transmigration von Leukozyten durch Endothel und Epithelien
  • c) "Homing" von Leukozyten in arteriosklerotische Plaques
  • d) "Priming" und Auslösen des oxidativen Burst in Phagozyten
  • e) Tissue Faktor Expression auf Monozyten, insbesondere Verstärkung der durch Lipopolysaccharid (LPS) ausgelösten Reaktion
  • - Schäden durch Entzündungsreaktion, Thrombosen, etc.
• 4. Aktivierung von glatten Muskelzellen (SMC's)
  • a) Proliferation von SMC's
  • b) Intimaverdickung in arteriosklerotischen Plaques
  • - Reocclusion nach Bypass, Stent, PTCA; Fortschreiten von Arteriosklerose
• 5. Stimulation der Renin-Freisetzung aus juxta-glomerulären Zellen
  • - Renin-abhängiger Bluthochdruck bei Nierenerkrankungen
• 6. Bildung von Schaumzellen über Stimulation der Aufnahme von co-inkubiertem LDL durch Makropinozytose
  • - Entwicklung/Verstärkung von Arteriosklerose
• 7. Apoptose von Gefäßzellen im Zentrum von arteriosklerotischen Läsionen
  • - Nekrose, Plaque-Ruptur
• 8. Stimulierung der Expression von Matrix-Metalloproteinasen in Endothelzellen
  • - Stimulation der Ruptur von arteriosklerotischen Plaques.

The following are examples of some reactions that are triggered not only by oxLDL, but also by other oxidized proteins via CD36:

• 1. Activation of platelets
  • - Thrombosis, heart attack, stroke on the one hand, but also hemostasis on the other hand
• 2. Damage to endothelial cells
  • - Ischemia, inflammatory reactions, edema formation, disruption of prostacyclin release
• 3. Activation of leukocytes, e.g. B.
  • a) Increased adhesion of leukocytes to endothelial cells
  • b) Promotion of transmigration of leukocytes through endothelium and epithelia
  • c) "Homing" of leukocytes in arteriosclerotic plaques
  • d) "Priming" and triggering the oxidative burst in phagocytes
  • e) Tissue factor expression on monocytes, in particular enhancement of the reaction triggered by lipopolysaccharide (LPS)
  • - Damage caused by an inflammatory reaction, thrombosis, etc.
• 4. Activation of smooth muscle cells (SMC's)
  • a) Proliferation of SMC's
  • b) Intimal thickening in arteriosclerotic plaques
  • - Bypass, stent, PTCA reocclusion; Progression of atherosclerosis
• 5. Stimulation of renin release from juxta-glomerular cells
  • - Renin-dependent hypertension in kidney diseases
• 6. Formation of foam cells by stimulating the uptake of co-incubated LDL by macropinocytosis
  • - Development / reinforcement of arteriosclerosis
• 7. Apoptosis of vascular cells in the center of arteriosclerotic lesions
  • - necrosis, plaque rupture
• 8. Stimulation of the expression of matrix metalloproteinases in endothelial cells
  • - stimulation of rupture of arteriosclerotic plaques.

In addition, it was found in this invention that not only IDAAT (immune defense activated antithrombin, patent application No. DE 100 45 047.4) binds to HIV GP120 and CD4, but also other oxidized Proteins.

It was also found in this invention that not only IDAAT, but other oxidized proteins also bind thrombospondin on cells such as platelets, monocytes, endothelial cells and T cells cause. It is therefore imperative to assume that oxidized proteins generally induce thrombospondin-mediated cell responses, such as that Inhibition of angiogenesis, the defense against HIV infection Regulation of inflammatory processes by e.g. B. "Downregulation" of IL-12 in monocytes and "upregulation" of IL-10. With that, too Oxidized proteins themselves therapeutically in certain disease processes have a useful effect.

Furthermore, it was found in the context of this invention that the pathological cell functions caused by oxidized proteins by substances that oxidized the interaction between CD36 and Inhibit proteins or compete with TSP bound to CD36 can be inhibited.

Soluble thombospondin-1 and the monoclonal anti CD36 antibodies clone 37, 13 manufactured by us and 7.

Examples 1. Isolation of human thrombospondin-1

Human thrombospondin-1 was isolated from platelets as described in Kehrel et al 1991. In deviation from this, the platelets were activated with thrombin and EDTA in the wash buffer was replaced by Na citrate (0.08 M). In addition, Ca ²⁺ was added to the aggregation buffer and all buffers for the subsequent purification steps in a concentration of 2 mM.

2. Hybridoma culture for the production of monoclonal antibodies

The monoclonal antibodies against CD36 were largely after the Instructions by Peters and Baumgartner (1990).

8-12 week old Balb / c female mice were immunized with purified CD36 (50 µg / Boost). The long immunization schedule (4 months) according to Baumgartner et al 1990 was used for the immunization. Approximately 14 days before the planned fusion date, blood was taken from the animals and the IgM and IgG titer against CD36 in the serum of the mice was determined. If the IgG titer clearly differed from the control at a dilution of 1: 100,000, the spleen cells were fused with Ag 8,653 cells. The Ag 8,653 cells were selected with 0.13 M 8-azaguanine in the culture medium. Lymphocytes from the spleen were prepared and fused with Ag 8.653. Fused cells (1.10 ⁶ spleen cells / ml) were incubated for 24 hours directly after the fusion in selection medium (culture medium with the addition of hypoxanthine, 27.2 μg / ml and azaserine (50 μg / ml)) in a cell culture bottle (75 ml). The macrophages from the spleen adhere to the plastic surface and no longer interfere with the actual culture of the hybridomas in a 96-well plate.

The cloning steps were carried out by the dilution method ("limited-dilution-cloning") with a sowing probability of 0.5 cells per hole of the 96-well plate according to Würzner 1990. Supernatants from hybridomas were tested for the production of IgG or IgM in the ELISA method. IgG-positive cell culture supernatants were tested for their specificity against CD36 using several methods:
19 clones specific for CD36 were obtained which did not react with CD36 deficient platelets (description: Kehrel et al 1993, Saelman et al 1994, Kehrel et al 1995), but showed a clear connection to control platelets. This has been demonstrated both by flow cytometry and by the dot blot method and immunoprecipitation. All antibodies recognized purified CD36.

Three of these clones (clones 37, 13 and 7) inhibited the reaction of oxidized Proteins with human cells (see below).

Isolation of CD36

CD36 was isolated as by our research group (Kronenberg et al 1998), on phase separation of the Membrane proteins with Triton X-114 and subsequent Affinity chromatography on cholesteryl hemisuccinate agarose.

Examples of the production of oxidized proteins

348 pg protein (commercial fibrinogen, human albumin, Bovine serum albumin (BSA) or antithrombin) were mixed with 832 µg NaOCl (Sodium hypochlorite) in 1 ml phosphate buffered saline with EDTA addition (0.1 mM) incubated on ice for 10 minutes. Protein and oxidant were immediately after the end of the reaction by gel filtration at 4 ° C. over Sepharose G25-Coarse (PD-10 column, Amersham Pharmacia) separated.

