KR100797567B1 - Protein for inhibiting secretion of platelet granules - Google Patents

Abstract

The present invention relates to a protein for inhibiting granule secretion of platelets. The protein of the present invention not only prevents platelet aggregation but also inhibits granule secretion of platelets, and is effective in various diseases such as cancer metastasis, asthma or arthritis, which may be caused by activated epithelial cells and immune cells.

Platelet granules

Description

Protein for inhibiting secretion of platelet granules

1 is a diagram showing the effect of platelet induced supernatant on HUVEC migration. The supernatants obtained after activating platelets with and without NWK were used as chemoattractants in the HUVEC migration experiment. Platelet-induced supernatants obtained after activation with 600 μl of 5 μM TRAP were placed under the transwell in the Boyden transwell chamber and 100 μl of HUVEC (10 ⁶ cell / ml) was placed on top of each well. The cells were reacted at 37 ° C. for 24 hours, and then fixed with 3.7% formaldehyde and stained with hematoxylin and eosin Y. The cells on the upper side of the filter were removed by wiping with a cotton swab, and then the bottom side was enlarged 40 times and the number of cells in each of three parts was counted. Each sample was tested in duplicate and each experiment was repeated three times.

2 shows the results of integrin GPIIbIIIa enzyme immunoassay experiments. HUVEC (10 ⁴ / 100μl) NWK is induced and that activated platelets in the absence of TRAP to put the M199 medium containing the supernatant, or a 10% FBS were grown for 24 hours. In order to confirm the HUVEC integrin expression changes avb3, a3b1, a5b1, a1, a4, a6, b3 , such as antibodies against various integrin 1: 150mM NaCl (100 in TBST containing _{2mM CaCl 2, 0.05% Tween-} 20, 0.002% NaN ₃ , 20 mM Tris, pH 7.5) and reacted with the cells for 2 hours at room temperature. After washing with TBST and reacted with a secondary antibody diluted 1: 3000 in TBST for 1 hour at room temperature. After washing three times with TBST, a 1-step ABST solution was added and the absorbance at 405 nm was measured with a LabSystems multiskin reader. Experimental results were expressed in% by the control group 100% HUVEC grown in M199 medium containing 10% FBS.

Figure 3 is a diagram showing the reduction of granule secretion by NWK. Washed platelets (3 × 10 ⁸ / mL) were pretreated with different amounts of NWK protein for 10 minutes and then activated with TRAP (5 μM). After 10 min activation, platelets were stained with Phycoerythin (PE) attached CD62P antibody (A) or fluorescein isothiocyanate (FITC) attached PAC-1 antibody (B) for 15 minutes at room temperature. PAC1 is a GPIIbIIIa specific monoclonal antibody that binds platelets in an activation dependent form and CD62P is an antibody that is the label of alpha granules. The reaction was stopped immediately by addition of 0.5% paraformaldehyde. Stained platelets were quantified using FACSCalibur (Becton Dickinson, San Jose, USA).

Figure 4 is a diagram showing the decrease in PDGF-AB secretion in platelets by NWK. Washed platelets (3 × 10 ⁸ / mL) were pretreated with different amounts of NWK protein for 10 minutes and then activated with TRAP (5 μM). After activation at room temperature for 2 hours, the amount of PDGF-AB contained in platelet-induced supernatant obtained by centrifugation at 1,500 xg for 5 minutes was measured by enzyme-immunoassay.

The present invention relates to a protein for inhibiting granule secretion of platelets.

Platelet activity is essential for hemostasis and thrombosis. It also actively influences inflammatory, immune, and atherosclerosis [Andrews & Berndt, 2004, Thromb Res., 114, 447-453; Gibbins, 2004, J Cell Sci., 117, 3415-3425; Weyrich et al., 2003, J Thromb Haemost., 1, 1897-1905. Major activators such as thrombin, ADP and collagen directly induce platelet activity, adhesion and even aggregation (Wagner & Burger, 2003, Arterioscler Thromb Vasc Biol., 23, 2131-2137). The activity of platelets causes the release of microparticles, which leads to the expression of chemokine receptors that recruit monocytes. Thus, the release of microparticles is also associated with the inflammatory response that occurs during the onset of blood vessels. Most chemokines expressed in atherosclerotic injury are known to be released from platelets, and sustained platelet activity causes vascular damage, supporting the need for chronic antiplatelet therapy. Chemokine secretion in vascular cells activated by stimulated platelets is an important component involved in initiating vasculitis in atherosclerotic vascular disease. In addition, supernatants derived from platelets induce the growth and metastasis of cancer cells [Janowska-Wieczorek et al., 2005 ,. Int J Cancer , 113, 752-760] also induce the migration of hematopoietic cells [Baj-Krzyworzeka et al., 2002]. The migration of mesenchymal progenitor cells extracted from bone marrow [Gruber et al., 2004, Platelets, 1, 29] The involvement of platelets in angiogenesis has been assumed by Pinedo [Pinedo et al., 1998, Lancet, 352, 1775-1777].

