Process for producing antithrombogenic vinyl acetate polymer or hydrolyzate thereof

A process for producing an antithrombogenic vinyl acetate polymer or a hydrolyzate thereof which comprises treating the vinyl acetate polymer or hydrolyzate with a solution of a fibrinolytic enzyme so as to fix the fibrinolytic enzyme to the polymer. The resulting antithrombogenic vinyl acetate polymer or hydrolyzate is advantageously used in a material which comes into contact with the blood in use and is especially useful in a surgical tube.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a process for producing an antithrombogenic vinyl 
acetate polymer or hydrolyzate. 
2. Description of the Prior Art 
A material obtained by fixing urokinase to the surface of a nylon tube is 
described in A. Sugitachi, K. Takagi, The International Journal of 
Artificial Organs, Vol. 1, No. 2, pp. 88-92 (1978) as having superior 
antithrombogenic properties and as being suitable for clinical 
applications such as in the form of surgical drains, intravenous 
catheters, etc. Sugitachi et al, supra, state that the nylon tube is 
somewhat non-elastic for clinical applications and to remedy this defect 
urokinase was fixed to a silicone tube which is softer but does not have 
as good antithrombogenic property. 
The inventors of the present application have previously developed a 
process for preparing an elastic antithrombogenic material by fixing a 
fibrinolytic substance to a polyester elastomer, e.g., as disclosed in 
Japanese patent application (OPI) No. 113194/78 (the term "OPI" as used 
herein refers to a "published unexamined Japanese patent application"). 
However, the resulting elastic antithrombogenic material is opaque, has a 
lower creep resistance than silicone, and is unable to accommodate a large 
volume of contrast media for X-ray photography such as barium sulfate 
which is detrimental to its softness. 
Two U.S. patent applications are now pending filed in the names of the 
present inventors. One is entitled "A Process for Producing an 
Antithrombogenic Material by Fixation of a Synthesized Fibrinolytic 
Compound", Ser. No. 844,856, filed Oct. 25, 1977, U.S. Pat. No. 4,273,873 
and the other is "A Process for Producing an Antithrombogenic Polyurethane 
by Fixation of a Fibrinolytic Enzyme to the Surface of Polyurethane", Ser. 
No. 928,496, filed July 27, 1978, now abandoned. 
SUMMARY OF THE INVENTION 
It is a primary object of this invention to provide an antithrombogenic 
material suitable for use in clinical applications which is flexible, 
transparent, and X-ray opaque. 
The present invention provides a process for producing an antithrombogenic 
vinyl acetate polymer or hydrolyzate by treating a vinyl acetate polymer 
or hydrolyzate with a solution of fibrinolytic enzyme so as to fix the 
fibrinolytic enzyme to the polymer. 
DETAILED DESCRIPTION OF THE INVENTION 
The term "vinyl acetate polymer" as used herein includes both homopolymers 
and copolymers of vinyl acetate. Examples of the suitable comonomers are 
ethylenically unsaturated compounds such as vinyl esters such as vinyl 
formate, vinyl propionate, vinyl benzoate, and vinyl stearate; olefinic 
hydrocarbons such as ethylene and propylene; chlorinated olefins such as 
vinyl chloride and vinylidene chloride; acrylic monomers such as methyl 
methacrylate, ethyl acrylate, and .beta.-hydroxyethyl methacrylate; allyl 
derivatives such as allyl alcohol and N-allyl urethane; and 
.alpha.,.beta.-unsaturated acid anhydrides such as maleic anhydride and 
itaconic anhydride. The vinyl acetate copolymer having these monomeric 
components may be a random copolymer, an alternating copolymer, a block 
copolymer or a graft copolymer. 
The vinyl acetate polymer is prepared by bulk, solution, emulsion or 
suspension polymerization of the corresponding vinyl monomers in the 
presence of a free radical initiator. Examples of suitable free radical 
initiators are peroxides such as benzoyl peroxide, lauroyl peroxide, 
acetyl peroxide, t-butylhydroperoxide, etc.; azo-compounds such as 
azobisisobutyronitrile; and redox initiators such as t-butyl 
perbenzoate-ascorbic acid. A suitable amount for the initiator ranges from 
about 0.0001 to about 5 wt%, preferably from 0.001 to 2 wt%, based on the 
vinyl monomer. A suitable polymerization temperature ranges from about 
-50.degree. to about 250.degree. C., preferably from about 10.degree. to 
180.degree. C. A suitable polymerization time is from about 1 minute to 
100 hours, preferably from about 5 minutes to 50 hours. 