Examples of the effect of the invention

• 1. Oxidized proteins (oxFibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) bind specifically to immobilized CD36.
• 2. Oxidized proteins activate platelets. This activation is inhibited by substances that inhibit the interaction of CD36 with oxidized proteins or compete with thrombospondin bound to CD36
  • 1. Oxidized proteins (oxFibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) increase the adhesion of platelets to adhesion proteins such as thrombospondin, vitronectin, fibrinogen, fibrin, fibronectin and collagen depending on the dose (see Fig. 2a).
    This increase in adhesion is inhibited by soluble thrombospondin-1 (see Fig. 2b). This adhesion enhancement is inhibited by the monoclonal anti CD36 antibodies clones 37, 13 and 7 (see Fig. 2c / d), while the VCTG peptide, which inhibits the binding of thrombospondin to CD36, has no influence (see Fig. 2e). The commercial anti CD36 antibody FA6 / 152 shows no inhibitory effect (see Fig. 2f).
  • 2. Oxidized proteins (oxFibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) induce fibrinogen binding to platelets in the presence of thrombospondin-1 (10 µg / ml) (see Fig. 3a). This activation is inhibited by soluble thrombospondin-1 in a concentration of> 20 µg / ml (see Fig. 3b).
    This activation is inhibited by the monoclonal antibodies against CD 36, clones 37, 13 and 7 (see Fig. 3c-e).
  • 3. Oxidized proteins (oxEibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) trigger platelet aggregation in the presence of thrombospondin-1 (10 µg / ml) (see Fig. 4a). This aggregation is inhibited in a dose-dependent manner by the monoclonal antibodies against CD 36, clones 37, 13 and 7 (see Fig. 4b).
  • 4. Oxidized proteins (oxEibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) trigger the procoagulant state of the platelets in the presence of thrombospondin-1 (10 µg / ml) (see Fig. 5a-c) and lead to the formation of microparticles (see Fig. 5d ). This activation is inhibited by soluble thrombospondin-1 in concentrations of ≥ (20 µg / ml) (see Fig. 5e).
    This activation is inhibited by the monoclonal antibodies against CD 36, clones 37, 13 and 7, depending on the dose (see Fig. 5f and g).
• 3. Oxidized proteins (e.g. oxFibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) activate monocytes. This activation is by substances such as. B. soluble thrombospondin or monoclonal antibodies against CD36, which inhibit the interaction between CD36 and oxidized proteins or compete with thrombospondin bound to CD36, inhibited in a dose-dependent manner.
  • a) Oxidized proteins (e.g. oxFibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) induce a Ca ²⁺ signal in monocytes (see Fig. 6).
  • b) Oxidized proteins (e.g. oxFibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) induce the oxidative burst in PMNL (see Fig. 7).
  • c) Oxidized proteins (e.g. oxFibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) induce increased transmigration of monocytes, PMNL and lymphocytes through endothelial monolayers (see Fig. 8a). This reaction is inhibited by substances that inhibit the binding of CD36 to oxidized proteins, such as soluble thrombospondin or monoclonal antibodies against CD36, clones 37, 13 and 7 (see Fig. 8b).
• 4. Oxidized proteins (e.g. oxFibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) are causally involved in the development of arteriosclerosis.
  • a) Oxidized proteins (eg oxEibrinogen, oxAntithrombin III, oxBSA, oxHumanalbumin) induce "homing" of macrophages in arteriosclerotic plaques. The "homing" was carried out according to Patel et al (Circulation 1998; 97, 75-81). C57BL / 6 mice were stimulated by intraperitoneal injection of thioglycolate to migrate monocytes / macrophages into the peritoneum. After 4 days the peritoneum was washed and activated peritoneal macrophages were obtained. The erythrocytes in the samples were lysed. Macrophages were taken up in RPMI 1640 medium and transferred to cell culture plates to allow them to adhere. Fluorescence-labeled microspheres (2 µm yellow-green fluorescent latex microspheres, Molecular Probes) were opsonized for 30 minutes with 50% mouse serum for better absorption by the phagocytes and then added to the plated macrophages. The adherent macrophages phagocytized the microspheres. Non-adherent cells and microspheres were removed by washing the plate. Labeled macrophages were detached from the plate on ice and resuspended in "Hanks balanced salt solution". 50-week-old ApoE-deficient mice were injected intraperitoneally 3 times with 50 µg of oxidized protein, here oxidized antithrombin III, or placebo (only HBSS); 6 hours before intravenous injection of labeled macrophages, 2 hours after macrophage injection and 10 hours after macrophage injection. 10 × 10 ⁶ macrophages were suspended in 0.2 to 0.3 ml HBSS and injected into the tail vein. The animals were sacrificed 24 hours after macrophage injection. The heart base and the ascending aorta were embedded in OCT, stored at -80 ° C and 7 µm thick cryosections made. The fluorescence-labeled macrophages from 140 serial sections with the mouse from a 1 mm wide area of the ascending aorta at the level of the Valsalva sin were counted. Compared to the placebo-treated ApoE ^{- / -} control mice, the migration of labeled macrophages into the arteriosclerotic plaques in mice treated with oxidized antithrombin III increased significantly from 100 ± 15% (n = 14) to 156 ± 9.2% (n = 5) p = 0.008) ("alternate which test"). Since this immigration is causally linked to the formation, progression and risk of rupture of the arteriosclerotic plaque, this result illustrates the danger of oxidized proteins with regard to arteriosclerosis ( Fig. 9).
  • b) Substances that inhibit the interaction between CD36 and oxidized proteins or compete with thrombospondin bound to CD36 prevent / reduce the proarteriosclerotic effect of oxidized proteins. Soluble thrombospondin inhibits the adhesion of macrophages to arteriosclerotic endothelial cells. This is the prerequisite for the immigration of macrophages into arteriosclerotic plaques. Murine immortalized endothelial cells were stimulated with β-VLDL (50 µg protein / ml) from plasma from ApoE-deficient mice for 6 hours and then with 2 × 10 ⁵ peritoneal monocytes / macrophages per ml at a shear rate of 400 s ^-1 in a flow chamber superfused. β-VLDL induced a significant relative increase in rolling leukocytes of 224 ± 44% compared to the control. Addition of 10 µg / ml soluble thrombospondin completely inhibited this increase in adhesion and also partially inhibited the basic adhesion of the macrophages to the endothelium (75 ± 37% adhesion compared to the control, n = 4-7; p <0.01) (see Fig. 10a) , The permanent adhesion of the macrophages clearly induced by the β-VLDL after a 5-minute washing period was also reduced by thrombospondin-1 (100 ± 21% control vs 300 ± 119% β-VLDL vs 157 ± 70% β-VLDL + TSP- 1; n = 4-7; p <0.01) (Fig 10b)..
• 5. Soluble thrombospondin-1 inhibits inflammatory reactions in vivo. An arthus reaction was triggered in the ear of Balb / c mice by local injection of anti-BSA at time 0 and simultaneous injection of FITC-coupled BSA into the peritoneum. Control animals (negative controls) were only injected with FITC (without BSA) into the peritoneum. After about 6 hours there was a pronounced inflammatory reaction with swelling of the ear (edema), FITC storage, immigration of PMNL and petechial bleeding into the tissue in the animals treated with anti-BSA and BSA-FITC. In addition, 18 mice were injected intraperitoneally with 50 µg thrombospondin-1 in buffer (PBS) at time 0 and after 0 + 3 hours. 16 control mice received only PBS at 0 + 3 hours. The arthus reaction was almost completely prevented by thrombospondin. Mice treated with thrombospondin showed significantly lower FITC deposits, significantly lower ear thicknesses (fewer edema) and almost no petechiae compared to PBS-treated control animals (see Fig. 11a-c).
• 6. Examples for the quantification of oxidized proteins Production of monoclonal antibodies that recognize epitopes on proteins or peptides that are directly or indirectly altered by oxidation processes. Human albumin, antithrombin III and fibrinogen were, as described in this invention, oxidized with HOCl and immunized 8-12 week old female Balb / c mouse. Hybridoma production and culture was carried out using the classic method. Supernatants from hybridomas were tested for the production of IgG or IgM. IgG-positive cell culture supernatants were tested for a positive reaction with oxidized protein and a negative reaction with the unchanged starting protein. Clones that produced antibodies against oxidized protein (oxHumanalbumin, oxAntithrombin III, oxFibrinogen) and at the same time did not react with unoxidized mother protein were tested for cross-reaction with oxidized other proteins and peptides.
  Quantification of oxidized proteins using monoclonal antibodies as described above:
  Such quantification is easily possible using methods such as ELISA, RIA, quantitative flow cytometry on cell surfaces and similar routine methods.
  z. E.g .: quantification of oxidized human albumin using ELISA. A rabbit polyclonal antibody to human albumin (production - routine method) was bound as a capture antibody to the bottom of an ELISA plate (Nung-Maxisorb). The plate was washed thoroughly with PBS pH 7.4, 0.5% Tween 20 and places on the plastic surface were blocked with 3% BSA for 1 hour at room temperature (RT). The plate was washed again and then incubated for 1 hour at RT with differently diluted plasmas, sera, supernatants from blood products (e.g. platelet concentrates, erythrocyte concentrates, FFP) or buffer solutions to which oxHumanalbumin was added in a defined amount. Sample material or standard solutions were removed, the plate was washed thoroughly and incubated with the monoclonal antibody described above, which recognizes oxidized human albumin and which had been labeled with biotin, in a dilution of 1: 15000 in PBS, 1% NGS (normal goat serum) , The plate was again thoroughly washed several times and incubated with streptavidin peroxidase (1 hour, RT). After washing the plate again, it was washed with substrate solution (100 μl / well) (20 mg ortho-phenyldiamine, 5% H ₂ O ₂ in a buffer of 12.15 ml 0.1 M citric acid and 12.85 ml 0.2 M Na ₂ HPO ₄ plus 25 ml H ₂ O dest.) Added. The absorbance at 405 nm measured in the ELISA photometer reflects the amount of oxidized human albumin. The reaction was stopped with 50 μl / well 4n H ₂ SO ₄ and the absorbance was measured at 490 nm.
  HOCl oxidized human albumin in the measurement sample was quantified using the calibration series. The procedure was analogous for HOCl-modified fibrinogen and HOCl-modified antithrombin III.
• 7. Example of the detection of the activation state of the receptor for oxidized proteins, CD36
  Production of polyclonal antibodies against threonine (92) phosphorylated CD36.
  The peptides (15 AS) (1) Lys, Gln, Arg, Gly, Pro, Tyr, Thr, Tyr, Arg, Val, Arg, Phe, Leu, Ala, Lys and (2) Lys, Gln, Arg, Gly, Pro, Tyr, PhosphoThr, Tyr, Arg, Val, Arg, Phe, Leu, Ala, Lys (like peptide (1) but phosphorylated on threonine) were synthesized and coupled to KLH (Keyhole Limpet Hemocyanin). Rabbit polyclonal antibodies were prepared with these coupled peptides by standard procedures. For this purpose rabbits (New Zealand, white) were immunized with 250 pg peptide 1-KLH or peptide 2-KLH plus addition of complete Freund's adjuvant sc and with 250 pg peptide 1-KLH or peptide 2-KLH with addition Alu-Gel-S (1.3% aluminum hydroxide in water, SIGMA) "boosted" 3 times. Blood was taken from the ear vein, serum was prepared and this was tested for antibodies against the peptides used for immunization. The antibody against the phosphorylated CD36 peptide was cross-absorbed on an affinity column with non-phosphorylated CD36 peptide, so that the antibody mixture obtained only contained antibodies which specifically recognize phosphorylated, unactivated CD36 (AK CD36P).
  (The production of specific monoclonal antibodies against threonine-phosphorylated / -dephosphorylated CD36 is possible with these peptides analogously to the production of anti-CD36 protein AK described).
  Both polyclonal antisera reacted with CD36 on the platelet surface in flow cytometry. While antibody CD36P against phosphorylated CD36 recognizes the CD36 preferentially on non-activated platelets, antibody "CD36 total" recognizes non-phosphorylated CD36 and phosphorylated CD36. When platelets are activated, CD36 is dephosphorylated and the binding capacity for antibodies "CD36P" decreases. Quantitative flow cytometry with both antibodies enables the proportion of activated CD36 to be calculated.
• 8. The organism of patients with type I diabetes is particularly sensitive to oxidized proteins:
  z. For example, platelets from patients with type I diabetes are more sensitive to oxidized protein than agonists than platelets from healthy control subjects. Activation-dependent fibrinogen binding can be carried out on platelets from diabetics with lower concentrations of ox. Trigger protein than on platelets from control subjects (see Fig. 13).
• 9. Oxidized proteins (oxAntithrombin III and oxHumanalbumin were tested) bind with high affinity to both HIV-GP120 and its receptor CD4.
  z. For example: The binding of oxidized antithrombin III and oxidized human albumin to HIV-GP120 and CD4 was demonstrated in the BIACORE 2000 system. Running buffer: 25 mM Tris; 100 mM NaCl pH 7.4; 1 mM CaCl ₂ ; 1 mM MgCl ₂ ; 0.005% surfactant P-20.
  Protein HIV-GP120: c = 100 µg / ml, 200 µl
  Storage buffer: Tris / HCl, NaCl pH 7.6
  Protein CD4: c = 63 µg / ml, 318 µl
  Storage buffer: 10 mM Tris; 300 mM NaCl pH 8
  Protein ox-antithrombin III: c = 1 mg / ml, 120 µl
  Protein ox-human albumin: c = 1 mg / ml, 120 µl
  C1 chip (BIACORRE AB)
  Amine coupling kit (BIACORE AB)
  HIV-GP120 and CD4 were immobilized on the C1 sensor surface. 10 mM NaAc pH 4 were used as the coupling buffer.
  Coupling conditions: HIV-GP120: 20 µl; 10 µg / ml GP120 in 10 mM NaAc pH 4; immobilized amount: 1158 RU, 300 pg
  CD4: 30 µl; 6.3 µg / ml CD4 in 10 mM NaAc pH 4; Immobilized amount: 568 RU, 148p.
  The chip surface was saturated with BSA and the binding of the oxidized proteins was examined. For this purpose, z. B. 50 ul oxidized antithrombin III injected in different concentrations at a flow rate of 20 ul / min. The protein solutions were diluted with the loading buffer. The resulting signals increase with increasing concentration of oxidized antithrombin III.
  14a) Overlay plot of 12 sensor programs, which show the binding of oxidized antithrombin III to immobilized HIV-GP120 and the dissociation.
  14b) Overlay plot of 12 sensor programs, which show the binding of oxidized antithrombin III to immobilized CD4 and the dissociation.
  The quantitative evaluation showed the binding of
  • 1. Oxidized antithrombin III on HIV-GP120:
    K _on (1 / Ms): 6.38 × 10 ⁵
    K _off (1 / s): 4.44 × 10 ^-4
    and a K _D [M] of 7.01 × 10 ^-10 ,
  • 2. Oxidized antithrombin III on CD4:
    K _on (1 / Ms): 7.13 × 10 ⁵
    K _off (1 / s): 1.12 × 10 ^-3
    and a K _D [M] of 1.63 × 10 ^-9 ,
  • 3. unmodified antithrombin III bound neither to HIV-GP120 nor to CD4. Oxidized human albumin bound to HIV-GP120 and CD4 with even higher affinity than ox. Antithrombin III. Unoxidized human albumin bound neither to HIV-GP120 nor to CD4.
• 10. Oxidized proteins induce TSP binding to cells Oxidized proteins (oxHumanalbumin, oxAntithrombin III and oxFibrinogen used here as an example) specifically and dose-dependently induce the binding of TSP-1 to cells containing CD36 (see Fig. 15a-d).
• 11. Since oxidized proteins induce TSP binding, medications according to claim 21-24 inevitably thus indirectly induce effects of TSP bound to cell surfaces, such as, for. B. the inhibition of angiogenesis (see Fig. 16a) or the inhibition of HIV infection. On the other hand, inhibiting the interaction between oxidized proteins and CD36 consequently suppresses the functions that are triggered by the reaction chain oxProtein-CD36 cell-bound TSP. Inhibitors according to claims 1-4 thus also inhibit TSP-mediated processes, such as the inhibition of angiogenesis, and are therefore proangiogenic (see Fig. 16b).

