IE84069B1 - Recombinant thrombin receptor and related pharmaceuticals - Google Patents
Recombinant thrombin receptor and related pharmaceuticalsInfo
- Publication number
- IE84069B1 IE84069B1 IE1992/0530A IE920530A IE84069B1 IE 84069 B1 IE84069 B1 IE 84069B1 IE 1992/0530 A IE1992/0530 A IE 1992/0530A IE 920530 A IE920530 A IE 920530A IE 84069 B1 IE84069 B1 IE 84069B1
- Authority
- IE
- Ireland
- Prior art keywords
- thrombin
- cha
- receptor
- peptide
- amino acid
- Prior art date
Links
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- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- APFVFJFRJDLVQX-UHFFFAOYSA-N indium Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 230000002427 irreversible Effects 0.000 description 1
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 230000000670 limiting Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 108010068488 methionylphenylalanine Proteins 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- WXEHBUMAEPOYKP-UHFFFAOYSA-N methylsulfanylethane Chemical compound CCSC WXEHBUMAEPOYKP-UHFFFAOYSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000004433 nitrogen atoms Chemical group N* 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 230000003287 optical Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
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- 238000003615 platelet activation assay Methods 0.000 description 1
- 230000001402 polyadenylating Effects 0.000 description 1
- 108091008117 polyclonal antibodies Proteins 0.000 description 1
- 230000017363 positive regulation of growth Effects 0.000 description 1
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- 125000001235 proline group Chemical group [H]N1[C@@](C(=O)[*])([H])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
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Description
RECOMBINANT THROMBIN RECEPTOR AND RELATED
PHARMACEUTICALS
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
COR THERAPEUTICS, INC.
Technical Field
The invention relates to materials involved in
the control of the cardiovascular system, and in
particular to activities mediated by thrombin and its
cellular receptor. More specifically, it concerns
recombinant materials useful for production of the
thrombin receptor, use of the receptor as a diagnostic
tool, and therapeutic agents which either stimulate or
block thrombin receptor activation and diagnostic
compositions relevant to the receptor.
Background Art
Thrombin is a powerful factor in regulating the
state of the cardiovascular system. It is clear that
thrombin aids in the formation of blood clots by
catalyzing the conversion of fibrinogen to fibrin, which
is an integral part of most clots. In addition, thrombin
is known to act directly on cells in the blood and in the
interior blood vessel wall, and specifically to activate
platelets to form clots. Thrombin-induced platelet
activation is particularly important for arterial
thrombus formation, a process that causes myocardial
infarction and some forms of unstable angina and stroke.
In addition, thrombin promotes inflammation and other
cellular activities. Thrombin is chemotactic for
monocytes, mitogenic for lymphocytes, and causes
endothelial cells to express the neutrophil adhesive
protein GMP—l4O on their surfaces and inhibits the growth
of these cells. Thrombin elicits platelet—derived growth
factor from the endothelium and is a mitogen for
mesenchymal cells.
Because thrombin is capable of direct
activation of cells, it is assumed that at least one
thrombin receptor exists. However, it has not been
possible to detect the presence of thrombin receptor by
traditional binding studies, since thrombin is capable of
binding a large number of materials present on cells
which do not directly mediate the cellular responses to
thrombin, and thus the background levels of binding are
prohibitively high.
protease, if the receptor were proteolytically cleaved by
(Gronke, et al.,
et al.,
the binding sites identified by
since thrombin is a
the interaction with thrombin, the receptor's ability to
bind tightly to thrombin would be decreased. All of the
foregoing factors suggest that traditional binding
studies in an effort to find a thrombin receptor might
ultimately be unproductive.
While it has been assumed that a thrombin
receptor might exist, it has been unclear, even from the
studies conducted so far, whether proteolytic cleavage by
thrombin is involved in its receptor activation. When
thrombin is treated with reagents which covalently modify
and render it proteolytically inactive, its ability to
stimulate platelets is abolished (Berndt, M.C., et al.,
"Platelets in Biology and Pathology" (1981)
Elsevier/North Holland Biomedical Press, pp. 43-74;
Martin, B.M., et al., Biochemistry (1975) ;g:1308-1314;
Tollefsen, D.M., et al., J Biol Chem (1974) ggg:2646-
2651; Phillips, D.R , Thrombin Diath Haemorrh (1974)
;g:2o7-215; Workman, E.F., et al., J Biol Chem (1977)
Some of
thrombin-induced platelet activation and one modified
nonproteolytic thrombin which does block platelet
activation, D-phenylalanyl—L-prolyl—L-arginyl
chloromethyl ketone (PPACK) thrombin fails to bind
substrate. Thus, it cannot be concluded that a lack of
protease activity accounts for failure to activate
platelets.
The identification and characterization of the
thrombin receptor, as described herein, permits the
design of systems and substances which can regulate thrombosis in the cardiovascular
system. In addition, new diagnostic reagents for assessing cardiovascular status are
provided by this work.
Disclosure of the Invention
We describe methods and materials useful in the regulation of the cardiovascular
system in mammals. The isolation, recombinant production, and characterisation of the
thrombin receptor associated with surfaces of cells activated by thrombin permits effective
regulation of these functions. In accordance with the present invention, there is provided a
DNA molecule encoding the human thrombin receptor having the amino acid sequence set
forth in Figure 1 herein.
In one aspect, the invention is directed to recombinant materials associated with the
production of thrombin receptor. In accordance with the present invention there is
provided a DNA molecule comprising an expression system which when transformed into
a recombinant host, produces human thrombin receptor at the cell surface of the host;
wherein said expression system comprises a nucleotide sequence encoding the human
thrombin receptor operably linked to a heterologous control sequence operable in said host,
and wherein said thrombin receptor is encoded by a nucleotide sequence comprising a
DNA molecule encoding the amino acid sequence of Figure 1 herein. The invention also
relates to transfected cells, which contain the expression system of the invention, which
can be cultured so as to display the thrombin receptor or their surfaces, and thus provide an
assay system for the interaction of materials with native thrombin receptor. These cells, or
peptides which represent relevant portions of the receptors, can be used as diagnostics to
determine the level of thrombin in samples, as well as screening tools for candidate
substances which affect thrombin activity ii_i_\/_i\/_o.
We also describe thrombin receptor agonists which mimic the activated form of the
extracellular portion ofthe receptor protein. These agonists are useful in encouraging
platelet aggregate formation, for example, in localized application at internal bleeding sites
of hemophiliacs. The agonists also mimic thrombin’s ability to stimulate fibroblast
poliferation and thus may be useful in promoting wound healing.
Tlirombin receptor antagonists are also described. These antagonists comprise
modified forms ofthrombin receptor
agonist peptides which lack the essential features
required for activation of the receptor. These
antagonists bind to receptor, do not activate it, and
prevent receptor activation by thrombin.
A second group of compounds are described,
that antagonize the action of thrombin are, in effect,
thrombin inhibitors.
receptor which would ordinarily represent cleavage and
This group includes mimics of the
thrombin-binding regions of the receptor, including
noncleavable peptides and peptides with enhanced binding
for thrombin. These peptides are capable of binding
directly to thrombin so as to diminish the levels of
thrombin capable of binding to receptor. They thus
diminish or prevent thrombin—mediated events such as
thrombin—induced platelet aggregation, fibrinogen
clotting and cell proliferation.
A third group of compounds which behave as
antagonists blocks the binding of thrombin to its
receptor by providing alternate anionic regions to
replace those of the thrombin receptor. These
antagonists are mimics of the anionic region included in
the thrombin-binding portion of the receptor. These
antagonists also bind to thrombin, thereby preventing
thrombin interaction with the intact receptor.
Conversely, alternate cationic regions which
mimic those present in the thrombin ligand can be
included in antagonists which occupy the binding region
of the receptor and thus prevent binding of thrombin.
A fifth group of antagonists are described that include
antibodies which are designed to bind specific regions of
receptor protein. In general, these are monoclonal
antibody preparations which are highly specific for any
desired region of the thrombin receptor. The
antibodies are also useful in immunoassays for the
receptor protein, for example in assessingc successful
expression of the gene in recombinant systems.
A sixth group of antagonists comprises modified
forms of thrombin lacking proteolytic activity.
In another aspect, the invention is related to
assay systems which utilize the hast cells of the
invention displaying recombinant thrombin receptor to
screen for agonists and antagonists. Some systems
include the use of the agonist peptides to screen for
antagonists which inhibit the agonistic effect.
These is also described diagnosis of cardiovascular
disease by detection, in fluids such as blood or urine,
of the peptide cleaved from the thrombin receptor when
activated as a measure of thrombosis. Another diagnostic
method is described including visualization of activated
forms of receotor‘ and detecting" clots in the body’ by
localizing and imaging these targets in situ using
antibodies specific to the activated receptor.
Pharmaceutical compositions are also described
containing the compounds described herein. The compounds
which serve as antagonists to the activation of the
thrombin receptor are useful as anti—thrombotics and are
helpful in a variety of clinical indications including
treatment of abrupt closure in the context of
angioplasty, the treatment of restenosis in the context
of angioplasty, the treatment of unstable angina, the
treatment of myocardial infarction, and treatment of
some forms of thrombotic or thromboembolytic stroke.
These compounds can be used alone or in combination with
other therapeutic agents such as urokinase and tPA.
Brief Description of the Drawings
Figure 1 shows the DNA and deduced amino acid
sequence of a human thrombin receptor.
Figure 2 shows a proposed model of thrombin
receptor activation based on the deduced amino acid
sequence.
Figure 3 shows a comparison of amino acid
sequences for the cleavage site and exosite binding
domains deduced from the CDNA encoding human thrombin
receptor and from the cDNA encoding murine thrombin
receptor. Also shown is the relevant portion of the
hirudin sequence.
Figure 4 shows platelet response to agonist
peptide.
Figure 5 shows the mitogenic effect of an
agonist peptide of the invention on fibroblasts.
Figures 6A, 6B and 6C show the effects of three
thrombin inhibitor peptides on thrombin—induced platelet
activation.
Figure 7 shows the effect of mutant thrombin on
platelet ATP secretion stimulated by thrombin.
Figure 8 shows the increase in thrombin needed
to overcome inhibition of platelet ATP secretion by
mutant thrombin.
Figure 9 shows the effect of thrombin on
platelet ATP secretion by varying concentrations of
thrombin mutant.
