TEST FONCTIONNEL POUR DETERMINER L'ACTION DE LA PROTEINE S

FUNCTIONAL ASSAY FOR DETERMINING THE PROTEIN S ACTIVITY

Abrégé anglais

- 1 -
BEHRINGWERKE AKTIENGESELISCHAFT HOE 9D/B 009 - Ma 819
Dr. Pfe/Zi
Abstract
Functional assay for determining the protein S activity
The invention relates to a method for the functional
determination of protein S in liquids, in particular in
plasma, and to a reagent suitable for this.

Revendications

- 12 - HOE 90/B 009 - MA 891
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for determining protein S by determining
the clotting time in a biological sample, wherein
the amount of added activators is adjusted in such
a way that the clotting time is prolonged beyond the
normal clotting time.
2. The method as claimed in claim 1, wherein an excess
of protein S-deficient plasma is added to the sample
and, after admixing a reagent comprising an
activator of clotting, calcium ions, phospholipids,
activated protein C, an inhibitor of heparin and, if
appropriate, a chromogenic peptide substrate for
thrombin, the time is measured until a defined
increase in extinction at the absorption optimum of
the free chromophor is reached or clotting occurs.
3. The method as claimed in claim 1, wherein the sample
is initially introduced together with an excess of
protein S-deficient plasma and, after separately
adding activated protein C by adding a reagent
comprising an activator of clotting, calcium ions,
phospholipids, an inhibitor of heparin and a chromo-
genic peptide substrate for thrombin, the time is
measured until a defined increase in extinction at
the absorption optimum of the free chromophor is
reached or clotting occurs.
4. The method as claimed in claim 1, wherein the
undiluted sample is admixed with 4 to 10 times the
volume of protein S-deficient plasma, the clotting
reaction is started by admixing (5 to 10 times the
sample volume) of a reagent comprising activated
protein C (1 to 50 pmol/ml), an activator of the
clotting system, preferably a thromboplastin or a
snake venom protease, phospholipids (5-300 ppm
(w/v)), e.g. cephalin, calcium ions (2-10 mmol/l)
preferably CaCl2, a heparin-neutralizing substance,

- 13 -
such as e.g. Polybren (0.1-10 µg/ml), and a chromo-
genic thrombin fiubstrate, such as e.g. Tos-Gly-Pro-
Arg-ANBA-IPA. The time from adding the reagent to
reaching a certain extinction (e.g. 0.1) at the
absorption optimum (e.g. 405 nm) of the liberated
chromophor is measured.
5. The method as claimed in claim 1, wherein the
clotting tIme is determined with the aid of a
chromogenic substrate.
6. The method as claimed in claim 1, wherein the
deficient plasma has a factor V content of 20 to
100%.
7. The method as claimed in claim 1, wherein the
protein C was activated by a snake venom protease
from the venom of the snake Agkistrodon contortrix.
8. A reagent for the functional determination of
protein S by determining the clotting time in a
biological sample, wherein the clotting time in the
absence of protein S is at least 50 seconds.
9. The reagent as claLmed in claLm 8, which contains,
inter alia:
activated protein C, an activator of the exogenous
or endogenous path of clotting, phospholipids,
calcium ions, if appropriate a chromogenic substrate
for thrombin, and a heparin-neutralizing supstance.
10. The reagent as claimed in claim 8, wherein the
activator is a protease from the venom of a snake or
the refractionated snake venom.
11. The reagent as claimed in claim 8, wherein a throm-
boplastin is used as activator.
12. The reagent as claimed in claLm 8, wherein the

- 14 _
activator is a sulfatide or a mixture of sulfatides.
13. The reagent as claimed in claim 8, wherein the
heparin inhibitor i Polybren.
14. The reagent as claimed in claim 8, wherein
chromogenic thrombin substrate used is a compound of
the formula I
<IMG> (I)
where R is C1-5-alkyl or -CH[CH(CH3)2]COOCH3 and
X is H-D-Phe-, Boc-Gly or Tosyl-Gly.

- 15 -
15. A method for determining protein S as claimed in claim 1
and substantially as described herein.