The vinyl acetate polymer is shaped into a tube, film, sheet, elastomer, 
fiber and other desired forms depending upon its use. Alternatively, the 
polymer is coated on the surface of a shaped article made of another 
material. A filler such as a fiber, non-woven cloth and glass fiber can 
optionally be incorporated into the vinyl acetate polymer in the course of 
its shaping in an amount ranging from about 0.1 to about 50 wt% of the 
polymer. For the production and shaping of the vinyl acetate polymer, 
reference may be had to "Vinyl Ester Polymers", Encyclopedia of Polymer 
Science and Technology, Vol. 15, p. 531, Interscience Publishers (1971) 
and Polyvinyl Alcohol, edited by C. A. Finch, John Wiley & Sons (1973). 
The vinyl acetate polymer may be converted to the corresponding vinyl 
alcohol polymer by alkaline hydrolysis, ammonolysis or acid hydrolysis. 
Alkaline hydrolysis is most commonly used on an industrial scale. Alkaline 
hydrolysis of the vinyl acetate polymer is performed in methanol, ethanol, 
water or a mixture thereof containing from about 0.1 to about 10 N, 
preferably 0.5 to 6 N, of an alkali such as sodium hydroxide, potassium 
hydroxide or sodium methoxide at a temperature ranging from about 
10.degree. C. to the boiling point of the solvent for a period of time 
ranging from about 0.5 minute to about 48 hours, preferably from 1 minute 
to 24 hours. By controlling the conditions under which the vinyl acetate 
polymer is hydrolyzed, hydrolyzates of the polymer can be produced in 
varying degrees of hydrolysis. Antithrombogenic articles may be prepared 
from the hydrolyzate or the surface of and article such as a tube, sheet, 
film, etc., into which the vinyl acetate polymer has been shaped may be 
hydrolyzed. When the vinyl acetate polymer is hydrolyzed under a 
homogeneous or suspended condition before shaping from the standpoint of 
preparing a uniform hydrolysis product, the hydrolysis degree is at least 
about 50% and preferably about 60% or more. For hydrolysis and shaping of 
the vinyl acetate polymer, reference may be made to "Vinyl Alcohol 
Polymers", Encyclopedia of Polymer Science and Technology, Vol. 14, p. 
149, Interscience Publishers (1971), and Polyvinyl Alcohol, supra. 
A surgical drain, catheter and other medical tubes are preferably prepared 
from a copolymer of ethylene and vinyl acetate. An ethylene-vinyl acetate 
copolymer may be prepared by copolymerizing ethylene and vinyl acetate as 
described in U.S. Pat. Nos. 2,395,381, and 2,703,794, and British Pat. 
Nos. 569,927, 835,466, 875,983, 807,112, 843,974 and 929,138. The 
ethylene-vinyl acetate copolymer exhibits different properties depending 
upon the vinyl acetate content. Since an antithrombogenic material for use 
in clinical applications should be flexible and strong at the same time, 
the vinyl acetate content is preferably about 3 to 45 wt%, more preferably 
about 5 to 40 wt%, of the polymer. If the vinyl acetate content does not 
reach 3 wt%, flexibility is impaired, and if it exceeds 40 wt%, toughness 
is compromised. 
An antithrombogenic material for use in clinical applications is preferably 
prepared from the ethylene-vinyl acetate copolymer for the following 
advantages: (i) because the copolymer is thermoplastic, the copolymer is 
adaptable for extrusion molding; (ii) it has high transparency; (iii) the 
polymer is particularly advantageous as a surgical drain or intravenous 
catheter because flexibility is achieved without a plasticizer; (iv) the 
polymer is highly stable during molding procedure which not only 
eliminates the chance of including a decomposition product harmful to the 
resulting shaped article but also obviates the need of adding an injurious 
heat stabilizer; (v) the polymer which possesses good adhesion is obtained 
in a molten state, which proves particularly advantageous in preparing a 
shaped article of intricate form; and (vi) the polymer is able to 
accommodate filler loadings without experiencing a significant change in 
its physical properties, that is, a medical tube made of the polymer does 
not lose its flexibility even if it is filled with a high concentration of 
a contrast medium such as barium sulfate when the X-ray opacity mark is 
placed in the medical tube. 
