Note: Descriptions are shown in the official language in which they were submitted.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
"Plasminogen activators having a reduced capacity to
bind lysine"
The invention relates to an advantageous use of
plasminogen activators and claims the priority of
German patent application 103 42 518.7, the content of
which is hereby incorporated by reference.
The establishment of thrombolysis as a therapy option
in connection with various thromboembolic diseases is
regarded as having been largely concluded. The main
interest in thrombolysis research is therefore directed
toward the further development and modification of
known thrombolytics and/or the improvement of
concomitant therapies.
A thrombolytic which is important for taking as a
starting point for developing new thrombolytics is
tissue plasminogen activator (t-PA), which has a higher
fibrin selectivity and a superior activity in
comparison with streptokinase or urokinase. The
deletion mutants reteplase and lanoteplase, as well as
tenecteplase, are further developments of this
plasminogen activator.
Tissue plasminogen activator (rt-PA) is a single-chain
glycoprotein composed of 527 amino acids. The molecule
which is initially single-chain (sct-PA) is cleaved by
proteolysis into a two-chain form (tct-PA). t-PA
possesses defined structural and functional domains.
Thus, the N-terminal constituent chain comprises the
finger domain (F, Serl-Lys49), the epidermal growth
factor domain (E, Ser50-Asp87) and two kringle domains
(K1, Thr88-Glyl76; and K2, Asnl77-Cys261). The C-
terminal chain, which includes a serine proteinase
domain (P), comprises amino acids Ser262 to Pro527. By
means of the amino acids His322, Asp371 and Ser478, the
C-terminal chain forms the active center.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 2 -
t-PA and its recombinant variants, and their
preparation, are the subject, for example, of US patent
4,766,075 and a large number of publications (e. g. Bode
and Renatus: Tissue-type plasminogen activator:
variants and crystal/solution structures demarcate
structural determinants of function, Current Opinion in
Structural Biology 1997, 7: 865-872) . A diagram of the
structure of t-PA is enclosed as Fig. 1.
Like most other trypsin-like serine proteinases, sct-PA
is converted into the tct-PA form by cleavage. The
cleavage takes place at the bond between Arg275 and
I1e276. After that, the two chains are then only held
together by way of a single disulfide bridge between
serine 264 and serine 395.
This numbering of the t-PA cleavage site corresponds to
that chosen by Bode and Renatus (loc. city. However,
other authors use a different numbering as a basis and
define the cleavage or activation site as R15-I16 or
8310-I311. However, the sites which are defined in this
way do not differ functionally or structurally.
Tissue plasminogen activator (t-PA; alteplase) is able
to activate plasminogen, which is transformed into
plasmin. However, it is evident from kinetic constants
that t-PA is only able to activate circulating
plasminogen weakly. The two t-PA forms, i.e. both the
single-chain molecule and the two-chain molecule,
exhibit what are in principle the same pharmacological
properties. However, fibrin-bound plasminogen is
activated with a catalytic potency which is three
powers of ten higher than that with which free
plasminogen is activated. Accordingly, the thrombolytic
property of tissue plasminogen activator is very
greatly augmented by the presence of fibrin.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 3 -
A factor of from 500 to 1000 is reported for the
relative fibrin selectivity of t-PA. The catalytic
effect can also be increased by interaction of
plasminogen activator with beta-amyloid or fibrinogen.
The ability of t-PA to be activated by fibrinogen is
comparable to its ability to be activated by beta-
amyloid.
The effect of plasminogen activator is controlled
physiologically by inhibitors, with plasminogen
activator inhibitor I (PAI-1) being the important
antagonist. The binding of PAI-1 to the light
constituent chain of the t-PA molecule alters the
structure of the catalytic center such that an
activation reaction with plasminogen can no longer take
place (Bennet WF, Paoni NF, Keyt BA, Botstein D,
Jones AJS, Presta L, Wurm FM, Zoller M: High resolution
analysis of functional determinants in human tissue
type plasminogen activator. Journal of Biological
Chemistry 1991; 266: 5191-5201).
Tissue plasminogen activator is metabolized rapidly via
the liver. Hepatic insufficiency in a patient therefore
prolongs the plasma half-life of the substance
(Emeis JJ, van den Hoogen CM, Jense D: Hepatic
clearance of tissue-type plasminogen activator in rats,
Thomb Haemostas 1985; 54: 661-664; Tiefenbrunn AJ,
Robison AK, Kurnik PB, Ludbrook PA, Sobel BE. Clinical
pharmacology in patients with evolving myocardial
infarction of tissue plasminogen activator produced by
recombinant DNA technology Circulation 1985; 71:
110-116) .
