JP2005137884A - Antithrombotic blood contact medical instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of forming an environment close to the lumen of a blood vessel of an organism on the surface of a blood contact medical instrument such as an artificial cardiopulmonary circuit by efficiently covering the surface of the blood contact medical instrument such as an artificial extracorporal circulation circuit with intended protein in a liquid phase in a short time while retaining its function. <P>SOLUTION: Erythrocytic membrane protein dissolved material is prepared so that DAF(CD55) and CD59 which are GPI anchor type protein existing in the erythrocytic membrane protein dissolved material is well adsorbed onto a hollow yarn for an artificial lung and an assembled artificial cardiopulmonary circuit is covered with erythrocytic membrane protein solution. A chimera GPI anchor type protein is manufactured by extracellular part of CR1 (1 type complement acceptor: CD35) manufactured by recombinant DNA technology, or extracellular part of thrombomodulin and C terminal 38 amino acid part which is GPI anchor adhesion region of DAF (CD 55). <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  In the medical field, the present invention provides a glucosylphosphatidylinositol-anchored protein (Protein with GPI anchor, which has a function of controlling the activation of blood components on the surface of a blood contact medical device such as an artificial extracorporeal circuit. Hereinafter, it is referred to as a GPI-anchored protein (see FIG. 1), a method for preparing an antithrombotic blood contact medical device coated with anti-thrombotic blood contact medical device obtained by this preparation method, and a recombinant DNA technique. The present invention relates to a novel chimeric GPI-anchored protein and the like.

  Conventionally, blood on artificial extracorporeal circuit surfaces such as blood circuit tubes, sampling monitor tubes, intra-aortic balloon pumps, artificial heart blood pumps, angiographic catheters, artificial lungs, venous reservoirs, artificial kidneys, arterial filters, etc. Has been developed and put to practical use to control the activation of blood components such as antithrombosis so that the blood passes in a normal state. A technique that has been studied at the center is a method of coating the surface of a blood contact medical device with heparin having an anticoagulant action or a derivative thereof to control the activation of blood components.

  With respect to such a heparin coating technique, various methods have been studied to improve the biocompatibility of the blood circuit (for example, see Patent Document 1 and Non-Patent Document 1). However, it has been reported that heparin coating cannot suppress the generation of thrombin in the extracorporeal circuit (for example, see Non-Patent Document 2).

  As a recent technology of this kind, a blood compatible composition comprising an ionic complex comprising an organic cation compound and heparin or a heparin derivative, wherein the organic cation compound is bonded to four aliphatic alkyl groups. A blood compatible composition containing an ammonium salt having a total number of carbon atoms of four aliphatic alkyl chains of 22 to 26 in the range of 5% to 80% of the total ammonium salt (for example, patents) Document 2) is known.

  Moreover, as an antithrombotic material using a physiologically active substance, a thrombomodulin immobilization material (for example, see Patent Document 3), a urokinase immobilization material (for example, see Non-Patent Document 3) at the contact site of a catheter, suppresses platelet adhesion. There are known prostacycling immobilization materials (see, for example, Non-Patent Document 4). However, it has been reported that the use of such physiologically active substances and the like is still limited although the activation can be suppressed to some extent (see, for example, Non-Patent Document 5).

  Furthermore, there is a report that suppresses complement activation by administering a synthesized CR1 (type 1 complement receptor) or an anti-complement monoclonal antibody (for example, see Non-Patent Document 6, Non-patent). Reference 7).

On the other hand, medical materials (for example, see Patent Documents 4 to 6) in which a peptide having a specific amino acid sequence is immobilized on a blood contact portion are known. (2R, 4R) -4-methyl-1- [N2-((RS) -3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl) -L-arginyl] -2-piperidine A polymer substrate comprising a step of applying a uniform solution containing a carboxylic acid hydrate, one or more polymer materials, and at least two organic solvents to the surface of the polymer substrate and removing the solvent. A method of forming an antithrombogenic film on the surface of a base material of a medical device comprising, for example, Patent Document 7; A medical device in which an antithrombotic film is formed by coating an antithrombotic polysaccharide that forms a blood contact surface via a linker molecule made of a chaotic polymer on the layer (for example, see Patent Document 8), or at least One kind of anti When coating the surface of an antibacterial antithrombotic material, which is a composition comprising a lipid-solubilized mucopolysaccharide composed of an ionic complex of mucopolysaccharide and quaternary onium, and an organic polymer material. A coating method of an antibacterial antithrombotic material using a mixed solvent of the fat-solubilized mucopolysaccharide and a good solvent of the organic polymer material and a non-solvent or a poor solvent of the organic polymer material as a coating solvent (for example, a patent However, none is effective as a material having a function of controlling the activation of blood components used for the extracorporeal circuit.
Japanese Patent Laid-Open No. 48-13341 JP 2001-204809 A JP-A-61-82760 JP-A-7-224095 JP-A-8-3191 JP-A-8-59694 JP 2000-60960 A JP 2000-279511 A JP 2001-535 JP Ovrum E et al. Ann Thorac Surg; 60: 365-371,1995 Wager RW et al. Ann Thorac Surg; 58: 734-741 1994 “Artificial Organ”, Vol. 10, p989-992, 1981 Thromb.Res., 46, p685-695,1987 Y JG et al. Ann Thorac Surg 1993; 55: 917-922 Gillinov AM et al. Ann Thorac Surg 1993; 55: 67-70 Rinder CS et al. J Thorac Cardiovasc Surg 1999; 118: 460-466

  Blood endothelial cells have a strong antithrombotic action, and this antithrombotic activity is mainly borne by four molecules. That is, thrombomodulin, heparin-like sugar chain molecule, PGI2, and plasminogen activator. In addition, blood cells have complement control proteins such as DAF (Decay accelerating factor: CD55), CR1 (CD35), and MCP (Membrane cofactor protein: CD46) in order to protect themselves from activation of complement. Thus, there is a mechanism in the blood vessel that controls the activation of the coagulation system and the complement system and maintains homeostasis.

