Biological activity of urokinase immobilized to cross-linked poly(2-hydroxyethyl methacrylate)

Biological activityof urokinase immobilized to cross-linked poly(2-hydroxyethylmethacrylate) LbShuLiu,Yoshihiro Ito andYukio Imanishi Depaflment

of Polymer

(Received

26

Chemistry,

October

1989;

Kyoto

accepted

University.

16 May

Yoshida

Honmachi,

Sakyo-ku.

Kyoto,

606

Japan

1990)

The fibrinolytic enzyme urokinase was immobilized by encapsulation or cross-linking to poly(2hydroxyethyl methacrylate) networks. The immobilized urokinase was more thermally stable, more stable against pH change and more resistant to inactivation by plasma protease inhibitors. These stabilities were improved with increasing degree of cross-linking. On the other hand, the enzymatic activity of immobilized urokinase decreased with increasing degree of cross-linking. A suitable degree of cross-linking is needed for the maintenance of high biological activity of immobilized urokinase over a long period. Keywords:

Urokinase

Urokinase,

,mmobdizatIon,

has been widely

pfHEMA),

used for the clinical treatment

thrombogenetic

disease and haemorrhoidal

organ

on which

materials

fibrinolytic

urokinase

urokinase

For example, by bonding

For other immobilized

urokinase

membrane. grafted

examples,

as cross-linking

Caper-Annonini

et

a/.6

immobilized made

and

urokinase

tube

with

2-hydroxyethyl diacrylate induced

urokinase

copolymer

and

membrane

to which

al. lo immobilized composite

such immobilized az-macroglobulin ethylene

in

of

(Japan).

the

radiationpolyethylene

grafted.

encountered

Watanabe

et

(a,M).

antithrombin

Remyand

urokinase

and

inhibitors

depression

In the present non-thrombogenicity

AND

of

a-casein

(human

(No. A-91 41,

lot 37F-9467)

Chemical

(USA).

Co.

(human serum

a

polyoxy-

accompanying activity.

in view of relatively urokinase

was

high

origin,

purchased was

Inc. (Japan).

proteins

further

from Nakalai Tesque

from

purifications.

Inc. (Japan).

purchased

MEBAAand

were

purchased

The vinyl monomers

were

as usual and used.

Tris(hydroxymethyl)aminomethane chloride saline

from

and peptides

HEMA.

(MDI)

Sigma

4-methyl-7-

(Glu-Gly-Arg-MCA) These

No.

and AT III

Glutamyl-glycyl-arginyl

Peptide

Institute

Code

lot 84F-9555). were

EC:

from Midori Juji

coumarinamide

and

prepared or

to Professor Y. Imanlshl.

0 199 1 Butterworth-Heinemann

of the

actrvrty were

urine origrn,

was purchased

(No. C-7891,

buffer

(PBS,

KH,PO,

(50

pH 7.2)

IrIM,

NaCl 8.0 g/l,

(Tris)-hydro-

and

phosphate-buffered

KCI 0.2 g/l,

0.2 g/l, CaCI, 0.1 g/l,

MgCI,

NazHP04 0.1 g/l,

1 .I 5 g/l,

pH 7.0) were

used.

Materials

synthesis

imImmobilization

Correspondence

attack

enzymatic

urokinase

Code No. 4015) Plasminogen

1089),

purified

of the soluble conjugate

of the enzymatic p(HEMA),

of

plasma

(AT Ill)

albumin

was increased

investigation,

against III

Poznansky”

serum

The conditions

from

METHODS

were used without

polymer

in the utilization

is the instability

as

and found that the stability

a simultaneous

losing

4,4’-diisocyanatodiphenylmethane

such

against the protease

for.

of urokinase

without

High-molecular-weight

glycol

to collagen-synthetic

urokinase

inhibitors

conjugate

was

searched

3.4.99.26,

material.

One of the problems protein

encapsulated

by low-temperature

urokinase

inhibitors

HEMA-N,N’-methylene network.

