U.S. patent application number 11/118396 was filed with the patent office on 2005-09-15 for protease for activating clotting factor vii.
This patent application is currently assigned to ZLB Behring GmbH. Invention is credited to Feussner, Annette, Romisch, Jurgen, Stohr, Hans-Arnold.
Application Number | 20050202002 11/118396 |
Document ID | / |
Family ID | 27545095 |
Filed Date | 2005-09-15 |
United States Patent
Application |
20050202002 |
Kind Code |
A1 |
Romisch, Jurgen ; et
al. |
September 15, 2005 |
Protease for activating clotting factor VII
Abstract
A protease for activating the blood clotting factor VII is
described, which (a) is inhibited by the presence of aprotinin, (b)
is increased in its activity by calcium ions and/or heparin or
heparin-related substances, and (c) in SDS-PAGE, on subsequent
staining in the non-reduced state, has one or more brands in the
molecular weight range from 50 to 75 kDa and kDa in the reduced
state has a band at 40 to 55 kDa and one or more bands in the
molecular weight range from 10 to 35 kDa. The proenzyme of this
protease is also characterized. Further, a process for obtaining
this protease and its use in hemorrhage prophylaxis or hemostasis
is described. Moreover, a test system for the qualitative and
quantitative detection of a protease which activates the blood
clotting factor VII is described.
Inventors: |
Romisch, Jurgen; (Marburg,
DE) ; Feussner, Annette; (Marburg, DE) ;
Stohr, Hans-Arnold; (Wetter, DE) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
ZLB Behring GmbH
|
Family ID: |
27545095 |
Appl. No.: |
11/118396 |
Filed: |
May 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11118396 |
May 2, 2005 |
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10319592 |
Dec 16, 2002 |
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6911334 |
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10319592 |
Dec 16, 2002 |
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09295316 |
Apr 21, 1999 |
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6528299 |
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Current U.S.
Class: |
424/94.64 ;
435/7.1 |
Current CPC
Class: |
C07K 14/745 20130101;
A61K 38/00 20130101; A61P 43/00 20180101; G01N 2333/96447 20130101;
A61P 7/02 20180101; C12N 9/6424 20130101; C12Q 1/56 20130101; A61P
7/00 20180101; A61K 38/57 20130101; G01N 33/86 20130101; A61L 15/38
20130101; A61P 7/04 20180101 |
Class at
Publication: |
424/094.64 ;
435/007.1 |
International
Class: |
A61K 038/48; G01N
033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 24, 1998 |
DE |
198 18 495 |
Jun 22, 1998 |
DE |
198 27 734 |
Nov 6, 1998 |
DE |
198 51 332 |
Nov 6, 1998 |
DE |
198 51 336 |
Nov 6, 1998 |
DE |
198 51 335 |
Feb 1, 1999 |
DE |
199 03 693 |
Claims
1.-27. (canceled)
28. A reagent for the immunological detection of a protease or its
proenzyme, wherein the protease a) activates blood clotting factor
VII, b) is inhibited by the presence of aprotinin, c) is increased
in its activity by the presence of at least one of the following:
calcium ions, heparin, or heparin related substances, and d) in
SDS-PAGE, on subsequent staining in the non-reduced state,
comprises one or more bands in the molecular weight range from 50
to 75 kDa; and in SDS-PAGE, on subsequent staining in the reduced
state, comprises a band at 40 to 55 kDa, one or more bands in the
molecular weight range from 10 to 35 kDa, and a band, which
corresponds to a proenzyme, in the molecular weight range from 60
to 65 kDa, and wherein the reagent comprises a polyclonal or
monoclonal antibody against the protease or its proenzyme.
29. (canceled)
30. An assay system comprising the protease of claim 28 or a
mixture of its proenzyme and appropriate proenzyme activators,
wherein the protease or the mixture substitute for tissue factor or
thromboplastin in assaying prothrombin time.
31. A pharmaceutical preparation, comprising the protease of claim
28 or a mixture of the protease and its proenzyme, in an amount
adequate for dissolution of fibrin-containing thrombi.
32. The pharmaceutical preparation as claimed in claim 31,
optionally further comprising single-chain or two-chain plasminogen
activators.
33. The pharmaceutical preparation as claimed in claim 31,
optionally further comprising anticoagulants.
34. The pharmaceutical preparation as claimed in claim 31,
optionally further comprising soluble calcium salts.
35. The pharmaceutical preparation as claimed in claim 31,
optionally further comprising heparin or heparin-like
substances.
36. A pharmaceutical preparation, comprising an inhibitor of the
protease of claim 28, wherein the protease inhibitor is aprotinin,
C1-inhibitor, .alpha..sub.2-antiplasmin,
inter-.alpha.-trypsin-inhibitor, or ATIII/heparin.
37. A process for preparing a pharmaceutical preparation comprising
a protease, wherein the protease a) activates blood clotting factor
VII, b) is inhibited by the presence of aprotinin, c) is increased
in its activity by the presence of at least one of the following:
calcium ions, heparin, or heparin related substances, and d) in
SDS-PAGE, on subsequent staining in the non-reduced state,
comprises one or more bands in the molecular weight range from 50
to 75 kDa; and in SDS-PAGE, on subsequent staining in the reduced
state, comprises a band at 40 to 55 kDa, one or more bands in the
molecular weight range from 10 to 35 kDa, and a band, which
corresponds to a proenzyme, in the molecular weight range between
60 and 65 kDa, and wherein the process comprises 1) preparing the
pharmaceutical preparation in a pH range from 3.5 to 8.0; 2) adding
one or more amino acids in an amount greater than 0.01 mol/L; 3)
optionally, adding a sugar or a combination of different sugars in
an amount greater than 0.05 g/ml; and 4) optionally, adding one or
more substances which are able to complex calcium ions.
38. (canceled)
39. (canceled)
40. A pharmaceutical preparation obtainable by the process as
claimed in claim 37.
41. A method for promoting wound healing and hemostasis in a
patient comprising administering to the patient the protease or
proenzyme of claim 28, optionally together with proenzyme
activators, wherein the protease or proenzyme is prepared from
blood plasma or prothrombin complex (PPSB) concentrates or
expressed recombinantly or transgenically.
42. A method for rapid wound closure comprising a) adding to at
least one of a fibrin adhesive, fleece, or other release system the
protease or proenzyme of claim 28, optionally together with
proenzyme activators, wherein the protease or proenzyme is prepared
from blood plasma or prothrombin complex (PPSB) concentrates or
expressed recombinantly or transgenically: and b) applying said
fibrin adhesive, fleece, or other release system to a wound.
43. A method for treating inborn or acquired deficiencies in a
protease or proenzyme of a patient, comprising administering to the
patient the protease or proenzyme of claim 28, optionally together
with proenzyme activators, wherein the protease or proenzyme is
prepared from blood plasma or prothrombin complex (PPSB)
concentrates or expressed recombinantly or transgenically.
Description
[0001] The invention relates to a protease for activating the blood
clotting factor VII, to a process for isolating it, detecting it
and inactivating it, and to medicinal preparations which comprise
this protease.
[0002] The blood dotting system comprises two different,
cascade-like pathways for activating clotting factors which are
present in the plasma. The intrinsic or the extrinsic pathway is
preferentially used for initiating clotting, depending on the
triggering mechanism.
[0003] When a tissue is damaged, thromboplastin (tissue factor, TF
with phospholipids) is exposed by the affected cells as the starter
of the extrinsic clotting pathway. The membrane-located
thromboplastin can bind both clotting factor VII (FVII) and
circulating, activated FVII (FVIIa). In the presence of calcium
ions and lipids, this TF-FVIIa complex leads to the binding of FX,
which is converted into its activated form (FXa) by limited
proteolysis. FXa in turn leads, by activating prothrombin to form
thrombin, to the formation of fibrin and thereby ultimately to
closure of the wound.