  On the other hand, blood contact medical devices such as cardiopulmonary circuits not only activate the coagulation system and complement system themselves, but also do not have such a control system, so the initial slight activation is a huge amount It causes activation of the coagulation system and complement system. This is followed by activation of platelets and leukocytes, inducing a systemic inflammatory response (SIRS) and damaging various organs. Thus, a technique for coating the surface of a blood contact medical device using a coating agent having antithrombotic properties such as heparin has been proposed.

Although sugar chains such as heparin are chemically strong and conformation (how molecules fold) does not significantly affect their functions, they can be chemically decorated and covered with artifacts. When it is easy to adapt to the extracorporeal circuit, and when trying to use the coagulation system control factor and the complement control factor which are bioactive substances, the bioactive substance is a protein. There was a drawback that the function was lost. In addition, as a method for imparting a control function to the extracorporeal circulation circuit, a method of coating the vascular endothelial cell itself is also conceivable, but there are problems of storage of the extracorporeal circulation circuit, a collection source of endothelial cells, infection problems, and the like. It wasn't the way.

  The object of the present invention is to preserve the function in a liquid phase in a short time and efficiently coat the target protein on the surface of a blood contact medical device such as an extracorporeal circulation circuit, which is an artifact, An object of the present invention is to provide a new method for forming an environment close to a cavity on the surface of a blood contact medical device such as an artificial cardiopulmonary circuit.

  The present inventors prepared an erythrocyte membrane protein lysate, and DAF (CD55) and CD59, which are GPI-anchored proteins existing in the erythrocyte membrane protein lysate, were well adsorbed on the hollow fiber for artificial lung and assembled. When the artificial cardiopulmonary circuit was coated with a erythrocyte membrane protein solution, it was found to have excellent antithrombogenicity, platelet protective action and complement activation inhibitory action. In addition, the extracellular part of CR1 (type 1 complement receptor; CD35) or the extracellular part of thrombomodulin and the C-terminal 38 amino acid part, which is the GPI anchor attachment region of DAF (CD55), produced by recombinant DNA technology It was found that the chimeric GPI-anchored protein also adsorbs well on the artificial lung hollow fiber. The present invention has been completed based on the above findings.

  That is, according to the present invention, a protein having a function of controlling blood component activation binds to a glucosylphosphatidylinositol anchor (GPI anchor) via an amide bond on the C-terminal side of the base material surface of a blood contact medical device. A method for preparing an antithrombotic blood contact medical device (Claim 1), which is coated with a glucosylphosphatidylinositol anchored protein (GPI anchor type protein), and a blood contact medical device, 2. The method for preparing an antithrombotic blood contact medical device according to claim 1 (claim 2), wherein the substrate is an artificial extracorporeal circuit, or the substrate is a polymer substrate A method for preparing an antithrombotic blood contact medical device according to claim 1 or 2 (Claim 3), or a GPI anchor protein in which a GPI anchor protein is present in a cell membrane A method for preparing an antithrombotic blood contact medical device according to any one of claims 1 to 3 (Claim 4), or a chimeric GPI produced by recombinant DNA technology, A method for preparing an antithrombotic blood contact medical device according to any one of claims 1 to 4, wherein the chimeric GPI-anchored protein is a GPI anchor existing in a cell membrane. 6. An antithrombotic blood contact according to claim 5, comprising an amino acid sequence of a GPI anchor-attachment region of a type I protein and an amino acid sequence of an extracellular region of a GPI anchor type protein other than the GPI anchor type protein. A method for preparing a medical device (Claim 6) and a protein having a function to control blood component activation are proteins having a function to control blood component activation of a coagulation system. A method for preparing an antithrombotic blood contact medical device according to any one of claims 1 to 6 (claim 7), or a protein having a function of controlling blood component activation of a coagulation system, The method for preparing an antithrombotic blood contact medical device according to claim 7 or a protein having a function to control blood component activation according to claim 7, wherein the protein has a function of controlling blood component activation. A method for preparing an antithrombotic blood contact medical device according to any one of claims 1 to 6 or activation of a blood component of the complement system, characterized in that the protein has a function of controlling crystallization. The protein having the function of controlling the protein is DAF and / or CD59, and the protein having the function of controlling the activation of blood components of the complement system (Claim 10), , CR1 The present invention relates to a method for preparing an antithrombotic blood contact medical device according to claim 9 (claim 11).