Legends for the illustrations Fig. 2 Oxidized proteins are hemostatic / prothrombotic - Increase in platelet adhesion / inhibition of this reaction

2a) Oxidized proteins increase the platelet adhesion to type I collagen. The platelet adhesion was determined according to Santoro et al. Carried out in 1994. A 96-well cell culture plate was coated with type I collagen (25 μg / ml; 100 μl / well) overnight at 4 ° C. and the plates were blocked with BSA. Human platelets were purified from plasma proteins by gel filtration in HEPES-Tyrode buffer pH 7.4 with the addition of 2 mM Mg ²⁺ , 1 mM Mn ²⁺ , 0.9% glucose and 0.35% BSA. 100 µl of gel-filtered platelets (300000 / µl) were incubated with and without oxidized proteins for 1 hour at RT in a moist chamber in the wells. Platelets that had not adhered were thoroughly washed out. The number of adhered platelets was determined by lysing the platelets with Triton X-100 and detecting the lysosomal enzyme hexosaminidase. To calibrate the adhesion assay, a calibration series of known increasing number of platelets was given on the microtiter plate and the absorbance of the converted substrate P-nitrophenyl-N-acetyl-β-D-glucosaminide in relation to the number of platelets was determined. Oxidized antithrombin III increased platelet adhesion depending on the dose.

2b) Platelets were in the adhesion assay described above used and activated with 50 µg / ml oxidized ATIII. Addition of soluble purified thrombospondin inhibited the oxidized ATIII increased platelet adhesion depending on dose.

2c) Platelets were in the adhesion assay described above used and activated with oxidized ATIII. Monoclonal antibodies that inhibit the binding of oxidized proteins to CD36 like clones 37, 13 and 7, inhibit the action of oxidized ATIII. All experiments on Influence of antibodies on platelet functions were in the presence satiating, completely blocking concentrations of Fab fragments an antibody to the FcRIIA receptor (clone IV.3) Exclude Fc receptor effects.

2d) This effect is dose-dependent.

2e) The inhibition of platelet adhesion by soluble Thrombospondin-1 is not directly linked to its binding site on CD36 (Peptide VTCG) mediated. VTCG shows no influence on the increase in Platelet adhesion through oxidized proteins (here oxidized ATIII).

2f) Antibodies against CD36, which oxidized the binding of CD36 to Proteins, like clone FA6 / 152, do not inhibit, however, do not call significant inhibition. All experiments on the influence of Antibodies to platelet functions were present satiating, completely blocking concentrations of Fab fragments an antibody to the FcRIIA receptor (clone IV.3) Exclude Fc receptor effects.

Fig. 3 Oxidized proteins are hemostatic / prothrombotic - Increase in fibrinogen binding to platelets / inhibition of these reaction

Gel-filtered platelets (50,000 / µl) in HEPES-Tyrode-BSA buffer became FITC conjugated fibrinogen and 10 µg / ml thrombospondin added. A part the samples were oxidized with ascending proteins Concentrations added. After incubation for 30 minutes at RT the fibrinogen binding determined in the flow cytometer.