Reagents

of polyvinyl-

They also synthesized

urokinase

protection

protease

in

copolymer

Takagi7

(HEMA)-tetraethylene

network

polymerization.

for an efficient

MATERIALS

reagent.

of a polyacrylamide

Sugitachi

to the surface

methacrylate

encapsulation (MEBAA)

and Tamenase5

chloride tube. Kaetsu eta/.g coated the surface of a polyvinylchloride

by

of

to the inner wall of a nylon tube. Sugitachi

et a/.8 immobilized

mobilized bisacrylamide

for its

of a tube

to the surface

Sugitachi

urokinase

et a/.3.4

surface

collagen fibrils using glutaraldehyde

of

for blood-compatible

Senatore

to the

antithrombogemcity

disease. Artificial

is immobilized

activity have been developed

materials’-4.

encapsulation,

buffered

solution

of

urokinase (200~11)

by

cross-linking.

or urokinase

(300

Tris-HCIIU),

HEMA

12 August

545

Ltd. 0142-9612/91/060545-05 Biomatenals

199 1, Vol

Urokinase immobilized to p(HEMA): L.-S. Liu et al.

Theplasminogen method. Urokinase converts plasminogen to plasmin, which in turn hydrolysesa-casein to yield tyrosin that can be determined by UV spectroscopy. The determination by the plasminogen method was carried out by the procedure reported by Johnson et al.13 A sample of immobilized urokinase was placed in Tris-HCI-buffered solution (1 .O ml) containing plasminogen (20 IU) at 37°C for 10 min.Then the supernatant (0.5 ml) was transferred to Tris-HCI-buffered solution (2.5 ml) of a-casein (1.4 wt%). The mixture was kept at 37°C for 30 min under stirring, and then a portion (2.0 ml) of the mixture was mixed with aqueous perchloric acid solution (0.5 M, 3.0 ml). After standing the mixture at room temperature for 30 min, it was centrifuged at 3500 rev min-’ for 15 min and the supernatant recovered. The optical density at 275 nm due to tyrosin produced in the supernatant determined the urokinase activity. A calibration curve was established using native urokinase (1 O-50 IU) under the same conditions.

(200~1) and acetone solution (20~1) of MDI (2.0 mg/ml) were mixed in an ampoule (1.5 ml capacity). The mixture was purged with nitrogen stream and polymerized by 6oCo irradiation, 330 rad/min at -78°C for 20 h (Figure IA). A cross-linked gel-like polymer was obtained, which will be represented as H M-UK. Immobilization of urokinase by encapsulation. Tris-HCIbuffered solution (200,uI) or urokinase (300 IU), HEMA (200~1) and a requisiteamountof MEBAAwere mixed in an ampoule (1.5 ml capacity). The mixture was purged with nitrogen stream and polymerized by 6oCo irradiation, 330 rad/min at -78°C for 20 h. Gelation of the mixture occurred during the polymerization and, ultimately, a cylindrical polymer (5 mm radius) was obtained. The products obtained with th,0 feed of fvlEBAA of 15, 10 and 5 my will be represented as HMe-UK-a, HMe-UK-b and HMe-UK-c respectively (Figure IS). The immobilized urokinase (cross-linked and encapsulated) was stored at 4°C and tested for biological activities.

The peptide method, Urokinase specifically hydrolyses the C-terminal amide bond of Glu-Gly-Arg-MCA to liberate 7-amino-4-methylcoumarin (AMC) which is fluorescent. To the Tris-HCI-buffered solution (1 .O ml) of Glu-GlyArg-MCA (0.1 mM), which was equilibrated at 37°C for 30 min, a sample of immobilized urokinase was added and reacted under stirring. After 10 min, the reaction was stopped by adding acetic acid solution (1 7 wt%, 1.5 ml). The urokinase activity was determined by the measurement of fluorescence intensity of AMC produced with excitation at 380 nm and emission at 460 nm14. Thecalibration curve for the determination was obtained by using native urokinase under the same conditions.

