Note: Descriptions are shown in the official language in which they were submitted.
2~
The invention relates to a method of assaying
fibrinogen by enzymatic conversion with thrombin,
prothrombin fragments having a thrombin-like activi-ty
or mixtures thereof and subsequent detection of the
fibrin or fibrinogen cleavage products formed.
Furthermore, the invention relates to a reagent for
assaying the fibrinogen content of an optionally
undiluted and optionally also heparin-containing ~lasma
sample.
The assaying of fibrinogen in plasma is very
important within the field of the investigation of
coagulation defects. For the assaying of fibrinogen,
both immunological and functional methods, such as,
e.g., coagulation tests, have been kno~n. In the
coagulation tests, the fibrinogen content is determined
by measuring the time of clot formation.
A common assaying method has been described by
Clauss (Acta HaematO 17, 237-246, 1957). Oxalate plasma
is diluted in a buffer of pH 7.~, and a concentrated
thrombin solution is admixed. Subsequen-tly, the time
until the end of coagulation is determined. Yet, the
coagulation time of the plasma sample which has been
reacted with -thrombin is only proportionate to the
fibrinogen cont~nt, if a sufficiently large excess of
thrombin relative to plasma is chosen, so as to
eliminate plasmatic interfering factors. Antithrombin
III (AT III) which effectively inhibits the action of
thrombin, primarily together with heparin, is
considered an interfering fac-tor. Heparin is a commonly
used anticoagulant and i5 used as addi-tive when taking
blood or as a therapeutic agent. A heparin content in a
plasma sample may lead -to extended coagulation times
and thus to falsified results.
The action of antithrombin III in the plasma here
is greatly reduced, on account of the high thrombin
concentration and on account of the dilution of the
plasma, and thus on account of the reduction oX the
concentrations of ATIII and heparin, respectively. If
plasma is not dilutPd, the antithrombin effect is
stronger by a multiple and the coagula-tion time is
extended.
A plasma dilution and the additional method step
involved therein is a substantial burden in routine
check-ups. Beside increased operational expenditures
and the additional handling of potentially infectious
plasma, each dilution in principle constitutes a source
of error. Furthermore, dilutions often have to be
carried out according to the fibrinogen content, and
this makes working with analysis automats more
difficult.
Usually veronal buffer is used to dilute the
plasma, because this buffer facilitates the formation
of measureable fibrin clots, which improves assaying
particularly with plasmas having low fibrinogen
contents. Yet, veronal is also a narcotic having the
character of an addic-tive druy, and -therefore use of
this substance should be avoided, if possible.
Further attempts to eliminate the interferîng
factors antithrombin III or heparin, respectively,
involve the neutralization or the removal of -the
heparin. In Clinical Chemistry 29, 61A-617 (1983), a
method is described according to which heparin is-
neutralized in a plasma sample by adding polybrene.
Plasma is diluted in a buffer having a pH of 7.4, and
after the addition of thrombin, it is transferred into
a photometer. Subsequently, the reaction speed is
determined nephelometrically.
By using snake venom enzymes having a thrombin-
like activity (batroxobin) instead of thrombin,
fibrinogen may be assayed in plasma independently of
the interfering factors mentioned, the reason for this
being that these snake venom enzymes are not inhibited
by AT III or heparin.
The use of proteins which are not considered to be
natural coagulation enzymes, such as snake venom
enzymes~ e.g~, in methods for assaying human proteins
does have its problems. The reactions hetween a non-
mammal enzyme and a human protein often are unspecific.
In accordance with the principle of biochemical
analytics, to compare and to assay only
s
things that are alike, one aims at using only mammalian
and, above all, human enzymes as the reaction par-tners
for assaying human proteins.
Thrombin may be produced by ac-tiva-tion of
prothrombin. As precursors, Meizothrombin (MT) and
Meizothombin (desF1) form during activation. Yet,
contrary to prothrombin, the double-chained -thrombin
in-termediates have a protease activity similar to-that
of thrombin ("thrombin-like activity"). Therefore, they
can also cleave substrates that can be reacted by
thrombin. Thus, a "thrombin-like activity" is a
protease activity which is capable of cleaving thrombin
suhstrates, whose reaction kinetics and reac-tion
param~ters, such as pH or ionic strength optima,
inhibitors, effectors, allosteric interaction ancl so
on, need not necessarily be equal to those of thrombin.
The activities of the bovine enzyme
meizothrombin, meizothrombin (desFl~ and thrombin are
compared in J. of Biol. Chemistry 265, 10693-:L0701
(1990) at a pH of 7.4. Meizothrombin exhibits only 2~
of the "fibrinogen clotting activity" of thrombin. This
slight activity may, however! also be due to a 10 to 15
~ content of MT(desFl) in the MT composition. For
MT(desF'1) an activity of 1~ of that of thromb:in was
found.
The test for enzymatically converting fibrinogen
into fibrin is usually carried out at a pH in the range
~ J
of from 7.4 to 8Ø This pH range is generally used in
enzymatic reactions of thrombin, since it corresponds
to the physiological pH range. The optimum pH for
thrombin activity is close to pH 8.
The invention has as its object to provide a
method of assaying fibrinogen by means of a coagulation
test, by which the disadvantages of the known methods
described above are avoided and which may particularly
also be carried out in undiluted plasma samples,
independetly of their AT III or heparin contents.