The present invention also relates to a glucosylphosphatidylinositol-anchored protein in which a protein having a function of controlling blood component activation is bound to a glucosylphosphatidylinositol anchor (GPI anchor) via an amide bond on the C-terminal side. An antithrombotic blood contact medical device (Claim 12) or a blood contact medical device characterized in that a base material surface of a blood contact medical device is coated with (GPI anchor type protein). 13. The antithrombotic blood contact medical device (Claim 13) or the base material according to claim 12, wherein the base material is a polymer base material. 13. The antithrombotic blood contact medical device according to 13, wherein the GPI-anchored protein is a GPI-anchored protein existing in a cell membrane. The antithrombotic blood contact medical device according to any one of claims 12 to 14 (claim 15) or the GPI anchor protein is a chimeric GPI anchor protein produced by a recombinant DNA technique. The antithrombotic blood contact medical device according to any one of claims 12 to 15, and the chimeric GPI anchor protein is an amino acid sequence of a GPI anchor attachment region of a GPI anchor protein existing in a cell membrane. And an amino acid sequence of an extracellular region of a GPI anchor type protein other than the GPI anchor type protein. 13. The protein having a function of controlling component activation is a protein having a function of controlling blood component activation of a coagulation system. The antithrombotic blood contact medical device according to any one of claims 17 (Claim 18) or the protein having a function of controlling the activation of blood components of a coagulation system is thrombomodulin. The antithrombotic blood contact medical device (claim 19) or the protein having a function of controlling blood component activation is a protein having a function of controlling the blood component activation of the complement system. Item 20. The antithrombotic blood contact medical device according to any one of Items 12 to 17, and the protein having a function of controlling the blood component activation of the complement system is DAF and / or CD59. The antithrombotic blood contact medical device according to claim 20 (claim 21) or the protein having a function of controlling blood component activation of the complement system is CR1. Antithrombotic The present invention relates to a sex blood contact medical device (claim 22).

  Furthermore, the present invention relates to an amino acid sequence of a GPI anchor-attached region of a GPI-anchored protein existing in a cell membrane and an amino acid sequence of an extracellular region of a GPI-anchored protein other than the GPI-anchored protein produced by recombinant DNA technology. The amino acid sequence of the GPI anchor-attached region of a chimeric GPI anchor type protein (claim 23) or a GPI anchor type protein existing in the cell membrane is the amino acid sequence of the GPI anchor-attached region of DAF 24. The chimeric GPI-anchored protein according to claim 23 (claim 24) or the amino acid sequence of the extracellular region of GPI-anchored protein is the amino acid sequence of the extracellular region of CR1 or thrombomodulin. 25. A chimeric GPI-anchored protein according to claim 23 or 24 25) and a chimeric GPI anchor incorporating a DNA encoding a GPI anchor-attached region of a GPI-anchored protein existing in a cell membrane and a DNA encoding an extracellular region of a GPI-anchored protein other than the GPI-anchored protein A chimeric GPI-anchored protein production method characterized in that a chimeric GPI-anchored protein expression vector is expressed in a GPI-anchor expression host cell, and a recombinant DNA technique is used. The amino acid sequence of the GPI anchor-attached region of the GPI-anchored protein existing in the cell membrane is linked to the amino acid sequence of the extracellular region of the GPI-anchored protein other than the GPI-anchored protein. Recombinant protein that regulates blood component activation 27) or the amino acid sequence of the GPI anchor-attachment region of the GPI-anchored protein existing in the cell membrane is the amino acid sequence of the GPI anchor-attachment region of DAF. 29. The blood component activation according to claim 27 or 28, wherein the amino acid sequence of the extracellular region of the replacement protein (Claim 28) or the GPI-anchored protein is the amino acid sequence of the extracellular region of CR1 or thrombomodulin. A regulatory recombinant protein (claim 29), a DNA encoding a GPI anchor attachment region of a GPI anchor type protein existing in a cell membrane, and a DNA encoding an extracellular region of a GPI anchor type protein other than the GPI anchor type protein An integrated chimeric GPI-anchored protein expression vector was constructed and this chimeric GPI The present invention relates to a method for producing a blood protein activation-controlling recombinant protein, characterized in that a NNKER protein expression vector is expressed in a host cell (claim 30).

  The anti-thrombotic blood contact medical device of the present invention having a substrate surface coated with a protein having a function of controlling blood component activation via a GPI anchor is an artificial extracorporeal that is very close to the actual intravascular environment. It can form a circulation circuit, has antithrombogenicity, can suppress not only complement activation but also blood coagulation activation and platelet activation, which is advantageous for artificial extracorporeal circulation circuit etc. Can be used. Further, as the chimeric GPI anchor type protein, for example, when the thrombomodulin that does not normally exist as a GPI anchor type protein or the chimeric GPI anchor type protein of the present invention in which the GPI anchor is attached to CR1, the thrombomodulin is used. In addition to being able to be coated with a CR1 regulatory protein, a large amount of GPI-anchored protein derived from the patient can be synthesized.