Modes of Carrying Out the Invention
The characteristics of the thrombin receptor
elucidated by the invention herein are summarized in
Figures 1 and 2. Figure 1 shows the complete DNA
sequence of the clone encoding the receptor along with
the deduced amino acid sequence. The entire amino acid
sequence contains 425 amino acids, including a 24 or
amino acid signal sequence which provides an
approximately 400 amino acid mature receptor protein.
Hydrophobicity/hydrophilicity plots of the
sequence shown in Figure 1 indicate that the mature
receptor is a member of the 7-transmembrane domain
receptor family and has a relatively long (approximately
75 amino acid) extracellular amino acid extension
containing several consensus sites for asparagine-linked
glycosylation. A disulfide bond linking cysteine—175 in
the first extracellular loop with cysteine-254 in the
second extracellular loop would be analogous to rhodopsin
and B-2 adrenergic receptor. A proposed model of the in
situ receptor is shown in Figure 2.
Referring again to Figure 1, the thrombin-
catalyzed cleavage site is represented by the Arg-Ser
linkage at positions 41 and 42. Cleavage at this site
results in the liberation of a peptide fragment of the
receptor designated an "activation peptide" extending
from position 1 of the mature peptide to the cleavage
site. The precise processing site of the receptor is not
known and thus the N-terminus of the mature protein is
somewhat uncertain. However, it is probably the arginine
residue at position 28. The "activation peptide" would
thus have the sequence RPESKATNATLDPR.
location of the N-terminus is unimportant for the design
The precise
of the compounds described below. This "activation
peptide“ is likely to be freely filtered by the kidney
and possibly concentrated in the urine, and can be used
as an index to platelet activation by thrombin.
The amino acid sequence destined to be cleaved
by thrombin--i.e., the cleavage site--binds to thrombin's
active site/"oxyanion hole" region which is contained in
an extended binding pocket. This oxyanion hole binds
large substrates via hydrophobic, hydrogen bonding, and
charge interactions. Typically, the sequence to be
cleaved interacts with the amino acids of the active
site, while sequences carboxyl to this cleavage site
interact with the more extended "anion binding exosite."
The thrombin receptor contains the anionic sequence
YEPFWEDEE at positions 52-60, as shown in Figure 1. This
region is just carboxyl to the cleavage site between
positions 41 and 42. The location and the composition of
this YEPFWEDEE sequence (aromatic/hydrophobic residues
and anionic residues) strongly suggest that this sequence
contains regions that mediate thrombin binding to the
receptor via thrombin's anion-binding exosite. This
hypothesis is confirmed hereinbelow by showing that
peptides representing at least a portion of this region
of the receptor bind thrombin and inhibit its actions.
This observation also predicts that polycationic
substances that bind to this portion of the receptor may
block thrombin binding and receptor activation.
Release of the activation peptide permits
refolding of the receptor protein to activate the
receptor. This is shown schematically in Figure 2, which
also shows that the conformational changes resulting from
the liberation of the activation peptide and refolding
results in an intracellular conformational change of the
receptor. This hypothesis is confirmed by the finding
that the thrombin receptor can be activated by a peptide
mimicking the new amino terminus created by the
activation. Accordingly, mimics of the N-terminus of the
new amino terminus on the activated receptor behave as
agonists therefor. The importance of the first two amino
acids in the newly created amino terminus in the receptor
for receptor activation has also been confirmed
hereinbelow. Switching the positions of the amino
terminal serine and phenylalanine results in complete
loss of agonist activity for the above agonist peptides.
Based on this information, and by analogy with the
mechanisms underlying trypsinogen activation to trypsin,
it appears that the positively charged amino group on
serine that is newly exposed when thrombin cleaves the
receptor plays an important role in receptor activation.
Peptides based on the agonist peptide sequence that bind
the thrombin receptor but are modified to be lacking the
a—amino group can function as antagonists of the thrombin
receptor. Thus, modifications of the agonist peptides
which lack the capacity for specific activating
interaction serve as thrombin receptor antagonists.
Compounds
The nomenclature used to describe the
peptide compounds follows the conventional
practice where the N-terminal amino group is assumed to
be to the left and the carboxy group to the right of each
amino acid residue in the peptide. In the formulas
representing selected specific embodiments of the present
invention, the amino- and carboxy-terminal groups,
although often not specifically shown, will be understood
to be in the form they would assume at physiological pH
values, unless otherwise specified. Thus,
H+2 and C—terminal O" at physiological pH are understood
the N-terminal
to be present though not necessarily specified and shown,
either in specific examples or in generic formulas. Free
functional groups on the side chains of the amino acid
residues can also be modified by amidation, acylation or
change the
other substitution, which can, for example,
solubility of the compounds without affecting their
activity.
In the peptides shown, each gene-encoded
residue, where appropriate, is represented by a single
letter designation, corresponding to the trivial name of
the amino acid, in accordance with the following
conventional list:
Amino Acid
Alanine
Arginine
Asparagine
Aspartic acid
Cysteine
Glutamine
Glutamic acid
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
One-Letter Three—letter
Symbol S o1
Ala
Arg
Asn
Asp
Cys
Gln
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Trp
Tyr
Val
< +< 2 +3 m 'u H: 3 :2 t'r4 m an m (3 0 t3 2 as v
The amino acids not encoded genetically are
abbreviated as indicated in the discussion below.
In the specific peptides shown in the present
application, the L-form of any amino acid residue having
an optical isomer is intended unless the D—form is
expressly indicated by a dagger superscript (T).
The compounds described herein are peptides
which are partially defined in terms of amino acid
residues of designated classes.
Amino acid residues can
be generally subclassified into four major subclasses as
follows:
Acidic: The residue has a negative charge due
to loss of H ion at physiological pH and the residue is
attracted by aqueous solution so as to seek the surface
positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium at
physiological pH.
Basic: The residue has a positive charge due
to association with H ion at physiological pH and the
residue is attracted by aqueous solution so as to seek
the surface positions in the conformation of a peptide in
which it is contained when the peptide is in aqueous
medium at physiological pH.
Neutral/nonpolar: The residues are not charged
at physiological pH and the residue is repelled by
aqueous solution so as to seek the inner positions in the
conformation of a peptide in which it is contained when
the peptide is in aqueous medium. These residues are
also designated "hydrophobic" herein.
Neutral/polar: The residues are not charged at
physiological pH, but the residue is attracted by aqueous
solution so as to seek the outer positions in the
conformation of a peptide in which it is contained when
the peptide is in aqueous medium.
It is understood, of course, that in a
statistical collection of individual residue molecules
some molecules will be charged, and some not, and there
will be an attraction for or repulsion from an aqueous
medium to a greater or lesser extent. To fit the
definition of "charged," a significant percentage (at
of the individual molecules are
The degree of attraction or
least approximately 25%)
charged at physiological pH.
repulsion required for classification as polar or
nonpolar is arbitrary and, therefore, amino acids
specifically contemplated herein have been
classified as one or the other. Most amino acids not
specifically named can be classified on the basis of
known behavior.
Amino acid residues can be further
subclassified as cyclic or noncyclic, and aromatic or
nonaromatic, self—explanatory classifications with
respect to the side chain substituent groups of the
residues, and as small or large. The residue is
considered small if it contains a total of 4 carbon atoms
or less, inclusive of the carboxyl carbon. Small
residues are, of course, always nonaromatic.
For the naturally occurring protein amino
acids, subclassification according to the foregoing
scheme is as follows.
Acidic: Aspartic acid and Glutamic acid;
Basicznoncyclicz
Arginine, Lysine;
Histidine;
Basiczcyclicz
Neutral olar small: Glycine, serine,
cysteine;
Neutral non olar small: Alanine;
Neutralzpolarzlargeznonaromatic: Threonine,
Asparagine, Glutamine;
Neutral/Dolar/large aromatic: Tyrosine;
Neutral/nonDolar/larqe/nonaromatic: Valine,
Isoleucine, Leucine, Methionine;
Neutralznonpolarzlargezaromatic:
Phenylalanine, and Tryptophan.
The gene—encoded secondary amino acid proline,
although technically within the group
neutral/nonpolar/large/ cyclic and nonaromatic, is a
special case due to its known effects on the secondary
conformation of peptide chains, and is not, therefore,
included in this defined group.
Certain commonly encountered amino acids, which
are not encoded by the genetic code, include, for
example, beta-alanine (beta-Ala), or other omega—amino
acids, such as 3-amino propionic, 4-amino butyric and so
forth, alpha-aminisobutyric acid (Aib), sarcosine (Sar),
ornithine (Orn), citrulline (Cit), t-butylalanine
(t—BuA), t-butylglycine (t—BuG), N-methylisoleucine
(N—MeIle), phenylglycine (Phg), and cyclohexylalanine
(Cha), norleucine (Nle), cysteic acid (Cya) 2-
naphthylalanine (2-Nal); 1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid (Tic); B—2—thienylalanine (Thi); and
methionine sulfoxide (MSO). These also fall conveniently
into particular categories.
Based on the above definitions,
Sar and beta—Ala and Aib are neutral/nonpolar/
small;
t-BuA, t-EuG, N-MeIle, Nle, Mvl and Cha are
neutral/nonpolar/large/nonaromatic;
Orn is basic/noncyclic;
Cya is acidic;
Cit, Acetyl Lys, and MSO are neutral/polar/
large/nonaromatic; and
Phg, Nal, Thi and Tic are
neutral/nonpolar/large/ aromatic.
The various omega—amino acids are classified
according to size as neutral/nonpolar/small (beta-Ala,
i.e., 3-aminopropionic, 4-aminobutyric) or large (all
others).
Other amino acid substitutions of those encoded in
the gene can also be included in peptide compounds and
can be classified within this general scheme according
to their structure.
All of the compounds described herein, when an
amino acid forms the C-terminus, may be in the form of
the pharmaceutically acceptable salts or esters. Salts
may be, for example, Na+, K+, Ca”, Mg” and the like;
the esters are generally those of alcohols of l—6C.
A. Agonists
The agonists described below comprise a series of
peptides of the formula
A155‘-AA}/'(AAi)n‘Z (1)
wherein AAX is a small amino acid or threonine,
preferably selected from ser, ala, gly and, and thr and
AAY is a neutral/nonpolar/aromatic amino acid residue or
is a neutral/nonpolar/large/nonaromatic amino acid
containing a cyclic portion (preferably a neutral/
nonpolar/aromatic amino acid residue);
wherein AA represents an amino acid residue and
the subscript i is an integer which denotes the position
of the referent amino acid residue downstream (N+C) of
the AAY residue of formula (1), and n is an integer of
2-12, with the proviso that if n=2, Z must comprise an
amidated C terminus of the formula NR'R' wherein at least
one R’ is alkyl containing at least one polar
substituent; and
in general, Z is a noninterfering substituent.
