Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
5 ~ 8
METI-IOD FOR DETECTING PROTEOLYTIC ENZYMES IN BLOOD
TECHNICAL FIELD
The invention relates to a method of detecting
proteolytic enzymes in blood by contacting the blood
with an adsorbent to adsorb the enzymes on said adsorb-
ent, and examining the presence of adsorbed enzymes. In
the present specification and claims the term blood is
used to indicate blood and fractions of blood, such as
blood plasma and solutions of purified blood plasma
pToteins.
BACKGROUND ART
It is previously known that blood coagulation en-
zymes can be adsorbed in their active form on surfaces
to which heparin or heparinoid substances have been
bound. Such adsorbed enzymes, however, have been entire-
ly inactivated upon contact with blood plasma or with
enzyme inhibitors isolated from blood plasma. Thus,
these known methods are not useful for detecting active
enzymes in blood by means of a direct contact between
the blood and an adsorbing surface, because the enzyme
inhibitors of the blood inactivate the adsorbed enzymes.
In simplified terms, the equilibrium state of
blood is regulated, in addition to cellular reactions,
by the capacity of certain substances to inhibit the ac-
tivity of said enzymes. From a diagnostic point of viewit is desirable to be able to detect enzymes in blood
and other body fluids, and preferably to measure the
content of such e~zymes. The inhibitor systems mcly be
affected by the addition of e.g. heparin, Therefore,
during an operation or in certain diseases, it is often
important to be able to rapidly establish the presence
of active enzymes in the blood, and, preferably, to de-
termine the quantity of said active enzymes in the
blood, in order to be able to determine the correct dose
1 ~0~8
of heparin to be given to the patient. Earlier attempts
to detect and measure free active enzymcs in blood have
been hampered by the fact that the inhibitor systems
rapidly inactivated the enzymes during the collection of
blood.
An enzyme usually occurs in blood in the form of
an inactive protein. ~hen being activated the protein is
converted into an active enzyme by having a portion of
the molecule split off. The enzymes are arranged in a
cascade sequence, in which one enzyme activates another
inactive protein so as to convert the latter into the
next enzyme. The sequence for the blood coagulation sys-
tem is illustrated in simplified form in the drawing.
The enzymes are referred to as Factor I to Factor XIII,
several enzymes also having other names. When reference
is made to the active enzyme the index "a" is added,
such as X ~ Xa. Of particular interest is Factor Xa
which is found in the intersection of the two pathways
of activation referred to as the intrinsic pathway and
the extrinsic pathway. Of particular interest is also
thrombin (Factor IIa) which is the enzym that finally
converts fibrinogen into insoluble fibrin. The drawing
also discloses that the first enzyme of the sequence,
Factor XIIa, also is involved in the activation of
additional enzyme systems. One of these systems results
in the formation of plasmin, which has a fibrinolytic
activity. Another system results in the formation of
kallikrein which is an intermediate product in the form-
ation of kinin, which acts, i.a. upon the permeability
of the blood vesscls.
TIIE INVENTION
The object of the invention is to adsorb enzymes
from blood under such circumstances that the enzymes have
a remaining activity after having been adsorbed on the
adsorbent. The invention aims at detecting proteolytic
l 160~8
enzymes, such as serine enzymes, in various enzy~e systems in blood, such as the
blood coagulation system, the fibrinolytic system, the kallikrein system. m e
method of the invention is characterized by c~ntacting the blocd with an adsor-
bent consisting of a carrier (substratum surface) having sulphate groups fixed
to its surfa oe in such a high density that the enzymes are adsorbed and remain
immDbilized in their active forms even after exposure to the natural enzyme
inhibitors existing in blood. The adsorbed enzymes can be detected by means of
existing techniques while they are still present on the adsorbent, or after they
have been dissolved by desorption. The invention is particularly devoted to
determining the quantity of active enzymes in blood, which is possible with the
enzymes referred to below.
We prefer to use an adsor~ent consisting of a polymer material
(plastic) which has been treated as described in the Swedish patent application
No. 7706746-0, made available to the public on Decemker 9, 1978. The surfa oe of
a polymer material consisting of polyethylene, polypropene, or polystyrene is
treated with an oxidizing agent dissolved in concentrated sulphuric acid. m e
surface is now preferably rinsed with water, and is subsequently contacted with
an aqueous solution of albumin. The oKidizing agent may be pokassium permangan-
ate, potassium dichr~mate, sodium chlorate, scdium perborate, hydrogen peroxide
or sodium peroxide. The content of oxidizing agent in the sulphuric acid is pre-
ferably from 1 millimole per liter up to a saturated solution. The polymer sur-
fa~e is preferably treated at room temperature for a time of a minute up to an
hour. We prefer to treat polyethylene with potassium permanganate dissolved in
concentrated sulphuric acid to a concentration of 12 millimoles per liter, for
two minutes at room te~,perature. The surface thus treated apFears to be some-
what unstable. Therefore, we prefer to coat the surface with a protein, thus
increasing the stability. The protein is suitably aLbumin, preferably human
--3--
~....