  As a method for preparing the antithrombotic blood contact medical device of the present invention, a protein having a function of controlling the activation of blood components is formed on the substrate surface of the blood contact medical device via an amide bond on the C-terminal side. The method is not particularly limited as long as it is a method of coating with a GPI anchor type protein bonded to a GPI anchor, and the antithrombotic blood contact medical device of the present invention has a function of controlling blood component activation. Is a GPI-anchored protein in which a CPI-terminal protein is bonded to a GPI anchor via an amide bond on the C-terminal side, and is particularly restricted so long as the base material surface of a blood contact medical device is coated Rather, the GPI-anchored protein refers to the total protein that is bound to the cell membrane by covalently binding to phosphatidylinositol (PI), which is a type of phospholipid. It is. FIG. 1 shows a GPI-anchored protein in which the protein is bound to the GPI anchor via an amide bond on the C-terminal side. The GPI anchor uses the hydrophobic part of PI as an anchor and is bound to the cell membrane, and there is a common oligosaccharide core structure between the amino acid residue at the C-terminal of the protein and the PI. Ethanolamine is bonded to the C-terminal carboxyl group of the protein with an amide bond, and the hydroxyl group is bonded to the 6-position of the non-reducing terminal mannose of the oligosaccharide by a phosphodiester bond. Hereinafter, mannose α1 → 2 mannose α1 → 6 mannose α1 → It is connected to 4-glucosamine α1 → 6 inositol.

  The type of blood contact medical device is not particularly limited as long as it is a medical device that comes into contact with blood, but for extracorporeal circuit, catheter, cannula, shunt, sampling monitor for cardiopulmonary and hemodialysis. Specific examples include tubes, intra-aortic balloon pumps, artificial heart blood pumps, catheters, blood reservoirs or blood chambers, arterial filters, guide wires, and stents. Among them, artificial extracorporeal circulation circuits are preferably exemplified. be able to.

  The base material constituting the blood contact medical device of the present invention is not particularly limited as long as a protein having a function of controlling blood component activation can be bound and adsorbed via a GPI anchor. , Polyethylene, polyvinyl chloride, polycarbonate, polyethylene terephthalate, polymethyl methacrylate, polyacetal, polystyrene, ABS resin, ethylene-propylene copolymer, ethylene-propylene-diene copolymer and other olefin elastomers, plasticized vinyl chloride, heat In addition to plastic polyurethane, ethylene-vinyl acetate copolymer, silicone rubber, thermoplastic polyester, silicone resin, fluororesin, various elastomers and polymer alloys such as polymer alloys can be mentioned. Doctor Substrate surface layer use the instrument is coated with a polymeric substrate, such as Teflon (registered trademark) may also be used.

  In addition, as a method of coating the base material surface of the blood contact medical device with a GPI anchor type protein, a solution containing one or more GPI anchor type proteins is contacted with the base material surface of the blood contact medical device, Although the method of making it adsorb | suck can be mentioned, it is not limited to this, Moreover, the predetermined part of the base-material surface of the blood contact medical device can also be coat | covered as needed.

  Examples of the protein having a function of controlling the activation of blood components include thrombomodulin, protein APC (Activated Protein C) downstream of thrombomodulin coagulation control, prostacyclin (PG12), plasminogen activator, heparin-like molecule, liver It binds to a protein having a function of controlling the activation of blood components of the coagulation system, such as an anticoagulant factor TFPI (Tissue Factor Passway Inhibitor), and Db (CD55), MCP, CR1 (CD35), CD59, and C3b. And proteins having a function of controlling the activation of blood components of the complement system such as factor H, MCP (Menbrane Cofactor Protein), and membrane protein that promotes inactivation of C3b and C4b by factor I.

  Examples of the GPI anchor type protein in the present invention include a GPI anchor type protein existing in a cell membrane and a chimeric GPI anchor type protein produced by a recombinant DNA technique. Examples of the GPI-anchored protein present in the cell membrane include GPI-anchored membrane proteins expressed in cell membranes such as blood cells, lymphocytes and vascular endothelial cells. In this case, a erythrocyte membrane protein lysate in which GPI anchor type membrane protein DAF and GPI anchor type membrane protein CD59 are mixed can be advantageously used as the GPI anchor type membrane protein. However, it is difficult to purify such existing GPI-anchored membrane proteins in large quantities. Because it is necessary to separate from the human cell membrane and it is desirable that it is the person's cell as much as possible in actual medical treatment. The ability to synthesize and store is essential for clinical application.

  The chimeric GPI-anchored protein produced by the recombinant DNA technology of the present invention includes a recombinant DNA molecule produced by fusing different DNAs derived in vitro using an enzyme or the like into a host cell and proliferated. Any non-naturally occurring chimeric GPI-anchored protein produced by recombinant DNA technology that synthesizes a predetermined substance in large quantities is not particularly limited, but the amino acid in the GPI anchor-attachment region of the GPI-anchored protein existing in the cell membrane Preferred examples include chimeric GPI-anchored proteins having a sequence and an amino acid sequence of an extracellular region of a GPI-anchored protein other than the GPI-anchored protein, and more specifically, a DAF GPI anchor attachment Of the C-terminal 38 amino acid part which is the region and the extracellular region of CR1 or thrombomodulin With a amino acid sequence, it can be exemplified chimeric GPI-anchored proteins.

  These chimeric GPI-anchored proteins comprise a viral vector comprising a DNA encoding a GPI anchor-attached region of a GPI-anchored protein existing in a cell membrane and a DNA encoding an extracellular region of a GPI-anchored protein other than the GPI-anchored protein. A chimeric GPI-anchored protein expression vector incorporated into an expression vector for mammals such as the above is constructed, and this chimeric GPI-anchored protein expression vector is transferred to GPI-anchor expression host cells such as CHO cells, for example, calcium phosphate transfection, DEAE- It can be created by introducing and expressing by dextran-mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation. That. A GPI-anchored protein is constructed by forming a complex between a protein part separately synthesized in the endoplasmic reticulum and a GPI anchor part.