Coating of immobilized urokinase on polyetherurethaneurea (PEUU) membrane. PEUU was ,.synthesized from polyoxytetramethylene, MDI and methyl L-lysinate as reported previously’2. PEUU membrane was cast on the inner wall of an ampoule from N,/V-dimethylformamide solution (10 wt%), and HEMA (200~1) was added. After 20 min. the mixture was y-irradiated with 6oCo. The urokinase-HEMA-MDI mixture or the urokinase-HEMAMEBAA mixture was added as described above. The mixture was again irradiated with 6oCo.The conditions of y-irradiation were the same as employed in the cross-linking and encapsulation experiments. HM-UK-coated PEUU membrane or HMe-UK-coated PEUU membrane was obtained, which will be represented as HM-UKP or HMe-UKP respectively (Figure IC).

Stability

The stability against the temperature, pH and protease inhibitors of the immobilized urokinase was investigated. Four measurements were performed for every sample and the average value was determined.

Assay of enzyme activity Activity of immobilized urokinase was assayed plasminogen or Glu-Gly-Arg-MCA as substrate.

A:

Thermal stability. A sample of immobilized urokinase was placed in the Tris-HCI-buffered solution (1 .O ml) of Glu-Gly-

using

-1

of immobilized urokinase

t

-7aoc,

MD1

h

HM-UK

y-==y -78OC,

MEBAA

B:

20

20

h

y-ray

c:

PEUU coating

t

-78OC,

HEMA

20

h

HEMA grafted PEUU

y-ray

[UK+

'[tHEMA]

HMe-UK (a,b,c)

t

MD1

$&=$

t

MEBAA -;"r;; 2o hb HMe-UKP

HM-UKP

(a,b,c) UK

: urokinase

HEMA MD1 MEBAA PEUU

: hydroxyethylmethacrylate

CH2=C(CH3)COOCH2CH20H

: 4,4'-diisocyanatodiphenylmethane

O=C=N@H2QN=C=0

: N,N'-methylenebisacrylamide : polvetherurethaneurea - _

CH2(NHCOCH=CH2)2 membrane

-$~(CH2)4CH~~{~QCH20~~l HOH

I HO H COOCHj

Figure 1

546

Scheme of urokinase immobilization.

Biomatarials

199 1, Vol 12 August

~~H~,401n~~~CHZ~$HO

OH

HO

lJrok/nase

Arg-MCA 70°C

(0.1

mrvt). After 0.5,

the sample

Tris-HCI-buffered

solution

and the reaction

1 .O, 1.5 and 2 h of stirring

was taken out and washed solution

three

times.

versus

liberated time

gave

species

conditions

at 70°C.

was

without

of urokinase

activity

It was confirmed

not

liberated

the immobilized

under

native

was added

A sample

that any the

above

urokinase.

In vitro clotting Citrated

0.05

as

buffered

solution,

solution, pH 10.

and

neutralized

After

keeping

the

solution

reaction

to pH 7.0.

urokinase

at different

that

of AMC without

reversibility hydrochloric

was

was

solution, activity

activity

and

solution,

membrane

was

The total volume

pH regions, in

1 N

for 6 or 12 h. solutron

solution.

To the

and the

of

enzymatic

determined. againstprotease

urokinase

inhibitors.

A sample of immobilized

was put in the Tris-HCI-buffered

of AT III (350pg/ml).

The mixture

stirring,

and

of the

suitable

time

aliquots intervals

and

solution

solution (1 .O ml) of Glu-Gly-Arg-MCA activity of the immobilized

solution

urokinase

were

fugation

5.

on

the

dried and weighed. sample

PEUU

gel.

CaCI,

was kept at

was then stopped formed

Four measurements

and

the

by

was fixed with

average

were

value

was

determined.