AA1 and AA2 must, therefore, be present in the
AAl and
AA2 are relatively precisely defined; however AA3-AA12
compounds of formula 1; AA3-AAl2 are optional.
are, generally, L—amino acid residues. The position of
AAI is also relatively tolerant; therefore,
AAl is a neutral or basic amino acid having a
free a=amino group in the L—configuration;
AA2 is a neutral or basic L-amino acid residue;
and
AA3-AAIZ are L—amino acid residues, wherein
preferably AA3 is a basic or neutral amino acid residue;
AA4 and AA6 are each independently
neutral/polar/large/nonaromatic amino acids or AA4 may be
a basic amino acid;
AA5 and AAll are each independently proline or
small amino acid residues;
AA7 and AAlO are each independently acidic
amino acid residues;
AA8 is a basic amino acid residue; and
AA9 and AAl2 are each independently neutral/
aromatic amino acid residues.
The peptide of formula 1 can be extended (shown
as included in Z) at the C—terminus (but not the N-
terminus) by further amino acid sequence to comprise a
noninterfering substituent.
At the C-terminus of the compounds of
formula 1, the carboxyl group may be in the underivatized
form or may be amidated; in the underivatized form the
carboxyl may be as a free acid or a salt, preferably a
pharmaceutically acceptable salt.
If the C-terminus is amidated, the nitrogen
atom of the amide group, covalently bound to the carbonyl
carbon at the C—terminus, will be NR’R’, wherein each R’
is independently hydrogen or is a straight or branched
chain alkyl of l—6C, such alkyls are 1-6C straight— or
branched-chain saturated hydrocarbyl residues, such as
methyl, ethyl, isopentyl, n—hexyl, and the like.
Representatives of such amido groups are: —NH2, -NHCH3,
_l7_
-N(CH3)2, -NHCHZCH3, —NHCH2CH(CH3)2, and
-NHCH2CH(CH3)CH2CH3, among others. Furthermore, either
or both R’ may in turn optionally be substituted by one
or more substituents such as, for example, -OR’, —NR’R',
-NR’CNR’NR'R’ and the like, wherein each R’ is as
Thus, Z may be -OH (or an
halo,
independently defined above.
ester or salt form), or -NR'R' wherein R’
defined.
is as above
Preferred embodiments of AAX-AAY include GF,
AF, sp, TF, G(pClPhe), A(pClPhe), S(pClPhe), T(pClPhe),
GThi, AThi, SThi, and TThi. Preferred embodiments of AAl
and AA2 are large nonpolar amino acids.
embodiments for the residues in the remainder of the
Preferred
compound of formula (1) are those wherein AAI and AA2 are
each independently Leu, Val, Ile, Cha, Phe, 2-Nal or Tic;
or AA3 is Arg, Lys, Orn, Har or Ala. For the remaining
amino acids, preferred are embodiments wherein AA4 and
AA6 are each independently Gln, Asn or Lys; or AA7 and
AAIO are each independently Asp or Glu; AA8 is Arg or
Lys; or AAl2 is Phe and AA9 is Tyr; or Z is OH, or NR’R’
wherein R’ is as defined above; or Z further includes
some or all of AAI3-AA17 as defined below.
preferred are compounds of formula (1) which are selected
from the group consisting of SFLLRNPNDKYE; SFLLRNPNDK;
SFLLRNPN; SFLLRNP; SFLLRN; SFLLR; GFLLR; TFLLRNPNDK;
S(pClPhe)LLR; S(Thi)LLR; SFFLR; SFFLRN; SF(Phg)LR;
sFL(Na1)RN; SFL(Cha)R; SF(Cha)(Cha)RN; SF(Cha)(Cha)RK;
SF(Cha)(Cha)LRNPNDK; SFLLKN; SFLL(Har)N; SFLLKN;
SFF(Cha)AN; and the amidated forms thereof.
Particularly
B. Antagonists
Compounds described below which interfere with
platelet activation and other cellular activities
mediated by the thrombin receptor include the following:
) Antagonists for the thrombin receptor which
represent modified agonist peptides lacking the N-
terminal serine residue;
2) Thrombin inhibitors which represent
noncleavable and/or enhanced binding forms of the
extracellular portions of the thrombin receptor which
behave as decoys for the circulating thrombin;
) Anionic and hydrophobic/anionic peptides
which mimic at least a portion of the YEPFWEDEE anionic-
binding exosite region and which also behave as decoys
for circulating thrombin;
) Cationic or extended cationic peptides
which mimic the anionic—binding exosite of thrombin
itself and bind to the receptor in competition with
thrombin;
) Antibodies which are immunoreactive with
various critical positions on the thrombin receptor; and
) Thrombin mutants lacking proteolytic
activity which compete with native thrombin for the
receptor.
Thrombin Receptor Antagonists
The antagonists of the first group--modified
agonists—-can be represented by the formula:
X-AAY-(AAi)n-Z (2)
wherein X is an amino acid residue other than Ser, Ala,
Thr, Cys or Gly or is a desamino or N—acylated amino
acid;
AAY is a neutral nonpolar large amino acid
residue containing a cyclic portion, preferably aromatic;
AA represents an amino acid residue and the
subscript i is an integer which denotes the position of
the referent amino acid residue downstream (N+C) of the
AAY residue of formula (2) and n is an integer of 4-12;
and
wherein AA1 and AA2 are each independently
neutral or basic L—amino acid residues wherein AAl has a
free a-amino group;
neutral amino acid residues;
and AA8 are each independently basic or
AA4 and AA6 are each independently basic or
nonaromatic amino acids;
AA5 and AA1l
residues or small amino acids;
AA7
amino acid residues;
AA9 and AAl2 are each independently neutral/
aromatic amino acid residues; and
are each independently proline
and AAlO are each independently acidic
Z is a noninterfering substituent.
Preferred embodiments of X include residues of
3-mercaptopropionic acid (Mpr), 3—mercaptovaleric acid
(Mvl), 2-mercaptobenzoic acid (Mba) and S-methyl—3—
mercaptopropionic acid (SMeMpr).
Preferred embodiments for this group of anti-
thrombin activity compounds include those wherein AAl and
AA2 are each independently Leu, Val, Ile, Phe, Cha, 2—Nal
or Tlc; or AA3 and AA8 are each independently Arg, Lys,
Orn or Har; or AA4 and AA6 are each independently Lys,
Arg, Orn, Har, Gly, Gln or Asn; or AA5 and AA1l are each
independently Pro or Ala; or AA7 and AAlO are each
independently Asp, Glu, B-Asp or 3-Glu; or AAl2 is Phe
and AA9 is Tyr; or Z is OH (or an ester or salt form),
NH2, or NR'R’ wherein each R’ is independently H or
straight- or branched-chain alkyl of 1—6C optionally
substituted as described above.
Particularly preferred embodiments are those
peptides wherein X is Mpr, S-Me Mpr or Mba, AAY is Phe,
AAl is Cha, and AA2 is Cha.
Particularly preferred are embodiments of AAl-
AA2 can each independently be Cha.
which are encoded by the gene, or wherein AAl and
Particularly
preferred among the antagonist peptides of this class are
those selected from the group consisting of
XFLLRNPNDKYEPF; XFLLRNPNDKYEP; XFLLRNPNDKYE; XFLLRNPNDKY;
XFLLRNPNDK; XFLLRNPND; XFLLRNPN; XFLLRNP; XFLLRN; XFLLR;
XFLL; XFL; X-F(Cha)(Cha)RNPNDK, X-F(Cha)(Cha)RNPNDKY,
XF(Cha)(Cha)RNPNDKYE—NH2, X-F(Cha)(Cha)RNPNDKY-NH2, X-
F(Cha)(Cha)RNPNDK-NH2, X-F(Cha)(Cha)RNPND-NH2,
X—F(Cha)(Cha)RN-NH2, X-F(Cha)(Cha)RAPNDK-NH2,
X-F(Cha)(Cha)RGPNDK-NH2, X—F(Cha)(Cha)RKPNDK-NH2,
X—F(Cha)(Cha)RNANDK-NH2, X-F(Cha)(Cha)RNPADK-NH2,
X-F(Cha)(Cha)RNPNDA-NH2, X—F(Cha)(Cha)RKPNEK-NH2, and
X-F(Cha)(Cha)RKPNDA-NH2; especially wherein X is Mpr.
Especially preferred are Mpr-F(Cha)(Cha)RNPNDK,
Mpr—F Cha (Cha)RNPNDKY, Mpr—F(Cha)(Cha)RNPNDKYE-NH2,
Mpr—F Cha (Cha)RNPNDKY—NH2, Mpr-F(Cha)(Cha)RNPNDK-NH2,
C
Mpr—F Cha (Cha)RAPNDK-NH2, Mpr-F(Cha)(Cha)RGPNDK—NH2,
Mpr-F Cha (Cha)RKPNDK-NH2, Mpr—F(Cha)(Cha)RNANDK—NH2,
Mpr-F(Cha)(Cha)RNPADK-NH2, Mpr-F(Cha)(Cha)RNPNDA-NH2,
Mpr-F(Cha)(Cha)RKPNEK—NH2, Mpr-F(Cha)(Cha)RKPNDA-NH2,
Mba-F(Cha)(Cha)RKPNDK-NH2, and SMeMpr—F(Cha)(Cha)RKPNDK—
NH2.
( )
( )
Mpr—F( ha)(Cha)RNPND-NH2, Mpr-F(Cha)(Cha)RN-NH2,
( )
( )
Thrombin Inhibitors
The thrombin inhibitors of group 2)
compounds that bind thrombin in competition with receptor
but are noncleavable and/or exhibit enhanced binding
These compounds are of the formula:
represent
properties.
J-AAy-(AAi)n-AA9-AA1O-AA1l-AA12-AAl3-z <3)
wherein J is a peptide extension of 2-5 amino acid
residues or an acylated or desamino form thereof.
In the compounds of formula (3), as above, AAY
is a neutral nonpolar large amino acid residue containing
a cyclic portion, preferably aromatic; and n is 8.
As above, AA represents an amino acid residue
and the subscript i is an integer denoting position
downstream from AAY.
As above, AAl and AA2 are each independently
neutral or basic amino acid residues;
AA3 and AA8 are each independently neutral or
basic amino acid residues;
AA4 and AA6 are each independently basic or
neutral nonaromatic amino acids;
AA5 and AAll are each independently proline
residues or small amino acids;
AA7 and AAlO are each independently acidic
amino acid residues;
AA9 and AAl2 are each independently neutral/
aromatic amino acid residues;
AA is an aromatic or small nonpolar amino
acid residue; and
Z is a noninterfering substituent.