3a) Oxidized proteins (here, as an example, oxidized fibrinogen, oxidized Human albumin and oxidized antithrombin III) enhance the Fibrinogen binding to platelets.

3b) Soluble thrombospondin-1 inhibits the dose caused by ox.

Protein-triggered fibrinogen binding.

3c) Antibodies which inhibit the binding of oxidized proteins to CD36 inhibit the fibrinogen binding to platelets induced by oxidized proteins in a dose-dependent manner
oxidized protein: oxidized ATIII;
Antibody: anti CD36 AK, clone 37.

3d) oxidized protein: oxidized fibrinogen;
Antibody: anti CD36 AK, clone 37.

3e) oxidized protein: oxidized human albumin
Antibody: anti CD36 AK, clone 37.

All experiments on the influence of antibodies on platelet functions were in the presence of saturating, completely blocking concentrations of Fab fragments of an antibody against the FcRIIA receptor (clone IV.3) carried out to rule out Fc receptor effects.

Fig. 4 Oxidized proteins are hemostatic / prothrombotic / induction of Platelet agareaation / inhibition of this reaction

4a) Oxidized proteins induce platelet aggregation. Platelet aggregation was carried out according to Born in 1962. To gel-filtered platelets (200000 / µl) in HEPES-Tyrode buffer pH 7.4 with 100 µg / ml fibrinogen was pipetted in an aggregation cuvette TSP-1 (25 µg / ml). Soluble TSP-1 alone did not trigger aggregation. Simultaneous administration of oxidized proteins (here oxidized fibrinogen or oxidized antithrombin III) led to a strong formation of aggregates.
Soluble thrombospondin inhibited the aggregation triggered by oxidized proteins in high concentrations> 50 µg / ml.

4b) Antibodies that bind oxidized proteins to CD36 inhibit, dose-dependent inhibit those induced by oxidized proteins Platelet aggregation. All experiments on the influence of antibodies Platelet functions became satiating, complete in the presence blocking concentrations of Fab fragments of an antibody against the FcRIIA receptor (clone IV.3) Exclude Fc receptor effects.

Fig. 5 Oxidized proteins are hemostatic / prothrombotic - Induction of the procoaaulant state of the platelets and the Microparticle formation / inhibition of this reaction

5a) Induce oxidized proteins (oxidized fibrinogen as an example here) binding of factor V / Va to platelets. The factor V / Va binding was as in Dörmann et al. 1998 described.

5b) Oxidized proteins (oxidized fibrinogen as an example here) induce the Binding of factor X / Xa to platelets. The factor X / Xa binding was as in Dörmann et al. 1998 performed by us.

5c) Oxidized proteins (oxidized fibrinogen as an example here) induce the Phospholipid flip-flop in the membrane and thus the binding of annexin V on platelets. Annexin V binding was carried out as in Dörmann et al. 1998 performed by us.

5d) Oxidized proteins (oxidized fibrinogen as an example here) induce the Platelet microparticle formation. Gel-filtered platelets (50000 / µl) were with oxidized protein for 30 minutes at RT under light Incubated panning. After that the platelets became and from platelets Microparticles formed for 30 minutes with the anti GPIX-PE antibody incubated and the number of microparticles formed in relation to 5000 counted particles measured in the flow cytometer.

5e) Soluble thrombospondin inhibits the oxidized proteins triggered microparticle formation. In this experimental example, the Microparticle formation from platelets by oxidized human albumin induced. Gel-filtered plates in Hepes-Tyrode buffer pH 7.4 were used 50 µg / ml of oxidized human albumin activated for 1 h at RT. The Platelet suspension was dissolved in the specified concentrations added. The microparticle formation was like described in 5d). Soluble thrombospondin inhibited the formation of the microparticles depending on the dose.

5f) Anti CD36 clone 37 inhibits the formation of the oxProtein induced procoagulant state of the platelets. The AnnexinV binding, as Indicator for the formation of a procoagulant membrane surface Platelets were measured as described under 5c). preincubation the plate with anti CD36 antibodies (30 minutes, RT), which the Binding of ox. Inhibiting proteins on CD36 inhibited the following Activation of these platelets by oxidized proteins (here as an example oxidized fibrinogen). All experiments on the influence of antibodies Platelet functions became satiating, complete in the presence blocking concentrations of Fab fragments of an antibody against the FcRIIA receptor (clone IV.3) Exclude Fc receptor effects.

5g) Anti CD36 AK 37 inhibits the oxidized proteins Platelet microparticle formation. The microparticle formation was as described under 5d). Pre-incubate the platelets with anti CD36 antibodies (30 minutes, RT), which oxidized the binding of Inhibit proteins on CD36 before activation with oxidized proteins (here as an example, oxidized fibrinogen) inhibits microparticle formation.

Fig. 6 Oxidized proteins activate leukocyte Ca ²⁺ signal

Oxidized proteins (shown here, oxidized antithrombin III) induce a Ca ²⁺ signal in monocytes. The Ca ²⁺ measurements were carried out according to Sozzani et al. Performed in 1993. Eluted monocytes (5 × 10 ⁶ / ml) were washed at RT with HEPES-Tyrode buffer pH 7.4 and then labeled for 15 minutes with 1 μM Fura2 / AM at 37 ° C., washed twice with HEPES-Tyrode buffer without Ca ²⁺ and then taken up in HEPES-Tyrode with 1 mM Ca ²⁺ . Ca ²⁺ signal, induced by oxidized proteins and substances which act as positive or negative controls, were determined fluorometrically in the Hitachi F-2000. Oxidized antithrombin III (100 µg / ml) activates monocytes and produces a clear Ca ²⁺ signal.

Fig. 7 Oxidized proteins activate leukocytes - oxidative burst

Oxidized proteins increase the activating effect of fMLF on the oxidative burst of PMNL and even solve it as independent, independent agonists too. The induction of the oxidative burst was essentially according to the manufacturer's instructions with the phagotest / burst test from Orpegen (Heidelberg) on Flow cytometer performed, but first the PMNL with the Incubated substrate DHR123 and then activated the PMNL. oxidized Antithrombin III increased the activating effect of fLMF and called even an ox. Burst reaction.

Fig. 8 Oxidized proteins activate leukocyte transmigration by the endothelium / inhibition of this reaction

8a) In "Transwell" cell culture chambers (Costar, Bodenheim), a "Transwell" insert covered with a microporous polycarbonate membrane was placed per one of the 24 wells. The polycarbonate membrane with a pore size of 5 μm was coated with fibronectin and then human microvascular endothelial cells (HMEC-1) were cultivated to confluence. Human monocytes isolated by density gradient centrifugation (200 μl with 2 × 10 ⁷ cells / ml in DMEM from the peripheral blood) were incubated at 37 ° C., 7% CO ₂ with the HMEC-1 monolayer. The number of monocytes in the lower "Transwell" compartment under the "Transwell" insert was determined as a measure of the transmigration rate. In order to investigate the influence of various oxidized proteins, thrombospondin or anti CD36 antibodies, the test substances were added to the medium in the upper "Transwell" chamber or the endothelial cells were pre-incubated with the test substances for 10 minutes and washed. After 4-7 hours of migration, the inserts were carefully removed, the cell culture plate was placed on ice for 30 minutes to detach the adhered monocytes and the number of transmigrated monocytes was counted. Oxidized protein (here oxidized ATIII) promoted the transmigration of monocytes through an HMEC-1 monolayer, while the non-oxidized starting protein did not show this reaction (transmigration time 4 h).

8b) by pre-incubation of the endothelial layer for 10 minutes with TSP-1, or addition of TSP-1 to the culture medium during the Transmigration attempt failed the transmigration of the monocytes are significantly inhibited (transmigration duration 7 h).

Fig. 9 Oxidized proteins induce processes that affect the Promote arteriosclerosis

Oxidized antithrombin III increased the "homing" of macrophages in arteriosclerotic plaques. The experiment was carried out in the Description of the example described in detail.

Fig. 10 Thrombospondin inhibits proarteriosclerotic processes

Thrombospondin inhibits macrophage adhesion arteriosclerotic endothelial cells. The experimental procedure is explained in detail in the description of the example.

10a) Thrombospondin-1 inhibits the transient by rolling Adhesion marked by macrophages, to arteriosclerotic changes Endothelium.

10b) Thrombospondin inhibits the permanent, stable adhesion of Macrophages on arteriosclerotic endothelium.