[0004] While the further activation of the thromboplastin-bound
FVII initially takes place autocatalytically, in particular, it is
supported, after the clotting cascade has been initiated, by FXa
and thrombin, in particular, leading to marked reinforcement of the
reaction cascade.
[0005] The administration of FVIIa or FVIIa-containing concentrates
is indicated in certain clinical situations. The so-called
FVIII-bypassing activity (FEIBA) of FVIIa is used in patients who
are suffering, for example, from hemophilia A and have developed
antibodies against FVIII as a consequence of the administration of
FVIII. According to presently available findings, FVIIa is well
tolerated in this context and, while it does not lead to any
tendency to thrombosis, it is suitable for ensuring that clotting
takes place to a limited but adequate extent. Recombinant FVIIa is
already being used therapeutically and prophylactically. FVII which
has been isolated from blood plasma can also be activated and then
used. Proteases such as thrombin can be used for this activation;
however, these proteases, as such, can themselves strongly activate
clotting and lead to the risk of a thrombosis. For this reason,
subsequent removal or inactivation of thrombin is necessary and
leads to yield losses. As a result of the risk of thrombosis which
is associated with it, the use of FXa or FIIa (thrombin) is
frequently contraindicated and only indicated in emergencies, e.g.
in association with extreme loss of blood and unstaunchable
hemorrhages.
[0006] FVIIa is found in very low concentrations in the plasma of
healthy subjects. Only very little is so far known about the
formation and origin of FVIIa which is circulating in the blood.
Traces of thromboplastin which has been expressed or released in
association with cell destruction might play a role in this
context. Although it is known that factor XIIa, for example, can
lead to FVII activation under certain conditions, the physiological
relevance of this reaction has not yet been clarified.
[0007] Surprisingly, a FVII-activating protease, which differs from
all the previously known proteases, has now been found in
connection with fractionating human plasma and certain prothrombin
complex concentrates. Investigations into this protease have shown
that it exhibits a particularly high amidolytic activity toward the
peptide substrate S2288 (HD-isoleucyl-L-prolyl-L-arginine-pNA) from
Chromogenix A B, Sweden. A particular feature of this protease is
that the amidolytic activity is efficiently inhibited by aprotinin.
Other inhibitors, such as the antithrombin III/heparin complex, are
also suitable for the inhibition. On the other hand, its activity
is increased by heparin and heparin-related substances such as
heparan sulfate or dextran sulfate and calcium ions. Finally, it
has been found that this protease is able, in a manner dependent on
time and on its concentration, to convert FVII into FVIIa. This
reaction, too, is inhibited by aprotinin.
[0008] Part of the subject matter of the invention is therefore a
protease for activating the blood clotting factor VII, which
[0009] a) is inhibited by the presence of aprotinin,
[0010] b) is increased in its activity by calcium ions and/or
heparin or heparin-related substances, and
[0011] c) in SDS-PAGE, on subsequent staining in the non-reduced
state, has one or more bands in the molecular weight range from 50
to 75 kDa and kDa in the reduced state has a band at 40 to 55 kDa
and one or more bands in the molecular weight range from 10 to 35
kDa.
[0012] In the following text, the activated form of the protease is
termed "protease" whereas the non-activated form is termed
"proenzyme".
[0013] Further investigations with this protease have shown that,
after enriching or isolation, it suffers from a rapid loss of
activity, which was observed in a solution containing 20 mM tris,
0.15 M NaCl at a pH of 7.5. The addition of albumin at a
concentration of 0.1% was not able to prevent the activity of the
protease from decreasing by 50% after one hour at room temperature.
On the other hand, very good stabilization of the protease was
observed in a solution which was buffered to a pH of 6.5 with 50 mM
Na citrate. If no particular stabilizers are added to the protease
solution, no, or only slight, losses in activity are observed if
the solution is adjusted to a pH of between 4 and 7.2, preferably
to a pH of between 5.0 and 7.0. However, it is expedient to add
stabilizers to the solution, with suitable stabilizers, apart from
citrate, being, in particular, glutamate, amino acids, such as
arginine, glycine or lysine, calcium ions and sugars such as
glucose, arabinose or mannose in quantities of 1-200 mmol/l,
preferably in quantities of 5-100 mmol/l. Efficient stabilization
was also achieved by adding glycols such as ethylene glycol or
glycerol, with quantities of 5-80% by weight, preferably of 10-60%
by weight, being used. The pH of the stabilized solution should
then be between the pH values 4-9.
[0014] While the novel protease, and also the proenzyme, can be
obtained by recombinant DNA methods or by production in e.g. the
milk of suitable transgenic animals, they can in particular be
obtained by fractionation of blood plasma or of prothrombin complex
(PPSB) concentrates. The starting material is then first of all
subjected to an anion exchange chromatography, which is followed by
an affinity chromatography of the eluate. A heparin which is
immobilized on a matrix, or a heparin-related substance such as
heparan sulfate or dextran sulfate, is particularly suitable for
the affinity chromatography. When such a chromatographic method is
used, the novel protease and/or the proenzyme can be selectively
bound and then eluted once again using known methods. The use of a
spacer is advisable for coupling the ligand to the matrix. A
heparin-lysine matrix has been found to be particularly suitable
for isolating the novel protease.
[0015] In SDS-PAGE with subsequent staining, the protease which has
been isolated by this method exhibits, in the non-reduced state,
one to several bands which lie closely together in the molecular
weight range of 55-75 kDa. Following reduction, one to several
bands were observed in the molecular weight range of 15-35 kDa and
one band was observed at 40-55 kDa. A further band between 60 and
65 kDa, which, after scanning and quantitative evaluation,
constituted 5-10% of the total protein, showed that non-activated
proenzyme was also present. This result was supported by
appropriate investigations using monoclonal antibodies against this
protease. It was therefore concluded that the proenzyme of this
protease can also be prepared, pasteurized and used by the method
according to the invention. Part of the subject matter of the
invention is therefore the proenzyme of the protease for activating
blood clotting factor VII. The proportion of the proenzyme is
indicated by the band between 60 and 65 kDa. Corresponding to the
amino acid sequence which constitutes the activation region of the
proenzyme, thrombin, kallikrein or FXIIa are, in accordance with
their substrate specificities, examples of suitable physiological
activators of the proenzyme.
[0016] Some of the properties of the novel protease which have been
described, namely the fact that it can be isolated from plasma or
from prothrombin complex (PPSB) concentrates which are derived from
plasma, the inhibition of its amidolytic activity by aprotinin and
the described migration behavior in SDS-PAGE, both in the reduced
and in the non-reduced states, are reminiscent of a protease which
was isolated by Hunfeld et al. (Ann. Hematol. 1997; 74; A87, 113;
Ann. Hematol. 1998; 76; A101, P294 and Etscheid et al. Ann.
Hematol. 1999, 78: A42) from a PPSB concentrate which was not
defined in any more detail. In that case, the preparation was
essentially achieved using an aprotinin matrix. As a result of the
amidolytic cleavage of certain peptide substrates, the activity was
described as being a thrombin-like activity. Hunfeld et al. did not
find any influence on global clotting parameters such as
prothrombin time, Quick or platelet aggregation.
[0017] The N-terminal sequencing of the protease described by
Hunfeld et al. shows concordances with a protein whose cDNA was
described by Choi-Miura et al. (J. Biochem. 119: 1157-1165 (1996)).
