Note: Descriptions are shown in the official language in which they were submitted.
~5~3t~
The present invention is concerned with a process for
producing Factor Xa which is employed in a reagent for the
determination of prothrombin in biological material, for
example, blood plasma.
This application is a divisional of Canadian Patent
~pplication, S.N. 318,481, filed December 2~, 197~.
The determination of the prothrombin level is an
important clinical parameter for the continuous monitoring of
anticoagulation therapy. Equally important is 1he detection
of a Factor-II deficiency which can not only be inherited but
can also be acquired, i.e., as a result of a primary funda-
mental disease.
Starting from natural thrombin-substrate fibrinogen,
in the course of the development of prothrombin determination
methods, use is now made of synthetic chromogenic substrates,
for example, peptide-E~nitroanilide derivatives, tissue thrombo-
plastin being used as activator. One disadvantage of this
method is, for example, the fact that the optimum activation
time varies with the prothrombin concentration~ Furthermore,
there is a lack of specificity. In the case of other sub-
strates it would be necessary to have lower plasma concen-
trations in the activation mixture in order to determine pro-
thrombin in normal plasma. However, due to the high plasma
dilution, it is not possible to achieve a complete activation
in plasmas with low prothrombin activity.
Staphylocoagulase and snake venoms have also been
used as activators. They activate prothrombin directly,
however, it has been found that these venoms at least partly
also activate the PIVKA-prothrombin which is formed during
treatment with oral anticoagulants. Staphylocoagulase also
reacts with the PIVKA-prothrombin.
Therefore, it is desirable to provide a process and
a reagent for the determination of prothrombin in plasma which
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permits the carrying out of a test with few sources of error,
increased precision and sensitivity and improved reprod~cability.
Such a test is to be specific, that is to detect prothrombin
but not PIVKA-prothrombin, All the prothrombin is to be con-
verted quickly and completely into thrombin without the
activator necessary for this purpose participating in any way
in the colour reaction,
There is disclosed a process for the determination
of prothrombin in biological material, for example, in blood
i~ plasma, by conversion o~ prothrombin into thrombin, enzymatic
fission of a thrombin substrate with the thrombin and measure-
ment of a fission product, wherein the test solution contain-
ing the prothrombin is incubated with the addition o~ Factor
Xa.
Although human Factor Xa has pro~ed to be best, it
is, however, also possible to use, for example, bovine Factor
Xa.
The principle of this method of determination can
be explained as ~ollows: from, for example, oligopeptides in
which E~nitroaniline is attached, as chromog~nic group, to
the carboxyl group of arginine by amide formation, thrombin
splits off E~nitroaniline,in a particular case this may be
represented by the following:
N-Tos-Gly-pro-Arg-pNA thHrO~bin~
N-Tos-Gly-Pro-Arg-OH ~ pNA
Examples of thrombin substrates which have proved
to be especially useful include N-~os-Gly-Pro-Arg-pNA and N-
Cbz-Gly-Pro-Arg-pNA (Chromozyme TH*, Boehringer Mannheim Gmbh),
as well as H-D-Phe-Pip-Arg-pNA (Substrate S-2238)** and Bz-Phe
Val-Arg-pNA (Substrate S-2160)** The yellow colour of the free
*trademark ** laboratory designation
.
~-nitroaniline can be measured photometrically at abou~ 390
to 410 nm, the amount of coloured material liberated per
unit time being proportional to the enzyme activity.
As buffer for carrying out the method, it is pre-
ferred to use tris and/or imidazole buffer with a pH of about
8 to 9, to which hydrochloric acid and/or sodium chloride can
be added~
Phospholipids and calcium chloride can be added as
co-reagents for the activation of the prothrombin.
The addition of the substrate takes place after com~
plete conversion of the prothrombin into thrombin in the test
solution, after the addition of Factor Xa.
The Factor Xa used is readily obtainable by a
simple process which is the subject of the present
invention.
;
According to this process, plasma and preferably
human plasma is treatèd with a prothrom~in activator, suspended
solids are removed, for example, by centrifugation, the
resulting plasma liquid is mixed with a protein adsorbent, the
precipitate obtained is eluted with a protein elution agent and
the eluate obtained is mixed with a Factor X activator and
optionally with a soluble calcium salt. The prothrombin acti-
vator used is advantageously Echis carinatus venom but there
can also be used other snake venoms, for example, Taipan snake
venom (O~Yuranus scutellatus) and trypsin.
The coagulate of suspended solids obtained after
treatment with the prothrombin activator, which is suitably
centrifuged off, consists partly of fibrinogen, antithrombin
and thrombin.