Platelets induce renal vascular response and platelet-induced supernatant (PDS) has recently been demonstrated to actually promote renal blood vessel production as well as chronic ischemia [Brill et al., 2005,. Cardiovasc Res., 67, 30-38. However, the effect of platelet-induced supernatants on endothelial cells is not elucidated at the molecular level.

Neovascularization is required for robust cancer growth and metastasis and for tissue recovery in various anemia conditions. Various growth factors, including VEGF, angiopoietins, TGF-b and PDGF, are essential for the generation of vigorous renal vessels [Ferrara, 1999; Hellstrom et al., 1999; Hirschi et al., 1998; Yancopoulos et al., 2000]. Renal angiogenesis is regulated by ECM molecules that carry signaling pathways through integrins as well as growth factors. av integrins, including avb3 and avb5, contribute to growth factor signaling regulated by bFGF and VEGF, respectively, in renal angiogenesis [Hood et al., 2003]. Expression of avb3 is increased in endothelial cells and cancer cells stimulated by neovascular growth factor [Brooks, 1994, Science, 264, 569-571].

Inhibition of platelet activity is observed by greatly reducing aggregation and secretion of their granule contents. Granule contents include proteins involved in hemostasis, thrombosis and inflammatory responses.

NWK, one of the derivatives of the human ADAM15 disintegrin-like domain, has similarities in including the Arg-Gly-Asp (RGD) sequence with saxatilin, and this sequence shows fibrinogen dependent platelet aggregation, cancer growth and metastasis, and bFGF. It is known to prevent neovascularization of endothelial cells that are induced in the human body (Kim et al., 2003, Cancer Res., 63, 6458-6462). NWK , a variant that replaces the three amino acids closest to the RGD tripeptide sequence from the RPT RGD sequence to NWK RGD, has greater binding to GpIIbIIIa. NWK originally has a greater inhibitory effect on human platelet aggregation than the ADAM15 disintegrin-like domain.

The present invention relates to a protein derived from a human ADAM15 disintegrin-like domain with platelet granule secretion inhibitory capacity, the total market size of anticoagulant and antiplatelet agent reached $ 148 billion in 2011, the new anticoagulant of 23.2% It is expected to occupy. Integrin activity control technology, which is used worldwide to selectively bind proteins or peptide ligands to target receptors on the platelet surface, to inhibit platelet aggregation, is a key technology of modern biotechnology. In addition to the core technology of developing a cardiovascular disease treatment, it is a core technology common to apply to the development of prevention and treatment of various diseases induced by integrin action such as cancer metastasis, asthma, arthritis, apoptosis. Therefore, the present invention binds to specific integrins of platelets to inhibit platelet aggregation, while inhibiting the secretion of granules accompanying platelet activation, thereby preventing side effects due to activation of immune and vascular endothelial cells. The technology does not have the side effects of conventional antiplatelet agents and can replace conventional platelet aggregation inhibitors.

The present invention has been made by the necessity of the above, an object of the present invention to provide a protein for inhibiting granule secretion of platelets.

In order to achieve the above object, the present invention provides a protein for inhibiting platelet granule secretion comprising a disintecrin-like domain.

In the present invention, the protein preferably has at least 80% or more homology with that described in SEQ ID NO: 1, more preferably 90% or more homology, and particularly in the sequence of SEQ ID NO: 52 to 54 Most preferred are proteins having homology with Asn, Trp and Lys, respectively.

In another aspect, the present invention provides a composition for treating cancer metastasis, asthma, or arthritis, comprising a protein for inhibiting platelet granule secretion comprising a disintechrin-like domain as an active ingredient.

In the composition of the present invention, the protein is preferably described in SEQ ID NO: 1.

The invention is briefly described below.

