JPH02305575A - Antithrombus material and manufacture thereof - Google Patents

Abstract

PURPOSE:To improve antithrombus properties, living body compatibility, and mechanical strength by a method wherein a polymerized protein layer not substantially containing at least erythrocyte and leukocyte is formed on the surface of a polymer material. CONSTITUTION:The surface of a polymer material is treated by means of a protein acting enzyme-contained solution (solution A) and then treated by a blood plasma forming component protein-contained solution (solution B). Protein acting enzyme is enzyme having a thrombin-like action, e.g. thrombin or lebutylase, and blood plasma forming component protein is preferably fibrinogen, cryoplecibtate or plasma. A protein layer polymerized by this treatment is formed by bringing a solution A into contact with a solution B, and a stabilized 'fibrin' not substantially containing erythrocyte and leukocyte. This antithrombus material has excellent antithrombus properties and organism adaptability and excellent mechanical performance, and thrombus is not increased even when the antithrombus material is used for a long time.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an antithrombotic material and a method for producing the same. especially,
The present invention relates to a medical polymer material with excellent antithrombotic properties and biocompatibility suitable for use in artificial blood vessels, artificial organs, etc., and a method for producing the same.

[Prior art] ``In the field of medical materials, many synthetic polymer materials such as polyester, polyethylene, polypropylene, and polyurethane are used and have a proven track record. Medical materials used, e.g.
When used as an artificial blood vessel, blood coagulates, causing thrombus formation. To solve this problem, various polymeric materials have been devised to have antithrombotic properties. For example,
A method of chemically bonding a natural anticoagulant such as heparin to the surface of a material (see Japanese Patent Publication No. 103190/1983);
Or synthesis of 1,2-diphenyl-3 or 5-dioxypyralizone derivatives! ! ! #A method of imparting an active compound to a polymer (see Japanese Patent Publication No. 142772/1983) is known. Currently used heparin has biocompatibility issues because it is an anticoagulant derived from non-human animals, mainly obtained from pig organs, and its activity decreases when it is covalently bonded to a polymeric carrier. Methods of binding heparin to carriers, such as loss, also leave an interlayer. In order to solve these problems, the surface of a polymer material was coated with a polymer containing heparin in an ionic bond, and a document showing good antithrombotic properties (Shoji Nagaoka et al., Artificial Organs, Vol. 17, No. 2, No. 598, 1988) 6
01) and polystyrene-PEO-heparin ternary polystyrene-PEO-heparin copolymers with heparin incorporated into one of the microdomains have been synthesized and tested for their antithrombotic properties (
Vulic, 1. Transactions
f 13th Annual Meeting of
5ociety for Biomaterials,
P, 81.1987), but it is still insufficient for practical use as an artificial blood vessel, and the ones that are satisfactory are:
Also, the thrombolytic enzyme urokinase has not been obtained.
Although it has been widely used clinically as a therapeutic agent for thrombosis, the action of urokinase cannot be suppressed because blood contains large amounts of plasmin inhibitors such as α8-plasmin inhibitor and α□-macroglobulin. However, the effects expected from in vitro activity have not been observed.

In addition to the above-mentioned method of imparting antithrombotic properties to polymeric materials, research on a series of segmented polyurethane systems is known as a method of making the material itself less likely to generate thrombi. By experimenting with research methods based on molecular design instead of conventional trial-and-error methods, the relationship between phase separation and antithrombotic properties of microdomain polymers is becoming clearer (Atsushi Takahara et al., 16th Medical High School Proceedings of the Molecular Symposium, p. 21, 1987), and a method using highly porous polyurethane as a material is being considered (Martz,
H, et al., Biosaterialg, 8.
3.1987: Reference), but in this case, neointimal formation does not occur or is very slow after transplantation, so these are still insufficient for practical use (Yasu Tamura et al., Artificial Organs , 16.1500.1987: Reference).