, i ~
5 4 8
albumin. The surface can be coated by being exposed to an aqueous solution of
the protein, preferably containing at least 100 grams protein per liter. The
surfa oe is preferably treated at roon temperature.
Other useful adsorbents are solid surfaces to which has been attached
heparin or heparinoid substr~n oe s, such as chondroitin sulphate, heparin sulphate,
dermatan sulphate, dextran sulphate, or other polysacch æ ides having a high ccn-
tent of sulphate groups in the molecule. When using heparin it is essential thatthe heparin is bound in a way ~hich d oe s not allow leakage of heparin from thesurface during the initial phase of the blood contact, because desorbed hep æin
will oecur in the layer of protein on the surfa oe, thus inactivating the adsorbed
enzyme. Therefore, such a surfa oe is not useful according to this invention. A
heparinized surface with no initial leakage of heparin which is useful accordingto the present invention can be prepared as diselosed in Larsson et al:
rrhrombosis Researeh, 15, 157, 1979.
A carrier having an insoluble surfaoe layer eontaining the sulphate-
containing polysaccharid~s mentioned above ean be produced by coating the surfa oe
with a complex eompound of the polysaech æide and a eationie surfactant in the
form of a primary amine. A useful coating prceess of this type has been des-
cribed for heparin and a primary amine in the Swedish patents Nos. 306,597,
315,362, 365,710, 7104506-6, 7503240-9 and in the Swedish patent applieation No.7708296-4/ made available to the publie on January 18, 1979. We prefer the eoat-ing method disclosed in said patent application, whieh comprises contacting the
surface of the carrier with a colloidal solution of a complex compound of the
polysaecharide and a eationic surfactant in the form of a primary amine. We pre-fer to use a colloidal solution in whieh the eolloidal partieles have a positivecharge. Such a colloidal solution can be prepared by pouring a comparatively
diluted aqueous solution of the polysaccha-
l 16U~48
ride into a comparatively concentrated aqueous solution
of a primary amine, while stirring vigorously, care being
taken that the mixture always contains primary amine in
excess. In order to achieve an effective deposition of
the positively charged colloidal particles on the surface
of the carrier, we prefer to pre-treat said surface so
as to introduce negative charges. This can be achieved by
choosing a carrier of polymer material, and by sulphating
the surface with the method described above with referen-
ce to the Swedish patent application No. 7706746-0
~ hen whole blood is used it is important that the
adsorbent has such surface characteristics that the blood
platelets (thrombocytes) are prevented from being activ-
ated and from adhering to the surface. The surface
according to the invention, having a high density of
surface-bound sulphate groups, has the capacity of ad-
sorbing a protein layer of such a selective composition
that the platelets do not adhere or activate. The likely
explanation is that fibrinogen does not adhere to the
surface. The adsorbed protein layer has the capacity of
binding proteolytic enzymes without said enzymes being
inhibited, with the exception of a special heparin sur-
face as mentioned above. The quantity of piatelets can
easily be assessed by means of bioluminiscense according
to a method described in Larsson et al: Thrombosis Re-
search, 11, 517, 1977.
According to the invention the surface shall con-
tain fixed sulphate groups in a high density. One way to
establish experimentally that said condition is mct, is
as folLows.
A number of test pieces, displaying the surracc to
be tested, are exposed to a solution of alhu~ for a
few minutes. The test pieces are now exposed to a solut-
ion of albumin and thrombin for a few minutes, suitably
15 minutes. The solution should have a thrombin activity
of approximately 20 units per milliliter, unit meaning a
5d.~
unit according to the NIH, i.e. the National Institute
of Health. The test pieces are now divided into two
groups. The pieces of the first group are incubated
with physiological saline, and those of the second
group with blood plasma having been defibrinogenated.
A suitable incubation time is 10 minutes. The surface
concentration of thrombin activity is now measured by
incubating the test pieces with a substrate specific for
Thrombin, viz. S-2238 from Kabi Diagnostica. The surface
thus tested is useful according to the invention if the
thrombin is not deactivated by the defibrinogenated
plasma. The measured value for the surfaces incubated
with defibrinogenated plasma shall not be lower than
50~ of the measured value for the surfaces incubated
with saline. The measured value for the surfaces incu-
bated with saline should be at least 5 x 10 4 units of
absorbance per square centimeter and second, measured
with the method described-in Example 1. As an example
of the required density of sulphate groups, a polyethy-
lene surface treated with potassium permanganate dis-
solved in concentrated sulphuric acid should have a
sulphate group density of at least 0.8 x 10 5 millimoles
per square centimeter.