Plasminogen activators are developed for treating
thrombotic diseases such as cardiac infarction and
stroke. t-PA is currently the only thrombolytic which
is approved by the Food and Drug Administration (FDA)
in the USA for treating stroke.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 4 -
Nevertheless, suspicions have increased in the past
that, while t-PA on the one hand exhibits the expected
positive thrombolytic effects in connection with
treating the stroke, it is on the other hand also
responsible for an undesirable destruction of tissue.
Thus, infusing t-PA into t-PA-deficient mice gave rise
to larger infarctions (Wang YF, Tsirka SE,
Strickland S, Stieg PE, Soriano SG, Lipton SA: Tissue
plasminogen activator (tPA) increases neuronal damage
after focal cerebral ischemia in wild-type and tPA-
deficient mice. Nat Med 1998; 4 (2) : 228-231) . It is
suspected that these enlarged lesions are due to
stimulation of the NMDA-dependent glutamate receptors
(Liberatore GT, Samson A, Bladin C, Schleuning WD,
Medcalf RL: Vampire bat salivary plasminogen activator
(desmoteplase): a unique fibrinolytic enzyme that does
not promote neurodegeneration. Stroke 2003; 34(2):
537-543). A proteolytic cleavage of the NMDA receptor
by t-PA could be responsible for this effect (Nicole O,
Docagne F, Ali C, Margaill I, Carmeliet P, MacKenzie
ET, et al: The proteolytic activity of tissue
plasminogen activator enhances NMDA receptor-mediated
signaling. Nat Med 2001; 7(1): 59-64).
The object of the present invention is therefore to
provide a novel therapeutic treatment of thrombotic
diseases, in particular of stroke.
This object is achieved by using a plasminogen-
activating factor which exhibits a capacity to bind
lysine which is reduced as compared with that of the
native plasminogen activator. In a particularly
advantageous embodiment, the plasminogen activator
exhibits a modified kringle domain, preferably a
kringle 2 domain which is modified as compared with
that of the native t-PA. This latter domain can be
entirely or partially deleted such that the capacity to
bind lysine is reduced.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 5 -
The K2 domain, or regions which are functionally or
structurally essentially homologous to it, can
advantageously be modified such that lysine residues no
longer bind or only bind with low affinity.
In a particularly advantageous embodiment, the
plasminogen activator which can be employed in
accordance with the invention is modified t-PA.
The significance of the K2 domain, including the
preparation of t-PA K2 deletion mutants, is described
in detail in Horrevoets AJ, Smilde A, de Vries C,
Pannekoek H (The specific roles of finger and kringle 2
domains of tissue-type plasminogen activator during in
vitro fibrinolysis: Journal of Biological Chemistry,
269, 17, 12639-12644, 1994).
Because their ability to bind lysine is lacking or
reduced, the plasminogen-activating factors which can
be used in accordance with the invention exhibit an
elevated fibrin selectivity and a reduced ability to be
activated by fibrinogen or beta-amyloid. This reduced
ability to be activated by fibrinogen is probably the
cause of the fibrin selectivity. The possession by the
plasminogen activator of a high fibrin selectivity is
of considerable importance in connection with treating
stroke, in particular, since, for example, the native
t-PA can be activated by the fibrinogen which overcomes
the damaged blood-brain barrier and then stimulates
glutamate-mediated excitotoxicity by subsequently
activating the NMDA receptors. Accordingly, the
reduction, in accordance with the invention, of the
ability of the plasminogen activator to be activated by
fibrinogen also leads to a reduction in neurotoxicity.
The importance of the lysine-binding sites on the t-PA
kringle 2 became clear, in particular, as a result of
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 6 -
comparative investigations of the structural and
functional domains of t-PA as compared with DSPA. DSPA
is a plasminogen activator which was originally
isolated from vampire bat saliva (in this regard, see
US patent 6,008,019; EP 0 383 417). DSPA can be
isolated in four isoforms, of which DSPAalphal can be
prepared recombinantly using CHO cells.
In contrast to t-PA, DSPA only has one kringle domain.
This domain corresponds functionally and structurally
more closely to the t-PA K1 domain than to the K2
domain and does not possess any lysine-binding sites
(Bringmann P, Gruber D, Liese A, Toschi L,
Kratzschmar J, Schleuning WD, Dormer P: Structural
features mediating fibrin selectivity of vampire bat
plasminogen activators; Journal of Biological Chemistry
1995; 270(43): 25596-25603). There are therefore some
statements in the literature to the effect that DSPA
does not possess any kringle 2 domain.
