Note: Descriptions are shown in the official language in which they were submitted.
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BRADYKININ ANALOGS AS
SELECTIVE INHIBITORS OF CELL ACTIVATION
Reference to Government Grant
The invention described herein was made, in part, in the course of
work supported by the National Heart Lung and Blood Institute under Grant Nos.
HL35553, HL55907, HL52779, and HL56415. The government has certain
rights in the invention.
Cross-Reference to Related Applications
This application claims the benefit of United States provisional
application Serial No. 60/046,085, filed April 23, 1997.
Field of the Invention
This invention relates to the inhibition of thrombin-induced cell
activation, and to the identification of compounds that inhibit thrombin-
induced
cell activation.
Background of the Invention
Bradykinin is a vasoactive peptide released from the precursor
plasma kininogens by kallikrein and other enzymes (Silva et al. , Amer. J.
Physiol.
156: 261-274 (1949)). Bradykinin has been described to have multiple
physiologic functions, including the stimulation of prostacyclin production
(Hong,
S. L. , Thromb. Res. 18, 787 ( 1980); Crutchley et al. , Biochim Biophy Acta
751,
99 (1983)) and the stimulation of the release of plasminogen activators (Smith
et
al. , Blood 66, 835 (1983)). Bradykinin induces superoxide formation and
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endothelium-dependent smooth muscle hyperpolarization (Holland, J.A. et al. ,
J.
Cell Physiol. 143, 21 (1990); Nakashima, M. et al. , J. Clin. Invest. 92, 2867
( 1993)). Along with acetylcholine, bradykinin is the major inducer of nitric
oxide
formation (Palmer, R.M.J. et al. , Nature 327, 524 (1987)). Bradykinin has
been
characterized to produce vasodilation in most vascular beds which in the
coronary
artery circulation results in increased blood flow (Line et al. , J. Mol. Cell
Cardiol. 24, 909 (1992)). These latter features have led some to characterize
bradykinin as a cardioprotective agent (Line et al. , supra; Gohlke et al. ,
Hypertension 23, 411 (1994); Parratt et al. , Cardiovascular Research 28, 183
(1994); Zanzinger et al. , Cardiovascular Research 28, 209 (1994)).
Bradykinin's
elevation by angiotensin converting enzyme inhibitors is believed to be the
mechanism by which these drugs promote their beneficial effects on heart
failure.
In addition to the delivery of bradykinin, its parent proteins, high
(HK) and low (LK) molecular weight kininogens, also have the ability to be
selective inhibitors of a-thrombin, inhibiting a-thrombin's ability to
activate cells
without interfering with its enzymatic ability (Meloni et al. , J. Biol. Chem.
266,
6786 (1991); Puri et al. , Blood 77, 500 (1991)). This activity was believed
to be
a unique function for the kininogens; one which had not been ascribed to other
proteins. Most naturally occurring human protein inhibitors of a-thrombin are
directed towards its protease activity. HK and LK are selective inhibitors of
thrombin's ability to activate platelets by blocking a-thrombin from binding
to the
platelet membrane (Meloni et al. , supra; Puri et al. , supra). This activity
of the
kininogens appeared to be localized to domain 3 on their heavy chain since
isolated domain 3 retains that activity (Jiang et al. , J. Biol. Chem. 267,
3712
(1992)).
Inhibition of platelet activation by domain 3 is observed by a
marked decrease in the platelet's ability to aggregate and secrete their
granule
contents. The granule contents comprise proteins which participate in
hemostasis,
thrombosis, and the inflammatory response. Inhibition of endothelial cell
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activation may similarly be observed by a decrease in secretion of endothelial
cell
contents such as tissue plasminogen activator and von Willebrand factor.
The isolated domain 3 from a tryptic digest of LK, like its parent
proteins HK and LK, functions to inhibit cell activation by blocking thrombin
binding to its target cells. This polypeptide is a selective inhibitor of
thrombin
induced platelet activation. Administration of domain 3 in vitro therefore
does
not impact on induction of platelet activation by physiological substances
other
than thrombin, such as, for example collagen, adenosine diphosphate,
epinephrine
and platelet activating factor.