The adsorbent may be shaped in any desired way. It
may, for example, consist of a test tube in which the
interior wall has been coated with a sulphated surface
according to the invention. The test tube is filled with
blood to be examined. After a certain time the test tube
is emptied and rinsecl with physiological salirle. The test
tube is now filled witl- a rea~ent speciric to the enzyllle
to be detected, preferably a synthetic substrate. In
another embodiment the adsorbent consists of a tube, both
ends open, in which the interior wall has been coated
with a sulphated surface according to the invention. The
tube is applied in an arteriovenous shunt in a patient,
for example during an operation. The tube can be rapidly
~ 16~541B
removed at any desired moment, and the presence of ad-
sorbed enzymes can now be examined. Alternatively, the
surface can be exposed to blood during the puncture o-f a
vein. If it is required that the adsorbent should have a
large surface, the adsorbent may have the form of small
particles, such as spheres, having a sulphated surface
according to the invention.
We prefer to detect adsorbed enzymes by means of
commercially available synthetic substrates. For example,
thrombin can be specifically detected by means of S-2238,
Factor Xa by means of S-2222, Kallikrein by means of
S-2302, and plasmin by means of S-2251, all these sub-
strates originating from Kabi Diagnostica. It is also
possible to detect adsorbed active enzymes in other ways.
For example, the so-called contact product, consisting
substantially of Factors XIIa, XIa and IXa, can be de-
tected in this way.
A background will first be given. If citrated blood
plasma is recalcified, the whole enzyme cascade will be
put into operation, to proceed at a speed which depends
on the degree of activation of Factors XII, XI and IX,
said Factors being activated independent l r calcium. The
time period from the addition of calcium to a detectable
formation of fibrin is called the recalcification time.
An established way of producing contact activation is to
incubate plasma in a glass tube, which results in a
strong reduction o~ the recalcification time.
Test pieces having a high density of sulphate groups
on their surfaces, and having preferably been pre-treated
witll albumi]l, are incubated for 10 millutes witll norlnal
plasma and with glass-activated plasma, and are subse-
quently rinsed with saline. All test pieces are now in-
- cubated with normal plasma, and the recalcification time
is determined. It wiil be found that the recalcification
time in those cases where glass-activated plasma was used
during the first incubation is shorter than in those
1 16~5~18
cases where normal plasma was used during the first in-
cubation. This proves that the enzymes forming the con-
tact product have been adsorbed on the sulphated surface.
Example 1
Pol~hylene tubings having a length of 1 meter and
an inner diameter of 1.8 millimeter were treated on the
inside with concentrated sulphuric acid containing
potassium permanganate in a concentration of 2 grams per
liter, for 2 minutes at room ten-perature. After having
been carefully rinsed with water the tubings were treated
with a solution cGntaining 4 per cent by weight albumin,
for 5 minutes, and were subsequently carefully rinsed
with physiological saline.
The tubings thus pre-treated were now tested as
follows.
Four tubings were rotated for 15 minutes on a
slanted turn-table. The tubings contained 1 milliliter
physiological saline containing 4 per cent by weight
albumin and 20 units per milliliter of thrombin. The
tubings were subsequently carefully rinsed with saline.
Two tubings were now rotated on the turn-table for 5
minutes with defibrinogenated plasma, and were subse-
quently rinsed with saline. The presence of surface-bound
thrombin activity was now investigated in this way. The
tubings were divided into pieces having a length of 45
cm. These shorter tubings were incubated with 0.7 milli-
liter of a solution of substrate S-2238 which is specific
to thrombin. The suhstrate had been dissolved in a Tris
~urr~r l~;lvil~ or~ to ~I~c rc~
by the manufacturer. The substrate solution was sucked
into the tubings by means of a reversible roller pump at
a velocity of 2.5 centimeters per second, and the solut-
ion was pumped forward and backward in the tubings by
repeatedly reversing the pump for a total time of 80
seconds. Subsequently, the solution was pumped down into
~ 16~54~
glacial acetic acid having a volume of 0.3 milliliter.
If thrombin is present the substrate will split
off paranitroaniline. The quantity of paranitroaniline
can easily be measured photometrically, and is proport-
ional to the thrombin activity. The reaction is discon-
tinued by changing the pil by means of the glacial acetic
acid.
The absorbance of the solution was measured in a
spectrophotometer using light having a wave-length of
405 nanometer and an optical pathway of 1 cm. The result
was:
- Group 1 ~rinsed with saline) 1.100
Group 2 (rinsed with saline and treat-
ed with defibrinogenated
plasma) 1.100
The values thus obtained can be compared with a
standard curve obtained by incubating substrate solutions
with various known amounts of thrombin. In this way the
values given above can be estimated to correspond to a
surface cGncentration of 0.6 unit of thrombin per square
centimeter.