The fibrinolytic enzymes used in this invention take part in the 
dissolution of fibrin. Examples of the suitable fibrinolytic enzyme are 
proteases useful as native enzymes such as urokinase, streptokinase, 
plasmin and brinolase. Urokinase extracted from human urine is 
advantageously used in view of effectiveness and safety. 
These fibrinolytic enzymes are fixed to the surface of the vinyl acetate 
polymer or hydrolyzate thereof by means of covalent bonding or adsorption. 
For the vinyl acetate polymer or its hydrolyzate to exhibits its 
antithrombogeneity over an extended period of time the fibrinolytic enzyme 
is desirably fixed by covalent bonding. However, fixation by adsorption 
serves the purpose of providing a polymer or hydrolyzate which is 
effective for a short period of time. 
One method of fixing a fibrinolytic enzyme by covalent bonding is by 
treating the surface of a vinyl acetate polymer or hydrolyzed vinyl 
acetate polymer containing a reactive functional group capable of forming 
a covalent bond with a solution of the enzyme. O. Zaborsky, Immobilized 
Enzymes, CRC Press (1973) describes a number of reactive functional groups 
which are capable of forming a covalent bond by reacting with an amino 
group, a carboxyl group, a sulfhydryl group, an imidazole group or the 
phenol group present in a fibrinolytic enzyme. Of these functional groups, 
isocyanate, epoxy, formyl, acid chloride, carboxylic acid anhydride, 
amino, carboxyl, azide and diazo groups are easily used for the purpose of 
this invention. These functional groups should be incorporated into the 
polymer in amounts such that the amount of the fibrinolytic enzyme 
immobilized provides a thrombus formation time of about 45 minutes or 
more. 
A vinyl acetate polymer or hydrolyzate thereof containing these reactive 
functional groups may be prepared by copolymerizing vinyl acetate with a 
comonomer containing a reactive functional groups. Suitable comonomers 
include acrolein (to introduce a formyl group), itaconic anhydride (to 
introduce an acid anhydride group), methacrylic acid (to introduce a 
carboxyl group). The most suitable comonomer is maleic anhydride; examples 
of the vinyl acetate copolymer having maleic anhydride as a 
copolymerizable component are a vinyl acetate-maleic anhydride copolymer 
and a vinyl acetate-maleic anhydride-vinyl chloride terpolymer. These 
copolymers can be prepared by reference to the methods disclosed in 
British Pat. No. 844,509 and U.S. Pat. Nos. 2,483,465 and 3,010,846. A 
suitable copolymerization amount for maleic anhydride is about 0.1 to 70 
mol%, preferably 0.5 to 60 mol%. 
Another method of producing a vinyl acetate polymer or its hydrolyzate 
having the above mentioned reactive functional groups is to hydrolyze the 
polymer first and then introduce the reactive functional groups into the 
hydrolyzate. 
An isocyanate group may be introduced into the hydrolyzate of vinyl acetate 
polymer by first dissolving a polyisocyanate such as hexamethylene 
diisocyanate, 4,4'-diphenylmethane diisocyanate, toluene diisocyanate, 
xylene diisocyanate or phenylene diisocyanate in a solvent composed of 
dimethylformamide, methyl ethyl ketone, tetrahydrofuran or a mixture 
thereof to provide a solution at a concentration of about 0.1 to 50 wt%, 
preferably about 0.5 to 20 wt%. Optionally a catalyst such as N-methyl 
morpholine or triethylene diamine may be present in an amount of about 
0.001 to 10 wt%, preferably about 0.01 to 1 wt%. The resulting solution is 
then used to treat the hydrolyzate of the vinyl acetate polymer at a 
temperature in the range of from about 20.degree. to 120.degree. C., 
preferably about 40.degree. to 90.degree. C., for a period of about 5 
minutes to 24 hours, preferably about 20 minutes to 10 hours. 
