JPS6023625B2 - antithrombotic material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antithrombotic material, and more specifically, the present invention relates to an antithrombotic material that suppresses the adhesion of platelets to the surface of the material by modifying a fibrillation system activating enzyme immobilized on a carrier. Regarding materials.

It is known that blood coagulation that occurs when using artificial organs, catheters, etc. is mainly caused by endogenous blood coagulation and adhesion of platelets.

Intrinsic blood coagulation, the initial reaction is initiated by the activation of coagulation system factors due to contact between blood and the surface of a foreign body, and as a result of the cascade activation of coagulation system factors, Xa, thrombin, etc. are generated, and the final reaction occurs. is caused by the formation of a fibrin network.

In addition, the adhesion of platelets to the material surface is such that when blood 4 or platelets come into contact with the material surface, they recognize a foreign object and adhere to the surface, forming a thrombus by involving red blood cells and fibrin. The above two causes act synergistically in thrombus formation. Therefore, an antithrombotic material having urokinase immobilized on its surface has been proposed in order to dissolve the formed fibrin network.

Urokinase is a fibrinolytic system activating enzyme that activates plasminogen in the blood and converts it into plasmin. Therefore, it has an excellent antithrombotic function that dissolves and removes blood clots. There is a problem of increased adhesion of platelets on the surface of the material due to the oxidized urokinase. As mentioned above, the adhesion of platelets on the material surface causes thrombus formation together with endogenous blood coagulation. Blood and plate adhesion caused by contact between blood and materials is an important reaction directly related to thrombus formation, but the reaction mechanism has not yet been fully elucidated.

On the other hand, it is true that several types of blood bran proteins are always adsorbed to the surface of the material, and the number of platelet adhesion is influenced by the type of platelet protein.
There is also no known material that specifically adsorbs only proteins with low platelet adhesiveness. Therefore, the present inventors
As a result of intensive research to solve the various problems mentioned above with conventional antithrombotic materials, we have found that the linear activity of urokinase can be reduced by modifying the sugar residues of urokinase immobilized on the material with a specific modification enzyme. without letting
The present invention was achieved by discovering that platelet adhesion to materials can be suppressed.

Therefore, the antithrombotic material according to the present invention is characterized in that urokinase immobilized on the material is modified with galactose oxidase.

The material used in the present invention does not have a substantially harmful effect on living organisms, and any material can be used as long as urokinase is immobilized thereon.

High molecular weight polymers are usually used as such materials, such as polyethylene, polypropylene, polyisobutylene, polymethylbentene, polystyrene, natural rubber,
Chloroprene rubber, polyvinyl chloride, polyvinyl fluoride, polytetrafluoroethylene, polyvinylidene chloride,
Polyvinyl acetate, polyvinyl alcohol, ethylene-vinyl alcohol copolymer, polyacrylonitrile, polyglycolic acid, polyvinylpyrrolidone, polyvinyl formal, polyvinyl butyral, acetal resin, acrylic resin, polyacrylamide, polycarbonate, polysulfone, polyethylene terephthalate, polyethylene naphthalate Examples include synthetic and natural polymers such as polyamide, polyimide, cellulose, nitrocellulose, cellophane, collagen, gelatin, and bran. The method for immobilizing urokinase onto the material is not limited in any way, and any conventionally known enzyme immobilization method can be used, and appropriate methods such as covalent bonding, ionic bonding, etc. can be used. For example, if the above enzyme is reacted with a material together with a carbodimide reagent or a Woodward reagent, it can be easily immobilized by a covalent bond, but it is not limited to these methods. Further, in order to ionically bond the enzyme to a material, the material may be soaked in an aqueous solution of the enzyme. There are no particular restrictions on the amount of urokinase immobilized on the material, but it is usually 0.1 to 200 urokinase per 1 material area.
If it is immobilized, it has good antithrombotic properties.

Furthermore, in the present invention, anticoagulants other than urokinase may be immobilized together with urokinase.
The antithrombotic material according to the present invention is obtained by modifying the sugar residues of urokinase immobilized on the material with galactose oxidase.