In contrast to these studies, initial thrombotic closure after graft grafting can be prevented and good short-term results are obtained, but long-term results lasting more than 1 month often result in closure, and the cause of this is neoplasia. It has been reported that this is due to intimal hypoplasia (Nobu Sato et al.: Journal of the Oral Society, pp. 89-109, 1988: Yoshinobu Kubo et al.; Angiology: 27.8.567-57011
987: Norio Morimoto et al.; Artificial organs; 14.941-94
4. 1985: Sakai, Fuko et al.; Artificial organs, 15.367
370, 1986), the limitations of providing both antithrombotic and early tissue healing properties using only synthetic polymeric materials have been pointed out. Under these circumstances, a method of forming a collagen layer on the surface of a polymeric material (
Japanese Patent Publication No. 61-58196), and a method of crosslinking natural blood vessels using polyglycerol, polyglycidel ether (PGPGE), which has epoxy groups as reactive groups in side chains and terminals (Chisato Nojiri et al.; Artificial organs, 16.147
Although attempts have been made to actively promote neointimal formation in the form of so-called hybrid artificial blood vessels, such as (see p. 0, 1987), there is still much work to be done in the future.

In addition, the conventional use of a tubular body made only of natural tissue requires
If left in the body for a long period of time, there is a mechanical problem of aneurysm-like expansion, and the use of heparin suppresses cell proliferation (Wolfgang et al.
Lankes, et al., Biochem, J.
.

251.831-842.1988: Reference), neointimal formation may be delayed.

[Problems to be Solved by the Invention] In view of the above circumstances, the present inventors have developed a material that eliminates the drawbacks of conventional antithrombotic polymer materials and has excellent antithrombotic properties and biocompatibility. In order to obtain a material with good mechanical strength, we conducted extensive research. That is, by creating an animal model of vascular intimal injury and conducting a detailed search for how natural blood vessels are repaired after injury,
The present invention was achieved by discovering the conditions that an artificial blood vessel should have and applying that knowledge. As a result, when the intima of a natural blood vessel was injured, a layer of fibrin thrombus that had formed on the exposed flesh elastic lamina was felt, and the neointima did not close after that, but the neointima formed in just one week. found that it is formed. Based on this knowledge, we focused on fibrin, which is temporarily formed on the elastic lamina after intimal injury, as a material that has both antithrombotic properties and early tissue healing properties, and completed the present invention.

[Means for Solving the Problems] The gist of the present invention is an antithrombotic material comprising a polymeric material provided with a polymerized protein layer substantially free of at least red blood cells and white blood cells on the surface thereof. . Here, 'substantially free of red blood cells and white blood cells' means '
This means that red blood cells and white blood cells have been removed to an extent that does not have a significant adverse effect on anti-+fit thrombosis. The present invention also provides an antithrombotic material in which a polymeric protein layer q is provided on the surface of a polymeric material by treating plasma constituent proteins with a protein acting enzyme. Preferably, the protein acting enzyme is an enzyme having thrombin-like action such as thrombin or reptilase, and the polymerized protein is buipurin.

The present invention also provides that the antithrombotic material is produced by treating the polymer surface with a protein-acting enzyme and then treating it with a solution containing plasma component proteins.
In this case, the protein acting enzyme is preferably an enzyme having a thrombin-like action such as thrombin or reptilase, and the plasma component protein is preferably fibrinogen, cryoprecipitate or plasma.

In the present invention, a polymerized protein layer is provided on the surface of a polymeric material by treating plasma constituent proteins with a protein-acting enzyme. That is, by forming an enzyme-treated polymeric protein layer on the surface of a polymeric material, antithrombotic properties and other excellent biocompatible properties are imparted. As mentioned above, fibrin is suitable as a substance that can be immobilized on the surface. According to the present invention, the desired antithrombotic material is obtained by treating the polymer surface with a solution containing a protein acting enzyme (solution A) and then with a solution containing plasma component proteins (solution B). is obtained.

The protein acting enzyme is preferably an enzyme having a thrombin-like action such as thrombin, reptilase or snake venom, and the plasma component protein is preferably fibrinogen, cryoprecipitate or plasma. That is, the plasma component proteins used in the present invention are all derived from plasma, and are referred to as "plasma".
refers to plasma obtained by anticoagulating whole blood with citrate or the like and removing blood cell components by centrifugation or the like. This "plasma" contains fibrinogen, but not at high concentrations, so the plasma is frozen and repeatedly subjected to cold centrifugation to increase the fibrinogen concentration.
The obtained product is called "cryoprecipitate" and is commercially available as a cryoprecipitate preparation from the Japanese Red Cross Society.