Fig. 11 Thrombospondin inhibits inflammatory processes in vivo

Soluble thrombospondin-1 inhibits the Arthus response in the ear of Balb / c mice

11a) Twice at 0 and 0 + 3 hours, 50 µg TSP-1 i.p. treated mouse - prevented arthus reaction in the left ear

11b) Twice at 0 and 0 + 3 hours with the control buffer i.p. treated mouse - arthus reaction in the left ear

11c) BSA-FITC stored in the ears as a measure of the Arthus reaction in mice treated with TSP-1 and control buffer - Arthus reaction in left ear.

Fig. 13

Patients with type I diabetes mellitus and control subjects became blood removed and this anticoagulated with citrate. By centrifugation platelet-rich plasma (PRP) was produced. The PRP of patients and healthy control subjects were treated with fibrinogen [150 µg / ml], which had been coupled to FITC and the platelets with oxidized protein (here oxidized human albumin) in ascending Concentrations activated for 30 minutes. The fibrinogen binding was like Measured in detail in the flow cytometer described above. The Figure shows a characteristic example of the at the same time Activation determinations carried out with PRP of patients with Type I diabetes mellitus compared to controls. Platelets from Patients with type I diabetes mellitus are particularly sensitive to that Activation with oxidized proteins.

Fig. 14

• a) Overlay-Plot von 12 Sensorgrammen, welche die Anbindung von oxidiertem Antithrombin III an immobilisiertes HIV-GP120 und die Dissoziation von oxidiertem Antithrombin III zeigen. HIV-GP120 ist immobilisiert (300 pg); die Konzentration an oxidiertem Antithrombin III wurde variiert (von unten nach oben: 0 nM; 1 nM; 5.1 nM; 10.2 nM; 17 nM; 20.4 nM; 23 nM; 34 nM; 40.8 nM; 51 nM; 85 nM; 119 nM). Mit steigender Konzentration an oxidiertem AT III nimmt das resultierende Signal zu.
  Die Interaktion zwischen oxidierten Proteinen und HIV-GP120 bzw. CD4 wurde wie in der Beschreibung des Beispiels im Detail erläutert mittels Plasmon Resonanz-Technik im BIACORE-System 2000 bestimmt.
• b) Overlay-Plot von 12 Sensorgrammen, welche die Anbindung von oxidiertem Antithrombin III an immobilisiertes CD4 und die Dissoziation von oxidiertem Antithrombin III zeigen. CD4 ist immobilisiert (148 pg); die Konzentration an oxidiertem Antithrombin III wurde variiert (von unten nach oben: 0 nM; 1 nM; 5.1 nM; 10.2 nM; 17 nM; 20.4 nM; 23 nM; 34 nM; 40.8 nM; 51 nM; 85 nM; 119 nM). Mit steigender Konzentration an oxidiertem AT III nimmt das resultierende Signal zu.

The interaction between oxidized proteins and HIV-GP120 or CD4 was determined, as explained in detail in the description of the example, using plasmon resonance technology in the BIACORE system 2000.

• a) Overlay plot of 12 sensor programs, which show the binding of oxidized antithrombin III to immobilized HIV-GP120 and the dissociation of oxidized antithrombin III. HIV-GP120 is immobilized (300 pg); the concentration of oxidized antithrombin III was varied (from bottom to top: 0 nM; 1 nM; 5.1 nM; 10.2 nM; 17 nM; 20.4 nM; 23 nM; 34 nM; 40.8 nM; 51 nM; 85 nM; 119 nM) , The resulting signal increases with increasing concentration of oxidized AT III.
  The interaction between oxidized proteins and HIV-GP120 or CD4 was determined, as explained in detail in the description of the example, by means of plasmon resonance technology in the BIACORE system 2000.
• b) Overlay plot of 12 sensor programs, which show the binding of oxidized antithrombin III to immobilized CD4 and the dissociation of oxidized antithrombin III. CD4 is immobilized (148 pg); the concentration of oxidized antithrombin III was varied (from bottom to top: 0 nM; 1 nM; 5.1 nM; 10.2 nM; 17 nM; 20.4 nM; 23 nM; 34 nM; 40.8 nM; 51 nM; 85 nM; 119 nM) , The resulting signal increases with increasing concentration of oxidized AT III.

Fig. 15

15a) Oxidized protein mediates TSP-1 binding to platelets. Gel-filtered human platelets were diluted to 50,000 / µl with Hepes-Tyrode buffer pH 7.4 and FITC-conjugated, purified thrombospondin-1 (50 µg / ml) was added. The platelets were incubated for 1 h at RT with oxidized protein (here oxidized fibrinogen) and the TSP-1 binding to the platelets was measured in the flow cytometer.
Oxidized proteins induce TSP-1 binding to platelets.

15b) Oxidized protein (here oxidized antithrombin III) induces the Binding of thrombospondin to endothelial cells. Human, microvascular Endothelial cells (HMEC-1) were prepared by the Cell culture plate detached, isolated and the suspension for 1 h at RT with incubated oxidized protein or oxidized protein plus TSP-1 additive. The Cells were washed and bound TSP-1 with mAK anti TSP-1 (Clone P10), coupled to phycoerythrin (PE), labeled and im Flow cytometer quantified. Oxidized protein induces TSP-1 Binding to endothelial cells.

15c) While without the addition of purified TSP-1 and without the addition of oxidized antithrombin III only about 1% of the eluted monocytes an antibody (clone P10) that recognizes TSP-1 on the cell surface, in Flow cytometers are detected, the number increases by adding purified TSP-1 (10 µg / ml) to approx. 5%. Addition of oxidized AT III (without the addition of exogenous TSP-1) mediates the binding of endogenous TSP-1 to the monocytes. Approximately 18% of the monocytes became TSP-1 positive. By simultaneous administration of TSP-1 and oxidized AT III almost all of the peripheral blood monocytes used were strongly positive for TSP-1.

15d) Oxidized protein induces the binding of TSP-1 to T cells. Cultivated human T cells (Jurkat cells) were used for 1 h at RT oxidized protein (here oxidized antithrombin III) or oxidized protein plus TSP-1 additive (25 µg / ml) incubated. TSP-1 bound to the T cells was by the monoclonal PE-conjugated anti TSP antibody (clone P10) marked and measured in the flow cytometer. Oxidized proteins induce the binding of endogenous and exogenous added TSP to T cells.

Fig. 16

This figure illustrates the mechanism through which medication inhibit functions according to claims 1-4 and according to claims 21-24 or mediate through the reaction of thrombospondin with CD36 are triggered (example angiogenesis). N. Bouck's group identified the thrombospondin-1 and derivatives thereof as a potent endogenous inhibitor of tumor angiogenesis (Good et al. 1990 PNAS 87 6624) and clarified that this reaction is mediated via CD36 (Dawson et al. 1997 J. Cell. Biol. 13813) 701-717; Jimenez et al. 2000 Nat. Med. 6 (1) 41-8).

16a) In this invention it has been demonstrated that oxidized proteins can Mediate binding of thrombospondin to CD36. Medication after Claims 21-24 thus induce reactions caused by this binding are triggered such. B. the inhibition of angiogenesis, a process which can be used therapeutically to fight tumors.