Furthermore, DSPA is always present as a single-chain
molecule since a plasmin activation site as in t-PA is
lacking. As compared with t-PA, the activity of DSPA is
stimulated about 45 000 times in the presence of fibrin
whereas, according to Gardell SJ, Duong LT, Diehl,
York JD, Hare TR, Register RB, Jacobs JW, Dixon RA,
Friedman PA (Isolation, characterization and c-DNA
cloning of a vampire bat salivary plasminogen
activator: Journal of Biological Chemistry 1989;
264(30): 17947-17952) this value is 205.
A diagram of the structure of DSPA is enclosed as
Fig. 2. Fig. 2b shows a comparison of the amino acid
sequence of t-PA with that of DSPA (SEQ ID Nos. 1+2).
In the past, the ability of t-PA to bind fibrin was
attributed functionally to the finger domain and
kringle 2 (van Zonnenfeld AJ, Veerman H, Pannekoek H:
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
_ 7 -
(1986) Proc. Natl. Acad. Sci. USA., 83: 4670-4674).
However, more recent publications also suggest that the
proteinase domain P may have a certain relevance in
this regard (Bennett loc. cit.). The functions of the
individual domains have also been investigated by
Bakker AHF, Jacoline E., Weening-Verhoeffet. D.,
Verheijen JH (The role of Lysyl-Binding site of tissue
type plasminogen activator in the interaction with a
forming fibrin clod: Journal of Biological Chemistry
270, 21, 12355-12360, 1995).
Bakker et al. prepared various modifications of t-PA,
for example two deletion mutants which lack either the
kringle 2, the finger domain or the epidermal growth
factor domain. These mutations were also combined and
some of them were additionally provided with a point
mutation in the kringle 2, namely involving the
substitution D236N. This selective amino acid
substitution leads to the replacement of the Asp in
position 236 with asparagine and thereby to the
deletion of the lysine-binding site (LBS in the K2
domain). For the preparation of the mutation, the
reader is expressly referred to the abovementioned
publication by Bakker et al including the references
which are cited therein.
In their investigations, Bakker et al. demonstrated
that occupation of the lysine-binding site in the K2
domain by EACA (s-aminocaproic acid) markedly
attenuated the binding of the native t-PA to fibrin.
The same applied to the modification of the LBS by the
D236N substitution except that the attenuation was
less. Even the deletion mutant, which consisted solely
of the K2 domain and the proteolytic C terminus, still
bound to fibrin if only to a slight degree. It was only
the K2P mutation, in which the LBS was deleted, which
no longer exhibited any fibrin binding.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
g -
While these results demonstrate, on the one hand,
the importance of the F and K2 domains as well as the
LBS in K2, they also demonstrate that the interaction
of t-PA of fibrin is not mediated exclusively by the F
and K2 domains. Rather, there is also the function of
the LBS in the K2 domain, with the LBS presumably being
responsible for stabilizing a confirmation of the t-PA
which is favorable for binding fibrin.
The importance of the K2 domain including its lysine-
binding site has also been made clear by investigations
carried out by Stewart RJ, Fredenburgh JG and Witz JI
(Characterization of the interactions of plasminogen
and tissue and vampire bat plasminogen activators with
fibrinogen, fibrin, and the complex of D-dimer
noncovalently linked to fragment E: Journal of
Biological Chemistry 273, 29, 18292-18299, 1998).
In binding studies, Stewart et al. investigated, inter
alia, the affinities of t-PA and DSPA for fibrin and
fibrinogen. In the studies, the affinities were
investigated in the presence or absence of the lysine
analog EACA in order to analyze the importance of the
kringle-dependent interactions. They concluded, from
their investigations, that DSPA is unable to bind to
fibrinogen because of the lack of the lysine-binding
sites in the kringle. They therefore attribute a
function, which is essential for the ability of t-PA to
bind fibrin, to LBS in the t-PA kringle 2 in
combination with the finger domain.
The experimental results reported in Example 1 confirm
the different affinities of DSPAalphal and recombinant
human t-PA for the cofactors beta-amyloid (1-42),
fibrinogen and fibrin. To obtain these data, the
kinetic parameters kcat and Km, and the kcat/Km ratio,
were determined for each plasminogen activator/cofactor
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 9 -
combination. In the absence of a cofactor, the
efficiency of DSPAalphal for plasminogen activation was
about 100 times lower than that of t-PA whereas the two
compounds were equally efficient in the presence of
fibrin. DSPAalphal was about 30 times less effective
than t-PA in the presence of the fibrinogen or beta-
amyloid cofactor. At a physiological plasminogen
concentration of 2 ~M, these data give a ratio for the
efficiency of fibrin as cofactor to that of fibrinogen
or beta-amyloid as cofactor to be 480 in the case of
DSPAalphal and 16 in the case of human t-PA.