According to the invention, this object is
achieved by a method of assaying fibrinogen largely
independently of thrombin inhibitors by enzymatic
conversion with thrombin, prothrombin fragments having
a "thrombin-like activity" or mixtures thereof, and
subsequent detection of the fibrin or fibrinogen
cleavage products formed, which is characterised in
that the enzymatic conversion of the fibrinogen is
carried out at a pH in the range of from 4 to 7.3, and
the detection of the fibrin formed is effected by
determining the coagulation time or the fibrinogen
cleavage products. Subsequently, the fibrin clot formed
is detec-ted. The detection may be effected by
determining the coagulation time photometrically or
turbidimetrically. Among the prothrombin fragments
having a "thrombin-like act:ivity" there are
particularly MT, MT(desFl) or mixtures thereof. The
I~J~Ç;J~ t~
concentrations and the mixing ratios of the enzymes
used are to be chosen such that a linear relation is
guaranteed over as broad a range of the fibrinogen
content in a sample as possible.
Also proteins produced by genetic engineering,
which have a thrombin-like activity on account of their
structure and which have a slighter affinity to
antithrombin III as compared to thrombin, are suited
for -the method according to the invention.
Carrying out the assaying method in ths pH range
indicated makes the latter independent of the above-
mentioned interfering factors AT III and heparin to
such an ex-tent that the fibrinogen assay may be
efected in a simple manner also from an undiluted and,
optionally also heparin-containing, plasma sample. The
additional me-thod step of diluting plasma can be
obviated in the me-thod according to the invention,
thereby improving the assay result by eliminating a
possible source of error.
The invention is based on the finding that the
inhibition of thrombin by AT III/heparin at a pH of
from 4 to 7.3 may be neglected, wherein -the thrombin
activity suffices for an enzymatic conversion of
fibrinogen, daspite suboptimal conditions. A somewhat
reduced activi-ty of thrombin even has the advantage
tha-t the coagulation times are not too short and can be
recorded more easily.
When using thrombin, prothrombin fragments having
a "thrombin-like activity" or mi~tures thereof, it has
surprisingly been found that the thrombin-like activity
in the pH range mentioned, preferably at a p~ of from 5
to 7, or, most preferred, at a pH of appro~imately 6,
is best suited for assaying fibrinogen independently of
thrombin inhibitors.
The enzymatic conversion of fibrinogen is
preferably carried out in a buffer having a molarity of
buffer salts in a range of from 0.02 to 0.5 M, most
preferred of 0.1 to 0.25 M, buffer. If desired, the
ionic strength may also be increased by the acldition
of salts, such as sodium chloride. It has been found
that, particularly at low ionic strengths, thrombin i5
insensitive to AT III inhibition. For carrying out the
method according to the invention, however, also an
environment having an equal or a higher buffer capacity
compared to the plasma sample is practicable to
maintain a certain pH during the assaying procedure.
Advantageously, fibrinogen assaying is ef~scted
from an undiluted and, possibly also heparin-
containing, plasma sample, i.e. a sample, in which
heparin is present and not neutralized.
The invention also relates to a reagent for
assaying the fibrinogen content largely independently
of thrombin inhibitors o* a possibly undiluted and
possibly also heparin-containing plasma sample,
s~
containing thrombin, prothrombin fragments having a
"thrombin-like activity" or mixtures -thereof, for
enzymatically converting fibrinogen in a buffer having
a pH in -the range of from A -to 7.3 and subsequ~nt
detection of the fibrin formad by determining -the
coagulation time or the fibrinogen cleavage products.
This reagent serves for the simple assaying of
fi~rinogen, without having to put up with a cumbersome
dilu-tion of plasma samples or neutralization or removal
of heparin from the plasma samples.
I'he invention will now be e~plained in more de-tail
by way of -the following examples.
Assaying of fibrinogen a-t pH 6 and pH 8:
For reasons of comparison, meizothrombin and
thrombin were used at pH 6 and pH 8, and the
coagulation times were determined in dependence on the
fibrinogen content in plasma samples. Meizothrombin was
produced by activating human prothrombin with
immobilized ecarin (Pentapharm) according to the
protocol of S-tocker and Muller (Thromb. Haemostasis 65
(6), Abstract 855 (1991)). Plasma-containing samples
having different fibrinogen contents were used for the
tests.
50 ~1 plasma sample were admixed with 100 ~1
buffer (0.2 M Tris HCl buffer, pH 8, or 0.2 M phosphate
buffer, pH 6) and 150 ~1 meizothrombin (15 NIH
analogous U/ml) or thrombin (10 NIH U/ml, Thrombin
Reagent, Immuno), and -the coagulation ~imes at 37C
were determined by means of a Schnitger-Gross
coagulometer (Amelung). The determining method was
carried out both in the presence and in the absence of
heparin (1 U/ml).
From the Tables it becomes apparP.nt that at pH 6,
the course of the calibration curves for fibrinogen is
independent of the heparin content in the sample..
However, by a content of heparin in the sample the
coagulation times are extended at pH 8, both, for
meizothrombin and for thrombin as coagulation enzymes.
Meizothrombin
pH 8 pH 6
Fibrinogen without withwithout with
content (mg/ml) hep. hep. hep. hep.
310 8.0 10.630.~ 30.1
238 11.1 15.150.7 49.1
94 26.0 35.5166.4 157.5
62.5 35.6 >250 >250 >250
Thrombin
pH 8 pH 6
Fibrinogen without withwithout with
content (mg/ml) hep. hep. hep. hep.
373 11.6 >20018.6 17.3
310 16.1 >20024.1 24.1
238 25.6 >20028.4 30.1
100 50.0 >20085.9 89.8
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