Furthermore, fibrinogen is commercially available as a preparation from various pharmaceutical companies.

In the present invention, a ``protein layer polymerized by treating plasma component proteins with a protein-acting enzyme'' on the surface of a polymeric material means ``a protein layer polymerized by treating plasma component proteins with a protein-acting enzyme''. Specifically, it is immobilized "fibrin," which is formed by fibrinogen consisting of α, β, and S chains, and contains thrombin, Ca, FXIl
[] was finally crosslinked to become insoluble buiplin. The main composition of liquid A is thrombin, reptilase, snake venom, etc.
It consists of a solution containing protein-acting enzymes, such as enzymes that have a binding-like effect. Furthermore, a calcium solution FXI or the like can be added to the A liquid as appropriate. Examples of the B fluid include fibrinogen, cryoprecipitate, and plasma constituent proteins such as plasma.

Furthermore, it is also possible to crosslink a protein immobilized on the surface of a polymeric material by a known method.

The so-called "fibrin" according to the present invention obtained in this manner differs from conventionally obtained fibrin in that it is uniform and forms a single layer with dense directionality when observed under an optical microscope, and is substantially composed of red blood cells and white blood cells. It does not include.

[Effect] Insoluble fibrin, which is the final product of blood coagulation used in the present invention, is important in the process of wound healing, and the formation of an insoluble fibrin network through crosslinking between buiprin bodies is essential for the formation of granulation tissue. (Maek
tl, W, et al, Thromb, Dia
thHaemorrh, 32.578-581'Lor
and,L,et al,Arch,Biochem,
Biophys, 105.58-67, 1964:
In addition, since blood clots generally grow as blood cell components are incorporated into the fibrin fiber network, there is a strong view that fibrin leads to the growth of blood clots.What should be noted here? The fundamental function of fibrin and fibers in which the cross-linking reaction is progressing is different from that of fibrin in which the cross-linking reaction has been completed. Also, unlike collagen etc., fibrin is a protein that does not exist physiologically and appears under non-physiological conditions, but when blood is injured,
Or, when an artificial material is applied inside a living body, it is a sensitive substance.

Through research to explore the tissue repair process in model animals, the present inventors have found that fibrin formation immediately after natural blood injury is the substance that can control the subsequent tissue repair process.

However, the so-called mixed thrombus containing a large number of blood cells contains many physiologically active substances such as blood-derived arachidonic acid metabolites, activated complement, and rhinosomal enzymes, and the present inventors' research It should be strictly distinguished from the fibrin that has arrived. Regarding the antithrombotic properties of fibrin that does not contain blood cells, the literature (Tomoyoshi Atsuta et al.: Artificial Organs, 19g0.96.932=935: HOvig, T.
at al.

J, Lab, & C11n, Med, ;71.
29.1968: Yutaka Izumi et al. 9 Artificial Organs, 12.1.
1983.217-220), and furthermore, in the presence of cross-linked V-purin, there is a literature that shows an enhancement effect on fibrinolytic activity (Yasuhiro Makino et al., Blood and Vascular Vessels, 1986.10).
9.109-118) are known, but when the polymer material comes into contact with blood, the coagulation system is activated as the next reaction, whereas in the presence of fibrin,
This suggests the possibility of inducing fibrinolytic reactions. Therefore, the coating of buiplin from which red blood cells and white blood cells have been substantially removed, as discovered in the present invention, and the fibrin containing red blood cells and white blood cells formed when the polymeric material comes into contact with blood are effective for subsequent use in biological systems. It is clear that they are fundamentally different in their impact. In addition, it has been shown that fibrin sites formed in model animal studies are completely organized by neointima in just one week, and that fibrin can form capillaries and migrate through endothelial thoracotomy in vitro. Literature that contributes to promotion (J, Volander, et al., J, Ce1l)
Physiol, 125.1-9.1985) is known, and furthermore, it is known that due to the fragmentation of V-purin, the degradation products at each stage have various physiologically active effects and influence the migration and proliferation of small breasts. There are great expectations for the various physiological functions of fibrin, such as (Ishida Teruyoshi et al., Arteriosclerosis, 8.605.1981), and the role of buiprin in the field of inflammation as a future medical material. There is.

