METHODE POUR LA DETERMINATION DES PLASMINOGENES

METHOD FOR THE DETERMINATION OF PLASMINOGEN

Abrégé anglais

Abstract of the disclosure
A method for the determination of plasminogen in body
fluids in the presence of streptokinase using a substrate
for plasmin, the action of plasmin on which results in a
cleavage product which can be measured optically, which
method comprises essentially simultaneous addition of
streptokinase and chromogenic or fluorogenic substrate for plasmin
to the body fluid, and determination of the amount of
the cleavage product formed in a defined time, or of the
rate of formation of the cleavage product, and from this
the concentration of plasminogen, is described. When the
amount of streptokinase which is used is such that the
plasminogen present is in molar excess, this method can
be used to determine the rate of activation of plasminogen.

Revendications

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for the determination of plasminogen in body
fluids in the presence of streptokinase using a sub-
strate for plasmin, the action of plasmin on which
results in a cleavage product which can be measured
optically, which method comprises essentially simul-
taneous addition of streptokinase and a chromogenic
or fluorogenic substrate for plasmin to the body
fluid, and determination of the amount of the cleav-
age product formed in a defined time, or of the rate
of formation of the cleavage product, and from this
the concentration of plasminogen.
2. The method as claimed in claim 1, wherein the body
fluid is plasma.
3. The method as claimed in claim 1, wherein the total
concentration of plasminogen is determined by use of
a molar excess of streptokinase over the amount of
plasminogen present.
4. The method as claimed in claim 1, wherein the rate of
activation of plasminogen is determined by use of an
amount of streptokinase such that the plasminogen pre-
sent is in molar excess.

Description

~3~ ~9~
A method for the determination of plasminogen
The invention relates to a method for the determination
of plasminogen using a chromogenic or fluorogenic su~strate andin the
presence of streptokinase.
eecause of the importance of this protein for fibrinolysis,
the determination of plasminogen is nowadays a normal con-
stituent of the diagnosis and monitoring of peopLe at
risk of thrombcsis. A deficiency of plasminogen often
results in a thrombotic event such as myocardial infarc-
tion, pulmonary embolism or deep vein thrombosis in the
legs. The therapy of thrombolysis with streptokinase or
urokinase, which is often carried out nowadays, is like-
wise dependent on an adequate concentration of plasminogencapable of being activated.
Apart from immunological determinations using nephelometry
or radial immunodiffusion, it is possible to carry out
2û functional determinations of plasminogen with fluorogenic
or chromogenic peptide substrates after activation w;th
streptokinase. These assays~utilize the fact that plas-
minogen, after activation to plasmin, remains bound to
streptokinase and, in plasma, this complex (activator)
is not, in contrast to free plasminr ;mmediately inhibi-
ted. After the introduction of chromogenic tripeptide
substrates, this technique was described for the first
time in Chromogenic Peptide Substrates, Chemistry and
~linical Usage, page 128~ Scully and Kakkar ed. Churchill
L;vingstone, Edinburgh and New York, 1979. However, in
this and other similar known methods, it is necessary to
preincubate the sample with streptokinase for a rela-
tively long time, about 10 min.~ in order to convert the
plasma plasminogen completely into the activaeor complex,
and the substrate is added separately.

1 3 ~
-- 2
It has been found, surprisingly, that plasminogen can
also he activated by streptokinase in the presence of a
chromogenic substrate for pLasmin. After a relatively
short lag period, plasmin is formed as a complex with
streptokinase, and the substrate is cleaved. The onset
of substrate hydrolysis is rapid even in plasma samples
with low plasminogen concentrations. The advantage of a
method of this type is that there is no necessity either
for a preincubation or for a second pipetting step to add
the substrate.
Hence the invention relates to a method for the determina-
tion of plasm;nogen in body fluids in the presence of
streptokinase using a substrate for plasmin, the action
of plasmin on which results in a cleavage product which
can be measured optically, which method comprises essenti-
ally simultaneous addition of streptokinase and a chromo-
genic or fluorogenic substrate for plasmin to thebody fluid, and deter-
m;nation ~f the amount of the cleavage product formed in
a defined ti~e, or of the rate of formation of the cleav-
age product, and from this ~he concentration of plasminogen.
This method can be carried out in the following manner:
the plasminogen concentration in a sample can be deter-
mined with a reagent which, in addition to a chromogenicsubstrate, contains an excess of streptokinase, preferably
1,000 U/ml. The plasminogen in the sample is thereby con-
verted into plasminogen-streptokinase complexes which
convert the chromogenic substrate. The evaluation in
3û this method is effected by determining the rate of forma-
tion (delta E/min) of the chromophore cleaved off the
chromogenic substrate. However, it it also possible to
measure the time required for the development of a defined
difference in extinction, preferably 0~1 E.
In both types of evaluation the plasminogen concentration
in the sample is proportional to the parameters measured.
Comparison of this method (embodiment 1) with conventional

