Fluorescent substrate assay for antithrombin III

FLUORESCENT SUBSTF%TE ASSAY FOR ANTITHROMBIN III

Gary A. Mitchell, Patricia M. Hudson, Rolf M. Huseby, Sharon P. Pochron and Robert J. Gargiulo DADE Division American Hospital Supply Corporation Miami, Florida, U.S.A.

(Received

3.11.1977.

Accepted

by

Editor

1i.H. Seegers)

ABSTRACT A sensitive method for the assay of antithrombin III activity is described using a synthetic fluorescent tripeptide, CBZ-gly-pro-arg4-methoxy-gNA, as a substrate for thrombin. The inhibitor activity in 0.5 ul of plasma (not defibrinated) is determined with assay precision comparable to clotting and immunologic methods. Substrate assay values for normal range samples correlate with immunologic test values while abnormally low inhibitor samples give smaller substrate assay results.

INTRODUCTION Recently several procedures have been reported for the functional assay of antithrombin III

using the synthetic chromogenic substrate benzoyl-

(AT-III)

phenylalanine-valine-arginine-p-nitroaniline (1,2,3).

The chromophore is

hydrolyzed from the substrate by thrombin and its release can be measured spectrophotometrically.

Plasma AT-III activity is determined indirectly by

incubation of thrombin with patient plasma followed by the addition of substrate and measurement of the residual thrombin activity. This paper reports the use of a fluorescent peptide substrate, carbobenzoxy-glycine-proline-arginine-4-methoxy-~-naphthylamide, for AT-III assay.

The

peptide portion of this substrate is identical to the amino acid sequence reported for the natural substrate, human fibrinogen, located at a site of thrombin catalyzed cleavage (4).

This substrate is considerably more sensitive

to the measurement of thrombin thereby eliminating some interferences present in the chromogenic assays.

Also, assay precision is superior to that reported 219

221)

hSTITHFtOYBIS

III

ASSAY

VOl.l2,NO.~

for chromogenic substrate inhibitor assays with precision values in the range of clotting and immunologic test methods (5).

MATERIALSAND METHODS Buffer-l: 0.25 M glycine in 0.03 M NaCl with 2 mM K,EDTA and 0.01% thimerosal, pH adjusted to 8.3 with 5 N NaOH.

Used for all dilutions except the thrombin.

Buffer-2: 0.25 M glycine in 0.15 M NaCl with 2 mM K,EDTA and 0.01% thimerosal, pH adjusted to 8.3 with 5 N NaOH.

Used only for thrombin dilutions.

Human thrombin: lyophilized, 100 Ill/vial,no. 70/157, from WHO International Laboratory for Biological Standards, London NW3 6RB, England.

Each vial of

thrombin was reconstituted with 5 ml buffer-2 and 0.25 ml aliquots stored at -20C in siliconized glass vials.

Before use, the thrombin was thawed and

diluted to 1 IU/ml buffer-2. Bovine mucosal heparin: lyophilized, 1,250 IU/vial, no. 63/10 from WHO International Laboratory for Biological Standards, London NW3 6RB, England. Each vial of heparin was reconstituted and diluted with buffer-l to 10 IU/ml and 0.20 ml aliquots stored at -20C in siliconized glass vials.

Before use,

the heparin was thawed and diluted to a working concentration of 2 IU/ml buffer-l.

This concentration was selected following heparin titration

experiments.

Thrombin was not inhibited in the absence of AT-III

when this

concentration was used and greater concentrations gave turbid reaction mixtures, CBZ-glycine-proline-arqinine(HC1)-4-methoxy-B-naphth,ylamide: mol wt 654.1, from Enzyme Systems Products, Inc.,

4155 Rolling Trails, Greenwood, Indiana 46142. A

stock solution was prepared in absolute ethanol at a concentration of 12 mg/ml and stored at 4C.

Before use, the ;tock solution was diluted in buffer-l to a

concentration of 150 mg/l (0.23 mM/l) and warmed to 37C.

A Km value of 8.3 x

10s5 M (37C) was determined within the assay system using the Lineweaver-Burk equation. Blood specimens were collected in plastic or siliconized glass tubes containing one volume of 3.8% trisodium citrate for each nine volumes of blood.

The

specimens were centrifuged at 1,500 to 2,000 g for 10 to 15 minutes to obtain the plasma which was stored at -20C. diluted 1:400 in buffer-l. M-Partigennf Antithrombin III

Before use, the plasmas were thawed and

Diluted plasmas were maintained at 4C until assayed. Kit: radial immunodiffusion test from Behring

Diagnostics, American Hoechst Corp., Somerville, N.J. 08876. Perkin Elmer Model MPF-EA fluorescence spectrophotometer: Norwalk, Connecticut, 06856, with a controlled temperature sample cell holder and recorder model QPD-33.