RESULTS AND DISCUSSION Enzymatic activities of native urokinase (N-UK), HM-UK and HMe-UK For reference

taken

under out

experiment,

actrvrty

was

at

followed.

synthesrzed urokinase about

are

immobilized

10%

2, the

the assay method. encapsulation

native

However,

when

activities

of

assayed

The

of MEBAA

(0.1

molecular-weight difficult,

by the

lattice

make

plasminogen

while it permits

content

peptide

in

method.

method,

decreased

the

wrth

from the different

to different

may decrease and

by

in-

In preparation.

results obtained

could be ascribed

is of

of that of

of the MEBAA

urokinases

of MEBAA

The different

65%

by the plasminogen

immobilized

content

immobilized

are about

for the assay

of

series)

and independent

of urokinase

series)

activrty

(HM-UK

urokinase

and independent

immobrlization

urokinases

in Table 1. The

The activities

(HMe-UK

native urokinase

methods

of the immobilized

by cross-linking

of that of native

lattices.

by the

activities

summarized

content

ITIM). The residual

in Figure

Mrad. The enzymatic

the

As shown

was y-Irradiated

change of enzymatic

1 5% of the orrginal activity at a radiation dose

lost

to Tris-HCI-buffered was determined

natrve urokinase

its size wtth

the

assay

sizes of cross-linking approach

increasing

of the

to the encapsulated

the small peptide

high-

urokinase

to go through

the

lattice.

The same experiment by using

AT III solution.

formation

and the thrombus

every

creasing

method.

conditions

or

and the mixture

repeated

(1 .O ml)

was kept at 37°C

added

with water

of 0.4

the same

(1 .O ml, pH 7.2)

added

membrane

M, 5 ~1) was added

for

previously’

urokrnase-immobilized

formaldehyde,

To check the

immersed

solution

with

for 20 min. Thrombus

dilution

under

of the neutralized

ml) was

(0.1

urokrnase

of the buffer

gel

coated

with 6oCo and radiation dose-dependent

by the deter-

under variable

uroknase,

canine whole blood (50 ,ul) was put on the

urokinase-immobilized

It was confirmed

ml) at room temperature

(0.1

for

three times

urokinase.

sample

the Tris-HCI-buffered

Stability

peptide

at 37°C

collected

determined

not observed

9.0 ml by the addition

Glu-Gly-Arg-MCA

were

in the solution.

urokinase

and neutralized. was

mixture

immobilized

Tris-HCI-

M

by the buffer

the immobilized

acid (5.0

M

(I .O ml). The washing

dilution

pHs

of enzymatic

an immobilized

0.05

ml. The activity of the immobilized

liberated

any fluorescence

conditions

pH 3.5;

0.05

solution

After

acid,

carbonate-buffered

M

the

ITIM)

containing

was taken out and washed

the total volume was 10.0 mination

solution, 0.05

(0.1

hydrochloric

pH 7.2;

the Tris-Hal-buffered

solution

dilute

pH 8.5;

10 min, the sample with

follows:

M citrate-buffered

Tris-HCI-buffered solution

urokinase

of the

used as standard.

test was carried out as reported

(10 wt%)

solution

to each of the five kinds of solutions

Glu-Gly-Arg-MCA pH 2.5;

of immobilized

tests

was

L -S. Lw et al.

clotting test

In vitro

37°C pH stability.

urokinase

to p(HEMA).

liquid

5 ml. The determination

the variation

of heating

fluorescent

The washing

For all stability

at

1 .O ml of

were mixed and the buffer solution

was added to make the total volume of AMC

with

unmob~llzed

The human

at 3000

was carried out under the same

1 .O ml of human plasma

rev min-’

for

plasma

instead

was prepared 5 min

of the

It is concluded can

be

that a frbnnolytic

immobilized

particularly

by centri-

coating

on membrane

human

without

serious

of titrated

by

enzyme,

to biocompatible

damage

of enzymatic

urokrnase,

encapsulation

or by

p(HfMA)

matrix

activity.

blood.