As has already been explained above, the plasminogen-
activating factor which is employed in accordance with
the invention is characterized by the fact that its
kringle 2 lysine-binding site is either absent or
modified. In one embodiment, the kringle 2 is deleted.
However, it is also possible, in another embodiment, to
retain the kringle 2 and to substitute the aspartic
acid at position 236 with asparagine. Accordingly, it
is not crucial for the invention, according to the
invention, of the modified t-PA for the kringle
structure to be lacking but only for the lysine-binding
site to be modified such that an interaction, which
increases the activity of the t-PA, with a cofactor is
no longer possible.
In another advantageous embodiment, the plasminogen
activator, in particular the modified t-PA, which is
used can be modified by the deletion of the kringle 1
such that the t-PA is no longer able to bind receptor.
As a result, the tissue plasminogen activator is no
longer metabolized in the native manner via the liver,
resulting in the in-vivo half-life being prolonged
(Rijken DC, Otter M, Kuiper J, von Berkel TJC:
Receptor-mediated endocytosis of tissue-type
plasminogen activator (t-PA) by liver cells. Thromb.
Res . 1990 ; Supel . X : 63 -71 ) . Deletion of the kringle 1
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 10 -
is known from reteplase and described, for example, by
Martin U, Bader R, Bohm E, Kohnert U, von Mollendorf E,
Fischer S, Sponder G (BM 06.022: A Novel recombinant
plasminogen activator. Cardiovascular drug reviews
1993; 11: 299-311). For this, use is made of mutants in
which amino acids 4-176 are deleted. Prolonging the
half-life makes it possible to administer the
plasminogen activator as a bolus.
In a preferred embodiment, the plasminogen activator
which can be used in accordance with the invention is
based on one of the amino acid sequences shown in
Figures 3, 11 or 13. Each of these amino acid sequences
constitutes a modified t-PA in which the kringle 2 is
deleted and a structural change has been made in the
region of the sequence for the t-PA activation cleavage
site. This change can either only consist in the
replacement of amino acids R and I in the activation
cleavage site (e.g. by HS; see sequence SEQ ID No. 3,
Fig. 3) or additionally affect adjacent amino acids
(e.g. replacement of FRIK with LHST in the sequence SEQ
ID No. 4, Fig. 11). The latter modification corresponds
to the structure of native DSPA at this point.
According to the invention, preference is given to
producing, and employing for stroke treatment, a
plasminogen activator which, particularly suitably,
combines the advantages of native t-PA, namely, in
particular, low immunogenicity when used in human
patients, with the advantages of DSPA, namely the lack of
neurotoxicity. In one embodiment of the invention, the
deletion of the t-PA kringle 2 is accordingly selected
such that a transition region to the downstream structure
is created, which transition region corresponds to the
comparable structure in DSPA. The transition region
between the remaining kringle 1 domain of the modified
t-PA and the downstream cysteine bridge is accordingly
advantageously formed by the sequence SKAT. In DSPA,
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 11 -
the sequence SKAT is located between the kringle and
the cysteine bridge. This advantageous structure is
realized, for example, in the sequence SEQ ID No. 4 as
shown in Fig. 11.
Fig. 12 (Multiple Sequence Alignment) shows a
comparison between t-PA and the two described
embodiments in accordance with the invention.
In another embodiment according to the invention, use
is made of a plasminogen activator as depicted in
Figure 13 (SEQ ID No. 5).
It is naturally also possible, in accordance with the
invention to use proteins which possess sequences which
are homologous with, or partially identical to, the
amino acid sequences depicted in Figures 3, 11 and 13.
Preference is given to homologies or identities of at
least 70, preferably between 80 and 95%. These
homologous or identical proteins exhibit the activity
of a plasminogen-activating factor (preferably
displayed as the release of pNA) and cause a blood clot
to the lyse in vitro (see Example 3).
The plasminogen activator which can be used in
accordance with the invention can be characterized by
the lack of the finger and epidermal growth factor
domains. This deletion substantially reduces binding to
the liver receptors and once again prolongs the half-
life (Larson GR, Timony GA, Horgan PG, Barone KM,
Henson KS, Angus LB, Stoudemire JB: Protein engineering
of novel plasminogen activators with increased
thrombolytic potency in rabbits relative to activase.