16b) In this invention, substances were presented that inhibit the interaction of oxidized proteins with CD36 and thus the processes that are triggered in the body by oxidized proteins via CD36. Medicaments according to claims 1-4 inhibit these reactions and counter the angiogenesis inhibition which is triggered by the reaction chain of oxidized proteins (CD36 conformational change-thrombospondin binding to CD36-CD36 → signal) for angiogenesis inhibition. This reaction can with desired angiogenesis, e.g. B. in the heart muscle during infarction, can be used therapeutically. Reference list 1. Abumrad N, Coburn C, Ibrahimi A: Membrane proteins implicated in long-chain fatty acid uptake by mammalian cells: CD36, FATP and FABPm. Biochim. Biophys. Acta 1441: 4-13, 1999
2. Abumrad NA, el Maghrabi MR, Amri EZ, Lopez E, Grimaldi PA: Cloning of a rat adipocyte membrane protein implicated in binding or transport of lang-chain fatty acids that is induced during preadipocyte differentiation. Homology with human CD36. J. Biol. Chem. 268: 17665-17668, 1993
3. Acton SL, Scherer PE, Lodish HF, Krieger M: Expression cloning of SR-Bf, a CD36-related class B scavenger receptor. J. Biol. Chem. 269: 21003-21009, 1994
4. Aiken ML, Ginsberg MH, Byers-VVard V, Plow EF: Effects of OKM5, a monoclonal antibody to glycoprotein IV, on platelet aggregation and thrombospondin surface expression [see comments]. Blood 76: 2501-2509, 1990
5. Alessio M, Roggero S, Bussolino F, Saitta M, Malavasi F: Characterization of the murine monoclonal antibody NL07 specific for the human thrombospondin receptor (CD36 molecule). Curr. Stud. Hematol. Blood Transfus. 182-186, 1991
6. Alessio M, Greco NJ, Primo L, Ghigo D, Bosia A, Tandon NN, Ockenhouse CF, Jamieson GA, Malavasi F: Platelet activation and inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07. Blood 82: 3637-3647, 1993
7. Asch AS, Barnwell J, Silverstein RL, Nachman RL: Isolation of the thrombospondin membrane receptor. J. Clin. Invest 79: 1054-1061, 1987
8. Asch AS, Liu I, Briccetti FM, Barnwell JW, Kwakye-Berko F, Dokun A, Goldberger J, Pernambuco M: Analysis of CD36 binding domains: ligand specificity controlled by dephosphorylation of an ectodomain. Science 262: 1436-1440, 1993
9. Bablor BM: Oxygen-dependent microbial killing by phagocytes (second of two parts). N. Engl. J. Med. 298: 721-725, 1978
10. Bablor BM: Oxygen-dependent microbial killing by phagocytes (first of two parts). N. Engl. J. Med. 298: 659-668, 1978
11. Barnwell JW, Asch AS, Nachman RL, Yamaya M, Aikawa M, lngravallo P: A human 88-kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on Plasmodium falciparum-infected erythrocytes. J. Clin. Invest 84: 765-772, 1989
12. Baruch DI, Ma XC, Pasloske B, Howard RJ, Miller LH: CD36 peptides that block cytoadherence define the CD36 binding region for Plasmodium falciparum-infected erythrocytes. Blood 94: 2121-2127, 1999
13. Berendt AR, Ferguson DJ, Gardner J, Turner G, Rowe A, McCormick C, Roberts D, Craig A, Pinches R, Elford BC: Molecular mechanisms of sequestration in malaria. Parasitology 108 Suppl: S19-S28, 1994
14. Berlett BS, Stadtman ER: Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272: 20313-20316, 1997
15. Boullier A, Gillotte KL, Horkko S, Green SR, Friedman P, Dennis EA, Witztum JL, Steinberg D, Quehenberger O: The binding of oxidized low density lipoprotein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein. J. Biol. Chem. 275: 9163-9169, 2000
16. Brown MS, Goldstein JL: A receptor-mediated pathway for cholesterol homeostasis. Science 232: 34-47, 1986
17. Calvo D, Vega MA: Identification, primary structure, and distribution of CLA-1, a novel member of the CD36 / LIMPII gene family. J. Biol. Chem. 268: 18929-18935, 1993
18. Carr AC, McCall MR, Frei B: Oxidation of LDL by myeloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection. Arterioscler. Thromb. Vasc. Biol. 20: 1716-1723, 2000
19. Catimel B, McGregor JL, Hasler T, Greenwalt DE, Howard RJ, Leung LL: Epithelial membrane glycoprotein PAS-IV is related to platelet glycoprotein IIIb binding to thrombospondin but not to malaria-infected erythrocytes. Blood 77: 2649-2654, 1991
20. Chisolm GM, Steinberg D: The oxidative modification hypothesis of atherogenesis: an overview [In Process Citation]. Free Radic. Biol. Med. 28: 1815-1826, 2000
21. Coburn CT, Knapp FF, Jr., Febbraio M, Beets AL, Silverstein RL, Abumrad NA: Defective uptake and utilization of long-chain fatty acids in muscle and adipose tissues of CD36 knockout mice. J. Biol. Chem. 2000
22. Daniel JL. Dangelmaier C, Strouse R, Smith JB: Collagen induces normal signal transduction in platelets deficient in CD36 (platelet glycoprotein IV). Thromb. Haemost. 71: 353-356, 1994
23. Dutta-Roy AK, Crosbie LC, Gordon MJ, Campbell FM: Platelet membrane glycoprotein IV (CD36) is involved in arachidonic acid induced-platelet aggregation. Biochem. Soc. Trans. 24: 167S, 1996
24. Endemann G, Stanton LW, Madden KS, Bryant cm, White RT, Protter AA: CD36 is a receptor for oxidized low density lipoprotein. J. Biol. Chem. 268: 11811-11816, 1993
25. Fadok VA, Warner ML, Bratton DL, Henson PM: CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (alphav beta 3). J. Immunol. 161: 6250-6257, 1998
26. Febbraio M, Abumrad NA, Hajjar DP, Sharma K, Cheng W, Pearce SF, Silverstein RL: A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism. J. Biol. Chem. 274: 19055-19062, 1999
27. Febbraio M, Podrez EA, Smith JD, Hajjar DP, Hazen SL, Hoff HF, Sharma K, Silverstein RL: Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice [see comments]. J. Clin. Invest 105: 1049-1056, 2000
28. Frieda S, Pearce A, Wu J, Silverstein RL: Recombinant GST / CD36 fusion proteins define a thrombospondin binding domain. Evidence for a single calcium-dependent binding site on CD36. J. Biol. Chem. 270: 2981-2986, 1995
29. Glaser CB, Morser J, Clarke JH, Blasko E, Molean K, Kuhn I, Chang RJ, Lin JH, Vilander L, Andrews WH: Oxidation of a specific methionine in thrombomodulin by activated neutrophil products blocks cofactor activity. A potential rapid mechanism for modulation of coagulation. J. Clin. Invest 90: 2565-2573, 1992
30. Greenwalt DE, Watt KW, So OY, Jiwani N: PAS IV, an integral membrane protein of mammary epithelial cells, is related to platelet and endothelial cell CD36 (GP IV). Biochemistry 29: 7054-7059, 1990
31. Greenwalt DE, Lipsky RH, Ockenhouse CF, Ikeda H, Tandori NN, Jamieson GA: Membrane glycoprotein CD36: a review of its roles in adherence, signal transduction, and transfusion medicine. Blood 80: 1105-1115, 1992
32. Hansson GK: Cell-mediated immunity in atherosclerosis. Curr. Opin. Lipidol, 8: 301-311, 1997
33. Hansson M, Asea A, Ersson U, Hermodsson S, Hellstrand K: Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites. J. Immunol. 156: 42-47, 1996
34. Hansson M, Hermodsson S, Brune M, Mellqvist UH, Naredi P, Betten A, Gehlsen KR, Hellstrand K: Histamine protects T cells and natural killer cells against oxidative stress. J. Interferon Cytokine Res. 19: 1135-1144, 1999
35. Hazell LJ, Arnold L, Flowers D, Waeg G, Malle E, Stocker R: Presence of hypochlorite-modified proteins in human atherosclerotic lesions. J. Clin. Invest 97: 1535-1544, 1996
36. Holvoet P, Collen D: beta-VLDL hypercholesterolemia relative to LDL hypercholesterolemia is associated with higher levels of oxidized lipoproteins and a more rapid progression of coronary atherosclerosis in rabbits. Arterioscler, thromb. Vasc. Biol. 17: 2376-2382, 1997
37. Holvoet P: Oxidative modification of low density lipoproteins in atherothrombosis. Acta Cardiol, 53: 253-260, 1998
38. Holvoet P, Vanhaecke J, Janssens S, Van de WF, Collen D: Oxidized LDL and malondialdehyde-modified LDL in patients with acute coronary syndromes and stable coronary artery disease. Circulation 98: 1487-1494, 1998
39. Holvoet P, Collen D: Oxidation of low density lipoproteins in the pathogenesis of, atherosclerosis. Atherosclerosis 137 Suppl: S33-S38, 1998
40. Holvoet P, Stassen JM, Van Cleemput J, Collen D, Vanhaecke J: Oxidized low density lipoproteins in patients with transplant-associated coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 18: 100-107, 1998
41. Holvoet P: Endothelial dysfunction, oxidation of low density lipoprotein, and cardiovascular disease. Ther. Apher. 3: 287-293, 1999
42. Holvoet P, Collen D, Van de WF: Malondialdehyde-modified LDL as a marker of acute coronary syndromes. JAMA 281: 1718-1721, 1999
43. Holvoet P, Van Cleemput J, Collen D, Vanhaecke J: Oxidized low density lipoprotein is a prognostic marker of transplant-associated coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 20: 698-702, 2000
44. Huang mm, Boten JB, Barnwell JW, Shattil SJ, Brugge JS: Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets. Proc. Natl. Acad. Sci. USA 88: 7844-7848, 1991
45. Kehrel B, Kronenberg A, Schwippert B, Niesing-Bresch D, Niehues U, Tschope D, from de LJ, Clemetson KJ: Thrombospondin binds normally to glycoprotein IIIb deficient platelets. Biochem. Biophys. Res. Commun. 179: 985-991, 1991
46. Kehrel B, Kronenberg A, Rauterberg J, Niesing-Bresch D, Niehues U, Kardoeus J, Schwippert B, Tschope D, von de LJ, Clemetson KJ: Plateiets deficient in glycoprotein IIIb aggregate normally to collagens type I and III but not to collagen type V. Blood 82: 3364-3370, 1993
47. Kieffer N, Bettaleb A, Legrand C, Coulombel L, Vainchenker W, Edelman L, Breton-Gorius J: Developmentally regulated expression of a 78 kDa erythroblast membrane glycoprotein immunologically related to the platelet thrombospondin receptor. Biochem. J. 262: 835-842, 1989
48. Kielbassa K, Schmitz C, Gerke V: Disruption of endothelial microfilaments selectively reduces the transendothelial migration of monocytes. Exp. Cell Res. 243: 129-141, 1998
49. Knowles DM, Tolidjian B, Marboe C, D'Agati V, Grimes M, Chess L: Monoclonal anti-human monocyte antibodies OKM1 and OKM5 possess distinctive tissue distributions including differential reactivity with vascular endothelium. J. Immunol. 132: 2170-2173, 1984