In its primary structure, the corresponding protein exhibits
homology with an enzyme termed hepatocyte growth factor activating
enzyme (HGFA).
[0018] When two bands which were isolated from SDS-PAGE under
reducing conditions were subjected to N-terminal sequencing, the
following concordances were established:
1 Molecular weight Amino acid range of the band sequence Author
10-35 kDa IYGGFKSTAGK present invention 30 kDa IYGGFKSTAG Hunfeld
et al. 17 kDa IYGGFKSTAGKH Choi-Miura et al. 40-55 kDa LLESLDP
present invention 50 kDa SLDP Hunfeld et al. 50 kDa SLLESLDPWTPD
Choi-Miura et al.
[0019] Concordances are also found in other test results such as
substrate specificity and the ability of the activity to be
inhibited. Despite this, it is still not possible at present to
assume with confidence that these proteins are identical. At any
rate, the previously investigated, abovementioned proteins have not
been reported to possess the property of activating FVII or
activating other factors (see below).
[0020] On the basis of its described properties, the novel protease
can be used diagnostically and therapeutically.
[0021] 1. Test Systems Using the Novel Protease
[0022] The novel protease can be used diagnostically in test
reagents. Thus, the presence of factor VII can be determined
qualitatively and quantitatively in a clotting test by adding the
novel protease.
[0023] Conversely, the test system developed for measuring FVII
activation can also be used for detecting and quantifying the
protease. For this, a solution containing the protease is mixed
with an FVII-containing solution and, after an appropriate
incubation time, the resulting quantity of FVIIa is quantified.
This can be carried out, for example, using the Staclot.RTM.
FVIIa-rTF test (Stago/Boehringer Mannheim). When a preferred
procedure is used, this test is not limited by the FVII
concentration supplied. If the quantity of protease in the form of
the proportion of total protein is known, which proportion can be
determined
[0024] in a pure protease preparation, by means of the Kjeldahl
method or by means of another protein assay with which the skilled
person is familiar, or
[0025] using an antigen test, for example based on specific
antibodies and an appropriate immunochemical determination method
such as ELISA, the specific activity of the protease preparation
can then be measured in a corresponding manner.
[0026] Surprisingly, a property has now been found, in association
with characterizing the protease further, which makes it possible
to carry out an additional determination method. In assocition with
incubation of the blood clotting factors VIII/VIIIa and V/Va with
said protease, and subsequent quantification, it became clear that
said clotting factors are inactivated in a manner which is
dependent on the protease concentration and on the length of the
incubation.
[0027] Another part of the subject matter of the invention is
therefore a novel test system for qualitatively and quantitatively
detecting the protease which activates blood clotting factor VII,
in which system the protease can be determined by its action
inactivating the blood clotting factors VIII/VIIIa or V/Va. This
test system is based on a solution containing the protease being
incubated with factor VIII/VIIIa or factor V/Va and the remaining
quantity of factor VIII/VIIIa or the remaining quantity of factor
V/Va being measured by means of a conventional activity test and
the amount of protease then being quantitatively determined from
this by comparison with a standard curve. In carrying out this
test, the incubation of the protease activity is inhibited, after
predetermined periods of time, by the limited addition of
aprotinin, which has the advantage that it has no effect, at these
concentrations, on the subsequent measurements of the test system.
After that, the remaining activities of the doting factors are
measured by means of a test which is familiar to the skilled
person. For this, a test system has, in particular, proved its
worth in which use is made of the so-called Coamatic.RTM. factor
VIII test (Chromogenix AB), which essentially contains factors IXa
and X, with the resulting amount of FXa being quantified, in the
presence of a thrombin inhibitor, by means of the conversion of a
chromogenic substrate (see last third of page 2). This amount is in
turn proportional to the quantity of FVIII or FVIIIa. Determining
the residual FVIII activity then makes it possible to deduce the
concentration of protease which is present.
[0028] The degradation of the FVIII/FVIIIa or the FV/FVa due to the
proteolytic effect can be clearly demonstrated by SDS-PAGE
Depending on the time for which the protease is incubated, for
example, with an FVIII concentrate, bands which are typical for
FVIII disappear while other, new bands emerge or weak bands
increase in intensity. Accordingly, the activity of the protease
can also be correlated by quantifying the decreasing or increasing
bands and consequently measured quantitatively, for example using a
protease standard. The changes in the band intensities on the
SDS-PAGE electropherogram or following other electrophcretic
methods can be quantified, for example, using a scanner, with which
a skilled person is familiar, and the appropriate program. In
addition to this, antibodies against said clotting factors can be
used for Western blotting and employed for evaluation in the manner
described. Antibodies which specifically detect the decreasing
bands or, in particular, the emerging bands are particularly
suitable. In this context, these antibodies can also be used for
establishing other immunochemical tests such as an ELISA.
[0029] The proteolytic inactivation which has been described in the
case of FVIII/FVIIIa is also observed when the protease is
incubated with factor V/Va, which exhibits a certain degree of
structural homology with FVIII. The degradation can be monitored in
suitable activity test systems and in SDS-PAGE/Western
blotting.
[0030] Despite the inactivations of FV and FVIII, it was now found
that adding the protease to blood, to platelet-rich plasma or
plasma shortened the clotting times, that is the procoagulatory
effect predominated in various so-called "global clotting tests".
These test systems are understood as being, for example, the
non-activated partial thromboplastin time (NAPTT), the prothrombin
time (PT) and the recalcification time. Since the shortening of
these times, as measured, for example, in so-called coagulometers,
by means of thromboelastography or else in chromogenic tests,
correlates with the concentration of a clotting-promoting
substance, the concentration of the substance in a sample can
conversely be deduced using a calibration curve of the clotting
time. The concentration of the "FVII activator" can correspondingly
be determined using selected global clotting tests.
[0031] It was also surprising to find that the "FVII activator" is
likewise able to bring about effective activation of single chain
urokinase (scuPA, single chain urokinase plasminogen activator) and
single chain tPA (sctPA, single chain tissue plasminogen
activator), that is can act as a plasminogen activator activator
(PAA). The activity of the activated PAs can be measured, for
example, using chromogenic substrates. Accordingly, this property
can therefore also be used for detecting and quantifying the "FVII
activator". The activation of the plasminogen activators can also
be determined in a coupled reaction in the presence of plasminogen,
either by the formation of plasmin itself or by the dissolution of
a fibrin clot which is brought about by plasmin.
[0032] In summary, therefore, it can be stated that the protease
can be both detected and quantified by incubating it with a
solution containing FVIII or FVIIIa and then determining the
remaining quantity of FVIII/VIIIa by means of a suitable activity
test. In the same way, FV or FVa can be incubated with the protease
and the remaining quantity of FV/FVa can subsequently be
quantified. The unknown protease concentration can be determined
quantitatively by comparison with a standard curve of increasing
quantities of protease which is included in the test. Various
global clotting tests are likewise suitable for the quantification,
with the protease concentration being read off a calibration curve
on the basis of the shortening of the clotting time. The PAA
activity of the protease can also be used for determination
purposes.
[0033] Another feature of these tests is that the FV and FVIII
inactivation and the PAA activity are displayed particularly well
in the presence of adequately high concentrations of calcium,
preferably >0.001 mM, particularly preferably >0.005 mM, e.g.
in the form of CaCl.sub.2. In contrast to the direct chromogenic
assay, in which, as has been described above, both heparin and
heparin-like substances and also calcium increase the protease
activity, the inactivation of FV/FVIII is not promoted, or only
promoted insignificantly, by heparin. By contrast, the PAA activity
is stimulated in the presence of both agents, that is by calcium
and/or heparin or heparin-like substances.