Description

21~7~3~
BEHRI~GW~RRE AXTI~NGRS~LL~CHAET HOE 90/B 009 - Na 819
Dr. P~e/Zi
Functional a~sayL~or determinin~ the ~rotein S activi~
The invention relates to a method for the functional
determination of protein S in liquids, in par~icular in
plasma, and to a reagen~ ~uitable for this.
Protein S is an inhibitor of blood clotting and acts as
a cofactor of activated protein C in its proteolytic
degradation of the clotting factors Va and VIIIa.
An inborn or acquired deficiency in protein S can lead to
thromboembolic complications.
Protein S is syn~hesized in the liver (5tern, D. et al.
(1986), J. Cell Biol. 102, 1971-1978). The biosynthesi~
is vitamin R-dependent and the concentration in the blood
is therefore reduced on treatment with vitamin R
antagonists. The star~ of the treatment can, in the
presence of a protein S deficiency, lead to serious
Lmpairment of the health of the patient (Grimaudo, V. et
al. (1989) BMJ 298, 233-234).
The activity may also be reduced in the case of dis-
seminated intravasal clotting or thromboembolic diseases.
It is clear from the abovementioned fin~ings that protein
S has a decisive influence on the capability of the
protein C/thrombomodulin inhibitor system, and that a
reliable diagnostic system is correspondingly required.
In plasma, only about 40~ of the protein S is prese~t in
the free form. The rest of about 60% is in a complex with
the C4b-binding protein (Dahlback, B. (1981), Proc. Natl.
Acad. Sci. 78" 2512-2516~.
This bound protein S is not available as cofactor for
activated pro1ein C and therefore has no anticoagulatory

~37~3~
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action. An excessive proportion of bound protein S can
therefore also be found as a functional deficiency, while
the antigen is found to be normal or nearly normal (Comp,
P.C. et al. (1986), Blood 67, 51)4-508; Girolami, 5. et
al. (1989), Thromb. H emost. 61, 144-147).
On the other hand, the acti~ity can be normal in the case
of reduced protein S antigen if only a very ~mall propor-
tion is present in the inactive bound form, as is
normally the case in newborns (Schwarz, H.P. et al.
(1988~, Blood 71, 562-565).
It is known to determine pro~ein S immunologically.
However, these methods do not provide any information on
the activity of the protein and are not a subject of the
invention.
Methods for determining the activity of protein S have
also been de~cribed.
DE 3724443 A1 describes a me~hod for determining the
activity of protein S. This assay requires relatively
complex reagents, such as a synthetic substrate plasma,
purified factor Xa and pro~hrombin, and is inconvenient
to carry out due to the incubation times. Also, it is
interfered with by, e.g., heparin in the sample.
~E 3607559 Al mentions a functional determination of
protein S in combination with protein C. It is necessary
to determine a reference value ~ithout protein C
activator for each sample. The use of an activator of
F VII or F II is also claimed but no further evidence is
shown. Data on the measurements to be expected or inter-
fering influences for all the test systems are not given.
A functional assay i6 described by Comp, P.C. et al. in
J. Clin. Invest:. 74, 2082-2088 (1984). Here the prolo~ga-
tion of the clotting tLme by activated protein C is
measured in the l-step factor Xa assay. $his assay is