Thermal and pH stability of immobilized urokinase Figure

3

shows

immobilized

80

the

urokinase

activity

of native

heatrng

and less than

change

of

at different

urokinase

enzymatic

actrvitres

heating time at 70°C.

(N-UK)

decreased

half of Its original

actrvity

quickly

1

Actiwty

of mmob!lned

urokinase’

i Plasmlnogen

Sample

60

1 (r-1

0

0.2 Dose

Figure

2

y-irradiation,

Dose-dependent “‘Co.

- 78°C:

actwty initnlactwty,

change 200

of

I

1

0.3

0.4

(Hrad) native

uroklnase

IU: dose rate, 2 x

lo4

HM-UK HMe-UK-a HMe-UK-b HMe-UK-c

Peptlde method

method

IU

%:

IU

YfJ

33 - 1 123 * 3 1651 5 198 +4

11 41 55 66

35 + 1 192 -4 198 t 3 196-3

12 64 66 65

under rad/h

*The actwry

of uroklnase

before immobll~zatlon

IS 300

IU

on

remained

! Table

of The

Urokinase jmmabjiized

to SCHEMA]: L.-S. Liu et ef.

Table 2

Activity of immobilized urokinase after treatment in 1 whydrochloric

acid*

Sample

Residual activity after 6 h IU

N-UK HM-UK HMe-UK-a HMe-UK-b HMe-UK-c

0 24 98 89 63

IU

%

+ k t *

0 69 51 45 32

2 3 3 2

Residual actiwty after 12 h

0 21 67 57 33

i 3 fr2 t 3 I 1

%

i k * *

0 60 35 29 18

1 2 3 2

+ i IL +I

3 3 2 2

*The activity of lmmobllized urokinase before the treatment: N-UK 200 IU, HM-UK 35 IU, HMe-UK-a 192 IU, HMe-UK-b 198 IU, HMe-UK-c 196 IV.

I

I

0.5

1 .o

Heating

I

I

time

1.5

2.0

(h)

Figure 3 Activity variation of native or immobilized urokinase under incubation in Trs-HCI-bufferred solution (pH 7.2) versus heating time at 70°C: A, HM-UK: A, HMe-UK-a; n , HMe-UK-b; 0, N-l/R 0, NY-UK

after the acid treatment for 12 h, indicating that the immobilization suppressed deactivation or decomposition of urokinase by hydrochloride acid. It can be concluded that the immobilization reduced the deactivation of enzyme by heating or pH change through the stabilization of protein conformation due to cross-linking and lattice formation, although the absolute activity of the enzyme was somewhat reduced by immobilization.

Stability of immobilized urokinase against protease inhibitors

0

4

8

12

PH Figure 4 pH-dependent activity change of native or immobilized urokinase incubated at 37°C for 30 mint A. HM-UK; A, HMe-UK-a; 0. N-UK

after 2 h. Irradiated native urokinase without immobilization (NY-UK) was less thermally stable than N-UK. The urokinase immobilized by cross-linking (HM-UK series) was very stable and did not change its activity on heating under the present conditions. The thermal stabilities of urokinases immobilized by encapsulation (HMe-UK series) were intermediate between HM-UK and N-UK or Ny-UK, and increased with increasing content of MEBAA in the immobilization reaction. Figure 4 shows the effect of pH on the activities of native and immobilized urokinase. They showed the same optimum pH value, but the activity/pH profiles for immobilized urokinase were less narrow than that for native urokinase. Native and immobilized urokinase were incubated in 1 N hydr~hloric acid at room temperature for 6 or 12 h. After neutralization with NaOH, the enzymatic activities were measured and are shown in Tabie2. The native urokinase (N-UK) lost its activity completely by the treatment, indicating the occurrence of deactivation or decomposition. On the other hand, about 60% and 15-35% of the original activity of HM-UK and HMe-UK, respectively, were retained