50. Kronenberg A, Grahl H, Kehrel B: Human platelet CD36 (GPIIIb, GPIV) binds to cholesteryl-hemisuccinate and can be purified by a simple two-step method making use of this property. Thromb. Haemost. 79: 1021-1024, 1998
51. Lehr HA, Olofsson AM, Carew TE, Vajkoczy P, by Andrian UH, Hubner C, Berndt MC, Steinberg D, Messmer K, Arfors KE: P-selectin mediates the interaction of circulating leukocytes with platelets and microvascular endothelium in response to oxidized lipoprotein in vivo. Lab Invest 71: 380-386, 1994
52. Leung LL, Li WX, McGregor JL, Albrecht G, Howard RJ: CD36 peptides enhance or inhibit CD36 thrombospondin binding. A two-step process of ligand-receptor interaction. J. Biol. Chem. 267: 18244-18250, 1992
53. Li WX, Howard RJ, Leung LL: Identification of SVTCG in thrombospondin as the conformation-dependent, high affinity binding site for its receptor, CD36. J. Biol. Chem. 268: 16179-16184, 1993
54. McGregor JL, Clemetson KJ, James E, Luscher EF, Dechavannne M: Characterization of human blood platelet membrane proteins and glycorproteins by their isoelectric point (pl) and apparent molecular weight using two-dimensional electrophoresis and surface-labeling techniques. Biochim. Biophys. Acta 599: 473-483, 1980
55. McGregor JL, Clemetson KJ, James E, Capitanio A, Greenland T, Luscher EF, Dechavanne M: Glycoproteins of platelet membranes from Glanzmanns thrombasthenia. A comparison with normal using carbohydrate-specific or protein-specific labeling techniques and high-resolution two-dimensional gel electrophoresis. Eur. J. Biochem. 116: 379-388, 1981
56. Nakata A, Nakagawa Y, Nishida M, Nozaki S, Miyagawa J, Nakagawa T, Tamura R, Matsumoto K, Kameda-Takemura K, Yamashita S, Matsuzawa Y: CD36, a novel receptor for oxidized low density lipoproteins, is highly expressed on lipid-loading macrophages in human atherosclerotic aorta. Arterioscler. Thromb. Vasc. Biol. 19: 1333-1339, 1999
57. Nguyen-Khoa T, Massy ZA, Witko-Sarsat V, Canteloup S, Kebede M, Lacour B, Drueke T, Descamps-Latscha B: Oxidized low density lipoprotein induces macrophage respiratory burst via its protein moiety: A novel pathway in atherogenesis ? Biochem. Biophys. Res. Commun. 263: 804-809, 1999
58. Nicholson AC, Frieda S, Pearce A, Silverstein RL: Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines. Evidence implicating the lipid moiety of the lipoprotein as the binding site. Arterioscler. Thromb. Vasc. Biol. 15: 269-275, 1995
59. Nicholson AC, Febbraio M, Han J, Silverstein RL, Hajjar DP: CD36 in atherosclerosis. The role of a class B macrophage scavenger receptor. Ann. NY Acad. Sci. 902: 128-131, 2000
60. Nozaki S, Kashiwagi H, Yamashita S, Nakagawa T, Kostner B, Tomiyama Y, Nakata A, Ishigami M, Miyagawa J, Kameda-Takemura K: Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects. J. Clin. Invest 96: 1859-1865, 1995
61. Nozaki S, Tanaka T, Yamashita S, Sohmiya K, Yoshizumi T, Okamoto F, Kitaura Y, Kotake C, Nishida H, Nakata A, Nakagawa T, Matsumoto K, Kameda-Takemura K, Tadokoro S, Kurata Y, Tomiyama Y, Kawamura K, Matsuzawa Y: CD36 mediates long-chain fatty acid transport in human myocardium: complete myocardial accumulation defect of radiolabeled long-chain fatty acid analog in subjects with CD36 deficiency. Mol. Cell Biochem. 192: 129-135, 1999
62. Ockenhouse CF, Magowan C, Chulay JD: Activation of monocytes and platelets by monoclonal antibodies or malaria-infected erythrocytes binding to the CD36 surface receptor in vitro. J. Clin. Invest 84: 468-475, 1989
63. Ockenhouse CF, Tandon NN, Magowan C, Jamieson GA, Chulay JD: Identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor [see comments]. Science 243: 1469-1471, 1989
64. Okamoto F, Tanaka T, Sohmiya K, Kawamura K: CD36 abnormality and impaired myocardial long-chain fatty acid uptake in patients with hypertrophic cardiomyopathy. Jpn. Circ. J. 62: 499-504, 1998
65. Oquendo P, Hundt E, Lawler J, Seed B: CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes. Cell 58: 95-101, 1989
66. Palinski W, Ord VA, Plump AS, Breslow JL, Steinberg D, Witztum JL: ApoE-deficient mice are a model of lipoprotein oxidation in atherogenesis. Demonstration of oxidation-specific epitopes in lesions and high titers of autoantibodies to malondialdehyde-lysine in serum. Arterioscler. Thromb. 14: 605-616, 1994
67. Patel SS, Thiagarajan R, Willerson JT, Yeh ET: Inhibition of alpha4 integrin and ICAM-1 markedly attenuate macrophage homing to atherosclerotic plaques in ApoE-deficient mice. Circulation 97: 75-81, 1998
68. Pearce SF, Roy P, Nicholson AC, Hajjar DP, Febbraio M, Silverstein RL: Recombinant glutathione S-transferase / CD36 fusion proteins define an oxidized low density lipoprotein binding domain. J. Biol. Chem. 273: 34875-34881, 1998
69. Podrez EA, Febbraio M, Sheibani N, Schmitt D, Silverstein RL, Hajjar DP, Cohen PA, Frazier WA, Hoff HF, Hazen SL: Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species [see comments] jpublished erratum appears in J Clin Invest 2000 May; 105 (10): 1483]. J. Clin. Invest 105: 1095-1108, 2000
70. Puente N, Daviet L, Ninio E, McGregor JL: Identification on human CD36 of a domain (155-183) implicated in binding oxidized low density lipoproteins (Ox-LDL). Arterioscler. Thromb. Vasc. Biol. 16: 1033-1039, 1996
71. Ricciarelli R, Zingg JM, Azzi A: Vitamin E reduces the uptake of oxidized LDL by inhibiting CD36 scavenger receptor expression in cultured aortic smooth muscle cells. Circulation 102: 82-87, 2000
72. Rigotti A, Acton SL, Krieger M: The class B scavenger receptors SR-BI and CD36 are receptors for anionic phospholipids. J. Biol. Chem. 270: 16221-16224, 1995
73. Roberts DD, Sherwood JA, Spitalnik SL, Panton LJ, Howard RJ, Dixit VM, Frazier WA, Miller LH, Ginsburg V: Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence. Nature 318: 64-66, 1985
74. Ryeom SW, Sparrow JR, Silverstein RL: CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium. J. Cell Sci. 109 (Pt2): 387-395, 1996
75. Saelman EU, Kehrel B, Hese KM, de Groot PG, Sixma JJ, Nieuwenhuis HK: Platelet adhesion to collagen and endothelial cell matrix under flow conditions is not dependent on platelet glycoprotein IV. Blood 83: 3240-3244, 1994
76. Samanta A, Das DK, Jones R, George A, Prasad MR: Free radical scavenging by myocardial fatty acid binding protein. Free Radic. Res. Commun. 7: 73-82, 1989
77. Schraufstatter IU, Revak SD, Cochrane CG: Proteases and oxidants in experimental pulmonary inflammatory injury. J. Clin. Invest 73: 1175-1184, 1984
78. Schraufstatter IU, Browne K, Harris A, Hyslop PA, Jackson JH, Quehenberger O, Cochrane CG: Mechanisms of hypochlorite injury of target cells. J. Clin. Invest 85: 554-562, 1990
79. Schuepp BJ, Pfister H, Clemetson KJ, Silverstein RL, Jungi TW: CD36-mediated signal transduction in human monocytes by anti-CD36 antibodies but not by anti-thrombospondin antibodies recognizing cell membrane-bound thrombospondin. Biochem. Biophys. Res. Commun. 175: 263-270, 1991
80. Shah MM, Aust SD: Oxidation of halides by peroxidases and their subsequent reductions. Arch. Biochem. Biophys. 300: 253-257, 1993
81. Siddiqui FA, Lian EC: Platelet-agglutinating protein p37 from a thrombotic thrombocytopenic purpura plasma forms complexes with platelet membrane glycoprotein IV (CD36). Biochem. Int. 27: 485-496, 1992
82. Silverstein RL, Baird M, Lo SK, Yesner LM: Sense and antisense cDNA transfection of CD36 (glycoprotein IV) in melanoma cells. Role of CD36 as a thrombospondin receptor. J. Biol. Chem. 267: 16607-16612, 1992
83. Smith MA, Rottkamp CA, Nunomura A, Raina AK, Perry G: Oxidative stress in Alzheimers disease. Biochim. Biophys. Acta 1502: 139-144, 2000
84. Stadiman ER: Protein oxidation and aging. Science 257: 1220-1224, 1992
85. Stadtman ER, Levine RL: Protein oxidation. Ann. NY Acad. Sci. 899: 191-208, 2000
86. Steinberg D: Low density lipoprotein oxidation and its pathobiological significance. J. Biol. Chem. 272: 20963-20966, 1997
87. Tanaka T, Okamoto F, Sohmiya K, Kawamura K: Lack of myocardial iodine-123 15- (p-iodiphenyl) -3-R, S-methylpentadecanoic acid (BMIPP) uptake and CD36 abnormality-CD36 deficiency and hypertrophic cardiomyopathy [ letter; comment]. Jpn. Circ. J. 61: 724-725, 1997
88. Tanaka T, Sohmiya K, Kawamura K: Is CD36 deficiency an etiology of hereditary hypertrophic cardiomyopathy? J. Mol. Cell Cardiol. 29: 121-127, 1997
89. Tandon NN, Kralisz U, Jamieson GA: Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion. J. Biol. Chem. 264: 7576-7583, 1989
90. Tandon NN, Ockenhouse CF, Greco NJ, Jamieson GA: Adhesive functions of platelets lacking glycoprotein IV (CD36). Blood 78: 2809-2813, 1991
91. Vega MA, Segui-Real B, Garcia JA, Cales C, Rodriguez F, Vanderkerckhove J, Sandoval IV: Cloning, sequencing, and expression of a cDNA encoding rat LIMP II, a novel 74-kDa lysosomal membrane protein related to the surface adhesion protein CD36. J. Biol. Chem. 266: 16818-16824, 1991
92. Volf I, Bielek E, Moeslinger T, Koller F, Koller E: Modification of protein moiety of human low density lipoprotein by hypochlorite generates strong plateiet agonist. Arterioscler. Thromb. Vasc. Biol. 20: 2011-2018, 2000
93. Watanabe K, Toba K, Ogawa Y, Kodama M, Hirono S, Ohkura Y, Hanawa H, Nakamura Y, Aoki Y, Fuse I, Aizawa Y, Miyajima S, Kusano Y, Nagatomo T, Hasegawa G, Naito M: Hypertrophic cardiomyopathy with type I CD36 deficiency.
Jpn. Circ. J. 62: 541-542, 1998
94. Witztum JL, Steinberg D: Role of oxidized low density lipoprotein in atherogenesis. J. Clin. Invest 88: 1785-1792, 1991
95. Yamaguchi A, Yamamoto N, Akamatsu N, Saido TC, Kaneda M, Umeda M, Tanoue K: PS-liposome and ox-LDL bind to different sites of the immunodominant domain (# 155-183) of CD36: a study with GS95, a new anti-CD36 monoclonal antibody. Thromb. Res. 97: 317-326, 2000.