[0034] The protease-mediated reactions can be very efficiently
diminished or prevented by incubating the protease with inhibitors,
particularly antithrombin III in the presence of heparin or
heparin-like substances (preferably in the presence of heparin),
C1-esterase inhibitor, alpha2-antiplasmin, inter-alpha-trypsin
inhibitor or known synthetic, low molecular weight protease
inhibitors such as Guanidinocaproic
acid-para-ethoxy-carbonylphenylester which is available under the
trademark FOY.RTM.. These substances can therefore be used for
stopping the reaction, in order, for example, to define incubation
times precisely or to increase the specificity of the test still
further. Decreasing the free calcium ions in the mixture with a
chelating agent, for example, can also be used for this
purpose.
[0035] 2. Stabilized Preparations of Factor V and Factor VIII
[0036] The further task now ensued, from the above-described
observations concerning the proteolytic actions of the novel
protease on clotting factors V and VIII, of inhibiting the protease
or reducing its activity in order to avoid losses of yield and the
formation of what might possibly be interfering protein fragments.
This is all the more relevant since FV and FVIII are usually
prepared from cryoprecipitates which have been obtained from plasma
and in the presence of calcium ions because the latter are required
for maintaining protein conformations.
[0037] Another part of the subject matter of the invention is
therefore a stabilized preparation of FV or FVIII which is free of
the factor V or factor VIII fragments formed due to proteolytic
degradation as a result of the fact that the protease activating
the blood clotting factor VII is inhibited. Since more detailed
investigations have shown that inactivation of factor V and factor
VIII by said protease takes place particularly efficiently in the
presence of calcium ion concentrations greater than 0.5 mM, the
factor V or VIII preparation can be effectively stabilized if, for
the inhibition of the protease activating the blood clotting factor
VII, the concentrations of calcium ions in the factor V or in the
factor VIII preparation are adjusted to less than 1.0 mM,
preferably to less than 0.5 mM. While the factor V- and factor
VIII-inactivating properties of the protease are markedly reduced
at these concentrations, the quantity of calcium ions is still
sufficient for stabilizing the conformations of the FV and FVIII
molecules. The abovementioned quantities of calcium ions should not
be exceeded, not merely in the end product but also in the
cryoprecipitate itself and in the following purification steps.
[0038] In accordance with the above-described affinity of the
protease or the proenzyme for heparin and heparin-like substances,
the protease/the proenzyme can be removed from the FVIII- or
FV-containing solution by incubating with immobilized heparin or
other suitable immune- or affinity-adsorbents. Polyclonal or
monoclonal antibodies, respective antibody-fragments that are
useful in preparing the immune adsorbents are readily available by
techniques known in the art in using all or part of the protease or
proenzyme as antigen.
[0039] However, natural or synthetic protease inhibitors can also
be employed, where appropriate in addition to decreasing the
quantity of calcium ions, for preventing the proteolytic
degradation of the FV or the FVIII. Proteins such as aprotinin,
alpha2-antiplasmin, C1-esterase inhibitor or inter-trypsin
inhibitor may be employed as inhibitors. Low molecular weight
substances which are known to the skilled person as synthetic
serine protease inhibitors can also be used in this context.
Inhibitors, such as antithrombin III, whose inhibitory potential is
increased by heparin or heparinoids can likewise be added. Thus, it
has been found, surprisingly, that while heparin on its own is able
to increase the amidolytic activity of the protease towards small
chromogenic substances, it does not support inactivation of
FV/FVIII.
[0040] 3. Pharmaceuticals Which Comprise the Novel Protease
[0041] The novel protease and/or its proenzyme can also be used
therapeutically.
[0042] They can be employed as a blood coagulating agent, either on
their own or together with substances which increase the activity
of the protease, such as heparin, or heparin-related substances,
such as heparan sulfate, and/or calcium ions, with it being
possible additionally to add factor VII as well, in its inactive
form, to this agent. The use of such an agent, in which its
FVIII-bypassing activity (FEIBA) is exploited, for example, can be
indicated when intolerances exist toward FVIII and/or FIX and/or
FXI and/or the contact phase proteins, such as FXII, for example on
account of the presence of antibodies, or when other types of
deficiency situation exist. In this connection, the FVII can be
activated either in vitro, in the plasma, in enriched fractions or
by acting on purified FVII. It is also possible to use the novel
blood coagulating agent ex vivo for general hemorrhage prophylaxis
or for staunching hemorrhages.
[0043] On the other hand, the observed inhibition of the novel
protease by aprotinin or the abovementioned inhibitors can be used
for developing an agent which comprises a protease inhibitor and
which diminishes the ability of the blood to coagulate. In addition
to this, the novel protease can also be used to identify
physiological or non-physiological factors, such as synthetic
peptides, which impair blood clotting because of their
protease-inhibiting effect. The peptide sequences of the
chromogenic substrates which are transformed particularly
efficiently, such as those of the S 2288 (see above for details),
can be used as a structural basis for this. The addition of
suitable inhibitors to clotting preparations, or during their
preparation, can be necessary if these preparations are to be free
of proteolytic activities.
[0044] Surprisingly, a property has now been found, in association
with characterizing the protease further, which opens up the
possibility of an additional use for the so-called "factor VII
activator" protease. When single chain plasminogen activators such
as prourokinase (single chain urokinase, scuPA, single chain
urokinase plasminogen activator) or sctPA (single chain tissue
plasminogen activator) are incubated, the "factor VII activator"
brings about activation of these plasminogen activators (PA). In
this connection, there is a limited proteolysis of the single chain
PAs, resulting in the formation of double chain proteases, which
are particularly suitable for activating plasminogen. The resulting
plasmin is the effector of fibrinolysis, that is the physiological
system which is responsible for dissolving thrombi. PAs, such as
prourokinase or tPA, are endogenous proteins which are released
when needed and which, as is known, are activated by plasmin or by
kallikrein (scuPA). The mechanism by which scuPA is activated in
the healthy state has not yet been fully clarified.
[0045] The plasminogen activators are employed therapeutically, as
isolated or recombinantly prepared proteins, in pharmaceutical
preparations in association with thromboembolic diseases or
complications, such as in leg vein thrombosis, cardiac infarction
or strokes.
[0046] In accordance with the properties of the "factor VII
activator" which have now been found, the latter can be used for in
vivo or ex vivo activation of plasminogen activators such as
prourokinase or sctPA. This activity can also be applied by using
said protease for the prophylaxis or therapy of thromboembolic
diseases, specifically in combination with single chain or double
chain plasminogen activators or anticoagulants as well. This
possible use is not contradictory to the fact that the protease is
also able to act in a procoagulatory manner. The question of which
of the two reactions predominates is probably resolved by the
availability of the physiological substrates. According to the
current state of knowledge, factor VII is activated moderately in
plasma and continuously maintains a certain concentration of FVIIa
in order to be able to counteract immediately any sudden vascular
damage. On the other hand, only nanogram quantities of tissue
plasminogen activator and urokinase plasminogen activator are
present in a milliliter of blood plasma. It is only when fibrin
deposition or thrombi occur that there is an increase in the
concentration, by secretion or synthesis, of plasminogen
activators, which then display their thrombolytic activity by
activating plasminogen after they have been activated locally, in
particular when bound to the thrombus. When single-chain PAs are
present, particularly in a locally restricted manner, their
activation might outweigh FVII activation, thereby making it
possible to adjust to the physiological situation. Accordingly,
this protease might also regulate hemostasis, thereby indicating a
replacement with the protease and/or the proenzyme in the case of
inborn and acquired deficiency states.