For these thrombin inhibitors which are of
group (2) above, wherein the peptide mimics the thrombin
receptor extracellular chain but lacks a proteolytic site
and/or has enhanced binding for thrombin, particularly
preferred embodiments are those which include downstream
anionic amino acid residues and wherein J is a peptide
extension of 4-5 amino acid residues. Particularly
preferred are those wherein the residues immediately
upstream of AAY have the sequence pro-arg-pro (PRP)
preceded by residues selected from the group consisting
of dipeptide sequences consisting of a
large/nonaromatic/nonpolar/neutral amino acid residue
conjugated through a peptide bond to an acidic amino acid
residue downstream. Particularly preferred embodiments
of this dipeptide sequence are ile—asp, val-asp, ile-
glu, and leu—asp, especially wherein said peptide
extension represented by J is selected from the group
consisting of LDPRP, LEPRP, IDPRP, IEPRP, VDPRP and
VEPRP.
In addition, where the peptide extension
includes the immediately upstream sequence pro-arg-pro,
an additional preferred upstream further extension is a D
amino acid. Particularly preferred are D amino acids
which are large/nonpolar/neutral/aromatic, particularly
tryptophan or phenylalanine, and in particular
phenylalanine.
Z is preferably OH (or an ester or salt form)
or NR’R’, where R’ is defined as above, which may
optionally be preceded by a peptide extension mimicking
the receptor sequence downstream from AAl3.
Particularly preferred compounds of formula (3)
are peptides which are selected from the group consisting
of LDPRPFLLRNPNDKYEPFWEDEEKNES;
LDPRPFLLRNPNDKYEPFWEDEEKNE; LDPRPFLLRNPNDKYEPFWEDEEKN;
LDPRPFLLRNPNDKYEPFWEDEEK; LDPRPFLLRNPNDKYEPFWEDEE;
LDPRPFLLRNPNDKYEPFWEDE; and LDPRPFLLRNPNDKYEPFWED, and
the amidated or acylated forms thereof. Also preferred
are those which are selected from the group consisting of
FTPRPFLLRNPNDKYEPFWEDEEKNES, FTPRPFLLRNPNDKYEPFWEDEEKNE,
FTPRPFLLRNPNDKYEPFWEDEEKN, FTPRPFLLRNPNDKYEPFWEDEEK,
FTPRPFLLRNPNDKYEPFWEDEE, FfPRPFLLRNPNDKYEPFWEDE, and
FTPRPFLLRNPNDKYEPFWED; and FTPRPFLLRNPNDKYEPFWEDEEKNES,
FTPRPFLRNPNDKYEPFWEDEEKNES, FTPRPFRNPNDKYEPFWEDEEKNES,
FTPRPFNPNDKYEPFWEDEEKNES, FTPRPFPNDKYEPFWEDEEKNES,
FTPRPFNDKYEPFWEDEEKNES, FTPRPFDKYEPFWEDEEKNES,
FTPRPFKYEPFWEDEEKNES, FTPRPFYEPFWEDEEKNES, and
FTPRPFEPFWEDEEKNES, and the amidated and acylated forms
thereof.
Anion Exosite-Binding Antagonists
Antagonists which represent peptides mimicking
the binding region of the receptor, YEPFW, optionally
including the anionic extension (EDEE) thereof (group 3),
are represented by the formula:
B—AA9—AA1O—AA1l—AAl2-AA13—Z (4)
wherein AA9, AAl2 and AAI3 are each, independently,
neutral aromatic or small amino acid residues, AAlO is an
acidic amino acid residue, AAl1 is proline or a small
amino acid residue; and wherein B and Z are
noninterfering substituents, typically peptide
extensions, but can also include noninterfering organic
radicals in general. B can also be H or acyl (including
said peptide extension if present); Z may also be OH (or
an ester or salt form thereof) or NR'R'(also including
said peptide extension if present), as set forth
hereinabove.
Preferred forms of compounds of formula (4) are
those wherein each of AA9, AA12 and AA13 is phe, trp, ala
or tyr; and AAIO is glu, asp, 6-glu or B-asp.
Particularly preferred are embodiments wherein AA9—AAl3
is YEPFW, FEPFW, YDPFW, YEPYW, YEPFY, YEPWY or WEPFW. Z
may include the peptide sequence EDEE, QDQQ, EDEQ, QDEQ,
QDEE, EDQE, EDQQ or QDQE.
Preferred embodiments of B include those
wherein B is H or a peptide extension of 1-4 amino acids
or the acylated form thereof.
These antagonists serve as decoys for thrombin,
thus lowering its effective concentration.
Anionic-Binding Exosite Mimics
The cationic peptides mimicking a portion of
the anionic-binding exosite of thrombin (group 4) are of
the formula:
B-AAa-AAb-AAC-AAd—AAe—Z (5)
wherein B and Z are defined as above, and wherein AAa and
AAe are each independently hydrophobic amino acids or
basic amino acids, and where each of AAb, AAC, and AAd is
independently a basic amino acid.
Preferred are compounds of formula (5) wherein
B comprises acyl or H; or Z comprises OH (or an ester or
salt) or NR'R’ wherein each R’ is defined as above; or
AAa and AAe are each independently selected from phe, trp
and ala; or AAb-AAd are each independently selected from
the group consisting of arg, lys and gln; especially
wherein AAa-AAe has the sequence WKKKK, KKKKW, QKQQW, or
WQKQQ.
The noninterfering substituents represented by
B and Z may be further peptide extensions which are
compatible with the binding pattern of the thrombin
anionic-binding exosite. As they mimic this capacity of
thrombin to bind its substrate, these antagonists are
operative by virtue of their ability to bind the relevant
regions of the thrombin receptor protein, and, in
particular, the region YEPFWEDEE at positions 52-60, as
shown in Figure 1.
Antibodies
Antagonists which are antibodies immunoreactive
with critical positions of the thrombin receptor
(group 5) are obtained by immunization of suitable
mammalian subjects with peptides containing as antigenic
regions those portions of the thrombin receptor intended
to be targeted by the antibodies. Critical regions
include the region of proteolytic cleavage, the binding
site at the YEPFWEDEE box, the segment of the
extracellular segment critical for activation (this
includes the cleavage site), and the portions of the
sequence which form the extracellular loops, in
particular, that region which interacts with the N-
terminus of the activated receptor extracellular region.
The agonist peptides of the invention may be used as
immunogens in this case.
Thus, suitable peptides to use as immunogens to
prepare the desired antibodies include those peptides
representing portions of the mature sequence of the
extracellular region from positions 28 to position 68, at
the C—terminal end of the YEPFWEDEE region. This region
has the sequence:
PESKATNATLDPRSFLLRNPNDKYEPFWEDEEKNESGLTEY
and peptides which include the sequence LDPRSFLL (which
includes the cleavage site) and YEPFWEDEE (which includes
the binding site) are particularly useful. Alternative
regions which are useful as immunogens include the
segment representing amino acids 161-176; 240-265; and
336-346. These peptides of the sequences, respectively,
YYFSGSDWQFGSELCR, KEQTIQVPGLNITTCHDVLNETLLEG, and
HYSFLSHTSTT, represent the proposed extracellular loops.
The antibodies are prepared by immunizing
suitable mammalian hosts in appropriate immunization
protocols using the peptide haptens alone, if they are of
sufficient length, or, if desired, or if required to
enhance immunogenicity, conjugated to suitable carriers.
Methods for preparing immunogenic conjugates with
carriers such as BSA, KLH, or other carrier proteins are
well known in the art. In some circumstances, direct
conjugation using, for example, carbodiimide reagents may
be effective; in other instances linking reagents such as
those supplied by Pierce Chemical Co., Rockford, IL, may
be desirable to provide accessibility to the hapten. The
hapten peptides can be extended at the amino or carboxy
terminus with a Cys residue or interspersed with cysteine
residues, for example, to facilitate linking to carrier.
Administration of the immunogens is conducted generally
by injection over a suitable time period and with use of
suitable adjuvants, as is generally understood in the
art. During the immunization schedule, titers of
antibodies are taken to determine adequacy of antibody
formation.
While the polyclonal antisera produced in this
way may be satisfactory for some applications, for
pharmaceutical compositions, use of monoclonal
preparations is preferred. Immortalized cell lines which
secrete the desired monoclonal antibodies may be prepared
using the standard method of Kohler and Milstein or
modifications which effect immortalization of lymphocytes
or spleen cells, as is generally known. The immortalized
cell lines secreting the desired antibodies are screened
by immunoassay in which the antigen is the peptide hapten
or is the thrombin receptor itself displayed on a
recombinant host cell. When the appropriate immortalized
cell culture secreting the desired antibody is
identified, the cells can be cultured either in gitgg or
by production in ascites fluid.
The desired monoclonal antibodies are then
recovered from the culture supernatant or from the
ascites supernatant. Fragments of the monoclonals or the
polyclonal antisera which contain the immunologically
significant portion can be used as antagonists, as well
as the intact antibodies. Use of immunologically
reactive fragments, such as the Fab, Fab’, of F(ab’)2
fragments is often preferable, especially in a
therapeutic context, as these fragments are generally
less immunogenic than the whole immunoglobulin.
The antibodies or fragments may also be
produced, using current technology, by recombinant means.
Regions that bind specifically to the desired regions of
receptor can also be produced in the context of chimeras
with multiple species origin.
Noncleavable Thrombin
In addition to the foregoing, antagonists
comprise thrombin mutants lacking proteolytic activity
that compete with native thrombin for the receptor
(group 6). As set forth above, it is understood that the
participants in the proteolytic cleavage site of thrombin
include the serine residue at B-chain position 205, the
histidine residue at position 57, and the aspartic acid
residue at position 99. Mutants of thrombin containing
replacements for these residues which render the thrombin
molecule proteolytically inactive are prepared using
standard site-directed mutagenesis techniques, and the
mutant genes used to produce the modified thrombin using
recombinant techniques. The relevant substitutions are
denoted by the position number preceded by the native
residue and followed by the substituted residue. Thus,
thrombin with serine at position 205 replaced by alanine
is denoted S205A.
Preferred Embodiments
In both the agonists and antagonists of groups
(1) — (4), some of the preferred embodiments of
the amino acid sequences are those wherein the amino
acid in the peptides are those encoded by the gene.