~3 ;7~3~
-- 3 --
relatively insensitive having a prolongation of only
24 sec for the maximum concentration of 50% which can be
measured with this method. It was not possible to measure
plasmas from patients under vitamin R antagonist treat-
S ment or ha~ing liver damage.
Bertina et al. (Thromb. Haemost. 1985, 53 (2), 268-272)
indicate a possibility of de~ermining functional protein
S in human plasma. However, the test cannot b~ evaluated
quantitatively for protein S insofar as there is no pos-
sib~e control of the endogenous factor VIII or factor Vin the plasma to be measured and the prolongations depend
decisively on these two factors. If these factors are
present in the sample in an increased or reduced con-
centration, findings which are correspondingly reduced
or increased result for protein S.
Van de Waart et al. (1987, Thromb. Res. 48, 427-437) use
a system comprising adsorbed substrate plasma, added
prothrombin, activated protein C, phospholipids and
calcium chloride. A 100% difference in the protein S
content of the sample only cause about 20 seconds o~
prolongation, i.e. the assay is likewise relatively
insensitive.
Suzuki K. and Nishioka J. (1988, Thromb. Res. 49, 241-
251) describe a further assay system. They use Protaca, an
activator for protein C from snake venom. This method is
also time-consuming and inconvenient. The sensitivity is
very low at a prolongation of 13 sec for a 100~ dif-
ference in acti~ity.
Kobayashi I. et al. (1989, Clin. Chem. 35, 1644-1648)
also propose a protein S assay which likewise represents
a determination via a modified aPTT determination.
The test methods known hitherto have in common that, in
addition to an inconvenient procedure because of the
multiplicity of reagents used, they only have a low

~ 4 - 2 ~ 3 ~
sensitivity which is measured by the prolongation of the
reaction time in relation ~o the degree of protein S
activity.
It was therefore an object of the invention to develop a
method and a reagent which allows the determination of
the activity of protein S in plaæma in both a simple and
a reliable, sensitive and specific way.
According to the inven~ion, a method for determining
protein S by measuring the clotting time of a biological
sample is now disclosed, wherein the amount of added
activators is adjusted in such a way that the clotting
time is prolonged beyond the normal clotting ~ime.
Prolonging the ~lotting time made it possible in a
surprisingly simple way to achieve a very great increase
in sensitivity which can, moreover, be adapted to the
requirements.
Methods for determining the clotting time are known per
se to those ~killed in the art. They can, inter alia, be
methods which determine the liberation of thrombin from
prothrombin via the formation of a clot or the conYersion
of a chromogenic substrate. The preferred method in this
- connection is the chromogenic one, with a ~ery particular
preference for the use of a chromogenic thrombin sub-
strate, in particular of Tos-Gly-Pro-Arg-ANBA-IPA.
In a preferred embodLment of the method according to the
invention, an excess of protein S-deficient plasma is
added to the undiluted sample and the clotting time is
then determined by admixing a reagent comprising
activated protein C, an activator of the exogenous or
endogenous path of clotting, phospholipids, Ca~, a
chromogenic substrate for thrombin and a heparin
neutralizing substance. The activated protein C can also
be added to the sample separately a short time before the
rest of the reagent.

2 ~ 3 ~
In a particularly preferred embodiment of the method
according to the invention, the undiluted sample i6
admixed with 4 to 10 times the volume of protein S-
deficient plasma. The clotting reaction is started by
S admixing (5 to 10 times the sample volume of) a reagent
comprising activated protein C ~1 to 50 pmol/ml), an
activator of the clotting system, preferably a thrombo-
plastin or a snake venom ]protease, phospholipids
(5-300 ppm (w/v)), e.g. cephalin, calcium ions
lQ (2-10 mmol/l), preferably CaCl2, a heparin neutralizing
substancer such as e.g. Polybren (0.1-10 ~g/ml) and a
chromogene thrombin substrate, such as e.g. Tos-~ly-Pro-
Arg-ANBA-IPA. The time from adding the reagent to reach-
ing a certain extinction (e.g. 0.1) at the absorption
optimum (e.g. 405 nm) of the liberated chromophor is
measured.
The activator concentration which guarantees a clotting
time (without protein S) of at least 50 sec under the
given reaction conditions, can in each particular case be
determined by simple experiments.
Very particularly preferred methods and reagents are
those described in the examples.
The clotting time is, when using a chromogenic substrate,
preferably detenmined by determining the time from adding
~5 the reagent to reaching a certain extinction at the
absorption optimum of the liberated chromophor. For the
evaluation, a calibration curve is advantageously pre-
pared by using dilutions of a pool plasma (e.g. 100~,
75~, 50%, 25%, 12.5%, 10~) in the assay and determining
the clotting tLmes.
The biological sample can preferably be plasma of human
origin, the use of undiluted samples being particularly
preferred in this case.
A suitable protein S-deficient plasma can be obtained by