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-- 3 --
methods revealed good agreement in 40 samples from
patients (Example 1).
However, another possible procedure is as follows: the
reagent used for this has a substantially lower strepto-
kinase concentration. This entails the plasminogen
content of a sample being established from the concentra-
tion of plasmin produced by the action of streptokinase-
plasminogen complexes on plasminogen. Measurement of the
rate of formation of plasmin makes it possible not only
to determine plasminogen quantitatively but also to gain
information on the ability of plasminogen to be activated
(embodiment 2). Evaluation is effected by determining
the t;me required after addition of the reagent for a
defined difference in ext;nction, preferably 0.1 E, to be
reached.
It was shown on 40 plasma samples from patients that the
results of embodiments 1 and 2 agreed well in 34 cases.
In 6 cases, normal plasmino~en concentrations were found
with embodiment 1, whereas determinations ~ith embodiment
2 resulted ;n figures below 50X of normal (Example 2).
The methocl according to the invention is expediently car-
ried out in buffered solution, for example using a phosph-
ate, tris, HEPES or acetate buffer with a pH of 6 to 9
preferably a phosphate buffer, especially 0.1 mol/l K
phosphate, pH 7.5.
All chromogenic plasmin substrates are suitable as chromo-
genic substrates:
U-D-CHA-NVA-Lys-pNa explanations:
H-D-Val-Leu Lys-pNA CHA cyclohexylalanine
35 H-D-NVA-CHA-Lys-pNA NVA norvaline
H-D-NLEU-CHA-Arg-pNA pNA para-nitroanilide
H-D-~ut-CHA-Lys-pNA NLEU norleucine
H-D-NLEU-CHA-Lys-pNA 8ut epsilon-aminobutyric acid
H-D-Phe-Tyr~Arg-pNA

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The examples which follow illustrate the invention.
Example 1
Streptokinase (aehringwerke AG, Marburg, Federal Republic
of Germany) was dissolved in 0.1 mol/l K phosphate buffer,
pH 7.5, in a concentration of about 1,000 internat;onal
units/ml. 4 ml of this solution were mixed with 0~1 ml
of a 10 mmol/l solution of the chromogenic substrate H-D-
1û cyclohexylalanyl-norvalyl-lysyl-para-nitroanilide
(Pentapharm AG, ~asel), and the solution was heated to
37C. 500 ~l of this solution were pipetted into 50 ~l
of plasma in a cuvette which had been preheated to 37C.
The absorption at 405 nm was measured. The relation be-
t~een the absorption and the time was virtually l;nearafter 30 sec. Delta E/min was determined from the l;near
part of the function.
Construction of reference plots:
Serial dilutions were prepared by dilution of plasma with
isotonic saline, and each dilution was treated as des-
cribed above. The reference plots were obtained by use
e;ther of the linear increase in extinction per minute
after a lag period of 40 sec, or of the time taken to
reach a defined difference in extinction~
The results are shown in the table which follows.
Concentration of delta E/min Reaction time for
30 plasminogen t%) delta E = 0.1, ;n sec
100 (initial) 0.680 18.0
0.360 31.2
0.188 55.8
12.5 0.098 112.8
0 O.OOQ
A straight line was obtained when delta E/min was plotted
against the concentration. Semireciprocal plotting of

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-- 5
the time taken to reach a difference in extinction of
0.1 E likewise resulted in a straight line.
Example 2
The plasminogen de~ermination was carriecl out as in
Example 1 but a reagent with a streptokinase concen~ration
of 40 lU/mL was used. The shape of the reference plot
changed as the streptokinase concentration decreased. In
particular, it is possible at a low streptokinase concen-
tration, fQr example 40 IU/ml, to gain information on the
ability of the plasminogen ~o be activated by streptoki-
nase by measuring the reaction time for a fixed delta Eof 0u1, for example. Information of this type is of
importance for patients with myocardial infarction.
~hen comparative plasminogen determinations on 40 samples
from patients were carried out by the mathods of Example
1 and Example 2, the plasminogen roncentration in a total
of 34 samples ~as found to be in the normal range, with
good agreement between the figures from the two methods.
Ho~ever, pathological figures (less than 50% of normal)
were found by the method of Example 2 in 6 samples.