Also included are those wherein one, two, three or
more of the amino acid residues is replaced by one which
is not encoded genetically.
In more detail, for these preferred
embodiments, preferred embodiments of AAl and AA2 are
leu, val, or ile; especially preferred is leu. Preferred
embodiments of AA3 and AA8 are arg or lys; especially
preferred are embodiments wherein AA3 is arg and AA8 is
lys. Preferred embodiments for AA4 and AA6 are gln or
asn, and especially asn. Preferred embodiments for AA7
and AA1O are asp or glu; particularly preferred are
embodiments wherein AA7 is asp and AAIO is glu. A
preferred embodiment for AA12 is phenylalanine, and of
AA9 is tyrosine.
Preferred acyl groups are of the formula RCO-
wherein R represents a straight or branched chain alkyl
of 1—6C. Acetyl is particularly preferred.
In all of the peptides described, one or
more amide linkages (-CO-NH-) may optionally be replaced
with another linkage which is an isostere such as
—CH2NH—, -CHZS-, —CH2CH2, —CH=CH- (cis and trans),
—COCH2—, -CH(0H)CH2- and -CHZSO-.
be made by methods known in the art.
This replacement can
The following
references describe preparation of peptide analogs which
include these alternative-linking moieties: Spatola,
A.F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide
(general review); Morley, J.S.,
cis and trans);
(~COCH2-); Jennings—White, C., et al., Tetrahedron Lett
(1982) ;;:2533 (—COCH2-); Szelke, M., et al.,
Application E? 45665 (1982) CA:21:39405 (1982)
(—CH(OH)CH2—); Holladay, M.W., et al., Tetrahedron Lett
(1983) 2_
Ci (1982) ;;:l89-199 (-CH2-S-).
European
Preparation of Peptide Agonists and Antagonists
The peptide agonists and antagonists described
herein can be prepared using standard solid phase (or
solution phase) peptide synthesis methods, as is known in
the art. In addition, the DNA encoding these peptides
may be synthesized using commercially available
oligonucleotide synthesis instrumentation and produced
recombinantly using standard recombinant production
systems. The production using solid phase peptide
synthesis is necessitated if non—gene-encoded amino acids
are to be included.
Recombinant Production of Thrombin Receptor
The invention provides recombinant materials
for the production of thrombin receptor for display on
the surface of recombinant cells. Production of the
receptor using these recombinant methods provides a
useful diagnostic reagent either to determine the level
of thrombin in biological samples or, more importantly,
as a reagent to screen candidate substances which affect
thrombin activity.
For this recombinant production, a DNA sequence
encoding the thrombin receptor, as set forth in Figure 1,
or its degenerate analogs is prepared either by retrieval
of the native sequence, as set forth below, or by using
substantial portions of the known native sequence as
probe, or can be synthesized de novo using standard
The DNA is ligated into expression vectors
procedures.
suitable for the desired transformed host and transformed
into compatible cells. The cells are cultured under
conditions which favor the expression of the thrombin
receptor encoding gene and the cells displaying the
receptor on the surface harvested.
Use of Recombinant Thrombin Receptor as a Diagnostic and
Screening Tool
The availability of the recombinant DNA
encoding thrombin receptor permits expression of the
receptor on host cell surfaces, thus making the cells
available as a tool for evaluating the ability of
candidate agonists or antagonists to bind to receptor.
In one type of easily conducted assay,
competition of a candidate antagonist for binding to the
receptor with either labeled thrombin, a thrombin agonist
The labeled
substance known to bind the receptor can, of course, be a
or known binding antagonist can be tested.
synthetic peptide. Varying concentrations of the
candidate are supplied along with a constant
concentration of labeled thrombin, thrombin agonist, or
antagonist, and the inhibition of a binding of label to
the receptor can be evaluated using known techniques.
In a somewhat more sophisticated approach, the
effect of candidate compounds on thrombin—induced
responses can be measured in the cells recombinantly
expressing the thrombin receptor as described below.
Assay systems for the effect of thrombin on these cells
include calcium mobilization and voltage clamp which are
further described in detail hereinbelow. other suitable
endpoints include thrombin-induced phosphoinositol
turnover and inhibition of adenyl cyclase. These assays
permit an assessment of the effect of the candidate
antagonist on the receptor activity rather than simply
ability to bind to thrombin.
Diagnosis of Cardiovascular Disease
In one embodiment, the availability of the
recombinant thrombin receptor protein permits production
of antibodies which are immunospecific to the activated
form of the receptor which can then be used for
diagnostic imaging of activated receptors in yigg. These
antibodies are produced either to the activated form of
the receptor produced recombinantly, or to the peptide
representing the "new amino terminal" peptide described
in Example 2 below. The resulting antibodies, or the
immunospecific fragments thereof, such as the Fab, Fab’,
Fab'2 fragments are then conjugated to labels which are
detected by known methods, such as radiolabels including
technetium and indium or other radioactive labels as
is known in the art. When injected in yiyg, these
antibodies home to the sites of activated receptor, thus
permitting localization of problem areas which are
subject to thrombosis.
In another embodiment of diagnosis, the
presence of the activation peptide in body fluids can be
detected and measured. Antibodies are made to the
activation peptide as described above and can be employed
in standard ELISA or RIA assays to detect excess amounts
of the activation peptide in, for example, urine.
Utility and Administration of Antagonists
The antagonists described herein are useful
therapeutically in the treatment of abrupt closure or
restenosis in the context of angioplasty; in the
treatment of unstable angina; and in the treatment of
myocardial infarction. The peptides which behave as
antagonists may be administered in conventional
formulations for systemic administration as is known
in the art. Typical such formulations may be
found, for example, in Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton PA, latest edition.
Preferred forms of systemic administration of
peptides include injection, typically by intravenous
injection. other injection routes, such as subcutaneous,
intramuscular, or intraperitoneal, can also be used.
More recently, alternative means for systemic
administration of peptides have been devised which
include transmucosal and transdermal administration using
penetrants such as bile salts or fusidic acids or other
detergents. In addition, if properly formulated in
enteric or encapsulated formulations, oral administration
may also be possible.
The dosage range required depends on the choice
of antagonist, the route of administration, the nature of
the formulation, the nature of the patient’s illness, and
the judgment of the attending physician. Suitable dosage
ranges, however, are in the range of 0.1-100 pg/kg of
subject. Wide variations in the needed dosage, however,
are to be expected in view of the variety of antagonists
available and the differing efficiencies of various
routes of administration. For example, oral
administration would be expected to require higher
dosages than administration by intravenous injection.
Variations in these dosage levels can be adjusted using
standard empirical routines for optimization as is well
understood in the art.
The agonists described herein are useful in the
treatment of wounds and in other contexts wherein
fibroblast proliferation is useful. Administration of
these compounds is generally topical and/or localized, in
the form of salves, pastes, gels and the like.
Assay Systems
Various assay systems may be used to measure
the interaction of thrombin with its receptor and the
affect of various candidate agonists and antagonists
thereon. The role of the thrombin receptor and thrombin
in platelet aggregation can be measured directly by
aggregometry or the effect on blood clotting involving
In addition, ATP uptake
Also useful as a measure
__—_——_é_..:._...
In this assay, washed
Charo,
induce aggregation, approximately 1-20 nM thrombin or
ECSO of an alternate agonist is used to stimulate
aggregation in control reactions; the results are
followed by lumiaggregometry. Candidate agonists at
various concentrations may be used in place of thrombin
to stimulate aggregation. Candidate inhibitors are added
to the reaction mixture in addition to the thrombin in
order to assess their ability to prevent aggregation.
Washed platelets are suspended in modified
Tyrode's buffer, pH 7.4 with 2 mM magnesium and 1 mM
calcium at a concentration of 108 platelets/ml. The
thrombin or test compound is added in a small volume
(about 20 uL) in 600 mM NaCl, 10 mM MES pH 6.0,
0.5% PEG 6000 buffer and incubated for 15 minutes at 37°C
with a platelet suspension.
Platelet Activation/ATP Secretion:
prepared as above in 480 pl of suspension are added to 20
Platelets
pl of phosphate buffered saline containing sufficient
thrombin to give a final concentration of about 10 nM, or
About 20 pl
Chromolume® reagent (Chronolog Corporation, Havertown,
an alternate agonist is added at its ECSO.
PA) is added. In addition to measuring aggregation, ATP
secretion is assessed. These results quantitated
independently measuring changes in luminescence and light
transmittance in a chronolog dual channel
Platelet ATP
secretion is measured in a lumiaggregometer as
lumiaggregometer (chronolog Corporation).
luminescence signal. Candidate antagonists which
putatively interact with thrombin are preincubated with
the thrombin in 20 pl PBS at room temperature for 5
minutes before addition to the platelets. Preincubation
is not necessary for testing agonists or antagonists
which interact directly with the receptor.
Platelet Aggregation Assav Usinq Microtiter
Plates: Thrombin- or agonist-mediated platelet
aggregation as measured with washed platelets in 96-well
microtiter plates was performed as described (Fratantoni,
J.C. et al., Am J Clin Pathol (1990) gg:613—617). The
ability of hybridoma supernatants, purified MoAbs or
peptide antagonists to block the thrombin receptor was
assessed in this assay with various concentrations of
antibodies or antagonists.
Fibrinoqen Clotting Assay: Fibrinogen clotting
reactions are performed in a total volume of 300 pl in
150 mM NaCl, 20 mM Tris, pH 7.4, 10 mM CaCl2, 0.5% PEG
6000 at 37°C and a final fibrinogen concentration of
3.3 mg/ml. Thrombin at 5 nM gives an approximately
second clotting time as measured by a standard
Fibrosystem@ coagulation timer (Fisher Scientific,
Springfield, NJ).
As described above, candidate agonists are used
in place of the thrombin to stimulate fibrant formation;
oocytes are washed, then incubated in MESH II without
Briefly, intracellular calcium
antibiotics for 90 minutes. Groups of 5 oocytes are
selected and placed in individual wells in a 24-well
tissue culture plate (Falcon 3047) containing 0.5 ml/well
MSH II without antibiotics. This medium is removed and
replaced with fresh medium every 10 minutes; the
harvested medium is analyzed by scintillation counting to
determine 45Ca released by the oocytes during each 10-
minute incubation. The 10-minute incubations are
continued until a stable baseline of 45Ca release per
unit time is achieved. Two additional 10-minute
collections are obtained, then test medium including
agonist is added and agonist-induced 45Ca release
determined.