In addition to the above-mentioned properties of fibrin, the present inventors further discovered that by treating plasma component proteins with protein-acting enzymes, the polymerized protein layer is composed of densely oriented fibers; , which was found to form a smooth surface.

The polymerized protein is preferably fibrin. In addition, the present invention provides that the antithrombotic material can be produced by treating the polymer surface with a protein-acting enzyme and then treating it with a solution containing plasma component proteins; is an enzyme having a thrombin-like action such as thrombin or reptilase, and the plasma component protein is preferably fibrinogen, cryoprecipitate, or plasma.

The method for producing the antithrombotic material according to the present invention is as follows.

Specifically, a method for immobilizing fibrin on a polymeric material in the form of a tubular body will be described below as an example.

For example, Ca” in a porous polyurethane lumen in the form of a tubular body.
A thrombin solution (solution A) containing the solution is placed in, for example, a syringe and injected into the lumen. At this time, when using a porous polymer material, it is desirable to apply pressure so as to spread throughout the pores.

After injecting solution A, add a fibrinogen solution of 6% or less (
Liquid B) is injected into the inner surface of the tubular body under pressure using a syringe, etc., and brought into contact with liquid A, and cross-linked on the inner surface of the tubular body, thereby allowing the formation of a single-layer V-Prin film and immobilization.

This is because if the fibrinogen concentration is 6% or more, it is poorly soluble, which is disadvantageous.

This reaction is preferably carried out in a near-neutral buffer solution. As this buffer solution, a HEPES buffer or the like is used.

The thrombin solution is used in an appropriate amount, and the thrombin solution is used in an amount of about 1!L or more per 1 g of fibrin, preferably about 50 to 500!. approximately 350 units/ml,
This is because when the physical strength reaches saturation and is about 5 feet or more, the effect will not improve even if the strength is increased, so 500 units or less is sufficient.

Ca""" solution, 5 molar equivalent or less per 100 ml of fibrinogen solution is used. That is, Ca1+ is 5
Since a saturated state is reached with mmol and adding more than this has no effect, the amount was set to 5 mmol equivalent or less.

In addition, it is also possible to use solution A (a protease inhibitor such as aprotinin), and an appropriate amount of FX■ can also be added.

Furthermore, instead of liquid B, proteins of plasma constituents such as cryoprecipitate and plasma can also be used. By forming a single immobilized protein layer substantially free of red blood cells and white blood cells on the surface of the polymeric material, the polymeric material is given antithrombotic properties and high biocompatibility.

The polymer forming the base material of the antithrombotic material of the present invention may be a synthetic polymer material with excellent mechanical performance such as nylon, polyester, polyethylene, polypropylene, polyurethane, silicone rubber, or a natural blood vessel, ureter, etc. Tissues, organs, etc. derived from living organisms can be used.

When using a highly porous polymer material, it is necessary to prevent blood leakage, to provide resistance to fibrinolysis, or to more firmly fix the V-Prin layer to the surface of the polymer material. In addition to bonding by physical adsorption, known methods such as crosslinking by reaction with a polyfunctional reagent can also be applied6. For example, after forming a fibrin film on the surface of a polymeric material, 0.01~
A material with excellent antithrombotic properties can be obtained by treating the buiplin layer with a physiological saline solution containing 2% by volume of glutaraldehyde. It is preferable to treat with physiological saline for washing.

In the manner described above, an antithrombotic material is obtained in which a layer of fibrin film substantially free of red blood cells and white blood cells is fixed to the inner surface of the polymeric material made of a tubular body.

The antithrombotic material of the present invention has excellent antithrombotic properties and biocompatibility, and has good mechanical performance, and can be used as a material for various medical devices used in areas that come into direct contact with blood, such as: Artificial blood vessels, vasculature, artificial kidney tubes,
It is extremely valuable as a material for artificial heart-lung machines, blood/vacuum tubes, artificial heart bombing chains/<-, balloon bombing, etc.

Since the antithrombotic material of the present invention is coated with fibrin that does not substantially contain red blood cells and white blood cells, there is no risk of thrombus growth even when used in vivo for a long time.
It is also highly safe.

Next, the production of the antithrombotic material of the present invention will be explained using specific examples, but the present invention is not limited to the following explanation.