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methods known per se to ~hose skilled in the art, e.g. by
immunoadsorption. Factor VIII is, if necessary, adjusted
by adding purified factor VIII. The factor V ~an/ if
necessary, bP adjusted by adding a protein S-deficient
rabbit plasma which contains large amounts of factor V.
The volume ratio of sample to protein S-deficient plasma
is preferably 1:4 to 1:10.
The deficient plasma preferably has a factor V content of
about 20 to 100%, particularly preferably of 50-80%.
Protein C can be purified from plasma by various pro-
cesses which have been describPd (e.g. Baja~ S.P. et al.
(1983) Preparative Biochemistry 13(3) 191-214) or can be
prepared by biotechnological processes. The purified
protein C can be activated by means of PROTA or thrombin
which have been coupled to a support material such as
Sepharose(R),or activated protein C obtained directly by
genetic engineering processes (Ehrlich H.J. et al.,
J. Biol. Chem. 264 (24) 14298-14309) can be employed. The
concentration of activated pro~ein C in the assay is
advantageously between 1 and 50 pmolJml.
A protein C acti~ated by a snake venom protease from the
venom of Agkistrodon contortrix can preferably be
employed.
Activators of the clotting system are known per se to
those skilled in the art. Activators for the purpose of
this invention can also be snake venom proteases and
activated factors of the clotting cascade, such as e.g.
factor VIIa, factor IXa and factor Xa. Preferably,
proteases from the venom of Vipera russellii, sulfatides,
ellagic acid, thromboplastins and/or silica particles can
be employed ~Shimada ~. et al. (1985) J. Biochem. 97,
429-439). The optimum concentration of the particular
activators can be determined by simple experiments.
Phospholipids are a known class of substances which can

- 7 - 2 ~3 ~ 3~
be prepared by methods known per se to those skilled in
the art or are commPrcially available. Concentrations of
5-300 ppm (w/v) in the test mixture are preferred. Ca
ions can advantageously be generated by adding CaCl2.
Concentrations of 2-lO mM in the test mixture are pre-
ferred.
Heparin-neutralizing substances are a class of compounds
known to those skilled in the art, such as e.g. Polybren,
protamine chloride, protamine sulfate.
The reaction can be carried out at 15~40~C, preferably at
20-40C, very preferably at 37C.
It has been found that the protein S present in the
plasma can be measured in a sensitive and specific way in
the assay described (Table 1). Changes in concentration
of factor VIII between 50 and 150%, of ~he vitamin ~-
dependent clotting factors, such as prothrombin or
protein C (50-150%), except for protein S itself, or the
presence of up to 0.4 U/ml heparin have no influence. The
factor V content of the sample, as expected, shows a
certain influence, since Yariations in the æample cannot
be completely balanced out due to the content in the
protein S-deficient plasma and the inhibition of this
factor is a measurement of the activity of the protein S.
The invention furthermore relates to a reagent for
datermining protein S by a method according to the inven-
tion, the clotting time of a æample without protein S
being at least 50 seconds.
Preferred reagents are, in this connection, those which
contain a protease from the venom of Vipera russellii as
activator, ancl those which contain a thromboplastin as
activator.
Preferred reagents are also those which contain a chromo-
genic thrombin substrate of the formula I

~3~
- 8 -
x-Pro-Arg-NH ~ N~2 ~I
~Nh~R
where R is Cl~-alkyl or -CH[CHtCH3~ 2 3COOCH3 and
~ is H-D-Ph~-, Boc-Gly s)r to~yl-Gly.
able 1: Specificity of the assay for the functional
determination of prot~in S
Changed I Concentration ¦ Protein S activity
parameter l lfound
I
Control l llOo,o %
Heparin ¦0.1 U/ml ¦102
Heparin ¦0 . 2 U~ml ¦ 97
Heparin ¦0. 3 U/ml ¦109 %
Heparin ¦0. 4 U/~l ¦109
Heparin ¦0. 5 U/ml 1I32
Heparin ¦ 1. 0 U/~D1 ¦ > 150 ~6
F V ~50 % ¦ 114 %
F V ¦75 % ¦ ~11 %
F Y ¦lOQ % ¦ 100
F V ¦125 ~ ¦ 96 %
F V ¦150 ~ ¦ 91 %
F VIII ¦S0 % ¦ 106 9
F VIII ¦75 ~ ¦ 99
F VIII ¦100 % ¦ 98
F VIII ¦125 % ¦ 103 ~6
F VIII ¦lS0 % ¦ 104 ~
Protein C 1 50 % 1 96 9~i
Protein C ¦100 ~ I 100 %
Protein C ¦ 150 % ¦ 103 ~6