548

Biomaterials

799 1, VoI 12 Augusz

Figure 5 shows the time-dependent activity change of native and immobilize urokinase when incubated in the presence of AT III at 37°C. The urokinase immobilized by~ross-linking (HM-UK series) is most resistant against the deactivation by AT III. The stability against AT Ill of the urokinase immobilized by encapsulation (HMe-UK series) was intermediate between those of HM-UK and native urokinase and increased with increasing content of MEBAA in the immobilization process. The effect on the deactivation by AT III is very similar to that on the enzymatic activity assayed by the plasminogen method. It is feasible to say that higher degree of crosslinking in the gel makes an access of AT III to the urokinase more difficult. Since the rate of activity loss of native urokinase by AT Ill was higher at a low concentration of urokinase (30 IU compared with 200 IU), the stability of the immobilized urokinase against AT III should come from the concentration effect. A very similar activity change of native and immobilized urokinases was observed when incubated in the presence of human plasma, as shown in Figure 6. However, comparison

0

0.3

1

0.6 Incubation time

2

3

tht

Figure 5 Activity variation of urokinase versus incubation time of AT JII at 37°C: A, HM-UK (35 IU); II, HMe-UK-a (192 IU); 0. HMe-UK-c (196 IU); l, N-UK-a (200 IU): 0, N-UK-b (30 IU).

Urokinase !mmob!lized to p(HEMAJ: L.-S. L/r/ et al.

-

4 *

1 4

7

9 10 11 12

0

0.5

1.5

1 .o

-

-

~

FIgwe 6 Actw~ty vanation of urokinase warsus incubation t,me of human plasma at 37°C: A, HM-UK (35 tU); a, HMe-UK-a /192 tl& n , HMe-UK-b ( 198 IU); 0. HMe-UK-C f 196 RJ); 0, N-UK-a (200 IU}: 0. N-UK-b (30 IU).

In vitro thrombus wwght on va~ous polymer membranes during 20mm contact with canine blood a? 37°C: 1, glass@andard): 2, PEUU; 3, HEMA-MDI copolymer; 4, HM-UK (35 1U.l;5. HM-UKP (35 IU); 6, NEMAME3~ copolymer; 7. HME-UK-a /792IUJ; 8, ~ME-UKP-a (1921Uj; 9, HME-UK-b (198 IU]; lo, HME-UKP-b (198 tU): 11. HME-UK-c (196 IU); 12, HM&UKP-c (196 IU).

of Figure 6 with

REFERENCES

Incubation

urokinase

Figure 5 indicates

in the presence

presence

as n,M

(mol wt

of HM-UK

presence

of human

plasma

than that in the

other

X 104)

The

activity

than

tothe

is more difficult

protease

of

1

inhibitors

in addition

reason

for the

HMe-UK

plasma is unclear,

some other proteases linking

deactivation

could have arisen from the

contains 72-82

X f03).

62-67

decrease

plasma

(mol wt

(h)

a faster

of human

of AT III. This difference

fact that human such

time

to AT III

much

faster

series

in the

because the access of

immobilized

urokinase

2

3

by cross-

than that by encapsulation

as shown

4

in Table 1. 5

In vitro clotting

test

Figure 7 shows

the

6

relative

immobilized

urokinase

immobilized

urokinase,

is also

reduced

found

encapsulation

that the

urokinase

immobilization decreased

When

these

the immobilized

nearly the same

are well correlated the plasminogen short-term

of

formation.

urokinases

urokinase

In the

case

results

by of

a long-term gated

of MEBAA. on PEUU

antithrombogenicity

protease

inhibitors

12

activities

assayed

concluded

from the deactivation of plasminogen. which

by

that the

urokinase

is

important.