The protein adsorbent used is preferably barium
sulphate, citrate or c~alate but there can also be used, for
~, . . .
~S~V7
example, aluminium hydroxide, DEAE-Sephadex* and QAE-Sephadex*
The protein elution agent used can be, for example,
an aqueous solution of trisodium c:itrate, a phosphate buffer
or an aqueous solution of sodium chloride.
Russell's viper venom is particularly preferred as
the Factor X activator.
As soluble calcium salt, it is preferred to use
calcium chloride.
If the eluate, after treatment with the protein
elution agent, contains components which have a negative effect
on the coagulation system, for example, calcium-binding com-
ponents, for example, citrate ions when the protein elution
agent is sodium citrate, it is necessary to introduce a
dialysis step. In the case mentioned, for example, dialysis
is carried out against a buffered physiological solution of
sodium chloride (M~chaelis buffer) at a low temperature,
i.e., at about 4C.
After the treatment with the Factor X activator and
the addition of the calcium ion-containing solution, the pre-
paration is, before use, left to stand at a low temperature
and preferably at about 4C. for the complete conversion of
Factor X into Factor Xa, Further steps can subsequently be
carried out in order to obtain highly purified Factor Xa.
These further purification steps can comprise gel filtration
with a molecular sieve which differentiates the range of from
50,000 to 100,000, for example Sephadex * G 100 or Biogel *
P 100, eluting with, for example, sodium acetate buffer (0~4
molar; pH 7). Furthermore, it is also possible to carry out
an ammonium sulphate precipitation (for example, with a
45 to 55% solution pH 6 to 8), an ion exchange chromatography
with DEAE-'Sephadex'* A 50, DEAE cellulose or QAE- Sephadex"*/
cellulose, using as buffer sodium potassium phosphate (for
*trademark
- 4 -
. '
607
example, 0.02 molar, pH 6.8) or a gradient of 0.1 to 1.0
molar aqueous sodium chloride solution. It is also possible
to carry out a treatment with hydroxyapatite (phosphate buffer
0.2 to 0~5 molar; pH 6,8), as well as preparative electro-
phoresis and possibly ultracentrifuging. The above-mentioned
methods are advantageously combined. Factor Xa purified in
this manner can be used in the test in the form of a solution,
for example, in veronal or Michaelis buffer, in physiological
sodium chloride solution or in the test buffer. However, for
use in the test according to the present invention, this puri-
fication procedure can be omitted and the Factor Xa pre-
p æ ation, enriched in the above-described manner, used.
The present invention also provides a reagent for
the determination of prothrombin, comprising a thrombin sub-
strate and a prothrombin activator, the prothrombin activator
being Factor Xa and preferably human Factor Xa~
This reagent preferably consists essentially of tris-
and/or imidazole buffer and human Factor Xa as prothrombin
activator. As co-reagents, it is advantageous to use phos~
pholipids from human brain and calcium chloride as well as a
synthetic thrombin substrate.
It will be understood that the individual components
of ~he reagent will be pre,sent in amounts to perform their
intended function. Thus, for example, there should be
sufficient thrombin substrate to react with all of the thrombin
formed from the prothromhin, and Factor Xa should be employed
in an amount effective to convert the prothrombin to thrombin.
Such a reagent preferably comprises:
0.3 to 6.5 ~g./ml. phospholipids,
0.7 to 10 m~M/litre calcium chloride and
150 to 380 ~M/litre substrate,
~5~)7
the Factor Xa concentration corresponding to 0.2 to 2.5%, by
weight, of the plasma extract. The reagent can be in dried
or dissoLved form.
The process and reagent for determination of pro-
thrombin permit a rapid and dependable determination of pro-
thrombin. The process is characterised by its specificity
because Factor Xa detects normal prothrombin but not PIVKA-
prothrombin. The previously employed activators certainly did
not act specifically. The addition of a standardised Factor
Xa preparation ensures that, in every case, a sufficient amount
of activator is present. It was very surprising that Factor
Xa, after the addition thereof, converted all of the pro-
thrombin rapidly and completely into thrombin without itself
~ participating in any way in the colour reaction, which is of
great importance. Thus, it is known that the Factor Xa has a
direct splitting action on, for example, synthetic peptide-
E~nitroanilide derivatives, for example, Chromozyme TH*. In
particular, it was also surprising that human Factor Xa could
be used more advantageously than, for example, bovine Factor
Xa, It has proved to ~e especially advantageous to add Factor
Xa in definite amounts; the activation of Factor Xa itself
present in blood plasma would here give rise to unsatisfactory
results. Furthermore, it was surprising that, by means of
such a simple preparative process, such as that according to
the present invention, an activator could be made available,
which ensures practicability and especially technical useful-
ness, and, having regard to its origin, namely human plasma,
could be sufficiently p~oduced at all.