The excitation and emission wavelength and slit width settings were

?‘-

j-3

,. z,

‘15

r,;;

6 x 12 nm respectively.

37s

13 x i2 x 45 mm rectangular 100 >l of diluted 130 ::1 of the

heoarin

>{armed to 37i.

100

After

incubation

rniniibited

plasma

thrombin

was

was

radial

over

the test their used

allowing

plasmas

normal

89 to 111% range

for

reported

performed value. Internal

the

mean

were

RIG

plasmas

with

and

were

sampie

-,

of

The

an_'

cuvette

zrd

cixec.

solution,

to mix

measurement

interval.

‘-i

used.

conten:s

substrate

inverted

and were

with

partial values

The

supplied

value value

performed

the

:23

reagents.

the release:

inhibitor

acti,iity

time

and

of 48 hours

normal

clotting

ranges,

as 80

RID

test

74

it 2 SD,

to 120% range

to 130%.

for

Dr.

of Michigan,

of thrombotic

Ann

at 25C.

for

the

A.

of 5y

the

samples two

*jere

tes:

substrate

ass?:; and

than

nor+-?1

the

determinations of the

Penner,

Arbor,

episodes

Eigh:

as judgel

These

is narrower All

John

to nanufacf;rer's

values

times.

as a percentage by

x 100

according

thromboplastin

calculated

University histories

was

diffusion

determined

test.

olank-plasma blank

test

by the manufacturer,

Medicine,

individuals

cuve tte was

=

patients

and

normal

ranges

in duplicate Other

the

rj-r

determinations)

to the

added

2.0 ml of

by kinetic

ted

(RID

from

prothrombin

The

blank

added was

a 2-4 minute

inhib

a minimum

were

to establish

methods.

4 ml capacity (for

25

;.;:j

follows:

inlmunodiffusion

instructions

and

determined

? thrombin The

were

1 IU/ml,

at 37C,

added

witn

or buffer

2 IU/ml,

60 seconds

4-methoxy-B-naphthylamine as

sample

solution,

to 37C)

kfas calculated

cuvettes

~1 of thrombin,

for

rrg/l (prewarmed

quartz

rc~c~c~r~~?r joe&

Tie

assay

were nor-ma? yean

Department

Michigan,

or suspected

of

and were ciotting

from problems

RESULTS The triplicate

substrate

assay

precision

assay

of plasma

samples,

ranged Table

from 1.

0.6

to 6.4X

as determined

51,

TABLE 1 Precision of Plasma Antithrombin III Measurements by Fluorescent Substrate Assay Plasma Sample

Relative Fluorescent Rates

: 3 4

0.83, 0.99, 0.89, 0.86 1.01 0.86, 0.90, 0.80 0.72, 0.72, 0.68

6.4 3.0 5.0 2.3

2

0.67, 0.63 0.69, 0.69, 0.65

;::

: 9 Yil

0.61, 0.61, 0.59 0.62, 0.56, 0.60 0.64, 0.59, 0.58 0.61, 0.53, 0.53 0.65, 0.60 0.59, 0.60, 0.57, 0.57

3.0 0.6 3.2 4.2 6.0

12

Precision, CV;:

1.7 x = + 3.4%

Assay specificity was determined by comparison of dilution curves for a pool of plasma from five patient samples with normal clotting times to a WHO purified human AT-III preparation, no. 75/564, Fig. 1.

The slopes of the

response curves are nearly identical. FIG.

1

60

1:&0 Plasma

1:soo or Purified

AT-II.f

1:iOO

I:;00

Dilutions

Fluorescent substrate assay response curves for dilutions of pooled normal plasma and purified antithrombin III.

The s:abi;ity of alasma AT-iii activity after f?eezjnl ;q?s a1j3 fluorescent titive

substrate

freezing

inhibitor

activity

I 2'; for

some

bias

low probability When are

than

gives

the

toward

for

abnormally

larger

analysis

2 and

the

The

pool

and

thawing

one

to three

values

in Tables

are within strates

after

AT-III

Plasma compared

assay.

at -20C

determined 3.

Mean

of normal

at 20-25C

freeze-thaw by RID

values

larger

RID values

low AT-III fluorescent

considerable

bias

samples

fluorescent normal

least

while

the

compared are

substrate

RID

subjected

to repe-

loss

substrate

range

souares paired

assay

samples, analysis

t-test

gives

Table

3, all

RID

results. values

and

The

least

Sample

2

Least

X Normal RID Test

3

Mean

AT-III FS Assay

117.5 114.5 107.1 104.5 104.2 101.1 100.7 97.9 96.7 93.7 92.3 92.2 83.9

116.0 111.7 107.5 105.5 104.1 100.3 108.7 91.7 86.9 97.1 90.4 80.6 84.9

si= 100.5

98.9

squares analysis slope, m = 1.07 y intercept, b = -8.2 t-test, paired P = 0.3 - 0.4 Correlation Coefficient, r = 0.89

values

squares

the difference

Compar -ison of Normal Range Antithrombin III Values Determined Radi al Immunodiffusion Test and Fluorescent Substrate Assay

Plasma

are

Table demon-

the results of the two test methods is significant by the t-test.