Claims (29)

1. Substances that bind oxidized proteins to CD36 or induced by interaction of CD36 with oxidized proteins Inhibit functions of CD36 as a medicine for humans and Animals. 2. Substances according to claim 1, in particular antibodies, which inhibit the binding of oxidized protein to CD36; very particularly monoclonal antibodies, including fragments F (ab) ₂ , F (ab), antigen recognition region. 3. Substances according to claim 1, in particular peptides from CD36, Peptide mimetics, analogues, characterized in that they are the Inhibit binding of CD36 to oxidized proteins (these can e.g. can be identified by using those in the invention mentioned monoclonal anti CD36 antibodies clone 37, 13 or 7 react). 4. Substances according to claim 1, in particular proteins that the Inhibit binding of CD36 to oxidized proteins or with CD36 bound thrombospondin compete; most notably soluble thrombospondin. 5. Substances according to claim 1, in particular peptides and Peptide mimetics, characterized in that they bind to CD36 and thus inhibit the interaction of CD36 with oxidized proteins (These can easily be done, e.g. with the phage display method be identified). 6. Substances according to one of claims 1-5 as a medicament for Prophylaxis of thrombosis, especially in inflammatory Diseases. 7. Substances according to one of claims 1-5 to support a antithrombotic therapy. 8. Substances according to one of claims 1-5 for preventing Transplant rejection. 9. Substances according to one of claims 1-5 for preventing transplant-related arteriosclerosis. 10. Substances according to one of claims 1-5 for preventing a High blood pressure in kidney disease, especially one Renin-related hypertension. 11. Substances according to one of claims 1-5 for preventing the Development and progression of arteriosclerotic (atherosclerotic) diseases. 12. Substances according to one of claims 1-5 for the treatment of chronic inflammatory reactions. 13. Substances according to one of claims 1-5 for preventing a early vascular reocclusion after bypass surgery, stent, PTCA, or similar. 14. Substances according to one of claims 1-5 for preventing a Vascular restenosis after bypass surgery, stent, PTCA, or like. 15. Substances according to one of claims 1-5 as a medicament Prevention of reperfusion damage, such as, but not limited to on, with myocardial ischemia, organ transplantation, stroke, peripheral occlusive disease, after surgery, Multi-organ failure after successful resuscitation. 16. Substances according to one of claims 1-5 as a medicament for Prevention of vascular damage, especially in patients with diabetes mellitus. 17. Substances according to one of claims 1-5 as a medicament Prevention of oxidized proteins via CD36 / TSP-1 triggered inhibition of endothelial proliferation and angiogenesis. 18. Substances according to one of claims 1-5 as medicaments for Support wound healing. 19. Quantification of ox. Proteins (especially ox human albumin, oxFibrinogen), individually or as a whole for the evaluation of the individual indication of therapy with medication after a of claims 1-5, for the diagnosis of diseases in which Inflammatory reactions play a role in how, but not limited on, arteriosclerosis, diabetic vasculopathy, rheumatic Arthritis, goodpasture syndrome, sepsis, ulcerative colitis, Graft-versus-host diseases, pemphigus, cancer, neurodermatitis, HIV infection, glomerulonephritis, reperfusion damage and Quality control of blood products. 20. Characterization of the activation state of CD36, the Receptor for oxidized proteins, as a diagnostic for diseases, in which inflammatory reactions play a role, like, but not limited to, atherosclerosis, diabetic vasculopathy, rheumatic arthritis, goodpasture syndrome, sepsis, colitis ulcerosa, graft-versus-host diseases, pemphigus, cancer, Neurodermatitis, HIV infection, glomerulonephritis, Reperfusion damage, or to monitor a CD36 / ox protein Inhibition therapy with medicaments according to one of claims 1-5 by detecting the state of phosphorylation of CD36. 21. Medicament containing oxidized protein / oxidized proteins, oxidized peptide, structural analogs or mimetics. 22. Medicament according to claim 21, characterized in that it further pharmaceutically acceptable auxiliary and / or carrier substances contains. 23. Medicament according to claim 21, characterized in that it for local, intradermal, superficial, intraperitoneal, formulated intravenous, oral or intramuscular administration or is administered via vesicles. 24. Medicament according to claim 21, characterized in that it other substances, such as B. antibiotics, immunosuppressants or Interaction partner of oxidized proteins in the body contains. 25. Process for the preparation of oxidized protein / peptide Reaction with HOCl or peroxynitrites. 26. Use of a medicament according to one of claims 21-24 for the prophylaxis or therapy of acute infections, for inhibition angiogenesis and to improve hemostasis. 27. Use according to claim 26, characterized in that as acute infection preventing or treating HIV infection. 28. Use according to claim 26, characterized in that through oxidized protein, via induction of TSP binding to CD36, tumor angiogenesis is inhibited. 29. Use according to claim 26, characterized in that it for hemostasis, especially in patients with congenital or acquired coagulation disorders, or congenital or acquired thrombocytopathies, under anticoagulation therapy or thromboprophylaxis, or in operations under Heart lung machine is used.