[Example 1] [Fibrin membrane fixation method] In a syringe A, 50μ/m containing 3mμ of Ca Ce * (pharmacopoeia: manufactured by Takeda Pharmaceutical Co., Ltd.) 1 5mffi of pharmacopoeial thrombin solution (manufactured by Mochida Pharmaceutical Co., Ltd.) was added to solution A. I prepared it as. Meanwhile, in syringe B, add 5 ml of 3% human fibrinogen solution (manufactured by Midori Juji Pharmaceutical Co., Ltd.) as liquid B.
The prepared zero order has the configuration shown in FIG. 2, that is, the syringe 1
The inner wall surface of the tubular body 3 made of porous polyurethane connected to the cylinder (cylinder) and the connector 2 is subjected to the treatment according to the present invention. First, liquid A was injected using cylinder 1 while applying pressure from above, and liquid B was similarly injected to form one layer of fibrin film on the inner cavity surface of polyurethane 3. After standing for 5 minutes, 20ml
Excess reaction solution was washed away with physiological saline to obtain a fibrin membrane-fixed tubular body.

[Example 2] [Crosslinking method of immobilized fibrin] In addition, to the fibrin membrane-immobilized tubular body shown in Example 1,
By immersion treatment in physiological saline containing 0.1% glutaraldehyde, a fibrin membrane-immobilized tubular body was obtained which allowed the fibrin layer to be firmly bonded. After thorough washing, the antithrombotic properties were tested in the circulation experiment for evaluating antithrombotic properties described below. After 3 hours of circulation testing, almost no red blood clots were observed. .

[Antithrombotic evaluation ex vivo circulation experiment] Healthy Japanese white rabbits with no clinical abnormalities (male and female, weight 2.3-3, 25 kg bentoparbital)
, 92 mg/kg was administered through the ear vein, and general anesthesia was administered. After fixing the patient in the supine position, the oblique vein was exposed and relocated using a technique similar to general surgery. The specimen was placed in the A-V shunt circuit shown in Figure 3, and after brimming with physiological saline, the exposed inclined vein 15
14G indwelling needle (manufactured by Chiru Shichisha: Surflow [registered trademark])
) and started the circulation test. That is, blood is introduced from the rabbit's carotid artery through the indwelling needle 8, through the connector 9, to the specimen 11, through the infusion set 12, through the tough tube 13 for sampling, and into the fetal cephalic vein 1.
5 is returned. Immediately after the start of the circulation test, blood was collected from the sampling tube 13 of the circulation circuit and injected into a blood collection tube (Venoject (registered trademark) manufactured by Chil-Nana) containing EDTA-2 as an anticoagulant. This anticoagulated blood was transferred to an ELT-8 (Ortho Instrument).
The platelet count was calculated using the following method (manufactured by Ho Instrument).

In addition, after 60 minutes, 120 minutes, and 24 hours of circulation, blood was collected in the same manner as described above, and the platelet count was calculated. ]X100, and expressed as a platelet reduction rate.

Further, after the completion of the 24-hour circulation test, the specimen was removed from the circulation circuit, washed lightly with physiological saline, and then fixed with a 10% neutral buffered formalin solution. After fixation, paraffin sections were prepared according to the general pathological tissue specimen preparation method (How to make pathological tissue specimens: Osamu Watanabe Yonosuke, Igaku Shoin, 6th edition, 1986), and HE staining was performed. Submitted for microscopic examination. As an evaluation method, the characteristics of the thrombus and the thickness of the formed thrombus were measured. Regarding the thickness of the thrombus, 8
A 90x microscopic photograph was taken of each site, and the thickness was measured on the photograph to determine the thrombus point. Also, 24
After the time cycle test, the state of thrombus adhesion was observed using a scanning electron microscope (manufactured by JEOL Ltd.; JSM-840).

[Example 3] Human fibrinogen preparation Fibrinogen HT-Midori (manufactured by Midori Juji Pharmaceutical Co., Ltd.) was dissolved in distilled water, and lysine-Sepharose (Lysine-5 epharos) was dissolved in distilled water.
e) Passed through a 4B column (Pharmacia) to remove plasminogen (plasmin). Test team (registered trademark) measures the amount of plasminogen in the solution after passing through the column.
It was measured using a PLG kit (manufactured by Daiichi Chemical Co., Ltd.) and confirmed that it was not detected.