An epoxy group may be introduced into the hydrolyzate by reacting the 
hydrolyzate either with a glycidyl halide such as epichlorohydrin or 
epibromohydrin or with a polyglycidyl ether of a polyol such as ethylene 
glycol, tetramethylene glycol, diethylene glycol or glycerin 
pentaerythritol in the presence of an aqueous solution of alkali such as 
sodium hydroxide. For detailed information on the conditions for such 
reaction, reference may be made to J. Porath, N. Fonstedt, Journal of 
Chromatography, 51, p. 479 (1970) and L. Sundberg, J. Porath, Journal of 
Chromatography, 90, p. 87 (1974). 
A formyl group may be introduced into the hydrolyzate of a vinyl acetate 
polymer by first dissolving a polyaldehyde such as glutaraldehyde, glyoxal 
or dialdehyde starch in an aqueous solution of an acid such as 
hydrochloric acid, sulfuric acid or acetic acid to provide a solution 
having a concentration of about 0.1 to 50 wt%, preferably about 0.5 to 20 
wt%. The resulting solution is used to treat the hydrolyzate of the vinyl 
acetate polymer at a temperature ranging from about -20.degree. to about 
100.degree. C., preferably from 0.degree. to 80.degree. C., for a period 
ranging from about 5 minutes to about 24 hours, preferably from 10 minutes 
to 10 hours. 
An acid chloride group may be introduced into the hydrolyzate of a vinyl 
acetate polymer by first dissolving a polyacid chloride such as adipoyl 
chloride, terephthaloyl chloride or cyanuric chloride in acetone, 
tetrahydrofuran, benzene, toluene, dimethylacetamide or a mixture thereof 
at a concentration of about 0.1 to about 50 wt%, preferably about 0.5 to 
20 wt%, and using the resulting solution to treat the hydrolyzate at a 
temperature in the range of from -20.degree. to about 90.degree. C., 
preferably about 0.degree. to 70.degree. C., for about 5 minutes to 24 
hours, preferably for about 30 minutes to 10 hours. 
An acid anhydride group may be introduced into the hydrolyzate of a vinyl 
acetate polymer by first dissolving a polycarboxylic acid anhydride such 
as a maleic anhydride/methyl vinyl ether copolymer, maleic 
anhydride/styrene copolymer or a maleic anhydride/ethylene copolymer in 
acetone, tetrahydrofuran, benzene, toluene, dimethylformamide, dimethyl 
sulfoxide or a mixture thereof in a concentration of about 0.1 to 30 wt%, 
preferably about 0.5 to 10 wt%, optionally in the presence of from about 
0.01 to about 10 wt%, preferably from 0.05 to 2 wt%, of a catalyst such as 
hydrochloric acid, sulfuric acid, acetic acid, etc., and using the 
resulting solution to treat the hydrolyzate at a temperature of about 
0.degree. to 100.degree. C., preferably about 70.degree. to 80.degree. C., 
for about 10 minutes to about 24 hours, preferably for about 30 minutes to 
10 hours. 
An amino group may be introduced into the hydrolyzate of a vinyl acetate 
polymer by first dissolving an amino acetal such as 
.beta.-aminoacetaldehyde dimethyl acetal or .beta.-aminoacetaldehyde 
diethyl acetal in from about 0.1 to about 12 N, preferably 0.5 to 10 N, 
hydrochloric acid or sulfuric acid, and other aqueous acidic solutions to 
provide a solution having a concentration of about 1 to 20 wt%, preferably 
about 2 to 10 wt%, and using the resulting solution to treat the 
hydrolyzate at a temperature of about 0.degree. to about 100.degree. C., 
preferably about 20.degree. to 80.degree. C., for about 1 to 48 hours, 
preferably for about 2 to 24 hours. 