  The blood component activation control recombinant protein of the present invention includes an amino acid sequence of a GPI anchor-attached region of a GPI anchor type protein existing in a cell membrane produced by a recombinant DNA technique, and a GPI anchor other than the GPI anchor type protein. The amino acid sequence of the GPI anchoring region of the GPI-anchored protein existing in the cell membrane is not particularly limited as long as it is a recombinant protein linked directly or via a linker to the amino acid sequence of the extracellular region of the type protein, A recombinant protein in which an amino acid sequence of an extracellular region of a GPI anchor type protein other than the GPI anchor type protein is linked directly or via a linker can be preferably mentioned. More specifically, a DPI GPI C-terminal 38 amino acid part which is an anchor attachment region, and CR1 or thrombomodulin It can be exemplified recombinant protein and the amino acid sequence of the extracellular region is linked directly or via a linker.

  These blood component activation control recombinant proteins can be prepared from the chimeric GPI-anchored protein by enzymatic treatment, but the DNA encoding the GPI-anchor attachment region of the GPI-anchored protein existing in the cell membrane and the GPI-anchor type A chimeric GPI-anchored protein expression vector was constructed by incorporating a DNA encoding the extracellular region of a GPI-anchored protein other than a protein into a mammalian expression vector such as a viral vector. Host mammalian cells, eg, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation Introduced in Deployment it can be produced by expressing. The blood component activation controlling recombinant protein can be used as a therapeutic agent for diseases that require structural analysis of GPI-anchored proteins and blood component activation control.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Preparation of erythrocyte membrane protein lysate (collection of existing GPI-anchored membrane protein)]
20 ml of human whole blood was collected and 100 U of heparin was added. Centrifugation was performed at 500 G and 4 ° C. for 10 minutes, and the serum portion was discarded. 30 ml of distilled water was added, and after stirring well, it was centrifuged at 500 G, 4 ° C. for 10 minutes. The supernatant was removed, 30 ml of distilled water was added again, and after stirring well, the mixture was centrifuged at 500 G, 4 ° C. for 10 minutes. The precipitate (erythrocyte membrane) was sufficiently washed with a phosphate buffer (hereinafter referred to as “PBS”) (pH 7.4). The precipitate (erythrocyte membrane) was dissolved in 10 ml of 1 w / v% octylglucoside, allowed to stand at room temperature for 30 minutes, and then centrifuged at 500 G, 4 ° C. for 10 minutes to remove insoluble components. About 10 ml of 1 w / v% octylglucoside lysate after centrifugation was used as the erythrocyte membrane protein lysate.

[Coating experiment of artificial lung hollow fiber with erythrocyte membrane protein (experiment as to whether CD55 or CD59, which is an existing GPI-anchored membrane protein, is adsorbed to the artificial lung hollow fiber)] Polypropylene artificial hollow fiber ( 10 cm was cut out and placed in 9 ml of PBS. To this was added 1 ml of the erythrocyte membrane protein solution prepared in Example 1, and left at room temperature for 30 minutes. To the control, 1 ml of 1 w / v% octyl glucoside was added and left at room temperature for 30 minutes in the same manner. After washing these well, 50% human serum was added and left standing at room temperature for 30 minutes for blocking. The membrane protein-reacted hollow fiber (RBC fiber) and the control hollow fiber (control) were labeled with phycoerythrosin-labeled anti-CD55 antibody (antiCD55-PE; manufactured by Phamingen) or fluorescein isocyanate Anti-CD59 antibodies (antiCD59-FITC; manufactured by Phamingen) were reacted with each other and observed with a fluorescent stereomicroscope (manufactured by Nikon). For comparison, the same operation was performed using DTAF (Dichlorotriazinylamino Fluorescein) -labeled alkyl heparin instead of erythrocyte membrane protein, and observation was performed with a confocal laser scanning microscope (manufactured by Bio Rad). Alkylheparin was prepared according to the method described by the present inventors (Biomacromolecules, 2, 1169-1177).

  A confocal laser scanning microscopic image (FIG. 2A) of the cross section of the hollow fiber for artificial lung showed a clear fluorescent activation layer by fluorescent dye-bound alkyl heparin. In addition, DAF (CD55) and CD59, which are GPI-anchored membrane proteins, were also well adsorbed on the artificial lung hollow fibers (FIGS. 2B and 2C). Adsorption of DAF (CD55) or CD59 on the artificial lung hollow fiber hardly desorbed even after washing several times with PBS.

[Assembly of an artificial cardiopulmonary circuit]
An artificial lung for animals (EC-30A, manufactured by Izumi Kogaku Medical Industrial Co., Ltd.) and a heat exchanger for small animals (HHE72, manufactured by Izumi Kogaku Medical Industrial Co., Ltd.) are combined with a vinyl chloride tube, and a roller pump (BPC-S, Tonokura Medical Industrial Co. As shown in FIG. 6, it was attached to a rabbit (body weight 3 kg) and subjected to normal temperature extracorporeal circulation. The extracorporeal circuit was filled with 100 ml of lactic acid Ringer solution.