Furthermore, the amount of plasminogen is measured by clotting time 41! (D
Measured by ata-Fi (registered trademark); Dito Inc.),
Concentrated by freeze-drying to remove plasminogen 3
% human fibrinogen solution was prepared and used as Solution B. On the other hand, pharmacopoeial thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) was added to 2-Fl caclm, 2mM MgCj! m, 150
5mM HEPES buffer containing raMNaCe (pH
Near 4), dissolve to about 50 m/ml,
This was called liquid A.

The porous polyurethane tubular body used in Example 1 (4 mφ
, 451 m) 3 in the configuration shown in Fig. 2, 3 ml of liquid A was pressurized with a syringe, and then 2.5 ml of liquid B was injected into the inner cavity of 3.
j! was similarly injected to form a single layer of fibrin film on the surface of the polyurethane lumen. Furthermore, 5 to 1
After standing at room temperature for 0 minutes, 10mF! The lumen was washed with physiological saline to obtain a fibrin-fixed tubular body. FIG. 1 shows the surface structure of a fibrin layer formed on a polyurethane surface according to the present invention, observed under a microscope.

[Example 4] The plasminogen-removed buiprinogen solution prepared in Example 3 was further mixed with gelatin-cellulofin (Gelatin-Cellulofin).
The mixture was passed through an in-Cellulofine column (manufactured by Seikagaku Corporation) to remove bupronectin.

The solution after passing through the column was subjected to 5DS-polyacrylamide electrophoresis* (manufactured by TEFCO), and it was confirmed that no fibronectin band was detected. Furthermore, by the same procedure as in Example 3, a 3% human fibrinogen solution from which plasminogen and fibronectin were removed was prepared.

Using the fibrinogen solution thus prepared, a polyurethane tubular body coated with V-purin was produced in the same manner as in Example 3.

When this fibrin-coated polyurethane tubular body wall was observed using an optical microscope and an electron microscope, it was confirmed that a single fibrin layer with a smooth surface similar to that in Example 3 was formed on the polyurethane surface.

[Example 5] Using the fibrinogen solution obtained in Example 3.4,
According to the present invention, the same in vitro circulation experiment as described in Example 2 was carried out on a tubular body in which a V-Purin membrane was immobilized on a polyurethane surface along with a comparative specimen as follows.

Namely, according to the invention, 3 samples (A) for the material obtained in Example 3 and 2 samples (B) for the material obtained in Example 4, in contrast to the porous polyurethane without V-Purin coating. Three specimens (C) and Boretex (Gore-ex [Registration direction]
) (manufactured by Gore-Tex) 3 samples (D)
[Boretex is a Teflon-based fiber material and is a commercially available material for artificial blood vessels that is generally said to have antithrombotic properties. ] and in contrast, a total of 13 samples, including 2 samples (E) only in the circuit without indwelling the sample in the circulation circuit,
A circulation experiment was conducted using 13 domestic rabbits. The results are shown in Figures 4 to 6 and Tables 1 and 2.

FIGS. 4A, B, C, and D are micrographs showing the structure of the inner wall surface, and A to D correspond to and show the structure of the inner wall surface. As shown in No. 4 C, on the polyurethane surface without fibrin coating, a fibrin network with a rough surface containing many red blood cells has been completely formed. On the other hand, as shown in FIGS. 4A and 4B, it was revealed that when the polyurethane surface was coated with fibrin in advance according to the present invention, no red blood clots were observed and the surface was smooth.

Figure 5 A, B, C, and D are electron micrographs showing the surface of the inner wall of the polyurethane tubular body. In the polyurethane without fibrin coating, a strong fibrin network containing many blood cell components is clearly observed. (Fig. 5C), whereas in the case of polyurethane with fibrin coating,
Only a thin fibrin membrane containing almost no red blood cells was observed (Fig. 5A, B).

The following Table 1 shows thrombus points indicating the amount of thrombus attached after a 24-hour circulation test. Groups 0, A1, B, and C1 correspond to the symbols A to D shown above, respectively. It is.