Journal of biological Chemistry 1991; 266: 8156-8161;
Smalling RW: Molecular biology of plasminogen
activators: what are the clinical implications of drug
design? Am J Cardiol. 1996; 78 (suppl. 12): 2-7).
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 12 -
Aside from deletion mutants, the plasminogen activators
which are employed in accordance with the invention can
also be only punctately modified by means of site-
directed mutagenesis. Thus, it is known, for example,
from rt-PA-TNK (tenecteplase) that three mutations at
the binding site for the plasminogen activator
inhibitor, on the one hand, and at the binding site by
which t-PA binds to liver cells, on the other hand,
lead to the catalytic activity being increased while,
at the same time, the ability to be inactivated by PAI
is decreased. This thereby gives tenecteplase a
catalytic conversion constant Kcat/Km which is
increased 100-fold (in this regard, see also Paoni NF,
Keyt BA, Refino CJ, Chow AM, Nguyen HV, Berleau LT,
Badillo J, Pena LC, Brady K, Wurm FM, Ogez J,
Bennett WF: A slow clearing fibrin-specific, PAI
resistant variant of t-PA (T103N, KHRR 296-299 AAAA).
Thromb Haemostas 1993; 70: 307-312 and Cannon CP,
Love TW, McCabe CH, Kirshenbaum JM, Henry T, Sequira R,
Schweifer M, Breed J, Cutler D, Tracy R, for the TIMI
investigators. TNK-tissue plasminogen activators in
myocardial infarction (TIMI) 10: Results of the initial
patients in the TIMI 10 pilot - a phase l,
pharmacokinetics trial. Circulation 1995; 92 (suppl.):
1-415) .
EXAMPLE 1:
1. Comparative analysis of the efficiencies of beta-
amyloid (1 to 42), fibrinogen and fibrin as
cofactors for plasminogen activation by DSPAalphal
and recombinant human t-PA.
Materials and methods
The following substances were used:
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
13
- DSPAalphal (prepared by Paion, batch No. 2DSA01,
12 December 2002, sample 10). 10 mg of the
substance were dissolved in 10 ml of sterile water
in order to obtain a final concentration of
1 mg/ml.
- Recombinant t-PA (Actilyse~, prepared by Paion,
batch No. 102572). 10 mg were dissolved in 10 ml
of sterile water to give a final concentration of
1 mg/ml.
- Human Glu-plasminogen, purified from human plasma
(prepared by Paion) and dissolved in PCLA buffer
at a concentration of 25 ~M.
- Human fibrinogen, essentially plasminogen-free,
was obtained from Sigma (catalog No. F4883, batch
12K7620). 25 mg were dissolved in 25 ml of PCLA
buffer to give a final concentration of 1 mg/ml.
- Human thrombin (Sigma; catalog No. T7009. Batch
61K7603). 100 U were dissolved in 10 ml of PCLA
buffer to give a concentration of 10 U/ml.
- Flavigen.pli color reagent (D-but-CHT-Lys-p-
nitroaniline-DHCL) from Biopool (catalog
No. 101353, batch 1512016). 100 ~M were dissolved
in 50 ml of PLCR buffer to give a final
concentration of 2 millimolar.
- beta-Amyloid protein (1-42) from Bachem (catalog
No. H-1368, batch 0535120). 4 mg were dissolved in
4 ml of 0.1 percent ammonium hydroxide to give a
final concentration of 1 mg/ml.
All reagents were divided into aliquots and stored at
-20°C for no more than two weeks.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 14 -
PLC Buffer (Jones 1990): 0.1 M NaC1.2H20 (Acros catalog
No. 20779); 0.03 M NaHC03 (Acros catalog No. 21712);
4 mM KCl (Fluka, catalog No. 60130); 1 mM CaC12.2H20
(Acros, catalog No. 207780); 1 mM Na2HP04.2H20 (Fluka,
catalog No. 71638); 0.3 mM MgC12.6H20 (Acros, catalog
No. 197530); 0.4 mM MgS04.7H20 (Fluka, catalog
No. 63140); 20 mM HEPES (Applichem, catalog No. A1969);
0.01% Polysorbate 80 (Fluka, catalog No. 93781).
Plasminogen activation assay:
The assay for plasminogen activation was carried out on
microtiter plates at a total volume of 0.15 ml, as
described by Bringmann et al. (loc. cit.). The reagents
were added as follows:
50 ~l of plasminogen (0-24 ~M); 15 ~1 of cofactor
(1 mg/ml); 10 ~l of plasminogen activator 7.5 nM and
75 ~l of Flavigen (2 mM) .