[Coating of cardiopulmonary circuit with erythrocyte membrane protein solution]
10 ml of the initial filling liquid was taken out, 10 ml of erythrocyte membrane protein lysate was added, and the mixture was circulated at room temperature for 30 minutes at a flow of 50 ml / min. Thereafter, all of the initial filling liquid was taken out, 100 ml of lactic acid Ringer solution was added, and it was circulated and washed at a flow of 250 ml / min. This washing operation was performed three times. After washing, it was again filled with 100 ml of lactic acid Ringer solution to form an erythrocyte membrane protein-coated circuit (CD55, CD59 coat). 10 ml of erythrocyte membrane protein lysate was not added as a control circuit (non-coat circuit). For comparison, 10 ml of 5% alkyl heparin lactate Ringer solution was used instead of 10 ml of erythrocyte membrane protein lysate. An alkylheparin coat was formed.

[External circulation method and sample collection]
100 ml of healthy human whole blood was collected with a 200 U heparin-containing syringe and placed in each of the three types of extracorporeal circulation circuits (erythrocyte membrane protein-coated circuit, control circuit, and alkyl heparin-coated circuit) formed in Example 4. It was circulated at a flow of 100 ml / min for about 1 minute and mixed well with the filling liquid. The final hematocrit value averaged 22%. Samples were collected immediately after mixing (Time 0). The sample was collected after 10 minutes, 30 minutes, 60 minutes, and 120 minutes by heating at 37 ° C. while being heated to 37 ° C. with a heat exchanger (Time 10, Time 30, Time 60, Time 120). ). Each of the control circuit, the erythrocyte membrane protein-coated circuit, and the alkyl heparin-coated circuit was performed four times. The levels of TAT (Thrombin-antithrombin complex), C3a, C5a, and β-thromboglobulin in plasma were measured (measurement was requested from SRL Fukuoka). Platelet count was counted with a hemocytometer (Sysmex K-4500).

[Evaluation of antithrombogenicity]
Antithrombogenicity was assessed by the generation of TAT, an indicator of thrombin generation. The results are shown in FIG. The TAT level increased with time in the three circuits, but in the erythrocyte membrane protein coat circuit (CD55, CD59 coat) and the alkylheparin coat circuit, compared with the non-coat circuit, It was found that there was a significant decrease in circulation for 120 minutes. Regarding antithrombogenicity, no significant difference was found between the erythrocyte membrane protein-coated circuit and the alkyl heparin-coated circuit, but the erythrocyte membrane protein-coated circuit seems to have a lower TAT level than the alkyl heparin-coated circuit.

[Evaluation of platelet protective effect]
The platelet protective action was evaluated by the production of β-thromboglobulin (β-TG), which is an indicator of platelet count and platelet activation. The results are shown in FIG. In the non-coat circuit, the platelet count decreased rapidly after one cycle, reached a minimum after 60 minutes, and then gradually recovered (FIG. 4A). In the alkylheparin coat circuit, the number of platelets decreased with the passage of the circulation time, but in the erythrocyte membrane protein coat circuit (CD55, CD59 coat), it hardly decreased even after 120 minutes of circulation (FIG. 4A). On the other hand, the production of β-TG increased rapidly from the start of circulation in the control circuit (FIG. 4B). In the erythrocyte membrane protein-coated circuit and the alkyl heparin-coated circuit, β-TG increased with the passage of the circulation time, but was significantly lower than that of the control circuit. The erythrocyte membrane protein-coated circuit had lower β-TG levels than the alkylheparin-coated circuit, but no statistically significant difference was observed.

[Evaluation of inhibitory effect on complement activation]
Complement activation inhibitory action was evaluated by the generation of complements C3a and C5a. The results are shown in FIG. The production of C3a increased with the passage of circulation time in the three circuits, but compared to the alkylheparin coat and the non-coat circuit, the erythrocyte membrane protein coat circuit (CD55, CD59 coat) The production amount of C3a was significantly low (FIG. 5A), and a significant difference was observed after 10 minutes of circulation. The production of C5a was also lower in the erythrocyte membrane protein-coated circuit compared to the alkyl heparin-coated circuit and the control circuit, but no statistically significant difference was observed (FIG. 5B). The C5a level in the erythrocyte membrane protein-coated circuit was below the detection limit.

  In the extracorporeal circuit in which DAF (CD55, CD59), which is a GPI-anchored membrane protein of the present invention, is adsorbed, the production of C3a is suppressed as compared with the extracorporeal circuit of the alkyl heparin-coated and non-coat circuit. (FIG. 5).

  From the above examples and the results thereof, the extracorporeal circulation circuit is coated using the existing GPI anchor type membrane protein, and the GPI anchor type membrane protein is efficiently adsorbed to the extracorporeal circuit and its function is exhibited. I was sure that.