[0047] Another part of the subject matter of the invention is
therefore a pharmaceutical preparation which comprises a quantity
of the blood clotting factor VII-activating protease and/or its
proenzyme form which is sufficient for dissolving fibrin-containing
thrombi. This preparation may additionally comprise single chain
plasminogen activators (PA) and/or anticoagulants. When the
proenzyme is present it is advantageous to comprise a suitable
activating agent within or together with the pharmaceutical
preparation above.
[0048] Since it has been found that the plasminogen
activator-reinforcing effect of the "FVII activator" is
particularly promoted by calcium and/or heparin and heparin-like
substances such as dextran sulfate, pharmaceutical preparations
which additionally comprise soluble calcium salts and/or heparin or
heparin-like substances may particularly advantageously be employed
for dissolving, in accordance with the invention, fibrin-containing
thrombi. In this context, the protease/proenzyme can be employed on
its own or in combination with single chain or double chain
plasminogen activators with or without substances which exhibit
particular affinities for the protease and thereby increase its
activity as carrier substances for pro-longing plasma half life or
as mediators to surfaces.
[0049] Pharmaceutical preparations which comprise the blood
clotting factor VII-activating protease can, because of its special
fibrinolytic effect, be employed for treating diseases which are
caused by fibrin-containing thrombi. Fibrinolytic processes are
also involved in wound healing processes. In this connection, said
protease and/or proenzyme can be administered intravenously or
locally, subcutaneously, intradermally or intramuscularly, or else
topically in the case of injuries and wounds, or bound to a
suitable carrier matrix. Both protease/proenzyme which has been
isolated from body fluids such as blood or plasma and
protease/proenzyme which has been prepared recombinantly or
transgenically can be employed in this context. The
protease/proenzyme is also suitable for use as a component of a
so-called fibrin adhesive, which should not then contain any
substance, such as aprotinin, which inhibits the
protease/proenzyme. In this case, use can be made of the
clotting-shortening properties of the protease.
[0050] The protease/proenzyme above may be used for inherited or
acquired hemostasis deficiencies, in (diffuse) bleeding occurencies
respective thrombosis associated complications. If used to treat
bleeding the combination of protease/proenzyme together with F VIII
optionally under addition of further clotting factors is
advantageous.
[0051] 4. Process for Pasteurizing the FVII-Activating Protease
[0052] As a protein which has been isolated from human plasma, the
novel protease and/or its proenzyme can only be employed as a
pharmaceutical preparation if it has previously been subjected to a
process for inactivating viruses. The pasteurization process is in
particular recognized as being the most important process for
inactivating viruses. However, heating at about 60.degree. C. for
up to 10 hours requires the protein which is to be treated to be of
adequate stability. The optimal stabilizers have to be determined
separately for each protein and their concentrations have to be
optimized.
[0053] In the case of the novel protease and/or its proenzyme,
conditions which stabilize the protein in solution, without any
pasteurization being performed, have already been mentioned above.
In this regard, a slightly acidic pH range has in particular proved
to be advantageous. However, when a pasteurization is carried out
under these conditions, the novel protease and/or its proenzyme as
a rule loses more than 50% of its original activity.
[0054] It has now been found that a pasteurization of a
pharmaceutical preparation comprising the novel protease and/or its
proenzyme ensures optimal stabilization results if the preparation
is prepared
[0055] a) in a pH range of from 3.5 to 8.0, preferably in a pH
range of from 4.0 to 6.8;
[0056] b) in the added presence of one or more amino acids in a
quantity of more than 0.01 mol/l, preferably more than 0.05 mol/l;
and/or
[0057] c) in the added presence of a sugar or of a combination of
different sugars having a total concentration of more than 0.05
g/ml, preferably more than 0.2 g/ml; and/or
[0058] d) in the added presence of one or more substances which are
able to complex calcium ions, such as citrate, oxalate,
ethylenediamine tetraacetic acid, etc.
[0059] Additives such as albumin, Haemaccel.RTM., heparin and
heparinoids, glycerol, glycol and polyethylene glycol, may also be
used separately or mixed together. After the pasteurization has
been completed, the sugars, amino acids and other additives which
have been added as stabilizers can be decreased, or removed
completely from the preparation, using methods with which the
skilled person is familiar. The results of the pasteurization
processes are given in Examples 12 and 13.
EXAMPLE 1
[0060] The Staclot.RTM. FVIIa-rTF test system (Stago/Boehringer
Mannheim) was used for demonstrating activation of FVII by the
prepared protease. This detection system is based on the particular
property of (recombinant) soluble tissue factor (rTF) which is only
able to use the preformed activated FVII (FVIIa) for initiating the
extrinsic clotting pathway. In contrast to the situation when
complete tissue factor is used, this makes it possible to determine
the real content of FVIIa precisely.
[0061] Isolated FVII (Enzyme Research Labs) was used for the
activation experiments. This FVII itself contains traces of FVIIa
since it is isolated from human plasma. The concentration was
adjusted to 0.05 IU of FVII/ml by diluting with buffer. The FVII
was incubated at room temperature for 10 min with the test
substances and then tested for the true FVIIa content. The FVIIa
contents were quantified using a reference curve which was
constructed in parallel.
[0062] It was ascertained in preliminary experiments, which are not
described here, that while, in the concentration employed,
aprotinin completely inhibited the activity of the prepared
protease, it had no direct effect on the FVIIa nor any significant
effect on the FVIIa-rTF test system.
[0063] The results given below relate in each case to triplicate
determinations.
[0064] The following experimental assays were accordingly set
up:
[0065] 1. FVII:
[0066] Result: 10 mlU of FVIIa/ml
[0067] Non-activated FVII was used as the control assay. This
already contains traces of FVIIa (see above) in the order of
magnitude of 10 mlU of FVIIa/ml.
[0068] 2. FVII+Aprotinin:
[0069] In this assay, FVII was incubated in the presence of
aprotinin and used in the FVIIa-rTF assay in order to demonstrate
that FVIIa itself was not inhibited, and nor was the test affected
by the aprotinin employed. This was confirmed (in comparison with
assay 1).
[0070] 3. Protease+FVII (Incubation), Followed by the Addition of
Aprotinin:
[0071] Result: 18 mlU of FVIIa/ml
[0072] In this case, the protease was given time to activate FVIIa.
Aprotinin was only added, in order to inhibit the protease, after
the 10-minute incubation had taken place. The resulting FVIIa was
quantified in the FVIIa-rTF assay. Subtracting the FVIIa base value
(assay 1), 8 mlU of FVIIa/ml have therefore been formed by the
action of the protease under the chosen conditions.
[0073] 4. Protease+Aprotinin, Followed by the Addition of FVII
[0074] Result: 11 mlU of FVIIa/ml
[0075] In this assay, the protease was inhibited with aprotinin
before contact with FVII. Neither the subsequent incubation with
FVII, nor the following FVIIa quantification led to any significant
increase in the FVIIa content (because of the range of variation in
the assay, 11 versus 10 mlU/ml in assay 1 is not to be regarded as
being significant).
[0076] 5. Protease
[0077] Result: 0 mlU of FVIIa/ml
[0078] This assay demonstrated that, at the concentration selected,
the protease did not itself have any effect on the FVIIa-rTF test
system.
[0079] In summary, it follows from the above that
[0080] the described protease activates FVII;
[0081] the activation of FVII by the protease takes place
"directly", that is independently of the presence of rTF;
[0082] the activation of FVII can be inhibited by aprotinin; at the
concentration selected, aprotinin itself does not have any
significant influence on the test system.