Platelet activation leads to platelet aggregation and granule secretion and platelet granules contain non-protein molecules, binding and healing proteins and hydrolases. Among them, the alpha granule contains growth factors including PDGF, TGF, and the like, when platelets are activated, alpha granules are secreted to activate surrounding epithelial cells and immune cells. Platelet induced supernatant (PDS) is known to contain large amounts of these substances. NWK, a derivative of the human ADAM15 disintegrin-like domain, inhibits platelet aggregation by antagonizing integrin GPIIbIIIa and this activity is known to be important for outside-in signaling outside of platelets. In the present invention, the effect of NWK on granule secretion of platelets was confirmed by activating platelets with thrombin receptor activating peptide (TRAP) and measuring the granule secretion marker. Platelet-induced supernatants induce umbilical cord venous endothelial cell (HUVEC) migration and infiltration, which is dependent on platelet-derived growth factor (PDGF) secreted from the alpha granules of platelets. On the other hand, alpha granule secretion by TRAP was inhibited in the presence of NWK, thereby reducing the angiogenetic activity of platelet supernatants. Therefore, NWK not only prevents platelet aggregation but also inhibits platelet granule secretion, thereby reducing various problems that may be caused by activated epithelial cells and immune cells.

In the present invention, "TRAP" is a thrombin receptor activating peptide; "HUVEC" refers to human umbilical vein endothelial cells; "FBS" refers to calf bobbin syrum; "ELISA" refers to enzyme immunoassay; "FITC" refers to fluorescein isothiocyanate; "PE" is Phycoerythin; "NWK" is Asn-Trp-Lys; "PBS" refers to phosphate buffered saline; "PDS" refers to platelet-derived-supernatant; "PRP" refers to platelet-rich plasma; "RGD" means Arg-Gly-Asp; "TACTS" refers to 20 mM Tris-HCl, pH 7.5, 0.02% Sodium Azide, 2 mM Calcium Chloride, 0.05% Tween-20, 150 mM Sodium Chloride.

Prostaglandin E1 used in the present invention was purchased from Sigma (MO, USA) and FBS from HyClone (Utah, USA). Anti-b3 and anti-avb3 monoclonal antibodies were purchased from Chemicon (Temecula, USA). PAC-1-FITC antibodies were purchased from Becton Dickinson (San Jose, USA) and CD62P-PE was purchased from Biolegend (San Diego, USA). PDGF-AB ELIZA was purchased from R & D biosciences (MN, USA). TRAP peptide was synthesized from Anygen (Korea). One-step ABTS was purchased from PIERCE (IL, USA) and Boyden chamber transwell from Costar (NY, USA). And HUVEC was obtained from BioBud (seoul, Korea).

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example 1:

ADAM15 Disintegrin  Expression for Similar Domain Proteins Plasmid  design

The disintegrin-like domain (A15Dis) of ADAM15 was cloned into human fetal liver using N-terminal primer 5'CTCGAGAAAAGAGAGCCGGGCGAGCAGT-3 '(SEQ ID NO: 2) and C-terminal primer 5'GCGGCCGCTCACCCTAGGCTGACATC-3' (SEQ ID NO: 3) Cloned by PCR, and the RPT RGD sequence in the disintegrin-like domain of ADAM15 was changed to NWK RGD via mutagenesis and named NWK.

The PCR products were cloned into pGEM-T Easy vectors (Promega, Madison, USA), sequenced and Pichia Transferred to pastoris expression vector pPIC9 (Invitrogen, Carlsbad, USA). The whole constructs were prepared. N-terminal primers include Xho I restriction enzyme sites and C-terminal primers include stop codons and Not I sites. Briefly describing the expression of cloned plasmids, pPIC9-A15Dis plasmids were treated with Sal I restriction enzymes and linearized plasmids were subjected to electroporation (Gene Pulser, Bio-Rad, Hercules, USA). Were transformed into competent GS115 cells. The methanol availability of His + pPIC9-A15Dis transformants was analyzed by means of refricar selection at minimal dextrose and minimal methanol plate. Finally His + and Mut + cells were collected for protein expression.