[Cloning of thrombomodulin, CR1 cDNA and connection of GPI anchor added sequence]
The cDNA (SEQ ID NO: 1) of the C-terminal 38 amino acid part (SEQ ID NO: 2), which is the GPI anchor attachment region of DAF (CD55), was cloned from human lymphocytes and incorporated into a mammalian cell expression vector pcDNA3 (Invitrogen) ( GPI anchor attachment vector G-1). The cDNA of CR1 (type 1 complement receptor; CD35) was cloned from human lymphocytes, and the thrombomodulin cDNA was cloned from the HUBEC cell line and incorporated into a mammalian cell expression vector pcDNA3 (manufactured by Invitrogen). The cDNA (SEQ ID NOs: 3 and 5) excluding the portion encoding the transmembrane portion of CR1 and thrombomodulin was incorporated into the GPI anchor attachment vector G-1 of the DAF, and the vector pcDNA3-GCR incorporating CR1. And vector pcDNA3-GTM incorporating thrombomodulin was constructed. These vectors were introduced into CHO cells to establish stable expression strains. The cells were mass-cultured in RPMI medium containing 10% fetal calf serum FCS. Each of these CHO cells was examined by flow cytometry, and the antigen expressed on the cell surface was bound to a FITC antibody having fluorescence to measure the amount of fluorescence, thereby confirming that the introduced gene was expressed (FIG. 7). ).

  Recombinant protein was separated and purified from the large-scale cultured cells using an affinity column. About 2 mg of GPI-anchored thrombomodulin (GPI-CR1), which is a chimeric GPI-anchored protein having a recombinant protein having the amino acid sequence represented by SEQ ID NO: 4, and the amino acid sequence represented by SEQ ID NO: 6 About 4 mg of GPI-anchored CR1 (GPI-TM), which is a chimeric GPI-anchored protein having a recombinant protein having the above, was purified.

[Antithrombogenicity of synthetic GPI-anchored thrombomodulin]
Crude purified GPI-TM (GPI-anchored thrombomodulin) was used to coat 20 cm of hollow fiber (polypropylene) for artificial lung. A 50 ml conical was filled with 25 ml of fresh human blood, and an untreated hollow fiber (control) and a coated hollow fiber were placed therein and gently shaken at 37 degrees. Before and after shaking, 2 ml of blood was collected at 15 minutes and 30 minutes after the start, and the TAT level was measured (FIG. 8). As a result, it was found that GPI-TM exhibits excellent antithrombogenicity.

[Inhibition of complement activation of synthetic GPI-anchored CR1]
Crude purified GPI-CR1 (GPI-anchored CR1) was used to coat 20 cm of hollow fiber (polypropylene) for artificial lung. A 50 ml conical was filled with 25 ml of fresh human blood, and an untreated hollow fiber (control) and a coated hollow fiber were placed therein and gently shaken at 37 degrees. Before and after shaking, 15 ml and 30 minutes after the start, 2 ml of blood was collected and C3a level was measured (FIG. 9). As a result, it was found that GPI-CR1 exhibits excellent complement activation inhibitory action.

It is a figure which shows frame | skeleton of GPI anchor type membrane protein. It is a figure which shows the coating result to the hollow fiber for artificial lungs of the labeled alkyl heparin of a comparative example, and the erythrocyte membrane protein of this invention. It is a figure which shows the measurement result of the TAT level in the erythrocyte membrane protein coating circuit (CD55, CD59 coat) of this invention, the alkyl heparin coating circuit (alkylheparin coat) of a comparative example, and a control circuit (non-coat circuit). * Indicates p <0.05 relative to the control circuit. The platelet count and β-thromboglobulin (β-TG) level in the erythrocyte membrane protein coated circuit (CD55, CD59 coat) of the present invention, the alkylheparin coat circuit of the comparative example and the non-coat circuit. It is a figure which shows a measurement result. * Indicates p <0.05 for the control circuit, and ** indicates p <0.01 for the control circuit. It is a figure which shows the measurement result of the complement C3a and C5a level in the erythrocyte membrane protein coating circuit (CD55, CD59 coat) of this invention, the alkylheparin coating circuit (alkylheparin coat) of a comparative example, and a control circuit (non-coat circuit). . * Indicates p <0.05 for the control circuit, and ** indicates p <0.01 for the control circuit. It is a figure which shows the outline of the coating | coated with the erythrocyte membrane protein solution in an artificial cardiopulmonary circuit. It is a figure of the flow cytometry which shows the expression of the chimeric GPI anchor type protein in a CHO cell. It is a figure which shows the measurement result of the TAT level in the synthetic | combination GPI anchor type thrombomodulin covering hollow fiber of this invention, and an untreated control hollow fiber. ** indicates p <0.01 relative to the control hollow fiber. It is a figure which shows the measurement result of the complement C3a level in the synthetic | combination GPI anchor type CR1 coating | coated hollow fiber of this invention, and an untreated control hollow fiber. ** indicates p <0.01 relative to the control hollow fiber.

Claims (30)