An amino group may also be introduced into the hydrolyzate of a vinyl 
acetate polymer by treating it with a polyfunctional reagent which has at 
least two functional groups capable of reacting with both a hydroxyl group 
and an amino group, and then treating the same with a polyamine. Examples 
of the polyfunctional reagent include polyisocyanates such as 
hexamethylene diisocyanate, xylene diisocyanate and phenylene 
diisocyanate; polyepoxides such as polyglycidyl ethers of polyols such as 
tetramethylene glycol, diethylene glycol, glycerine and pentaerythritol; 
polycarboxylic acid anhydrides such as a maleic anhydride/methyl vinyl 
ether copolymer, maleic anhydride/styrene copolymer, and a maleic 
anhydride/ethylene copolymer; polyaldehydes such as glutaraldehyde, 
glyoxal and dialdehyde starch; and polyacid chlorides such as adipoyl 
chloride, terephthaloyl chloride and cyanuric chloride. The hydrolyzate of 
vinyl acetate polymer can be treated with these polyfunctional reagents 
under the same conditions as used to introduce an isocyanate, an epoxy, a 
formyl, an acid chloride, or a carboxylic acid anhydride group into the 
hydrolyzate as already discussed. The hydrolyzate of the vinyl acetate 
polymer thus-treated with the polyfunctional reagent is then treated with 
a polyamine such as ethylene diamine, hexamethylene diamine, diethylene 
triamine, triethylenetetramine, polyvinylamine, polyethyleneimine, 
polyaminostyrene, polylysine, or poly(p-aminophenyl alanine). The 
hydrolyzate may be treated with the polyamine per se if the latter is 
liquid or alternatively with a polyamine dissolved in methanol, ethanol, 
propanol, dioxane, tetrahydrofuran, water or a mixture thereof at a 
concentration of at least about 0.1 wt%, preferably about 1 wt%. A 
suitable temperature for the treatment ranges from about -20.degree. to 
about 100.degree. C., preferably from about 0.degree. to 90.degree. C. A 
suitable treatment time is about 10 minutes to 24 hours, preferably about 
30 minutes to 5 hours. If the hydrolyzate of vinyl acetate polymer treated 
with a polyamine contains a formyl group, it may optionally be subjected 
to reduction using sodium borohydride or other suitable reducing agent. 
An isocyanate group, an epoxy group, a carboxylic acid anhydride group, a 
formyl group and other reactive functional groups capable of reacting with 
an amino group can be introduced into a hydrolyzate of a vinyl acetate 
polymer containing an amino group by treating the hydrolyzate with a 
polyfunctional reagent having at least two such reactive functional 
groups. Examples of the suitable polyfunctional reagents are 
polyisocyanates such as hexamethylene diisocyanate, xylene diisocyanate 
and phenylene diisocyanate; polyepoxides such as polyglycidyl ethers of 
polyols such as tetramethylene glycol, diethylene glycol, glycerine and 
pentaerythritol; polycarboxylic acid anhydrides such as a maleic 
anhydride/methyl vinyl ether copolymer, maleic anhydride/styrene 
copolymer, and a maleic anhydride/ethylene copolymer; polyaldehydes such 
as glutaraldehyde, glyoxal, and dialdehyde starch; and polyacid chlorides 
such as adipoyl chloride, terephthaloyl chloride and cyanuric chloride. 
The hydrolyzate of the vinyl acetate polymer containing an amino group is 
treated with a solution of these polyfunctional reagents in a suitable 
solvent at a concentration ranging from about 0.05 to 30 wt%, preferably 
from about 0.1 to 10 wt%, at a temperature ranging from about -20.degree. 
to 80.degree. C., preferably from about 0.degree. to 60.degree. C., for 
about 1 minute to about 24 hours, preferably for about 5 minutes to 10 
hours. 
A carboxyl group may be introduced into the hydrolyzate of a vinyl acetate 
polymer by dissolving .alpha.-haloacetic acid such as .alpha.-chloroacetic 
acid, .alpha.-bromoacetic acid or .alpha.-iodoacetic acid in about 0.1 to 
about 5 N, preferably about 0.5 to 4 N, aqueous solution of alkali such as 
sodium hydroxide or potassium hydroxide to provide a solution at a 
concentration ranging from about 1 to about 50 wt%, preferably from about 
2 to 20 wt%, and then using the resulting solution to treat the 
hydrolyzate at a temperature ranging from about 0.degree. to about 
100.degree. C., preferably from about 20.degree. to 80.degree. C., for a 
period ranging from about 1 to 24 hours, preferably from about 2 to 12 
hours. 
The hydrolyzate of vinyl acetate polymer into which a carboxyl group has 
thus been introduced can have an azo or azide group introduced thereinto 
in accordance with the method described in W. E. Hornby, H. Filippusson, 
Biochimica et Biophysica Acta, 220, p. 343 (1970) by first treating the 
hydrolyzate with benzidine or hydrazine, then with nitrous acid. 