Example 2:

ADAM15 Disintegrin  Expression and Purification of Similar Domains and Their Derivatives

Transformed cells were grown at 30 ° C. in glycerol final medium (without 1.34% yeast nitrogen base with ammonium sulfate, 4 × 10 ⁻⁵ M biotin and 1% glycerol and amino acids). After 48 hours, cells were collected by centrifugation and devoid of methanol minimal medium (1.34% yeast nitrogen base with ammonium sulfate, 4 × 10 ⁻⁵ M biotin and 0.5% methanol and amino acid in 100 mM potassium phosphate, pH 6.0). ) And grown at 30 ° C. A total of 0.5% (v / v) methanol was added every 12 hours for 3 days to induce A15Dis protein expression. Culture medium containing derived proteins were collected after centrifugation. Ammonium sulfate was added to the collected supernatant up to 1.5 M and then loaded onto a phenyl-sepharose column to proteinize with 1.5 M-0 M ammonium sulfate gradient. The A15Dis protein expressed by P. pastoris was further purified by Superdex G-75 gel filtration chromatography. The freshly derived A15DIS protein was purified and monitored by SDS-PAGE analysis. N-terminal sequencing of recombinant A15Dis was performed by an automated Edman digestion procedure to ensure the identification of purified proteins.

Example 3;

Cell culture

HUVECs were cultured on 5% CO ₂ in M199 medium containing 20% FBS and 100 unit / ml penicillin, 100 μg / ml streptomycin, 10 ng / ml basic fibroblast growth factor, 5 units / ml heparin, and cells 5-7 passages. Were used in the experiment.

Platelet induction Supernatant  Ready

Blood was collected from a healthy volunteer with a syringe containing citric acid and then whole blood was centrifuged at 120 xg for 15 minutes at room temperature to obtain PRP. The obtained PRP was centrifuged at 1,200 xg for 10 minutes in the presence of 1 μM prostaglandin E1 and then resuspended in modified Tyrode's buffer (140 mM sodium chloride, 2.9 mM potassium chloride, 1 mM magnesium chloride, 5 mM glucose, 10 mM HEPES (pH 7.4)). The washed platelets were activated with 5 μM TRAP after 10 minutes at room temperature with or without NWK After activation at room temperature for 2 hours, the reaction solution was centrifuged at 1,500 × g for 5 minutes at 4 ° C. and the obtained supernatant was tested. The platelet-induced supernatant was used for PDGF-AB ELIZA, chymotaxy analysis, integrin ELIZA and the like.

Example 4;

Chemotaxis analysis ( chemotaxis  assay)

Transfer experiments of HUVECs were carried out using a Boyden transwell chamber with an 8 μm-pore polycarbonate filter. Platelet-induced supernatants obtained after activation with 600 μl of 5 μM TRAP were placed under the transwell and 100 μl of HUVEC (10 ⁶ cell / ml) was placed on top of each well. The cells were reacted at 37 ° C. for 24 hours, and then fixed with 3.7% formaldehyde and stained with hematoxylin and eosin Y. The cells on the top side of the filter were removed by wiping with a cotton swab, then magnified the bottom side by 40 times and counted in each of the three sections. Each sample was tested in duplicate and each experiment was repeated three times.

Example 5:

Integreen GPIIbIIIa  Enzyme immunoassay experiment

HUVEC (10 ⁴ / 100μl) was grown overnight in 96 well plates and platelet-induced supernatant was added for 24 hours. In order to confirm the HUVEC integrin expression changes avb3, a3b1, a5b1, a1, a4, a6, b3 , such as antibodies against various integrin 1: 150mM NaCl (100 in TBST containing _{2mM CaCl 2, 0.05% Tween-} 20, 0.002% NaN ₃ , 20 mM Tris, pH 7.5) and reacted with the cells for 2 hours at room temperature. After washing with TBST and reacted with a secondary antibody diluted 1: 3000 in TBST for 1 hour at room temperature. After washing three times with TBST, 1-step ABST solution was added and the absorbance at 405 nm was measured with a LabSystems multiskin reader. The experiment was conducted three times in triplicate.

Example 6:

Flow cytometry

Membrane surface expression of PAC-1, CD62P on resting and activated platelets was analyzed by flow cytometry. Whole blood was centrifuged at 120 xg for 15 minutes at room temperature and PRP was removed. After 5 min incubation with 1 μM prostaglandin E1, PRP was centrifuged at 1,200 × g for 5 min and resuspended in modified Tyrode's buffer. Washed platelets (3 × 10 ⁸ / mL) were pretreated with different amounts of NWK protein for 10 minutes and then activated with TRAP (5 μM). After 10 minutes of activation, platelets were stained for 15 minutes at room temperature with fluorescein isothiocyanate (FITC) attached PAC-1 or Phycoerythin (PE) attached CD62P antibody. GPIIbIIIa specific monoclonal antibody that binds PAC1 to platelets in an activation dependent form and CD62P is an antibody that is the label of alpha granules. The reaction was stopped immediately by addition of 0.5% paraformaldehyde. Stained platelets were quantified using FACSCalibur (Becton Dickinson, San Jose, USA).