Voltage Clamp:
currents are measured in voltage-clamped oocytes
expressing thrombin receptor encoding CRNA essentially as
previously described (Julius, D., et al, Science (1988)
241:558-563) except that the single electrode voltage-
Agonist-induced inward chloride
clamp technique is employed.
The following examples are intended to
illustrate but not to limit the invention.
Example 1
Preparation of CDNA Encoding Thrombin Receptor
In summary, the human cell lines HEL
(Papayannopoulou, T., et al., J Clin Invest (1987)
1g:859-866) and Dami cells (Greenberg, S.M., et al.,
glppg (1988) 1g:1968-1977) were stimulated with phorbol
12-myristate 13-acetate (PMA) before isolation of mRNA
for microinjection into Xenopus oocytes. The oocytes
which had been injected with these mRNA samples were then
assayed for cellular calcium mobilization to detect those
eggs which were expressing the thrombin receptor encoded
by the RNA at their surfaces.
the mRNA, a 40 kb mRNA fraction was used for preparation
After size selection of
of a cDNA library. The library was assayed by conversion
of plasmid DNA, cloned in E. coli, into capped CRNA in an
in vitro system, and injection of the capped CRNA into
the oocytes. An insert in a positive clone was sequenced
to obtain the CDNA and deduced amino acid sequence shown
in Figure 1.
penicillin, 100 pl/ml streptomycin, and 50 ug/ml
A Practical
gentamicin).
Dumont stage V oocytes were selected and
microinjected with 50 ml of the mRNA to be tested (1
pg/pl in 10 mM Hepes, pH 7.0); 5 ng of CRNA transcribed
from a cDNA encoding a secreted form of alkaline
phosphatase (generously provided by Dr. S. Udenfriend)
was coinjected with all mRNA or cRNA samples as an
internal standard for selection of healthy oocytes (Tate,
s.s., et al., FASEB J (1990) 3:227-231).
oocytes were cultured for 48 h at 18°C in MESH II in
Microinjected
individual wells in 96-well culture plates; the oocyte-
conditioned medium was then assayed for alkaline
phosphatase activity as described (Tate et al., (supra))
and the "best-expressing" oocytes were selected for
functional assays.
Cytoplasmic and poly A+ RNA were prepared from
HEL and Dami cells by standard techniques (Sambrook, J.,
et al., Molecular Cloning, 1989, Cold Spring Harbor
Laboratory Press, New York). Poly A+ RNA was
fractionated by size by centrifugation through a 10-30%
sucrose density gradient exactly as described by
Sumikawa, K., et al., Nucl Acids Res (1982) ;g:5809-
5822. Aliquots of each gradient fraction were analyzed
for size by glyoxal gel electrophoresis.
of each fraction was twice ethanol precipitated, and RNA
dissolved at 1 pg/pl in 10 mM Hepes, pH 7.0.
each fraction were assayed in the oocyte system described
The remainder
Aliquots of
above for thrombin receptor activity.
the rare cutter Mlul next to the NotI site. pFROG placed
the cDNA under the transcriptional control of the SP6 RNA
polymerase promoter and directed the synthesis of a
hybrid mRNA containing the 5’—untranslated region of
Xenopus globin followed by message encoded by the cDNA
insert.
All pools were screened using both the voltage
clamp and 45Ca release assay. Of the first five pools
screened, all showed some thrombin receptor activity; in
the 45Ca release assay, thrombin-induced increases in
45Ca release ranged from two— to six-fold. The most
active pool was replated at approximately 2000 clones per
plate and rescreened in the oocyte system. Two of 10
pools screened were positive for thrombin receptor
activity. The most active of these was replated at 300
clones per plate and the pools rescreened. By
progressive selection and subdivision of active pools, a
single clone was identified.
The 3480-nucleotide cDNA insert was subcloned
into the XhoI site of pBluescript. Restriction fragments
of the insert were subcloned into M13. The cDNA sequence
was determined twice in each direction (three times for
the coding region) by dideoxy sequencing. The results
are shown in Figure 1.
Figure 1 shows both the nucleotide sequence and
the deduced amino acid sequence for the thrombin receptor
protein. Hydrophobic regions, including a putative
signal sequence and seven transmembrane spans are
After processing of the signal sequence by
it is probable that additional
overlined.
signal peptidase,
processing by proline-directed arginyl cleavage occurs
between the arginines at positions 27 and 28, which is
marked on the Figure. Thus,
mature protein begins RPESK....
the amino terminus of the
Possible asparagine—
linked glycosylation sites are underlined, and consensus
polyadenylation regions are in bold. The putative
thrombin receptor cleavage site at position R41/S42 is
also marked.
As set forth above, Figure 2 provides a diagram
of the disposition of the thrombin receptor in the cell
membrane. As shown in Figure 2, the amino terminal
extracellular extension of the intact and unactivated
thrombin receptor is cleaved by thrombin, exposing a new
amino terminus and releasing the short receptor fragment
designated the "activation peptide" herein. The newly
exposed amino terminus then functions as an agonist,
Vbinding to an as yet undefined region of the thrombin
receptor and activating it. The thrombin receptor is
thus activated by a mechanism analogous to zymogen-enzyme
conversion. Thus, the thrombin receptor, like other
receptors which contain seven transmembrane regions,
contains its own ligand with the N-terminus in the native
form of S42/F43.
The availability of the human cDNA encoding
thrombin receptor permitted the retrieval of the
corresponding murine form. A high degree of homology is
shown at the cleavage site and anion exosite binding
domain. The homology of these sequences with each other
and with the anion exosite binding domain of hirudin is
shown in Figure 3.
Example 2
Synthesis of Ser-Phe-Leu—Leu-Arq-Asn-NH2
(SFLLRN-NH2_)_
Starting with paramethylbenzhydrylamine resin -
HCl (0.5 mmol synthesis, 0.77 meq/g, Applied Biosystems,
Foster City, CA) was subjected to neutralization with
diisopropylethylamine (DIEA) in N—methylpyrolidinone
(NMP), followed by washings and addition of the required
amino acids coupled as 1-hydroxybenzotriazole esters and
introduced in order using an Applied Biosystems 431A
peptide synthesizer. The Boc-amino acids had the
following sidechain protection: Ser (OBzl) and Arg
(Tos).
achieved with HF/anisole/methylethylsulfide (56/6/1
(v/v)) to afford the crude peptide which was purified by
Cleavage of the completed peptide resin was
C18 reversed-phase liquid chromatography using a gradient
of acetonitrile in water containing 0.1% trifluoroacetic
acid (TFA).
Example 3
Agonist Activity of a "New Amino-Terminal"
Peptide On Oocvtes Expressing Wild-Tvpe
and Mutant Thrombin Receptor cRNA
Oocytes were microinjected with 5 ng wild—type
thrombin receptor CRNA (WT) or with 5 ng cRNA encoding a
mutant thrombin receptor with the amino acid substitution
R4lA (R4lA).
thrombin as set forth above——alanine replaces arginine at
position 41. Uninjected oocytes or oocytes expressing
thrombin receptor cRNAs were then cultured for 48 hr and
thrombin or peptide—induced 45Ca release determined as
The notation is analogous to that for
described above. Candidate agonists were added at
saturating concentrations: thrombin at 250 pM and the
"new amino-terminal" peptide SFLLRNPNDKYEPF (SFLL
peptide) at 25 pM. The control peptide FSLLRNPNDKYEPF
(FSLL peptide) was added at 100 pM and elicited no
response. The data shown in Table 1 represent the mean
+/- SEM of three replicate determinations; these results
are representative of those obtained in three or four
separate experiments.
Table 1
Fold increase
Receptor Aqonist in 45Ca
WT Thrombin 26
WT "SFLL" peptide 40 pM 32
WT "SFLL" peptide 200 pM 42
R4lA Thrombin 0
1241A "SFLL" peptide 200 pM 53
The agonist SFLL peptide has no activity on uninjected
oocytes (not shown). Qualitatively identical results
were obtained when agonist-induced inward current in
voltage-clamped oocytes was used as an endpoint rather
than agonist-induced Ca release.
Example 4
Agonist Function of the "New Amino-Terminal" Peptide
for Platelet Secretion and Aggregation
and Mitogenic Effects
Washed human platelets were prepared as
described by Baenzinger, N.G., Meth Enz (1974) ;;:149—
155; and Charo, I.F., et al., J Clin Invest (1977)
Platelet aggregation in response to 1, 10, 20,
100 or 200 pM peptide SFLLRNPNDKYEPF "SFLL" peptide or to
nM thrombin was measured in a lumiaggregometer, and
Agonist-induced responses were assessed as
the results are shown in Figure 4A.
Platelet ATP secretion in response to the
indicated final concentrations of "new amino-terminal“
peptide was also followed by lumiaggregometry, and the
results are shown in Figure 4B.
The data shown in Figure 4 are raw tracings
representative of aggregation or secretion responses
obtained in triplicate for each agonist concentration,
and are representative of results obtained in more than
five separate experiments. 100% aggregation is
arbitrarily defined as that occurring in response to a
saturating concentration of thrombin at one minute. 100%
secretion is arbitrarily defined as the maximal response
occurring in response to a saturating concentration of
thrombin. The “new amino terminal" peptide is comparably
active to 20 pM thrombin at concentrations of 100 pM in
both assays as shown in the figure. The control peptides
FSLLRNPNDKYEPF and LLRNPNDKYEPF were both without
activity at concentrations as high as 200 pM (not shown).
In an additional determination, the mitogenic
effects of the agonist peptide were demonstrated using
CCL-39 cells. The fibroblast cell line CCL—39 was made
quiescent in serum-free medium and then treated for 48
hours with the candidate agonist in the presence of
tritiated thymidine. The incorporation of label into DNA
was then determined as TCA-insoluble activity, shown as
cpm in Figure 5 using standard techniques. The data
shown in the figure represent the mean of six replicate
determinations plus or minus 95% confidence.
The agonists Shown in the figure were:
None (serum—free);
% fetal bovine serum (10% FCS);
nM a-thrombin (a-T);
, 10 or 100 pM agonist peptide of the sequence
SFLLRNPNDKYEPF (NTP);
pM "scrambled" agonist peptide, which is
the foregoing with the N—terminus scrambled
to FS (FSLL).
As shown in Figure 5, the NTP at 100 uM gives
significant stimulation of growth. Merely switching the
positions of the first two residues of the agonist caused
loss of activity. Thus, the agonist peptide not only
simulates platelet aggregation, but also is useful in
stimulating fibroblast proliferation, which is useful in
wound—healing applications.