The surface of the vinyl acetate polymer or its hydrolyzate now containing 
the reactive functional groups is treated with a solution of a 
fibrinolytic enzyme so as to fix the enzyme to the surface by means of 
covalent bonding. The fibrinolytic enzyme should be dissolved in water or 
a mixture of water and methanol, propanol, acetone, tetrahydrofuran, 
dioxane, dimethyl sulfoxide or dimethylformamide, with water being present 
in an amount of at least 50 wt%, and the surface of the vinyl acetate 
polymer or the hydrolyzed vinyl acetate polymer should be treated with the 
resultant enzyme solution. A suitable temperature for this treatment is 
from about -20.degree. to about 50.degree. C., preferably from about 
0.degree. C. to 30.degree. C., for about 30 minutes to 48 hours, 
preferably for about 1 hour to 24 hours. In fixing the fibrinolytic 
enzyme, the pH must be maintained at about 3 to 10, preferably at about 4 
to 9. A buffer such as a phosphate buffer or an acetate buffer, an acid 
such as hydrochloric acid or an alkali such as sodium hydroxide can be 
used for this purpose. If desired, a protein such as albumin and gelatin, 
e.g., in an amount of about 10 g/l or less, or a salt such as sodium 
chloride, e.g., in an amount of about 2 mol/l or less, can be added to the 
enzyme solution as a stabilizer. 
A dehydrating condensation agent such as N,N'-dicyclohexyl carbodiimide, 
1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate, 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 
N-cyclohexyl-5-phenylisoxazolium-3'-sulfonate, 
6-chloro-1-p-chlorobenzenesulfonyloxybenzotriazole, or diphenylphosphoryl 
azide may optionally be added to the enzyme solution in a concentration 
ranging from about 0.5 to about 200 g/l, preferably from 1 to 100 g/l. 
Since the fibrinolytic enzyme can be adsorbed on the vinyl acetate polymer 
or hydrolyzed vinyl acetate polymer, it can also be fixed to the surface 
of the polymer by means of adsorption. Adsorptive methods can most 
effectively be used to fix a large volume of fibrinolytic enzyme to a 
vinyl acetate polymer or hydrolyzate having a cation exchange group such 
as a carboxyl group, with the adsorbed enzyme retaining its activity for a 
relatively long period of time. 
To prepare an antithrombogenic material, a fibrinolytic enzyme such as 
urokinase or streptokinase must be fixed to the surface of the vinyl 
acetate polymer or hydrolyzed vinyl acetate polymer in an amount 
sufficient to delay formation of a thrombus for at least 45 minutes. For 
this purpose, the enzymatic activity of the solution of urokinase or 
streptokinase to be used for the fixing treatment must be from about 20 to 
about 100,000 international units/ml, preferably from 100 to 5,000 
international units/ml and that of plasmin or brinolase must be from about 
10 to about 50,000 casein units/ml, preferably from 50 to 2,500 casein 
units/ml. 
In addition to fixing a fibrinolytic enzyme such as urokinase to the 
surface of the vinyl acetate polymer or hydrolyzed vinyl acetate polymer, 
a nitrophthaloyl group or other groups which have an affinity for certain 
substances present in blood which inhibit fibrinolytic enzymes can 
optionally be introduced into the polymer or its hydrolyzate by adding 3- 
or 4-nitrophthaloyl anhydride to a polycarboxylic anhydride solution and 
thereby introducing an acid anhydride group into the polymer. The 
inhibiting substances are attracted to the nitrophthaloyl group and the 
more the inhibiting substance bonds to the nitrophthaloyl group, the less 
the fibrinolytic enzyme is inhibited by the inhibiting substance. 
The antithrombogenic vinyl acetate polymer or its hydrolyzate prepared 
according to the process of this invention has the following advantages 
that make it suitable for use as a material which comes into contact with 
the blood during use: (1) Because of its high moldability, it can be 
shaped into various forms for various clinical applications; (2) The 
material prepared from the polymer or its hydrolyzate exhibits high 
antithrombogenaity over an extended period of time.

This invention is now described in greater detail by reference to the 
following Examples which are given here for illustrative purposes only and 
are by no means meant to limit the scope of this invention. 