A glucosylphosphine in which a protein having a function of controlling blood component activation is bound to a glucosylphosphatidylinositol anchor (GPI anchor) via an amide bond on the C-terminal side of the base material surface of a blood contact medical device A method for preparing an antithrombotic blood contact medical device, characterized in that it is coated with a phatidylinositol-anchored protein (GPI-anchored protein). The method for preparing an antithrombotic blood contact medical device according to claim 1, wherein the blood contact medical device is an artificial extracorporeal circuit. The method for preparing an antithrombotic blood contact medical device according to claim 1 or 2, wherein the substrate is a polymer substrate. The method for preparing an antithrombotic blood contact medical device according to any one of claims 1 to 3, wherein the GPI anchor type protein is a GPI anchor type protein existing in a cell membrane. The method for preparing an antithrombotic blood contact medical device according to any one of claims 1 to 4, wherein the GPI-anchored protein is a chimeric GPI-anchored protein produced by recombinant DNA technology. The chimeric GPI anchor type protein comprises an amino acid sequence of a GPI anchor attachment region of a GPI anchor type protein existing in a cell membrane and an amino acid sequence of an extracellular region of a GPI anchor type protein other than the GPI anchor type protein. A method for preparing an antithrombotic blood contact medical device according to claim 5. The antithrombotic blood contact medicine according to any one of claims 1 to 6, wherein the protein having a function of controlling blood component activation is a protein having a function of controlling blood component activation of a coagulation system. Method of preparation of the device. The method for preparing an antithrombotic blood contact medical device according to claim 7, wherein the protein having a function of controlling the activation of blood components of the coagulation system is thrombomodulin. The antithrombotic blood contact medical treatment according to any one of claims 1 to 6, wherein the protein having a function of controlling blood component activation is a protein having a function of controlling blood component activation of the complement system. Method of preparation of the device. The coating method according to claim 9, wherein the protein having a function of controlling the activation of blood components of the complement system is DAF and / or CD59. The method for preparing an antithrombotic blood contact medical device according to claim 9, wherein the protein having a function of controlling activation of blood components of the complement system is CR1. Glucosylphosphatidylinositol-anchored protein (GPI-anchored protein) in which a protein having a function of controlling blood component activation is bound to a glucosylphosphatidylinositol anchor (GPI anchor) via an amide bond on the C-terminal side ), Wherein the base material surface of the blood contact medical device is coated. 13. The antithrombotic blood contact medical device according to claim 12, wherein the blood contact medical device is an artificial extracorporeal circuit. The antithrombotic blood contact medical device according to claim 12 or 13, wherein the base material is a polymer base material. The antithrombotic blood contact medical device according to any one of claims 12 to 14, wherein the GPI anchor type protein is a GPI anchor type protein existing in a cell membrane. The antithrombotic blood contact medical device according to any one of claims 12 to 15, wherein the GPI anchor type protein is a chimeric GPI anchor type protein produced by a recombinant DNA technique. The chimeric GPI-anchored protein comprises an amino acid sequence of a GPI anchor-attached region of a GPI-anchored protein existing in a cell membrane and an amino acid sequence of an extracellular region of a GPI-anchored protein other than the GPI-anchored protein. The antithrombotic blood contact medical device according to claim 16. The antithrombotic blood contact medicine according to any one of claims 12 to 17, wherein the protein having a function of controlling blood component activation is a protein having a function of controlling blood component activation of a coagulation system. Appliances. 19. The antithrombotic blood contact medical device according to claim 18, wherein the protein having a function of controlling the activation of blood components of the coagulation system is thrombomodulin. The antithrombotic blood contact medical treatment according to any one of claims 12 to 17, wherein the protein having a function of controlling blood component activation is a protein having a function of controlling blood component activation of the complement system. Appliances. 21. The antithrombotic blood contact medical device according to claim 20, wherein the protein having a function of controlling activation of blood components of the complement system is DAF and / or CD59. 21. The antithrombotic blood contact medical device according to claim 20, wherein the protein having a function of controlling the activation of blood components of the complement system is CR1. Amino acid sequence of GPI anchor-attached region of GPI-anchored protein existing in cell membrane and amino acid sequence of extracellular region of GPI-anchored protein other than GPI-anchored protein prepared by recombinant DNA technology A chimeric GPI-anchored protein characterized by The chimeric GPI-anchored protein according to claim 23, wherein the amino acid sequence of the GPI anchor-attached region of the GPI-anchored protein existing in the cell membrane is the amino acid sequence of the GPI anchor-attached region of DAF. The chimeric GPI-anchored protein according to claim 23 or 24, wherein the amino acid sequence of the extracellular region of the GPI-anchored protein is the amino acid sequence of the extracellular region of CR1 or thrombomodulin. Construction of a chimeric GPI anchor protein expression vector incorporating a DNA encoding a GPI anchor attachment region of a GPI anchor protein in a cell membrane and a DNA encoding an extracellular region of a GPI anchor protein other than the GPI anchor protein A method for producing a chimeric GPI anchor type protein, comprising expressing the chimeric GPI anchor type protein expression vector in a GPI anchor expression host cell. The amino acid sequence of the GPI anchor-attached region of the GPI-anchored protein existing in the cell membrane and the amino acid sequence of the extracellular region of the GPI-anchored protein other than the GPI-anchored protein produced by recombinant DNA technology is linked. A blood protein activation-controlling recombinant protein characterized by 28. The blood component activation controlling recombinant protein according to claim 27, wherein the amino acid sequence of the GPI anchor-attachment region of the GPI-anchored protein existing in the cell membrane is the amino acid sequence of the GPI anchor-attachment region of DAF. 29. The blood component activation controlling recombinant protein according to claim 27 or 28, wherein the amino acid sequence of the extracellular region of the GPI-anchored protein is the amino acid sequence of the extracellular region of CR1 or thrombomodulin. Construction of a chimeric GPI anchor protein expression vector incorporating a DNA encoding a GPI anchor attachment region of a GPI anchor protein in a cell membrane and a DNA encoding an extracellular region of a GPI anchor protein other than the GPI anchor protein And a method for producing a blood component activation controlling recombinant protein, characterized in that the chimeric GPI-anchored protein expression vector is expressed in a host cell.

Images