Interventional procedures for coronary artery disease such as
coronary thrombolysis or percutaneous transluminal coronary angioplasty have
reduced mortality from acute coronary thrombosis. However, after intracoronary
thrombolysis with lytic agents, the reocclusion rate is high. The major cause
for
reocclusion is platelet thrombus. When artificial dacron crafts are
anast~m~sed
to human arteries, up to 30% of all patients will develop a platelet
thrombosis
within hours of surgery. This expected high complication rate frequently
requires
an additional operation with attendant complications. Thus, additional
therapies
are needed to prevent these reocclusion events due to platelet thrombi.
Two competing classes of antiplatelet agents for the prevention of
coronary thrombosis are being considered. One class of agents, including
monoclonal antibody 7E3, aims to block the final common pathway of platelet
activation by inhibiting glycoprotein IIb/IIIa (GPIIb/IIIa), integrin a"b/3~.
7E3 is
an effective agent, but it is a murine antibody and is antigenic in humans. A
second class of antiplatelet agents inhibit a presumed, primary initiating
agent of
platelet activation, a-thrombin. Infusions of Phe-Pro-Arg-chloromethylketone
(PPACK), a potent inhibitor of a-thrombin's proteolytic activity, prolongs the
bleeding time, a crude measure of platelet function (Hanson, S.R. et al. ,
Proc.
Natl. Acad. Sci. 85, 3184-3188 (1988)). The first generation of potent a-
thrombin proteolytic inhibitors to enter into clinical trials is a recombinant
product
derived from medicinal leeches, hirudin. This compound, which is a small
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molecular mass and is not considered to be antigenic, is a potent anti-
thrombin.
A synthetic analog of hirudin, hirulog, combines the anion exosite I binding
properties of hirudin with the proteolytic inhibitory activity of PPACK. In
Phase
III clinical trials, both drugs were effective inhibitors of platelet
activation. The
tradeoff for effective anticoagulation, however, was increased hemorrhage into
brain leading to the termination of three clinical trials. These non-selective
inhibitors of a-thrombin have an antithrombotic efficiency dose close to their
toxicity dose and are not clinically tolerated and, thus, may never have
commercial significance.
An ideal anti-thrombotic to prevent arterial thrombosis would be
one which prevents platelet and endothelial cell activation without preventing
the
proteolytic activity of a-thrombin to clot fibrinogen and activate protein C,
factor
XIII, and factors V and VIII. Only two known proteins, high molecular weight
(HK) and low molecular weight (LK) kininogens, are naturally occurring
selective
anti-thrombins (Meloni, F.J. et al. , J. Biol. Chem. 266; 6786-6794 (1991);
Puri,
R. N. et al. , Blood 77:500-507 ( 1991 )). Both low and high molecular weight
kininogens have identical amino acid sequences from their amino-terminus
through 12 amino acids beyond the carboxy-tet~ninus of bradykinin. LK and HK
share a common heavy chain (62 kDa), the bradykinin (BK) moiety (0.9 kDa),
and the first 12 amino acids of the amino terminal portion of each of their
"light
chains" (Takagaki, Y. et al., J. Biol. Chem. 260:8601-8609 (1985}; Kitamura,
N. et al. , J. Biol. Chem. , 260:8610-8617 (1985)). This identity corresponds
to
residues 1 through about residue 383. See Salveson et al. , Biochem J. 234,
429
(1986); Kellerman et al., Eur. J. Biochem. 154, 471 (1986). They diverge in
the
size of their light chains; the light chain of LK is 4 kDa; that of HK is 56
kDa.
Takagaki et al. , supra; Kitamura et al. , supra.
There is a need for improved methods of identification, as well as
the identification of new compounds which specifically inhibit thrombin-
induced
platelet or other cell activation, and for compounds which prevent platelet
aggregation.
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Summary of the Invention
The invention comprises a method of inhibiting thrombin-induced
platelet or other cell activation comprising administering to an individual in
need
of such treatment an effective amount of a compound which inhibits thrombin
activation of platelets or other cells which express the thrombin receptor,
wherein
said compound has the formula:
X, -Arg-Pro-Pro-X, (I)
wherein:
X, is from zero to thirty natural or synthetic amino acids; and
X, is from zero to thirty natural or synthetic amino acids,
provided that the N-terminal amino acid of X~ is not glycine.