In the Examples, fibrinolytic activity was measured using a fibrin plate 
having a thickness of about 2 mm which was prepared by adding a 
physiological saline solution of thrombin from human plasma (25 units/ml) 
to an aqueous solution of human fibrinogen (0.5 g/ml) in a ratio by volume 
of 0.2:10. A sample was placed on the fibrin plate and allowed to stand at 
37.degree. C. for 24 hours and the fibrinolytic activity was evaluated by 
the extent of dissolution of the fibrin membrane around the sample. 
Antithrombogeneity of a sample in film or sheet form was determined in the 
following manner. 0.05 ml of citrated plasma (serum to which sodium 
citrate was added) and 0.05 ml of 0.025 M calcium chloride solution were 
separately added dropwise onto the sample and mixed together. The mixture 
was spread over the sample and allowed to stand in a wetting box at 
37.degree. C. The time required for the fibrin to precipitate was measured 
as the thrombus formation time. 
The antithrombogeneity of a tubular sample was evaluated by measuring the 
thrombus formation time using the Chandler rotary tube method as disclosed 
in A. B. Chandler, Laboratory Investigations, 7, p. 110 (1958). 
EXAMPLE 1 
Two films of polyvinyl alcohol (50.mu. thick), one cut into a circle 5 mm 
in diameter and the other into a square (3.times.3 cm), were immersed in a 
4 wt% solution of maleic anhydride-methyl vinyl ether copolymer in acetone 
at 50.degree. C. for a period of 10 hours. The films were then washed with 
acetone and dried. The films now containing a carboxylic acid anhydride 
group were left to stand in a physiological saline solution of urokinase 
(600 units/ml) at 7.degree. C. for 24 hours, then washed with a 
physiological saline. The circular film thus treated was subjected to 
measurement of its fibrinolytic activity wherein a circular dissolution 
zone 17 mm in diameter appeared around the film. The square film was used 
to measure the thrombus formation time which was more than 45 minutes. 
EXAMPLE 2 
Two films of polyvinyl alcohol (50.mu. thick), one cut into a circle (5 mm 
in diameter) and the other into a square (3.times.3 cm), were immersed in 
a dehydrated tetrahydrofuran solution having dissolved therein 5 wt% of 
4,4'-diphenylmethane diisocyanate and 0.1 wt% of N-methyl morpholine, 
respectively, and heated at the boiling point (66.degree. C.) of 
tetrahydrofuran for a period of 5 hours. The films were then washed with 
tetrahydrofuran and dried. The films now containing an isocyanate group 
were left to stand in a 2 wt% aqueous polyethylene-imine solution at 
30.degree. C., water washed and dried. The films thus treated to contain 
an amino group were immersed in a 4 wt% solution of a maleic 
anhydride-methyl vinyl ether copolymer in acetone at 20.degree. C. for a 
period of 2 hours, then washed with acetone and dried. The thus obtained 
films containing a carboxylic acid anhydride group were left to stand in a 
physiological saline solution of urokinase (600 units/ml) at 7.degree. C. 
for a period of 24 hours, then washed with a physiological saline. The 
circular film thus treated was subjected to measurement of its 
fibrinolytic activity wherein a circular dissolution zone 21 mm diameter 
appeared around the film. The square film was used to measure the thrombus 
formation time which was more than 45 minutes. 
EXAMPLE 3 
The inside of a tube (I.D. 3 mm, O.D. 5 mm) of an ethylene-vinyl acetate 
copolymer (containing 19 wt% of vinyl acetate) was treated sequentially as 
follows: 
(1) The inside surface of the tube was partially hydrolyzed by circulating 
through it a solution of 20 wt% sodium hydroxide in 20 vol% water 
containing methanol at 60.degree. C. for a period of 3 hours, then a 
solution of 0.1 vol% acetic acid in methanol was circulated to neutralize 
the sodium hydroxide solution, followed by washing with water; 
(2) A formyl group was introduced into the copolymer by circulating through 
the tube a solution of 5 wt% dialdehyde starch (degree of oxidation: 45%) 
in 1 N hydrochloric acid at 50.degree. C. for a period of 4 hours, 
followed by washing with water; 
(3) An amino group was introduced into the copolymer by circulating through 
the tube a 2 wt% aqueous polyethylene-imine solution at 30.degree. C. for 
a period of 1 hour, followed by washing with water; 
(4) The copolymer was reduced by circulating through the tube a 0.05 wt% 
aqueous sodium borohydride solution at room temperature for a period of 1 
hour, then washed with water and dried; 
(5) A carboxylic acid anhydride group was introduced into the copolymer by 
circulating through the tube a 4 wt% solution of a maleic anhydride/methyl 
vinyl ether copolymer in acetone at 20.degree. C. for a period of 3 hours, 
washed with acetone and dried; 
(6) The tube was filled with a physiological saline solution of urokinase 
(600 units/ml), left to stand at 7.degree. C. for a period of 4 hours, and 
washed with a physiological saline. 