Platelet Aqqreqation Aqonists:
Using the platelet aggregation assay described
above, the concentration of various peptides required to
elicit a 50% maximal aggregation was determined. The
values obtained, shown as ECSO, are shown in Table 2 in
micromolar units.
Table 2
Agonist Peptides
Peptide
SFLLRNPNDKYE 6 6
SFLLRNPNDK 6 3
SFLLRNPN 7 6
SFLLRNP-NH2 4.5
SFLLRN-NH2 l 6
SFLLR—NH2 7 5
SFLL-NH2 146
Ac-SFLLRNPNDYKE
Ac-FLLRNPNDKYEPF
FLLRNPNDKYEPF
Edesaminoserl-FLLR-NH2
[desaminoAsn]-FLLR—NH2
[nethythioacetyll-FLLR-NH2
[3-Tetrahydrofuranoyll-FLLR-NH2
S(N-MePhe)LLRNPNDKYE
DFLLR-NH2
KFLLR-NH2
FFLLR-NH2
[Acm-Cys]-FLLR—NH2
[Va1ery1]-FLLR—NH2
[2-MeButyry1]-FLLR-NH2
Edesaminoornl-FLLR—NH2
[N-MeSer]-FLLRNPNDKYE
[D-Ser]—FLLRNPNDKYE
CFLLR-NH2
(S-MeCys)FLLRN—NH2
[b-Ala]-FLLR-NH2
GFLLR-NH2
TFLLRNPNDK
AFLLRNPNDKYE
SALLRNPNDKYE
S(D-Phe)LLRNPNDKYE
SLLLR—NH2 ‘
SYLLR-NH2
S(NO2Phe)LLR-NH2
S(homoPhe)LLR-NH2
S(Phg)LLR-NH2
S(Tic)LLR-NH2
S(Cha)LLR-NH2
S(Na1)LLR—NH2
S(OMeTyr)LLR-NH2
S(pC1Phe)LLR-NH2
Inactive
796
Inactive
920
237
366
Inactive
Inactive
Inactive
Inactive
WA
2000
1500
WA
850
172
193.0
129.2
99
7.3
8.5
12.9
Inactive
Inactive
Inactive
288
250
Inactive
Inactive
Inactive
140
42
46
8
.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
S(Thi)LLR—NH2
SF(D-Leu)LRNPNDKYE
SF(D-A1a)LR—NH2
SF(b-A1a)LRN-NH2
SF(Aib)LRN-NH2
SFDLR-NH2
SF(N-MeLeu)LR-NH2
SFHLRN-NH2
SFALRNPNDKYE
SFWLR-NH2
SFFLR-NH2
SFFLRN-NH2
SF(Phg)LR-NH2
SFPLR-NH2
SFGLR—NH2
SFRLR-NH2
SFYLRN-NH2
SFILR—NH2
SF(Cha)LR—NH2
SF(Cha)LRN—NH2
SF(Tic)LRN-NH2
SFL(D-Leu)RNPNDKYE
SFLARNPNDKYE
SFLPR-NH2
SFLER-NH2
SFLAR-NH2
SFLQRN-NH2
SFLIRNfNH2
SFLFR-NH2
SFLRR-NH2
SFL(Na1)RN-NH2
SFL(Cha)R—NH2
SF(Cha)(Cha)RN—NH2
SF(Cha)(Cha)LRNPNDK
SFLLDN—NH2
.6
Inactive
Inactive
Inactive
Inactive
Inactive
>1000
. SFLL(D-Arg)—NH2 594
79. SFLLA-NH2 137
80. SFLLLN-NH2 44_9
81. SFLLQN—NH2 2o_2
82. SFLLKN—NH2 11_1
83. SFLLHarN-NH2 3_3
84. SFF(Cha)RA-NH2 1.4
85. SF(Cha)(Cha)RK-NH2 , 0.82
Example 5
Inhibition of Thrombin-Induced Platelet Activation
by Thrombin Inhibitor Peptides
Three antagonist peptides,
LDPRPFLLRNPNDKYEPFWEDEEKNES (LDPRP peptide),
FTPRPFLLRNPNDKYEPFWEDEEKNES (FTPRP peptide) and LDPRPFLL
(shortened LDPRP peptide), were tested for their ability
to inhibit thrombin-induced platelet activation.
Thrombin was incubated with the candidate inhibitory
peptide for 5 minutes, then the mixture was added to
washed platelets and platelet activation was followed as
platelet ATP secretion by lumiaggregometry. The mixtures
were added in a total volume of 20 #1 phosphate buffered
saline to 480 #1 of platelets prepared and suspended as
described in the description under the heading "Assays"
hereinabove. Various concentrations of the candidate
peptides were used. The results are shown in Figures 6A,
6B and 6C. The ATP secretion is expressed as a
percentage of the mean luminescence signal generated by
nM thrombin in the absence of the candidate peptides;
the figures shown are representative of the results of
three replicate experiments.
Figure 6A shows the results for the LDPRP
peptide, which shows an IC5O of approximately 500 nM.
The LDPRP peptide contains sequences which are
representative of both the cleavage site and the putative
thrombin binding site. Figure 6B shows the results
obtained for the shortened LDPRP peptide; the ICSO is now
approximately 200 uM.
However, as shown in Figure 6C, the FTPRP
peptide which contains an alternate form of the putative
cleavage site as well as the putative binding site has an
ICEO of approximately 200 nM; this peptide is thus a more
effective antagonist than either the LDPRP peptide or its
shortened form.
Example 6
Preparation of Thrombin Receptor Antagonist Peptide:
Synthesis of Mpr-Phe-Cha—Cha—Arq~Asn-Pro—Asn—Asp—LVs—OH
Starting with N—a-Boc—e-(Cl-CBZ)—Lys—O—Pam—
Resin (0.5 mmol, 0.70 meq/g, Applied Biosystems, Foster
City, CA), the Boc group was removed with TFA,
neutralized, washed and the required amino acids were
added in sequence by coupling as l—hydroxybenzotriazole
esters employing an Applied Biosystems 431A peptide
synthesizer. The peptide was cleaved from the resin and
purified by reversed—phase chromatography as described in
Example 3.
Candidate peptides analogously synthesized were
tested in the platelet activation/aggregation assays
described above and added at various concentrations in
the presence of thrombin. The concentration which
resulted in 50% inhibition of activation or aggregation
was designated the ICSO and is shown for the various
peptides tested in Table 3 in micromolar units.
Table 3
Antaqonist Activity
Mpr—FLLRNPNDK
Mpr—FLLRNPNDKYE—NH2
Mpr-FLLR—NH2
Mpr-FLLRC—NH2
Mpr-FLLRNC-NH2
Mpr-FLLRNPNC-NH2
Mpr-F(Cha)(Cha)RNPNDK
Mpr-F(Cha)(Cha)RNPNDKY
Mpr-F(Cha)(Cha)RNPNDKYE-NH2
Mpr—F(Cha)(Cha)RNPNDKY-NH2
Mpr—F(Cha)(Cha)RNPNDK—NH2
Mpr—F(Cha)(Cha)RNPND-NH2
Mpr-F(Cha (Cha)RN-NH2
)
Mpr—F(Cha)(Cha RAPNDK—NH2
Mpr—F(Cha)(Cha RGPNDK—NH2
Mpr-F(Cha)(Cha RFPNDK-NH2
Mpr—F(Cha)(Cha RKPNDK—NH2
(
Mpr-F(Cha) Cha RNANDK-NH2
Mpr—F(Cha)(Cha RNPADK—NH2
Mpr—F(Cha)(Cha)RNPNAK—NH2
Mpr-F(Cha)(Cha)RNPNDA-NH2
Mpr-F(Cha)(Cha)RKPNEK-NH2
Mpr-F(Cha)(Cha)RKPNDA-NH2
[SMe-Mpr]-FLLR-NH2
[Cam-Mpr]-FLLR-NH2
Mvl-FLLR-NH2
Pivaloyl-FLLR-NH2
(SMeMpr)—F(Cha)(Cha)RKPNDK—NH2
(2-Mba)-F(Cha)(Cha)RKPNDK-NH2
Mpr-F(Cha)(Cha)RKPND-OH
-400
500-1000
500-1000
As shown in Table 3, substitution of the amino
acid Cha for the leucine and Lys for Asn residues
improves the antagonist activity.
Example 7
Generation of Active-site Thrombin Mutants
After confirmation by DNA sequencing, DNA
determined by ELISA and Western blots and the highest
yielding clones were grown to confluence in a 24,000 cm2
surface cell "factory" (Nunc, Inter Med, Naperville, IL)
in MEM a-nucleoside—deficient medium with 80 nM
methotrexate, 100 units/ml penicillin, 100 pg/ml
streptomycin, 25 mM Hepes buffer, 5 pg/ml vitamin K,
0.2 mg/ml proline, and 10% dialyzed bovine calf serum.
Upon reaching full confluence, all medium was removed,
all growing surfaces washed six times with phosphate-
buffered saline to remove contaminating bovine
prothrombin and thrombin, and cells were grown in MEM
a-nucleoside—deficient medium containing 100 units/ml
penicillin, 100 pg/ml streptomycin, 25 mM Hepes buffer,
pg/ml vitamin K, 0.2 mg/ml proline, 1 pg/ml insulin and
pg/ml transferrin for 36-48 hours.
Conditioned medium was cleared of cellular
debris by centrifugation and filtration, diluted 1:1 with
water, made to 10 mM Tris-HCl, pH 7.4, and 20 mM citrate
(final concentration) and stirred overnight at 4°C with
1% (v/v) S—Sepharose beads were removed by
centrifugation and the conditioned medium was refiltered
(V/V)
Q-Sepharose was then collected in a 10 ml column and
eluted in 1 ml fractions with 600 mM NaCl, 10 mM Tris-
HCl, pH 7.4, 0.5% PEG 6000 and positive fractions
containing recombinant prothrombin identified by Western
S-Sepharose.
and stirred overnight at 4°C with 1% Q—Sepharose.
blot using anti—human thrombin antiserum.
pH was then changed to 7.0
,000-fold molar excess of (p-amidinophenyl)-
methanesulfonyl fluoride (APMSF) to inhibit Factor Xa and
any bovine thrombin that might contaminate the
preparation. APMSF is a serine—dependent irreversible
thrombin antagonist that rapidly inactivates native
thrombin at pH 7.0 but has a half-life of only 10'3 sec
at pH 8.0. For this reason, the pH of the APMSF-treated
mutant thrombin preparation was then changed to 8.0 for
min to eliminate all APMSF.