A similar experiment was made with polyethylene
tubings which had not been pre-treated. The resulting
absorbance was nil for both groups of tubings.
The experiments of this Example illustrate that
thrombin is bound to the albumin l~yer and is withdrawn
from being inactivated by the thrombin inhibitors in
plasma.
Exalllple 2
Polyethylene tubings having a length of 1 meter
and an inner diameter of 1.8 millimeter were treated on
the inside with concentrated sulphuric acid containing
potassium permanganate in a concentration of 2 grams per
liter, for 2 minutes at room temperature. After having
been carefully rinsed the tubes were rotated with 1
1 160~
milliliter of citrated plasma free from platelets for 60
minutes. Subsequently the tubes were rinsed carefully
with physiological saline.
The tubings thus pre-trea-ted were tested as described
in Example 1. The resulting absorbance was:
Group 1 (saline) 1.090
Group 2 (defibrinogenated plasma~ 1.120
The Example illustrates that the sulphated surface,
having been contacted with blood plasma, can bind throm-
bin according to the invention.
Exam~le 3
' Polyethylene tubings having a length of 90 centi-
meters and an inner diameter of 3.7 millimeters were
treated on the i~nside with concentrated sulphuric acid
containing potassium permanganate in a concentration of
2 grams per liter, for 2 minutes at room temperature.
After having been carefully rinsed the tubings were
treated with a 4 per cent by weight solution of albumin
and were subsequently rinsed with physiological saline.
The tubings thus pre-treated were tested in vivo in
this way. The tubings were applied as arteriovenous
shunts in dogs, viz. between Arteria and Vena femoralis.
The tubings were compressed to produce a blood flow of
approximately 40 milliliters per minute. After 20 minutes
of exposure the tubings were removed, and were immediately
rinsed with saline. A 30 centimeter length of each tube
was now tested consecutively with four different enzyme
suhstrates, in the way disc]oscd in l~amplc l, the buffer
solutiol~l)cil~g clloscn as rccomlllcll~lcd by tl~c malluf~cturc
of the substrates.,'l'he substrates and the resulting
absorbance values were:
' _bstrate Absorbance
S-2302 (kallikrein) 0.342
S-2251 (plasmin) 0.325
S-2222 (Factor Xa) 0.239
S-2238 (thrombin) 0.388
l 160~48
11
This Example illustrates that said enzymes are ad-
sorbed, without being inactivated, on a surface according
to the invention upon contact with circulating blood in
vivo.
Example 4
Polyethylene tubings having a length of 1 meter and
an inner diameter of 1.8 millimeter were treated on the
inside with concentrated sulphuric acid containing potas-
sium permanganate in a concentration of 2 grams per liter,
for 2 minutes at room temperature. After having been care-
fully rinsed with water the tubings were treated with a
solution containing 4 per cent by weight albumin, for 5
minutes, and -were subsequently carefully rinsed with
physiological saline.
One group of the tubings thus pre-treated were rotat-
ed with normal citrated plasma. A second group were rotat-
ed with citrated plasma which had been activated by rotat-
ion in a glass tube for 10 minutes at 37C. All tubings
were now carefully rinsed with physiological saline, and
were rotated with normal citr~ted plasma for 10 minutes.
The plasma samples from the last mentioned rotation, and
control samples, were finally tested in a recalcification
test. These results were obtained:
Plasma sample: Recalcification
time, seconds:
Normal plasma, not rotated 517
Normal plasma, activated in a glass
tube 148
Normal pl~sma, rotatecl ;11 a sul~h~ted tuh-
ing prc-rillsccl witl~ nor3llal c-itratcd plas~ 5(1
Normal plasma, rotatccl in a sulpllatecl tul)-
ing pre-rinsed l~ith glass-activated plasma 340
The Example illustrates ~hat a sur~ace having a
layer of albumin according to the invention can adsorb
the active enzymes Factors XIIa, XIa, IXa (the contact
36 product)produced when citrated plasma is activated by
contact with glass.
, ,
~ 16~35~8
12
Example 5
Polyethyl.ene tubings hav:ing a length of 1 meter and
an inner diameter of 1.8 millimeter were treated on the
inside with concentrated sulphuric acid containing po-
tassium permanganate in a concentrati.on of 2 grams per
liter, for 2 minutes at room temperature, and were sub-
sequently carefully rinsed with water.
The tubings .thus pre-treated were tested in the
way described in Example 1, with the difference that the
thrombin was dissolved in saline free from albumin. The
~ollowing absorbance values were found:
Group l.(saline) 1.110
Group 2 (defibrinogenated plasma) 1.080
The Example illustrates that the pure sulphated
surface can bind thrombin.