EXAMPLE 2
[0083] This example describes how FVII is activated in a reaction
which is dependent on the concentration of the protease and on the
time over which the protease is incubated with FVII.
[0084] Test systems and reagents were selected to correspond with
the conditions described in Example 1. In a first series of
experiments, the initally introduced FVII was preincubated with
different dilutions (1:5, 1:10 and 1:20) of the protease-containing
solutions (5 min at RT), then treated with aprotinin (to inhibit
the protease) and subsequently tested for its content of FVIIa in
the FVIIa-rTF assay.
[0085] Once again, the parallel assays, in which the protease had
been inhibited by aprotinin before contact with FVII, served as
control assays.
[0086] The results are given as activation factors, i.e. correspond
to x times the value which was measured in the abovementioned
control assay:
2 Assay Control Protease + FVII Protease + aprotinin Incubation +
Aprotinin Incubation + FVII Dilution of the protease solution
Activation factor 1:5 2.6 1.0 1:10 2.0 1.0 1:20 1.6 1.0
[0087] The activation factor 1:0 of the control assays corresponds
to the control, which was additionally included and in which only
the test buffer, containing the FVII employed, was treated under
identical incubation conditions and tested. That is, no significant
activation took place in the control assays.
[0088] It follows from this that FVII is activated by the protease
in a manner which is dependent on the concentration of the
protease.
[0089] It was similarly demonstrated that, when the concentrations
of the coreactants are kept constant, the FVII is activated by the
protease in a manner which is dependent on the length of the
incubation.
[0090] When equal volumes of a solution containing 0.2 IU of
FVII/ml and a 1:10-diluted protease solution were incubated
together, the following contents of FVIIa were obtained after
incubating for the relevant times and subsequently adding aprotinin
(in order to stop the activation):
3 Length of incubation Activation factor 0 min 1.0 2.5 min 1.3 5.0
min 2.0 10.0 min 2.8 40.0 min >3.8
[0091] It follows from this that FVII is activated by the protease
in a time-dependent manner.
EXAMPLE 3
[0092] Using this example, it will be demonstrated that activation
of FVII by the protease is increased in the presence of calcium
ions and heparin.
[0093] 25 .mu.l of the protease-containing solution were mixed with
50 .mu.l of
[0094] buffer (control)
[0095] 15 mM CaCl.sub.2
[0096] 50 USP units of heparin/ml
[0097] Pathromtin (lipid mixture, aliquot dissolved in accordance
with the manufacturer's instructions) at room temperature for 5
min, and then treated with 150 .mu.l of a tris/NaCl buffer solution
(pH 8.2) and 25 .mu.l of the chromogenic substrate S2288 (3 mM);
the time-dependent change in the extinction at 405 nm was then
measured (at 37.degree. C.). The activation factors, related to the
buffer control (x times), are given in the following table.
4 Activation factor Assays (x times buffer control) Buffer control
1.0 +CaCl.sub.2 3.6 +Heparin 2.6 +Lipid 0.9 +CaCl.sub.2 + heparin
4.3 +CaCl.sub.2 + lipid 3.3 +Heparin + lipid 2.7 +CaCl.sub.2 +
heparin + lipid 3.7
[0098] Under the conditions used in this example, marked increases
in the activity of the protease can be observed in the presence of
calcium ions and/or heparin.
EXAMPLE 4
[0099] In each case, 25 .mu.l of a solution, containing 10, 1 or
0.1 .mu.g of protease/ml, were mixed with 25 .mu.l of FVIII (2
IU/ml), after which 25 .mu.l of CaCl.sub.2 (25 mM) and 25 .mu.l of
Pathromtin.RTM. (Dade Behring GmbH) were added. After incubating at
37.degree. C. for 0, 3, 10 and 20 min, the reaction was stopped by
adding 400 .mu.l of aprotinin (500 KIU/mg). A sample in which
aprotinin was introduced initially served as a control.
[0100] Each sample was diluted in tris-buffer/BSA. In each case, 50
.mu.l of this solution were mixed with 50 .mu.l of the factor
reagent (essentially composed of FIXa, FX and a thrombin inhibitor,
appropriately modified in accordance with the Coamatic.RTM. FVIII
test, Chromogenix AB) and incubated at 37.degree. C. for 10 min.
After 50 .mu.l of substrate (e.g. S 2765,
N-a-Cbo-D-Arg-Gly-Arg-pNA) had been added, the reaction was stopped
after a predetermined period of incubation by adding 50 .mu.l of
acetic acid (50%), and the OD405nm was then measured. A standard
curve for FVIII was used for determining the concentration in the
sample.
[0101] Results:
[0102] In a first assay, the time for which the protease was
incubated with FVIII (2 IU/ml) was kept constant (10 min) but the
concentration of the protease was varied (0.1, 1 and 10 .mu.g/ml).
The reaction was stopped and the residual concentration of active
FVIII was determined. As the protease concentration increased,
correspondingly more FVIII was inactivated (FIG. 1).
[0103] The protease content of a sample can be quantified using an
appropriate standard curve.
[0104] In a second assay, the concentration of the protease was
kept constant (10 .mu.g/ml) but the time of incubation with FVIII
(2 IU/ml) was varied. A marked reduction in the residual
concentration of active FVIII was seen as the length of incubation
increased (FIG. 2).
EXAMPLE 5
[0105] The influence of the "FVII activator" on the activity of
factor V was investigated: 25 .mu.l of protease-containing solution
(0-100 .mu.g/ml) were incubated with 50 .mu.l of FV (5 IU/ml) and
25 .mu.l of 25 mM CaCl.sub.2 (0-20 min) and, after that, 400 .mu.l
of buffer containing 100 KIU of aprotinin/mi were added.
[0106] In each case, 100 .mu.l of each incubation assay were then
incubated with 100 .mu.l of FV-deficient plasma at 37.degree. C.
for 1 min, after which 200 .mu.l of Thromborel S.RTM. were mixed in
and the clotting times were determined in a Schnitger and Gross
coagulometer. The residual activities of FV were determined.
[0107] Results:
5 Residual FV activity Time for which protease Protease
concentration incubated with FV (min) (.mu.g/ml) 0 10 20 10 93 91
100 30 100 93 28 100 100 29 13
[0108] This example demonstrates that FV was inactivated by the
protease over time.
EXAMPLE 6
[0109] The influence of the "FVII activator" on clotting times in
so-called global tests was investigated using Schnitger and Gross
coagulometers. All the difference values listed correspond to the
clotting times which were shortened by this amount.
[0110] NAPTT (Non-Activated Partial Thromboplastin Time)
[0111] The protease-containing solution was diluted with buffer
down to 100, 30, 10 and 3 .mu.g/ml. 100 .mu.l of each of these
solutions were incubated, at 37.degree. C. for 2 min, with 100
.mu.l of citrate plasma (standard human plasma pool or individual
donors) and 100 .mu.l of Pathromtin.RTM., after which 100 .mu.l of
25 mM CaCl.sub.2 were added; the clotting times were then
determined. The differences between these measured values and the
corresponding clotting times obtained with buffer solution instead
of the protease were determined.
6 Clotting time differences (buffer minus sample) (sec) Protease
concentration (.mu.g/ml) 0 3 10 30 100 Standard human plasma (213
sec) Sample No. 0 13 20 42 43 1 0 20 33 42 41 2 0 27 31 45 47 3 0
13 14 23 29 4 0 18 37 51 50 5 0 25 49 54 46
[0112] The addition of FVII-acitvator resulted in a concentration
dependent shortening of NAPTT.