The modification treatment method is not particularly limited as long as the conditions are mild so that urokinase is not inactivated, but usually, immobilized urokinase is soaked in a buffer solution and heated at 10 to 40 °C at a pH of 7 to 9.
The reaction may be carried out for about 1 to 1 hour at a temperature of In addition to galactose oxidase, 8-galactosidase and neuraminidase are known as enzymes that modify the bran residue of enzymes, but unlike modification with galactose oxidase, even if urokinase is modified with these,
No effect on suppressing platelet adhesion to the material was observed, and
Modification of enzyme sugar residues with periodic acid is also known;
When applied to urokinase, it has been observed that the enzyme activity of urokinase is reduced, so it is thought that the effect of suppressing platelet adhesion to materials without reducing enzyme activity is unique to galactose oxidase.

The antithrombotic material according to the present invention can be formed into particles, sheets, or
It can be prepared in any form such as a tubular shape. As described above, the antithrombotic material according to the present invention maintains the fibrinolytic activity of urokinase while suppressing platelet adhesion caused by urokinase immobilized on the material, so it can be used in direct contact with blood in blood circulation circuits, reactors, etc. When used in artificial organs, it can effectively prevent thrombus formation associated with activation of the blood coagulation system.

The present invention will be explained below with reference to Examples, but the present invention is not limited to the Examples in any way. EXAMPLE An aromatic polyamide film (approximately 1 inch) was washed with water and then activated by immersion in an IN hydrochloric acid/methanol solution at a temperature of 3000° C. for 30 minutes, followed by partial hydrolysis.

After washing with water, immersion in an acetate buffer (5 M, pH 4.8) containing 10 mmol of ethyldimethylaminopropylcarbodiimide hydrochloride (EDC) for 15 minutes in the ground, followed by urokinase 500. Urokinase was directly immobilized on the polyamide by adding 25 volumes of potassium phosphate buffer containing 250 mg of potassium phosphate and allowing it to stand overnight. This urokinase-immobilized polyamide (hereinafter abbreviated as 1-UK).

) was soaked in the above phosphate buffer, 10 units of galactose oxidase was added, and the mixture was reacted at a temperature of 160°C for 4 hours.
8M) and thus modified with galactose oxidase 1-UK (hereinafter abbreviated as 1-UK-GO).
I got it. Platelets (platelet-rich blood clots) were brought into contact with the surface of this 11 UK-GO for 15 minutes, and the number of platelets adhesion was examined using a 60-sound microscope.

As a control, 1-UK was soaked in Tris-HCl buffer containing no galactose oxidase. The results are shown in Table 1. Next, the enzyme activities of the modified immobilized urokinase obtained above and the control were measured by a synthetic substrate method.

GIu pine ryl-GIycyl-~gn as a synthetic substrate
Using yl-me ratio yl-coumaryl-amide, it was added to 5 nM Tris-HCl buffer (0.1 MN
aC1, 1 skin M CaC12, pH 8.0) (final concentration 0.1 mM). Immobilized urokinase was immersed in this solution 2 and reacted at a temperature of 370°C for 30 minutes, followed by a 1% by weight acetic acid aqueous solution 3. The reaction was stopped by adding 1000 m, and the reaction was measured using a fluorescence spectrophotometer (excitation wavelength: 38 m, reflection wavelength: 46 m).
Amino-methyl-Com released in
The concentration of marine (AMC) was measured. The results are shown in Table 1. As is clear from the above results, platelet adhesion to the material can be significantly reduced by modifying 1-UK with galax oxidase without reducing the enzymatic activity of urokinase.

Comparative Example Immobilized urokinase obtained in the same manner as in Example 1 was soaked in a 0.1N periodic acid aqueous solution at room temperature for 2 hours, and then soaked in a 1 hM Tris-HCl buffer (NaCIO).
．． 149M, pH 7.4) to remove periodic acid.

Next, using the same machine as in Example, the number of platelets adhering to the material and enzyme activity were examined. Table 2 also shows the number of platelet adhesion and enzyme activity of the immobilized urokinase before and after treatment with galactose oxidase. From the above results, it is clear that by treating immobilized urokinase with periodic acid Table 2 Reverse B*periodic acid treated product, the number of platelets adhering to the material decreases, but the enzyme activity is completely lost. be.

Claims (1)

[Claims] 1. An antithrombotic material characterized by being modified with urokinase or galactose oxidase immobilized on a carrier.