TABLE

in

results,

compared,

assay

to larger

was

no detectable

5.i

cycles.

the

The

methods.

in the

and

for

two test

a difference

plasma

with

d>termireti

by

in

2,

\‘01.12,s0.2

TAaLE 3 Comparison of Abnormally Low Antithrombin III Values Determined by Radial Immunodiffusion Test and Fluorescent Substrate Assay

Plasma Sample 1 2 3 4 5

X Normal Mean AT-III RID Test FS Assay 74.3 70.6 66.4 65.4 50.6

65.6 52.8 60.1 27.9 30.0

x = 65.5

47.3

Least squares analysis slope, m = 1.41 y intercept, b = -45.1 t-test paired P = 0.02 - 0.05 Correlation Coefficient, r = 0.73 DISCUSSION Defibrinated plasma, 30 to 100 ~1, is used in some chromogenic substrate assays for AT-III fibrinogen (1,3).

since clotting would result from the use of plasma \:ith The clotting interference is reduced but not eliminated when

the plasma (not defibrinated) is reduced to 13 ul and a high pH and ionic strength buffer is used (2).

The fluorescent substrate used in our assay is

more sensitive to the detection of thrombin and, as a result, only 0.1 IU thrombin and 0.5 Pl plasma (not defibrinated) are employed in each test.

Since

the plasma dilution in the incubation and measurement steps is 1:800 and 1:4,800 respectively, defibrinated plasma is not required in the fluorescent assay. Interference from most icteric, hemolyzed or colored samples is eliminated by the higher plasma dilution.

Mean fluorescent assay precision is 3.4% as

compared to 5.5% for inhibitor assays performed with the chromogenic substrate (5).

The increased precision may be attributed to the elimination of some

plasma interferences as well as the greater specificity of fluorescent measurements as compared to spectrophotometric measurements. The normal range AT-III results, Table 2, show good comparison for the two test methods although there is a slight bias toward higher RID values. The low AT-III samples, Table 3, were abnormal by both test methods, but all RID test results are higher and there is poor correlation between the results of the two methods.

Similar results were reported recently in a comparative

study of immunologic and chromogenic substrate assay values of AT-III

where

the abnormal low samples gave higher values in the immunologic test (6).

Other

reportj

have

SiJgge

jted

the

izminologic tests (7,8).

detection

of

f>;ric:jq2p.a;

: , i~.a~tj!~e

rii-1:

I

by

Our results are in aagreezent with those reports

indicating that measurement of AT-III concentration may be less useful than the &termination

of AT-III activity. REFERENCES

1.

BERGSTRtiM, K. and TAHNGORG, G. The effect of major surgery low doses of heparin and thromboembolism on plasma antithrombin. Comparison of immediate thrombin inhibiting capacity and the antithrombin content. Thromb.Res. 5, 223, 1975.

2.

ODEGARD, M. TIE and ABILDGAARD, U. Heparin cofa ctor activity measured ,:ii:h an amidolytic method. Thromb.Res. 6, 237, 1975.

3.

VINAZZER, H. Photometric assay of antithrcmbin III with a chromogenic substrate. Haemostasis 2, 101, 1975.

4.

BLOMBACK, B., BLOMBkK, M., HESSEL, B. and IWANAGA,S. Structure of Titerminal fragments of fibrinogen and specificity of thrombin. Nature 215, 1445, 1967.

5.

BLOMBkK, M., BLOMBACK, B., OLSSON, P. and SVENDSEN, L. The assay of antithrombin using a synthetic chromogenic substrate for thrombin. Throm.Res. 5, 621, 1974.

6.

IlOrlTGOMERY, B. Automated test measures functional antithrombin, coagulation enzymes. J.Amer.Med.Assoc. 238, 1005, 1977.

7.

SAS, G., BLASK6, G., BANHEGYI, D., JAKO, J.A. and PALOS, I.A. antithrombin III (antithrombin III 'Budapest') as a cause of thrombophilia. Thromb.Diath.Haemorrh. 32_, 105, 1974.

8.

DONATI, M.B., VANDENBERGHE, K., MOLLA, A., deRO0, M., and VERSTRAETE, M. Postoperative antithrombin III, Lancet i, 726, 1973.

Abnormal a familial