The result of the above example is as follows.

Platelet-induced supernatants are known to possess chemotaxis of human hematopoietic stem cells and bone marrow induced mesenchymal progenitor cells. Therefore, to determine whether platelet-induced supernatant induces vascular endothelial cell migration and chemotactic metastasis, platelet-induced supernatant was placed under a Boyden Chamber and vascular endothelial cell migration experiments were performed (FIG. One). Platelet-induced supernatants activated by TRAP increased the migration of vascular endothelial cells as compared to controls containing only cell culture. Platelet-induced supernatants have a variety of growth factors, especially PDGF, which is contained in alpha granules that are released when activating platelets. Therefore, the neovascularization response induced by platelet-induced supernatants is thought to be due to the secretion of PDGF.

In addition, to determine how platelet-induced supernatants are involved in the neovascularization of vascular endothelial cells, platelet-induced supernatants activated by TRAP using enzymatic immunoassay are placed in vascular endothelial cells and expressed in the integrin The amount of v3 protein was measured. 1.5 times more integrin v3 expression was observed compared with the platelet-induced supernatant treated with the activated platelet-induced supernatant but not activated. On the other hand, the other integrins a5b1, a3b1, a1, a4, a6, and b3 expressed in vascular endothelial cells did not show any difference between the experimental and control groups. However, integrin v3 expression was decreased in vascular endothelial cells treated with platelet-induced supernatants treated with NWK for 2 hours.

In addition, the platelet-induced supernatant during NWK treatment decreased the growth factor secretion, which investigated whether NWK is involved in the activation of granule secretion when TRAP is applied to platelets, thereby preventing granule secretion. TRAP was activated on platelets while NWK was added with platelets at room temperature for 10 minutes, and then the amount of CD62P present on the surface of platelets was measured to investigate whether NWK influences platelet secretion of alpha granules. In addition, it was investigated whether PAC-1 antibody, which is a monoclonal antibody to integrin GPIIbIIIa, a fibrinogen receptor that is importantly involved in outside-in signaling upon platelet activation, adheres to platelets. As a result, it was confirmed that the exposure of the CD62P protein that labels the secretion of alpha granules during TRAP treatment was inhibited by NWK (FIG. 3). CD62P (P-selectin) exposed to activated platelets can bind to P-selectin glycoprotein ligand 1 (PSGL-1) of endothelial cells and immune cells, and these bindings cause activation of these endothelial cells and immune cells. . Therefore, it can be seen that platelet-induced supernatants obtained from NWK-treated platelets reduce angiogenesis-induced responses due to the role of NWK in reducing the activation of GPIIbIIIa, including platelet activation.

NWK, an ADAM15 disintegrin analog variant, also has the activity of inhibiting platelet aggregation in vitro. In the present invention, the platelet-induced supernatant was obtained by treating NWK prior to activating platelets with TRAP and then treating TRAP. As the amount of NWK increased, the effects of neovascularization measured by vascular endothelial cell migration by TRAP-activated platelet-induced supernatant decreased. However, NWK protein itself has no effect on the migration of vascular endothelial cells. In addition, to investigate whether NWK is involved in platelet activation, the amount of PDGF-AB secretion in platelets activated with TRAP and platelets activated with TRAP after NWK was measured. As a result, PDGF-AB secretion was increased in platelets activated with TRAP, while PDGF_AB secretion was decreased dose-dependently in TRAP-activated platelets after NWK treatment (FIG. 4).

As can be seen from the above configuration, the protein of the present invention not only prevents platelet aggregation but also inhibits granule secretion of platelets, and thus, various diseases such as cancer metastasis, which can be caused by activated epithelial cells and immune cells. It is effective for asthma or arthritis.

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Claims (8)

delete delete delete delete A composition for inhibiting platelet granule secretion comprising a protein comprising a disintechrin-like domain described in SEQ ID NO: 1 as an active ingredient. The composition of claim 5, wherein the composition is a composition for treating asthma or arthritis. 6. The composition of claim 5, wherein the 52-54th sequence of SEQ ID NO: 1 is substituted with asparagine, tryptophan, and lysine, respectively. The composition of claim 5, wherein the protein is expressed in yeast.

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