The final concentrations were as follows:
plasminogen activator: (t-PA or DSPAal): 0.5 nM; Glu
plasminogen; 0.0625, 0.125, 0.25, 0.5, 1, 2, 4 and
8 ~M; FlavigenPli: 1 mM and cofactors: 100 ~g/ml.
The cofactors beta-amyloid (1-42), fibrinogen and
fibrin were compared with the control without cofactor.
For the control, the reaction mixture contained a
further 0.13 units of human thrombin/ml.
Directly after the addition, the microtiter plate was
introduced, at 25°C, into a molecular device ThermoMax
microplate reader.
Stirring was carried out from time t - 0.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 15 -
The optical density at 405 nm and 490 nm was measured
at regular intervals. The optical densities at 490 nm
were subtracted from the optical densities at 405 nm in
order to eliminate the differences due to the movement
of the liquid. All the experiments were carried out in
three repeats using each plasminogen concentration and
plasminogen activator concentration.
The Km and kcat values were determined (Figs. 4-6).
Figures 4
a) Determining the molar extinction of the pNA.
b) Conversion of the Flavigen.pli by 10 nM plasmin as
a function of the Flavigen.pli concentration (in
~M ) .
c) Michaelis-Menten plot of the conversion of
Flavigen by 10 nM plasmin. The initial rates from
Fig. 4b were converted into pNA/s.
Figures 5
Curves of the optical density against the time for four
different combinations of plasminogen activators and
cofactors. Plasminogen concentrations of 8; 4; 2; l;
0.5; 0.25; 0.125 and 0.0625 ~M were used for this
purpose.
a) t-PA without cofactor
b) t-PA with beta-amyloid (1-42) as cofactor
c) t-PA with fibrinogen as cofactor
d) t-PA with fibrin as cofactor
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 16 -
e) DSPAalphal without cofactor
f) DSPAalphal with beta-amyloid (1-42) as cofactor
g) DSPAalphal with fibrinogen as cofactor
h) DSPAalphal with fibrin as cofactor
Figures 6
Plot of a ~= ks.kcat.PA.E.P./(Km + P)} against the
plasminogen concentration
a) Plots for t-PA without cofactor or beta-amyloid,
fibrinogen or fibrin as cofactor
b) Plots for DSPAalphal11.38 without cofactors
Results:
Table 1
kcat ) Km(~M) kcat/Km
(s-1
t-PActrl n.d. n.d. 1272 224
t-PAamyl n.d. n.d. 9307 579
t-PAfbg n.d. n.d. 10204 1546
t-PAfibrin 0.351 0.35 0.178 0.017 1 990 000 327
000
DSPActrl n.d. n.d. 12.5 0.9
DSPAfbg 1.649 0.110e-32.465 0.426 690 181
DSPAamyl 0.957 0.199e-30.853 0.316 1304 785
DSPAfibrin 0.532 0.136 1.098 0.382 506 000 120 000
Table l: Values for kcat/Km for the activation of
plasminogen by recombinant human T-PA or DSPAalphal
using beta-amyloid (1 to 42), fibrinogen or fibrin as
cofactors.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 17 -
The results are given as the mean ~ standard deviation
of three independent experiments.
It was not possible to determine the precise values for
kcat and Km since the Km values were higher than the
maximum plasminogen concentration (8 ~M) which it was
possible to use.
The derivation of the kinetic constants is not on its
own sufficient for comparing the efficiencies of the
different cofactors since the reaction rates at
plasminogen concentrations close to or below the Km
values are dependent on the plasminogen concentration.
For this reason, the reaction rates are calculated,
using Michaelis-Menten kinetics, at physiological
plasma plasminogen concentrations at 2 ~M. The relative
efficiencies of the different cofactors for plasminogen
activation by t-PA and DSPAalphal (Table 2) are
determined on this basis.
Table 2
cofactor DSPAal t-PA
none 1 101.8 17.9
DSPAamyloid 27.3 7.7 744.6 23.6
DSPAfbg 29.8 5.0 816.3 123.7
DSPAfibrin 13 560 2360 12 880 1360
Relative rates of plasminogen activation at the
physiological plasminogen concentration of 2 ~M, as
calculated using the kinetic parameters in Table 1. The
combination of the DSPA control is 1.