[0113] Plasma Recalcification Time
[0114] The protease-containing solution was diluted with buffer
down to 100, 30, 10 and 3 .mu.g/ml. 100 .mu.l of each of these
solutions were incubated with 100 .mu.l of citrate plasma (standard
human plasma pool or individual donors) at 37.degree. C. for 1 min,
after which 100 .mu.l of 25 mM CaCl.sub.2 were added; the clotting
times were then determined. The differences between these measured
values and the corresponding clotting times obtained with buffer
solution instead of protease were determined.
7 Clotting time differences (buffer minus sample) (sec) Protease
concentration (.mu.g/ml) 0 3 10 30 100 Standard human plasma (283
sec) Sample No. 0 17.2 15.1 30.5 50.4 1 0 29.8 51.7 60.3 90.1 2 0
25.2 51.7 69.5 101.3 3 0 28.0 -- 39.0 74.6 4 0 27.3 42.7 55.6 91.8
5 0 44.3 69.1 101.2 134.2
[0115] PT (Prothombin Time)
[0116] The protease-containing solution was diluted with buffer
down to 100, 30, 10 and 3 .mu.g/ml. 100 .mu.l of each of these
solutions were incubated with 100 .mu.l of citrate plasma (standard
human plasma pool or individual donors) at 37.degree. C. for 1 min,
after which 200 .mu.l of Thromborel S.RTM. (Dade Behring GmbH) were
added; the clotting times were then determined.
[0117] The differences between these measured values and the
corresponding clotting times obtained with buffer solution instead
of protease were determined.
8 Clotting time differences (buffer minus sample) (sec) Protease
concentration (.mu.g/ml) 0 3 10 30 100 Standard human plasma (13.6
sec) Sample No. 0 1.0 1.7 1.5 2.4 1 0 0.7 1.3 2.4 2.7 2 0 0.3 0.4
1.7 3.1 3 0 0.4 0.7 1.5 1.8 4 0 0.1 0.7 1.8 3.1 5 0 0.3 0.5 1.2
2.8
[0118] The clotting times in the above global tests were shortened
in a manner which was dependent on the concentration of the
protease. In a corresponding manner, it was possible, after
"calibrating" a plasma which was used with a known quantity of the
"FVII activator", to determine the protease concentration in a
sample by reading off from a standard curve.
EXAMPLE 7
[0119] The plasminogen activator-activating properties of the "FVII
activator" were investigated using single chain urokinase (scuPA)
and single chain tPA (sctPA).
[0120] Assay:
[0121] 0.1 ml of PA solution (20 .mu.g of scuPA/ml or 100 .mu.g of
sctPA/ml)
[0122] +0.1 ml of test buffer or
[0123] 100 U of heparin/mi in test buffer or
[0124] 20 mM CaCl.sub.2 in the test buffer
[0125] +0.5 ml of test buffer
[0126] +0.1 ml of protease/sample (increasing concentrations:
[0127] 2-10 .mu.g of scuPA/ml or
[0128] 50-200 .mu.g of sctPA/ml)
[0129] Incubation at 37.degree. C.
[0130] +0.1 ml of 100 KIU of aprotinin/ml in test buffer
[0131] Incubation at 37.degree. C. for 2 min
[0132] +0.1 ml of substrate S-2444 (3 mM)
[0133] As a control, aprotinin was introduced initially, instead of
the plasminogen activator (PA), prior to the first incubation, and
carried through in each case. In return, PA was not added until
later, in place of the aprotinin.
[0134] The Difference of the measurements (.DELTA.)
.DELTA.OD.sub.405 nm was determined photometrically. The control
values which were obtained were subtracted from the sample/protease
values and in this way the PA activity which was caused by the PM
activity was determined (in mlU/min).
[0135] Results:
[0136] scuPA Activation (20 .mu.g of scuPA/ml, 2-10 .mu.g of "FVII
activator"/ml)
9 Resulting PA activity (.DELTA. mIU/min) Incubation time "FVII
activator" (.mu.g/ml) (min) 2 5 10 A. Stimulant: none 2 25 60 117 5
79 179 165 10 186 449 517 B. Stimulant: heparin 2 190 332 425 5 330
455 458 10 417 462 460 C. Stimulant: CaCl.sub.2 2 255 370 401 5 338
424 438 10 416 445 448
[0137] The tables illustrate the fact that scuPA was activated in a
manner which was dependent on the concentration of the "FVII
activator" and on the length of the incubation. At the same time,
both heparin and calcium had a stimulatory effect on the activation
of the PA which was brought about by the protease.
[0138] sctPA Activation(100 .mu.g of sctPA/ml, 50-200 .mu.g of
"FVII activator"/ml)
[0139] Since the turnover rate of the activated tPA only increases
by a factor of 3-4 as compared with the tPA proenzyme (while that
of uPA increases by a factor of 1000-1500), higher concentrations
of the two coreactants (see above) had to be selected in order to
obtain an analyzable measurement signal.
10 Incubation time Resulting PA activity (.DELTA. mIU/min) (min)
"FVII activator" (200 .mu.g/ml) 1 10.2 2 16.8 5 38.8 10 60.2 20
73.3 B. Dependence on the concentration of the "FVII activator"
(incubation time: 20 min, at 37.degree. C.), stimulant: heparin
(100 IU/ml) "FVII activator" (.mu.g/ml) PA activity (.DELTA.
mIU/min) 50 33.6 100 51.0 200 71.9 C. Stimulants (period of
incubation: 20 min, at 37.degree. C.) Stimulant PA activity
(.DELTA. mIU/min) None 5.9 CaCl.sub.2 25.3 Heparin 63.8
[0140] The tables demonstrate that sctPA was also activated in a
manner which was dependent on the concentration of the protease and
on the incubation time. Both heparin and calcium ions had a
stimulatory effect on the PA-activating ability of the "FVII
activator".
EXAMPLE 8
[0141] Two FVIII-containing solutions, one of which was essentially
free of von Willebrand Factor while the other contained vWF, were
incubated with the abovementioned protease in the presence of
calcium. After predetermined times, the residual FVIII activities
were determined by means of a chromogenic test and related to the
control assays without protease.
[0142] For this, 25 .mu.l of a solution containing 0.1 IU of
FVIII/ml were treated with the same volume of the protease solution
(10 .mu.g/ml) and the whole was mixed with 25 .mu.l of CaCl.sub.2
(25 mM). After incubation periods of 0, 5, 10 and 20 min at
37.degree. C., the assays were in each case treated with 400 .mu.l
of a solution containing 200 KIU of aprotinin/ml in order to stop
the proteolytic activity of the protease. Preliminary experiments
had shown that this concentration of aprotinin had no significant
interfering effect on the FVIII activity test described below
(assays 1+3). In assay 2, the protease was incubated with aprotinin
prior to contact with FVIII, after which the procedure was as
described above.
[0143] In each case, 50 .mu.l of the stopped sample (or after
further dilution) were then treated with the so-called factor
reagent, essentially composed of FIXa, FX and a thrombin inhibitor,
and incubated at 37.degree. C. for 10 min. Following the addition
of 50 .mu.l of a chromogenic substrate which is cleaved by
activated FX, the reaction was stopped after 5 minutes of
incubation by adding 50 .mu.l of acetic acid (50%); the
.DELTA.OD.sub.405 nm was then measured. The FVIII activity (mlU)
was ascertained with the aid of a standard curve which was
constructed using a dilution series which was prepared from the
FVIII concentrate and which was included in the test.
[0144] The FVIII activities are given in percentages of the
controls to which protease was not added.