The fol:Lowing Examples are given for the purpose of
illustrating the present invention:
*trademark
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07
Example 1
Production of a Factor Xa preparation
.
Normal plasma is obtained from the blood of healthy
donors with an average age of 30 years, half of the donors
being male and half being female. The plasma contains 25 mM
sodium citrate. After a first centrifuging for 15 minutes
at 1500 g and at ambient temperature, the plasma is subjected
to a second centrifuging at 4C, for 30 minutes at 20,000 g.
Per 4 ml. normal plasma, there are added l ml. l/30 molar
aqueous calcium chloride solution and 2 drops of Echis
carinatus venom (Sigma V 8250* fundamental solutions in each
case l mg./ml. water3 and left to stand for about 2 hours in
a waterbath at 37C. The resulting coagulate is centrifuged
o~f and 150 mg. barium sulphate is added to the supernatant
obtained from each 4 ml. of normal plasma. The mixture is
stirred for 30 minutes at ambient temperature and then
centrifuged. The supernatant is discarded and the precipitate
washed four times with approximately 4 ml. amounts of physio-
logical sodium chloride solution. The centrifugate is eluted
with 2 ml. of a 0.2 molar trisodium citrate solution (pH 7.0).
After again centrifuging, the supernatant is dialysed against
physiological sodium chloride solution at 4C. The dialysate
is stored in small amounts of about 250 ~l. at -20C, To
each-portion is added, at ambient temperature, 30 ~l. of a
0.1 molar calcium chloride solution and l ul. Russell's viper
venom (Vipera russelli, The Wellcome Foundation Limited) and
the mixture is left to stand for about 14 hours at 4C. This
preparation can be used in the test, 2 ~l. corresponding to
1% plasma extract. It c~n also be lyophilised. Further puri-
fication can also be carried out in the manner described in
detail hereinbefore.
*trademar]c
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6C~7
Example 2
Method for the determination of prothrombin and thrombin,
Test mixtures were prepared which contained the
following components:
Substance ~ Volume Final
Concentration
buffer solution 400 ~1., minus
the other
amounts added
test plasma 1 to 5 ~1. 0~25 to 1.25%
phospholipids 2 ~1~ 1.25 ~g./ml.
calcium chloride
solution 40 ~1. (0.1 m) 10 mM/l.
activator ~
(Factor Xa pre- Corresponding
paration accord- ¦ to 1% plasma
ing to Example 1) 1 2 ~1. fraction
The bu~fer solution is prepared as follows:
Stock solution A: O.lM/l. tris + 0.1 M/l imidazole
0.1 M/l. hydrochloric acid
Stock solution B: 0.1 M/l. tris + 0.1 M/l. imidazole +
0~1 M/l. sodium chloride
Stock solution C: 0.2 M/l. sodium chloride.
The buffer used (pH 8.4) is prepared by combining
the stock solutions in the volume ratio'A:B:C of 1:1:2.
The phospholipids used were an acetone/diethyl ether extract
of human brain obtained by Bell and Altonis method.
The components of the test mixture are pipetted
directly in the given order into a microcuvette with a layer
thickness of 1 cm. of an "Aminco DW 2"* spectrophotometer at
37C. The incubation time for Factor Xa is 120 seconds. There
are then added th~ereto 50 ~1~ (1.5 mM/l.) substrate (Chromozyme
TH*; Boehringer Mannheim GmbH or Substrate-2238**, Kabi), the
end concentration thereby being 187.5 ~M/l. for Chromozyme TH*
*trademark ** laboratory designation
-- 8 --
6~7
and 148 ~M/l. for Substrate-2238*, After thoroughly mixing
as quickly as possible, commencement of measurement begins
immediately.
The absorption per unit time is recorded directly
with an indicator rate in minutes per cm. in the horizontal
and an absorption in mm. per mm. full scale in the vertical.
Various absorption sensitivities and running speeds can hereby
be selected. Therefore/ the change of absorption per minute
can be caiculated according to the following formula:
measured absor~tion in mm
x absorption sensitivity
total scale ln mm
-
indicator rate in min./cm. x measurement stretch
Example for measurement stretch 10 cm., vertical
43 mm., full scale 252 mm., absorption sensitivity
0.5 and running speed 0.033:
; 43
~52 x 0.5 = 0.258 abSorption chanqe
0,033 x 10 min./cm mlnute
*laboratory designation
_ g _