EXAMPLE 2:
The prion diseases, i.e. the spongiform
encephalopathies, are fatal neurodegenerative phenomena
which are characterized by an accumulation of the
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 18 -
abnormal isoform of the prion protein (PrPs°) in amyloid
deposits. The normal prion protein (PrP°) is expressed
in many tissues, in particular in the brain, in which
it is concentrated in the synapses. The mechanism of
the change in the conformation of PrP° to give PrPs° is
still not well understood and nor is the mechanism
which leads to neurodegeneration by way of prps°
formation. A possible mechanism could lie in an effect
on PrPs° in connection with the activation of
plasminogen by t-PA.
We have already shown, using a recombinant protein,
that PrP can increase t-PA-catalyzed plasminogen
activation 300-fold (Ellis V, Daniels M, Misra R,
Brown DR (2000): Plasminogen activation is stimulated
by prion protein and regulated in a copper-dependent
manner, Biochemistry 41: 6891-6896).
The ability of PrP to stimulate plasminogen activation
was dependent on the conformation of the PrP, with the
conformation being influenced by the presence or
absence of Cu2+. The latter form contains a large number
of beta structures which correspond to the PrPs° isoform
and stimulate plasminogen activation. These data agreed
with earlier observations that, while plasminogen bound
to PrPs° which was isolated from scrapie-infected mouse
brains, it did not bind to PrP° as normal brain cells
(Fischer M, Roeckl C, Rarizek P, Schwarz HP, Aguzzi A
(2000): Binding of disease-associated prion protein to
plasminogen. Nature 408: 479-483).
Furthermore, our experiments have shown that the
mechanism of PrP-stimulated plasminogen activation also
encompasses the binding of t-PA to apoPrP (i.e. PrP
without Cu2+). The structures which are required for
this binding have not yet been evaluated in detail.
However, the binding is specific since it was prevented
by DFP-inactivated t-PA.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 19 -
Other methods:
The PrP batches which were used in these investigations
were in each case derived from recombinant murine PrP
isolated from E. coli. Following the purification of
the His-labeled PrP, the protein folds once again, in
the absence or presence of copper chloride, in order to
form either a bolo-PrP or an apo-PrP. Aggregated forms
of these proteins are in each case produced by rapid
dissolution in water. The protein was collected by
centrifugation. The material is resistant to proteinase
K digestion. A mutant 451-90 form, which does not
comprise the Cu2+ binding site, was prepared in the same
way. The effect of these different forms of PrP on
plasminogen activation by DSPA was investigated by
incubating the PrP (0-100 ~g/m) with Lys-plasminogen
(25 nm) and DSPA (0.25 nm). Plasminogen activation is
determined by hydrolysis of the plasmin-specific dye
H-d-Val-Leu-Lys-7-amido-4-methylcoumarin at 37°C using
the SPECTRAmax Gemini-lourescence microplate reader.
The controls of these experiments contain sc-t-PA, as a
positive control for the effect of the PrP on
plasminogen activation, and fibrin fragments, as a
positive control for stimulation of the t-PA and DSPA
activities.
The specificity of the plasminogen binding was
determined by competition with DFP-inactivated t-Pa.
The lysine analog EACA was also used as a competitive
inhibitor.
Results:
The results of the experiments are presented in Table 3
and Figs. 7 to 10.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 20 -
Fig. 7, in particular, provides informative insight.
This figure makes clear that PrP specifically only
activates t-PA and not DSPA. It was furthermore made
clear that the lysine-binding site of the kringle 2
must, as the essential difference between DSPA and
t-PA, be involved. Table 3
Reaction rates, M-ls-1
minus plus PrP + fibrin
PrP PrP Hep
PrP preparation #1
PrP preparation #2
Fold stimulation
PrP preparation #1
PrP preparation #2
EXAMPLE 3:
tPA 7200 1 800 000 2 000 000
DSPA 15.3 9.77 2 000 000
tPA 7020 624 000 2 912 000
DSPA 15.75 162.75 5 565
minus plus PrP + fibrin
PrP PrP Hep
tPA 1 250 2 777 778
DSPA 1 0.638562 130 719
tPA 1 88.88889 414.8148
DSPA 1 10.33333 353.3333
a. Preparing and purifying humanized DSPA (humDSPA)
(amino acid sequence as depicted in Fig. 11)
Recombinant human DSPA baculovirus DNA was prepared
using the Bac-to-Bac system (Invitrogen) and the
purified virus DNA was transfected into Sf9 insect
cells. The recombinant baculovirus which was produced
was amplified using Sf9 cells. HumDSAP was expressed
using High Five insect cells in serum-free medium in
suspension culture. The cell culture supernatant
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 21 -
containing the recombinant protein was frozen at -20°C
until it was subjected to further working up.