[0145] Results:
11 FVIII activity (%) Incubatin period (min) Assay 0 5 10 20 1.
FVIII 97 27 11 <1 2. FVIII/aprotinin 98 97 97 96 3. FVIII/vWF 98
16 14 1
[0146] In the presence of CaCl.sub.2 (in this case 6.25 mM), FVIII
was inactivated by the protease in a manner which was dependent on
the length of the incubation. The vWF did not protect the FVIII
from inactivation by the protease. Inhibition of the protease with
aprotinin prior to contact with FVIII prevented the latter from
being inactivated.
EXAMPLE 9
[0147] This experimental series was carried out as described in
example 1/assay 1, but in this case the concentrations of calcium
in the mixtures of protease and FVIII were varied. For this,
CaCl.sub.2 was added, from the stock solution of calcium, up to the
final concentrations shown in FIG. 3.
[0148] Results:
[0149] If the concentration of calcium in the assay is decreased
below 1 mM, approx. 50% of the FVIII is then spared under these
conditions. Below 0.5 mM calcium, the percentage spared is more
than 60% (FIG. 1).
EXAMPLE 10
[0150] The influence of the "FVII activator" on the clotting times
in so-called global tests was investigated by means of
thromboelastography.
[0151] The change in the shear elasticity or the strength of the
relevant blood clot was recorded continuously using a Hilgard TEG
meter (from Hellige). The so-called r and k values are,
respectively, the times from the beginning of blood withdrawal and
from the start of the clotting reaction, and, in the case of
citrate blood plasma, the time of recalcification until the TEG
curve has been broadened by 1 mm and the time from the endpoint of
the r value until the curve has been broadened to 20 mm (clot
formation time).
[0152] For this, aliquots of 150 .mu.l of blood or plasma from 5
donors were in each case incubated in the measuring cuvettes at
37.degree. C. for 2 min, after which 50 .mu.l of sample (protease)
were mixed in. The reaction was started by adding 100 .mu.l of 25
mM CaCl.sub.2. The final concentration of the "FVII activator" in
the assay was 15 .mu.g/ml. The shortening of the r time was
measured in relation to the assay which contained buffer instead of
the sample.
[0153] Results:
12 r time k time r + k time Blood No. Sample (min) (min) (min) 1
Protease 5.2 3.4 8.6 1 Buffer 7.8 5.6 13.4 2 Protease 5.2 5.1 10.3
2 Buffer 6.8 7.1 13.9 3 Protease 4.0 5.2 9.2 3 Buffer 6.5 6.3 12.8
4 Protease 4.5 4.8 9.3 4 Buffer 4.8 6.0 10.8 5 Protease 4.2 3.8 8.0
5 Buffer 7.0 5.8 12.8 Plasma No. Sample r time (min) 1 Protease 9.0
1 Buffer 11.3 2 Protease 9.2 2 Buffer 12.5 3 Protease 9.5 3 Buffer
9.6 4 Protease 8.2 4 Buffer 12.1 5 Protease 9.7 5 Buffer 14.1
[0154] This example makes clear that, in almost all cases, addition
of the protease resulted in a marked shortening of the clotting
time. In this present instance, the fibrinolytic properties of the
"FVII activator" receded into the background. A reason for this is
that in "normal subjects", the concentrations of plasminogen
activator in the plasma lie in the nanogram region and do not have
any effect in the in-vitro clotting test.
EXAMPLE 11
[0155] The FVIII-bypassing activity of the protease was
demonstrated by the following experimental assay:
thronmboelastography was used as the measuring technique. The r
time was evaluated (see Example 10). A sample of whole blood was
incubated with a monoclonal antibody, whose
FVIII-activity-inhibiting properties were known, in order to
simulate the presence of a naturally occurring FVIII inhibitor
(antibody against FVIII). This sample was compared with the whole
blood sample control (buffer instead of Mab). The FEIB activity of
the protease was tested by adding the protease (final concentration
17 .mu.g/ml) to the whole blood sample which had been inhibited by
the Mab. Protease was added to a further sample, and the effect of
the protease, on its own, on the r time was determined.
[0156] Results:
13 r time Whole blood control 8.0 Whole blood + mAb 11.0 Whole
blood + mAb + protease 8.0 Whole blood + protease 3.5
[0157] The lengthening of the r time, caused by the anti-FVIII mAb,
was normalized once again by the presence of the protease, thereby
illustrating the FEIB activity of the protease. On its own, the
protease shortened the clotting time, as already demonstrated
above.
EXAMPLE 12
[0158] The following substances were added to a solution, which
contained 50 .mu.g of the FVII-activating protease/ml, to give the
corresponding final concentrations:
[0159] 25 mM Na citrate
[0160] 25 mM HEPES
[0161] 100 mM arginine
[0162] 0.75 g of sucrose/ml
[0163] The solution was divided into portions and the aliquots were
in each case adjusted to different pH values of from 5.0 to 8.6 and
then heated at 60.degree. C. for 10 hours.
[0164] The activities of the heated protease solutions were
determined in a chromogenic test, with the time-dependent
amidolysis of the chromogenic substrate S2288
(H-D-lle-Pro-Arg-pHA.times.2 HCl, Chromogenix AB, Sweden) being
recorded. This activity was expressed as a percentage of the
aliquots which were unheated and were measured in parallel:
[0165] Results:
14 Assay Activity (%) Starting material 100 pH 5.0 76 pH 5.5 65 pH
6.1 81 pH 6.5 50 pH 7.1 43 pH 7.5 46 pH 8.1 46 pH 8.6 32
[0166] This series of experiments makes clear that the
stabilization, particularly in the acid pH range, has markedly
reduced the inactivation of the protease. The slight "breakthrough"
at pH 5.5 can be explained by the fact that the isoelectric point
of the protease is in this range. Na citrate prevents a loss of
activity of >50% occurring in the preferred pH range.
EXAMPLE 13
[0167] The assay at pH 6.1 (Example 1) showed the best
stabilization of the protease. Accordingly, different additives
were tested at pH 6.0 and evaluated as described in Example 1:
[0168] The following final concentrations were set, with the
concentration of the protease being 50 .mu.g/ml:
[0169] 50 mM Na citrate/50 mM NaCl, pH 6.0
[0170] 0.75 g of sucrose/ml
[0171] 100 mM glycine
[0172] 100 mM arginine
[0173] Results:
15 Assay Activity (%) Starting material 100 Na citrate/NaCl 54 Na
citrate/NaCl/sucrose 85 Na citrate/NaCl/sucrose/glycine 92 Na
citrate/NaCl/sucrose/arginine 97
[0174] Marked stabilization of the protease was demonstrated by
adding sucrose and in each case one amino acid.
Sequence CWU 1
1
6 1 11 PRT Unknown Description of Unknown Organism Illustrative
peptide 1 Ile Tyr Gly Gly Phe Lys Ser Thr Ala Gly Lys 1 5 10 2 10
PRT Unknown Description of Unknown Organism Illustrative peptide 2
Ile Tyr Gly Gly Phe Lys Ser Thr Ala Gly 1 5 10 3 12 PRT Unknown
Description of Unknown Organism Illustrative peptide 3 Ile Tyr Gly
Gly Phe Lys Ser Thr Ala Gly Lys His 1 5 10 4 7 PRT Unknown
Description of Unknown Organism Illustrative peptide 4 Leu Leu Glu
Ser Leu Asp Pro 1 5 5 4 PRT Unknown Description of Unknown Organism
Illustrative peptide 5 Ser Leu Asp Pro 1 6 12 PRT Unknown
Description of Unknown Organism Illustrative peptide 6 Ser Leu Leu
Glu Ser Leu Asp Pro Trp Thr Pro Asp 1 5 10
* * * * *