The mutant thrombin-containing solution was
then changed to pH 6.0 by addition of 1N HCl and stirred
(V/V)
S—Sepharose was collected in a 10 ml column, washed with
150 mM Nacl. 10 mM MES. pH 6.0 and subsequently eluted
with 600 mM NaCl, 10 mM MES, pH 6.0, 0.5% PEG 6000 in
Positive fractions were identified by
V€rni9ht at 4°C with 1% S—Sepharose. The
ml fractions.
Western blot with anti—human thrombin antiserum and the
concentration and purity of recombinant S205A or
D99N/S205A thrombin preparations were determined by
Coomassie and silver-stained SDS-PAGE gels. The mutant
thrombin preparations used in these studies appeared
homogeneous on silver—stained SDS-PAGE gels.
Example 8
Fibrinogen Clotting Assay
Fibrinogen clotting activity was measured by a
standard Fibro System“ coagulation timer (Fisher
Scientific, Springfield, NJ) as the time required for
varying thrombin concentrations to generate a fibrin
clot. All fibrinogen clotting reactions were performed
in a total volume of 300 pl, in 150 mM NaCl, 20 mM Tris,
pH 7.4, 10 mM CaCl2, 0.5% PEG 6000 at 37°C with a final
fibrinogen concentration of 3.3 mg/ml. Both standard WT
and recombinant WT showed identical curves—-e.g., about
second clotting times at 5 nM. Neither S2OSA nor
D99N/S205A were able to induce clotting.
Example 9
Platelet ATP Secretion and Aggregation Studies
Washed platelets were prepared as described
above and suspended in modified Tyrode' buffer, pH 7.4
with 2 mM magnesium and 1 mM calcium at a concentration
of 108 platelets/ml. All platelet studies were performed
in a total volume of 500 #1 with 20 pl Chromolume®
Platelet
reagent (Chronolog Corporation, Havertown, PA).
ATP secretion and aggregation were quantitated
independently by measuring changes in luminescence and
light transmittance, respectively, in a Chronolog dual-
channel lumiaggregometer (Chronolog Corporation,
Havertown, PA). Platelets were stirred at 300 rpm to
ensure rapid and uniform distribution of agonist.
500 pl of platelets were incubated for 15
minutes at 37°C with 18 pl of diluted S205A stock in 600
mM NaCl, 10 mM MES, pH 6.0, 0.5% PEG 6000 buffer to give
the desired final concentrations, or 18 #1 of buffer
alone and then challenged with native thrombin (1 mM
final concentration). Platelet ATP secretion and
aggregation were followed for 30 seconds after thrombin
addition.
percentage of maximum, defined as the luminescence signal
Platelet ATP secretion data are expressed as a
obtained 30 seconds after addition of 1 mM native
thrombin to buffer—pretreated platelets. The results are
shown in Figure 7. Each point represents the mean of
three replicate determinations, and are representative of
three replicate experiments. As shown, increasing
concentrations of S205A thrombin cause increasing
inhibition of thrombin-induced platelet secretion.
Similar results were obtained using the D99N/S205A mutant
thrombin.
In an additional determination it was shown
(Figure 8) that 400 nM SZOSA thrombin right-shifts the
dose response of platelets to native thrombin by
approximately 1 log. In this determination, 18 pl of
S205A in 600 mM NaCl, 10 mM MES, pH 6.0, 0.5% PEG 6000
buffer to give a final S205A concentration of 400 nM) or
an equal volume of buffer alone (solid lines) were
incubated with 500 pl of platelets for 15 minutes at
37°C.
final concentrations of a—thrombin; platelet ATP
Platelets were then stimulated with the indicated
secretion and aggregation were followed for 30 seconds
after thrombin addition. The date shown reflect the
maximum initial rate of platelet ATP secretion,
specifically, the maximum rate of platelet ATP secretion
occurring within 30 seconds of agonist addition and
before any aggregation was detected. Thus, the platelet
ATP secretion rates reported represent only agonist-
induced and not aggregation-induced responses. Curves
from three replicate experiments are shown in Figure 5.
One arbitrary unit corresponds to 33 pmoles of ATP
released per second based on calibration with ATP
standards.
An additional experiment shows S205A thrombin
inhibits the extent of native thrombin—induced platelet
secretion. Platelets were preincubated with various
concentrations of S205A, then stimulated with native
thrombin (1 nM final concentration). To prevent
aggregation-induced secretion, platelets in these
experiments were suspended to a final concentration of
2 x 107 platelets/ml and were not stirred after the
addition of native thrombin. Under these conditions,
platelets did not aggregate but did secrete ATP in
response to thrombin. Platelet secretion rate is
expressed in arbitrary units as defined above. Figure 9
shows tracings of platelet secretion curves, and are
representative of the results obtained in three replicate
experiments. The decrease in luminescence seen in the
control curve (0 nM S205A thrombin) is characteristic of
the assay and may represent end—product inhibition of
luciferase.
However, S205A thrombin does not inhibit ATP
secretion induced in platelets by stimulation with
agonist peptide or a calcium ionophore.
Example 10
Preparation of Antibodies
The peptides representing portions of the
thrombin receptor amino terminal extension were used as
immunogens to prepare polyclonal antisera and monoclonal
antibodies.
The peptide PESKATNATLDPRSFLLC (the cleavage
site peptide) and the peptide YEPFWEDEEKNESGLTEYC (the
anion exosite domain peptide) were used to generate
antibodies. These antisera were tested as antagonists in
the platelet activation assay described above. Both were
effective in blocking activation. The polyclonal
antibody preparation which is immunoreactive with the
anion exosite domain peptide, AblO47, was incubated with
the platelets prior to the addition of thrombin at a 1 nM
concentration was added. The inhibition was reversed by
the addition of the peptide binding Ab1047,
360." Ab1047 at a 1:100 dilution almost completely
inhibits the aggregation and activation of the platelets.
The peptide PESKATNATLDPRSFLLRNPNDKYEPFWEDE
EKNESGLTEC which contains the cleavage site and the
"peptide
proposed anion binding exosite of the receptor was also
used to prepare potent receptor blocking monoclonal
antibodies. This 40 residue peptide which has a Cys
residue added at the carboxyl terminus of the native
sequence was covalently attached to keyhole limpet
hemocyanin (KLH) through the Cys residue using the thiol-
specific reagent, m-maleimidobenzoyl-N-hydroxysulfo—
succinimide ester (Sulfo-MES, Pierce Chemical Co.).
Following dialysis of the peptide-KLH conjugate, this
material was used to immunize 3 BALB/c mice. Spleen
cells obtained from each of the mice were fused with P3X
cells to form a panel of hybridomas.
Supernatants from these hybridomas were assayed
for their ability to crossreact with the native
residue peptide used for the immunization as well as 15-
residue peptides which span the length of the 40-residue
sequence in ELISA assays. Only IgG-specific clones were
investigated further. Positive hybridomas were then
tested for their ability to block thrombin-induced
platelet aggregation in the microtiter plate shaker
assay. Finally, positive hybridomas were reassayed with
the ELISA assay using the 40-residue peptide under
increasing salt washing conditions to choose 6 hybridomas
with apparent high affinity. The 6 hybridomas were
subcloned by limiting dilution resulting in clones 4-2,
-6, 31-2, 33-1, 61-1, and 62-5.
Each of the clones was used for the production
of ascites fluid by intraperitoneal injection of 1 x 107
cells/mouse cells. Ascites fluid rich in IgG was
purified on protein A—sepharose, as the therapeutic
The ability of
each of these purified monoclonal antibodies to inhibit
thrombin-induced platelet aggregation (using thrombin as
agonist) was evaluated in washed platelets and is shown
in Table 5. The IC5Os for these MoAbs ranged between
.5-20 yg/ml of purified IgG.
potential of IgG is greater than IgM.
Table 5
Inhibition of Platelet Aqqreqation by Antibodies
LQSO fggzml Washed
MoAb Platelets)
4-2 10-20
-6 >20
31-2 2.5-5.0
33-1 2.5-4.0
61-1 2.5-5.0
62-5 10-20
Claims (1)
1. A DNA molecule comprising a nucleotide sequence encoding the human thrombin receptor, said thrombin receptor having the amino acid sequence set forth in
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USUNITEDSTATESOFAMERICA19/02/19916 | |||
US07/657,769 US5256766A (en) | 1991-02-19 | 1991-02-19 | Recombinant thrombin receptor and related pharmaceuticals |
US07/789,184 US5688768A (en) | 1991-02-19 | 1991-11-07 | Recombinant thrombin receptor and related pharmaceuticals |
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IE84069B1 true IE84069B1 (en) | |
IE920530A1 IE920530A1 (en) | 1992-08-26 |
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ID=27097486
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IE053092A IE920530A1 (en) | 1991-02-19 | 1992-02-19 | Recombinant thrombin receptor and related pharmaceuticals |
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US (8) | US5688768A (en) |
EP (2) | EP0572553B1 (en) |
JP (2) | JP3556215B2 (en) |
AT (1) | ATE247165T1 (en) |
AU (1) | AU665752B2 (en) |
CA (1) | CA2104394A1 (en) |
DE (1) | DE69233157T2 (en) |
IE (1) | IE920530A1 (en) |
NZ (1) | NZ241666A (en) |
WO (1) | WO1992014750A1 (en) |
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-
1991
- 1991-11-07 US US07/789,184 patent/US5688768A/en not_active Expired - Fee Related
-
1992
- 1992-02-19 JP JP50733192A patent/JP3556215B2/en not_active Expired - Fee Related
- 1992-02-19 EP EP92907700A patent/EP0572553B1/en not_active Expired - Lifetime
- 1992-02-19 WO PCT/US1992/001312 patent/WO1992014750A1/en active IP Right Grant
- 1992-02-19 CA CA002104394A patent/CA2104394A1/en not_active Abandoned
- 1992-02-19 AT AT92907700T patent/ATE247165T1/en not_active IP Right Cessation
- 1992-02-19 NZ NZ241666A patent/NZ241666A/en not_active IP Right Cessation
- 1992-02-19 IE IE053092A patent/IE920530A1/en not_active IP Right Cessation
- 1992-02-19 DE DE69233157T patent/DE69233157T2/en not_active Expired - Fee Related
- 1992-02-19 AU AU14568/92A patent/AU665752B2/en not_active Ceased
- 1992-02-19 EP EP03016689A patent/EP1378524A3/en not_active Withdrawn
-
1993
- 1993-02-17 US US08/018,760 patent/US6197541B1/en not_active Expired - Lifetime
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