As shown in Table 1, Group A1 and Group B show the thrombus points for fibrin-coated polyurethane, and the adhesion of thrombus is significantly suppressed, and the amount of adhesion is different from that of Voortex. It is clear that this is significantly less.

Table 1 Blood clot point measurements Nagaya Group A 81B Group Kou 172
1-6 2 * 18 3 12 3 *1
9 18 11 3 *20
11 13 9 *21
23 12 1 *22
3 12 1 *23
3 3 16 *24 1
4 14 1 *Average value 14
．． 5 8.8 4.7 5.0 standard deviation 1.
96 0.79 0.76 0.93 Number of measurements 24
24 24 16 Next, for the samples A to D mentioned above, blood was collected at the start, at 60 minutes, at 120 minutes, and at 24 hours to observe a decrease in the number of platelets in the blood after the start of the circulation experiment. The number of platelets was then counted, and the results are shown in Table 2. That is,
The number of platelets at the start is assumed to be 100%, and the number of platelets over time is shown below as a percentage of the number of platelets at the start.

A 100 117.0±0 116. , o±5.9
87.5±0.5B 100 98.0±o
tto, o±2.0 67.0±7.5C10081,
3±11.5 88.3±6.2 76.0±7. O.D.
100 83.5±9.3 70.0±2.0 5
6.0±4. OB 100 103.5±3.5 9
6.5±0.5 98.5±17.5 Note that E indicates a circulation experiment using only a circuit in which no specimen was placed.

FIG. 6 is a graphical representation of Table 2, that is, a graphical representation of the rate of decrease in platelet count with respect to the elapsed time of the circulation test for the samples A to D.

That is, in the case of fibrin immobilization (curve 1iA, B) of Fig. 6, the decrease in platelet count was more suppressed than in the case of no immobilization, and furthermore, the decrease in platelet count was more suppressed than in the case of Voortex (curve D). It was clear that the decrease in platelet count was small.

[Effects of the Invention] The present invention provides a novel antithrombotic material, which can be used as a polymeric material with mechanical strength as a material used in areas that come into direct contact with blood in the medical field. , an antithrombotic material that has mechanical strength, antithrombotic properties, and biocompatibility by fixing a fibrin film formed substantially free of red blood cells and white blood cells on the material surface through an enzymatic reaction. In other words, a protein substantially free of red blood cells and white blood cells is immobilized on the surface, which is polymerized by an enzyme having a thrombin-like action such as thrombin or reptilase using a starting material such as propinogen, cryoprecipitate, or plasma. To do this, to provide an antithrombotic material with excellent biocompatibility, etc. Written by 17 'r'it, which has achieved great technical effects.
Ru.

[Brief explanation of the drawing]

Figure 1 shows the surface state (particle structure) of a single-layer V-Purin membrane immobilized on the surface of porous polyurethane according to the present invention.
is shown in the ψ-micrograph. FIG. 2 is a cross-sectional view showing a structure used in the method of immobilizing buiprin on a tubular body according to the present invention. Figure 3 shows a circulation circuit diagram for a 24-hour circulation test in domestic rabbits for the antithrombotic material of the present invention. Fig. 5 shows an optical micrograph of a tissue section showing the thrombus properties and thickness after a 24-hour circulation test. The figure shows the variation in platelet count in a 24-hour circulation test4
- There are 771' groups.

Claims (6)

[Claims] (1) An antithrombotic material comprising a polymeric protein layer provided on the surface of a polymeric material with a polymerized protein layer substantially free of at least red blood cells and white blood cells. (2) An antithrombotic material formed by providing a polymerized protein layer on the surface of a polymeric material by treating plasma constituent proteins with a protein-acting enzyme. (3) The protein acting enzyme is an enzyme having a thrombin-like action such as thrombin or reptilase;
The antithrombotic material according to claim 2, wherein the polymerized protein is fibrin. (4) A medical device formed from the antithrombotic material according to any one of claims 1 to 3. (5) A method for producing an antithrombotic material, which comprises treating the surface of a polymer with a protein-acting enzyme, and then treating the surface with a solution containing plasma constituent proteins. (6) The protein acting enzyme is an enzyme having a thrombin-like action such as thrombin or reptilase;
6. The method for producing an antithrombotic material according to claim 5, wherein the plasma component protein is fibrinogen, cryoprecipitate, or plasma.