13

by protease However,

of urokinase

14

for

has not been investi-

of deactivation

may become

11

as

on antithrombogenicity

of immobilized

antithrombogenicity

here, the suppression

10

the thrombus

were coated

It is therefore

but with activation

8 9

inhibitors that should be equally accessible to the immobilized urokinase,

7

It

themselves.

antithrombogenicity

not related with protection

urokinase

urokinase

content

with the enzymatic method.

on

than that by cross-linking

decreasing

experimental

the

of urokinase.

by encapsulation,

with

immobilized

film, they showed These

immobilization

thrombus

formation

formed

for immobilizing

was more effective

in suppressing

of thrombus

by the immobilization the

on

coated with the

It is shown that the thrombus

on the matrix polymers

was strongly

weight

membrane

the amount

glass being taken as standard. formation

thrombus

or PEUU

by

15

Kusserow, B.K., Lattow. R.W. and Nochols, J-E., The surface bonded, covalently crossltnked uroktnase synthettc surface: ,n virro and chrontc in viva and in viva studtes, Trans. Am. Sot. Artif. Int. Organs 1973, 19, 8-12 Woman, B. and Wallem. P.. Actrvatton of human plasmtnogen by an tnsoluble derwatwe of urokmase, Ear J B&hem. 1973, 36. 25-31 Senatore, F.. Bernath, F. and Mewer, K., Cltntcal study of uroktnasebound fibrocollagenous tubes, J Biomed Mater Res, 1986, 20, 177-188 Senatore. F. and Bernath. F., Lysts bme and FDP tmmunoprectpttatton by soluble and tmmobtltzed uroktnase, J Eiomed. Mater. Res. 1986, 20, 189-203 Caper-Annonw F.C. and Tamenase, J., Ktnebc studtes on soluble and insoluble uroktnase, Can. J 3io~hem. 1975, 53, 890-894 Sugttacht. A., Kawahara, T., Kotama. J.. Kikkawa, Y. and Takagt, K., Anttthrombogenictty of tmmobtltzed urokmase and Its cltntcal stgntftcance. Blut 1978. 37, 31-36 Sugttacht. A. and Takagt. K.. Anttthrombogentctty of tmmobtltzed uroktnase -clinical apphcatton, ht. J. Artif Organs 1978,1,88-92 Sugttacht, A., Tanaka, M.. Kawahara, T., Kttamura, N. and Takagt, K., A new type of dram tube, Artificial Organs 198 1, 5. 69 Kaetsu. I., Kumakura, M., Asano, M., Yamada, A. and Sakurat, Y,, lmmobtltzatton of enzymes for medtcal uses on plasttc surface by radtatton-tnduced polymerwatton at low temperature. ./. Biomed Mater. Res. 1980, 14, 199-2 10 Watanabe, S.. Shmtzu, Y.. Teramatsu. T , Murachi, T. and Htno. T.. The m vitro and in v&o behawor of urokinase tmmobihzed to collagensynthettc polymer composrte m&err&J Biomed Mater Res. 198 1, 15. 553-563 Remy, M.H. and Poznansky, M.J., lmmunogentctty and anttgenrctty of soluble cross-ltnked enzyme/albumtn polymers: advantages for enzyme therapy, Lancer 1978, ii, 68-70 ito, Y., Swdo, M. and Imancsht. Y.. Syntheses, antrthrombogentctty, and wtteraction wtth plasma proteins of ankx~tc polyurethanes and hepann-bound polyurethanes. J. Biomed. Mareei Res. 1986, 20. 1157-1178 Johnson, A.J.. Kline, D.L. and Alkjaerstg. N.. Assay methods and standard preparatton for plasminogen and urokmase rn purlfled systems, Thromb. Diath. tiaemorrh. 1969, 21, 259-272 Monta. T., Kate. H., Iwanaga, S.. Takata, K. and Sakakibara, S., New fluorogenrc substrates for a-thrombtn. factor Xa. kallikretns, and uroktnase, J Biochem. 1977, 82, 1495 1498 Ito. Y.. Stsrdo, M. and Imantsht, Y.. In vitro antithrombogentctty of and platelet adheston onto ~omcally or covalently heparwzed polyetherurethaneurea. Biomaferials 1988, 9. 235-240

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