The cell culture supernatant was thawed slowly at RT on
a shaker and then centrifuged at 4000 x g for 1 hour.
The protein solution (1.5 1) was equilibrated to pH 6.0
with 50 mM ammonium acetate, loaded onto an SP-
sepharose XL (Amersham) ion exchange column (350 ml bed
volume) and washed with 5 column volumes of 50 mM
ammonium acetate solution. The bound protein was eluted
in a gradient of 0-100% 1M ammonium acetate, pH 6, over
10 column volumes.
HumDSPA-containing fractions were identified by Western
blotting; following protein determination, these
fractions were subjected to an activity test, frozen at
-80°C and subsequently lyophilized.
In contrast to the standard activity assay, 50 ~l of
eluate (= humDSPA-containing fraction), 50 ~l of 0.2 M
Tris, pH 8.0, and 100 ~l of 2 mM S-2288 in PBS were
used in this experiment.
b. Assay of DSPA activity
The activity assay employed is the determination of the
rate at which a plasminogen activator converts the
colorless substrate S-2288 into a colored product. The
assay is a standard method for determining the
proteolytic activity of plasminogen-activating factors.
The activity is visualized by determining the release
of a chromogen (p-nitroaniline, pNA) by the test
substance. The Chromogenix company offers the chromogen
S-2288 for this purpose.
In this assay, the rate of reaction is not determined
directly (e.g. in catal units); instead, comparison is
made with a standard which is defined as 100% activity.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 22 -
The reaction rate was measured in a buffer having the
following composition: 25 mM Tris-HCl, pH 8/0.1%
albumin/100 mM NaCl/l.l mM glycine/1.2 mM
mannitol/2.5 mM S-2288.
The measurement was carried out in a 96-well plate
using blank values, standards and samples containing
different concentrations of protein.
The increase with time in the absorption at 405 nm was
recorded using a photometer. The slope of the linear
portion of the curve was determined using Excel. This
gives the activity.
Fig. 14 shows the activities of the fractions
(expressed in S-2288 activity). The silver-stained gels
can be seen in Figs. 15a and 15b. Figs. 16a and 16b
show the Western blots. The protein detected in the
Western blot corresponds to the protein marked with the
arrowhead in the silver-stained gel.
[L = load; T=flow-through; W = wash; 0-40% B, A2, A3 ...
fractions]
EXAMPLE 4:
Clot lysis (thrombolytic activity)
The residue of the humDSPA-containing fraction B6 was
dissolved in 15 ml of PBS and the solution was
centrifuged at 4000 x g for 15 min. This fraction was
selected since it exhibited the highest purity. An
aliquot was removed and used for the activity
determination. The catalytic activity of this sample
was roughly comparable to the catalytic activity of
5 ~g of activase/ml.
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 23 -
The blood clots which were used were derived from a
normal blood sample taken 24 h earlier and were in each
case formed from 2 ml of blood which was transferred
into polypropylene tubes and coagulated under natural
conditions. The lysis was carried out in PBS.
The humDSPA-containing fraction B6 (from the SPXL-
sepharose capture step) was first of all lyophilized.
The residue was dissolved in 15 ml of PBS and
centrifuged at 4000 g for 15 min. A sample of this
solution was removed for an S-2288 activity
determination before the blood clot was added. The
catalytic activity of this sample corresponded
essentially to the catalytic activity of 5 ~g of
activase/ml.
Figures 17a to 17d show the results of the
chronological course of the clot lysis at 0 h, 3 h, 4 h
and 24 h after adding the humDSPA. The left-hand
experimental assay in each case shows the control. PBS
containing humDSPA in the above-described quantity is
present in the right-hand assays.
The blood cells slowly sediment out of the
disintegrating clot. A type of mesh, which can already
be discerned after 4 h, remains after 24 h. In the
assay, detached blood cells were aspirated after 4 h
using a syringe in order to view the structure of the
remaining clot.
Consideration has to be given to the fact that the
fibrinolysis slows down when the clot is no longer
hanging in its own lysate. This is the reason why any
clot at all is still present after 24 h.
It is not possible to quantify this reaction. However,
the humDSPA exhibits a marked increase in activity as
compared with native DSPA since at least 4 times the
CA 02537696 2006-03-02
Konig Szynka von Renesse 46 029 K
- 24 -
quantity of DSPA has to be used in order to dissolve a
clot of the same size.