9 ~7~3~
The following examples are intended to illustrate ~he
in~ention and chall not restrict it in any way.
Example 1
Preparation of a ready-to-use reagent based on z~ aP~T
reagent
0.5 units of a~ivated protein C ~nd 10 ~9 polybrene were sdded to an aPTT
reagent (Partochrom~, Behringwerke AG, D-3550 MQxburg),
comprising phospholipid, ~ulfatide, ~olybren, a chromo- -
genic thrombin substrate (Tos-Gly~-Pro-~rg-AN~A-isoprop~
amide) and ~epes, pH 7.6, and the mi~ture i6 heated at
37C. The reagen~ is then ready to use.
ExEmple 2
Prep~ration of a ready-to-use reaqent based on a PT
reagent
0.5 units of activated protein C are added to a PT
reagent (Behringwerke AG, D-3550 Marburg) comprising
phospholipid, thromboplastin at a low concentration, a
chromogenic thrombin substrate (~os-Gly-Pro-Axg-ANBA-
isopropylamide) and Hepes, p~ 7.4, and the mixture is
heated at 37C. The reagent i8 then ready to use.
Example 3
Preparation ~f a ready-to-use reagent based on an
activator from a snake venom
40 ng/ml of a snake venom from Vipera ru~sellii are added
2S to a buffer comprising phospholipid, a chromogenic
thrombinsubstrate(Tos-Gly-Pro-Arg-AN~A-isopropylamide),
a heparin ~ntagonist (Polybren), zodium chloride and
Hepes, pH 7.0l and the reaction mi~ture i8 heated at
37C. ~he rea~ent i~ then ready to use.

3 ~
Example 4
Determination of the protein S conten~ in plasma
For the calibration, 100%, 75%, 50~, 25%, 10% and 0%
dilutions of a healthy-donor plasma are prepared in
phosphate-buffered isotonic saline. The measurement of
the standards and the samples is carried out as described
below:
10 ~1 of sample
50 ~1 of protein S-deficient plasma
500 ~1 of reagent according to a) Example 1
b) Example 2
are pipetted into a cuvette.
When adding the reagent, a clock is started and the
extinction at 405 nm i~ monitored until an increase in
the ab~orption by a certain value (e.g. of 0.1) has been
reached. ~he prolongation of the clotting time beyond the
value for 0~ protein S is proportional to the concentra-
tion of protein S in the sample (Fig. 1).
The data for the determination according to the method
described in DE 37 24 443 have been taken from German
Offenlegungsschrift DE 37 24 443 Al, Figure 1.
Example 5
Determination of the protein S content in plasma when
separately adding activated protein C
For the calibration, 100%, 75%, 50%, 25%, 10% and 0%
dilutions of a healthy-donor plasma are prepared in
saline. The measurement of the standards and the samples
is carried out as described below:

3 ~
10 ~1 of sample
50 ~1 of pxotein S-deficient plasma
25 ~1 of activated protein C
500 ~1 of reagent according to Example 3
are pipetted into a cuvette.
On adding the reagent, a clock .is started and either the
extinction at 405 nm is monito:red until an increase in
absorption by a certain value (e.g. of 0.1) has been
reached, or the occurrence of clot formation is measured.
The prolongation of the clotting time beyond the value
for 0% protein S is proportional to the concentration of
protein S in the sample (Fig. 2). ,-