A circular dissolution zone 14 mm in diameter appeared around a round slice 
of the tube to which urokinase was fixed by the procedures described 
above. The thrombus formation time measured on the tube was more than 45 
minutes. 
EXAMPLE 4 
A mixture of 65 g of vinyl chloride, 25 g of vinyl acetate, 10 g of maleic 
anhydride and 1 g of lauroyl peroxide was polymerized in 150 g of ethyl 
acetate at 50.degree. C. for a period of 40 hours. The resulting copolymer 
was isolated through addition of ethanol. A 5 wt% solution of the 
copolymer in methyl ethyl ketone was sprayed on a circular nylon film (5 
mm in diameter and 200.mu. thick) and dried at 90.degree. C. for 5 hours. 
The film was then left to stand in a physiological saline solution of 
urokinase (600 units/ml) at 7.degree. C. for 24 hours, and washed with a 
physiological saline. A circular dissolution zone 19 mm in diameter 
appeared around the film to which urokinase was fixed. The thrombus 
formation time was more than 45 minutes. 
EXAMPLE 5 
The inside of a tube (I.D. 3 mm, O.D. 5 mm) of an ethylene-vinyl acetate 
copolymer (containing 19 wt% of vinyl acetate) was treated sequentially as 
follows: 
(1) The inside surface of the tube was partially hydrolyzed by circulating 
through it a solution of 20 wt% sodium hydroxide in 20 vol% water in 
methanol at 60.degree. C. for a period of 3 hours, then a solution of 0.1 
vol% acetic acid in methanol was circulated to neutralize the sodium 
hydroxide solution, followed by washing with water; 
(2) An amino group was introduced into the copolymer by circulating through 
the tube a 2 vol% solution of .beta.-aminoaldehyde diethyl acetal in 1 N 
hydrochloric acid at 55.degree. C. for a period of 24 hours, and washed 
with, in the order written, water, 0.01 N sodium hydroxide and water; 
(3) A physiological saline solution having dissolved therein 600 units/ml 
of urokinase and 10 mg/ml of 
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate 
was circulated through the tube at 7.degree. C. for a period of 5 hours, 
and washed with a physiological saline. 
A circular dissolution zone 11 mm in diameter appeared around a round slice 
of the tube to which urokinase was fixed by the procedures described 
above. The thrombus formation time was more than 45 minutes. 
EXAMPLE 6 
The procedure of Example 5 was repeated to hydrolyze the surface of a tube 
of ethylene-vinyl acetate copolymer and introduce an amino group into the 
copolymer by means of treatment with .beta.-aminoaldehyde diethyl acetal. 
Acetone having dissolved therein 4 wt% of a maleic anhydride-methyl vinyl 
ether copolymer and 0.8 wt% of 3-nitrophthalic anhydride was circulated 
through the tube at 20.degree. C. for a period of 3 hours, washed with 
acetone and dried. The tube was then filled with a physiological saline 
solution of urokinase (600 units/ml), left to stand at 7.degree. C. for 24 
hours, and washed with physioligical saline. 
A circular dissolution zone 16 mm in diameter appeared around a circular 
cross-section of the tube to which urokinase was fixed by the procedures 
described above. The thrombus formation time was more than 45 minutes. 
COMATIVE EXAMPLE 
Control films or tubes were prepared as in Examples 1 to 6 without 
treatment with fibrinolytic enzyme. No dissolution zone appeared. The 
thrombus formation time for these control films or tubes ranged from 15 to 
20 minutes. 
While the invention has been described in detail and with reference to 
specific embodiments thereof, it will be apparent to one skilled in the 
art that various changes and modifications can be made therein without 
departing from the spirit and scope thereof.