One embodiment of the invention comprises administering a
compound according to formula I, wherein X, is zero to seven amino acids and
X, is zero to nine amino acids.
A preferred embodiment of the invention comprises administering
a compound according to formula I, wherein X, is from zero to thirty amino
acids
from amino acids 1-30 of SEQ ID NO:1.
A most preferred embodiment of the invention comprises
administering a compound according to formula I wherein the compound has the
formula Arg-Pro-Pro. Another most preferred embodiment of the invention
comprises administering a compound according to formula I wherein the
compound has the sequence Arg-Pro-Pro-Ala-Phe (SEQ ID N0:6).
The invention also comprises a method of inhibiting thrombin
induced platelet or other cell activation comprising administering to an
individual
in need of such treatment an effective amount of a compound which inhibits
thrombin activation of platelets or other cells, wherein said compound
comprises
two or more segments having the amino acid sequence X,-Arg-Pro-Pro-X,_, and
the compound has the formula:
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L-(X,-Arg-Pro-Pro-XZ}~ (II)
wherein:
L is a linker comprising a covalent bond or chemical group;
X,, which may be the same or different in each segment, is from
S zero to thirty natural or synthetic amino acids;
X,, which may be the same or different in each segment, is from
zero to thirty natural or synthetic amino acids;
the N-terminal amino acid of X~ is not glycine; and
n is an integer from two to twenty.
One embodiment of the invention comprises administering a
compound according to formula II, wherein X, is zero to seven amino acids and
X, is zero to nine amino acids.
Another embodiment of the invention comprises administering a
compound according to formula II wherein the compound has the formula:
1 S L-(Arg-Pro-Pro-XZ)
A preferred embodiment comprises administering a compound
according to formula II wherein the compound has the formula:
Arg-Pro-Pro-Lys
Arg-Pro-Pro-Asp
Another preferred embodiment comprises administering a
compound according to formula II wherein the compound has the formula:
Arg-Pro-Pro
Lys
?S Arg-Pro-Pro
Lys-/3Ala (III)
Arg-Pro-Pro
Lys
Arg-Pro-Pro
The invention further comprises a method for preventing platelet
aggregation comprising administering to an individual in need of such
treatment
an effective amount of a compound according to formula I, II, or III.
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The invention further comprises a method for inhibiting ADP-
induced platelet activation in vivo comprising administering to an individual
in
need of such treatment an effective amount of a compound according to formula
I, II, or III.
The invention also comprises a compound according to formula I,
II, or III. In a preferred embodiment the compound has the formula:
Arg-Pro-Pro-Lys
Arg-Pro-Pro-Asp.
Another preferred embodiment is a compound according to formula
III.
The invention further comprises a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a compound according to
formula I, II, or III.
The invention also comprises the use of a compound according to
formula I, II, or III for the preparation of a medicament for inhibiting
thrombin-
induced platelet or other cell activation, or for preventing platelet
aggregation.
The invention further provides a method for identifying compounds
that selectively inhibit thrombin-induced platelet and other cell activation
comprising measuring the ability of the compounds to bind to the thrombin
cleavage site on the thrombin receptor. In a preferred embodiment the
compounds are present in a combinatorial library. In a more preferred
embodiment the method further comprises (a) measuring the ability of the
compounds to inhibit thrombin-induced platelet aggregation; and (b) measuring
the ability of the compounds to inhibit thrombin-induced calcium mobilization
in
fibroblasts.
Description of the Figures,
Figure 1 shows the prolonged inhibition of ~y-thrombin-induced
platelet aggregation of rabbit platelets ex vivo after the in vivo infusion of
RPPGF
(SEQ ID N0:7).
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Figure ? shows aggregometer tracings of y-thrombin induced
aggregation of human platelets in platelet-rich plasma treated with y-thrombin
alone (control), and with the BK analog Arg-Pro-Pro (50 ~cM).
Figure 3 shows aggregometer tracings of y-thrombin induced
aggregation of human platelets treated with y-thrombin alone (control), with
the
peptide Arg-Pro-Pro-Gly-Phe {SEQ ID NO: 7, 125 ~,M), with a RPP heterodimer
peptide (75 ~,M), and with a RPP MAP-4 peptide (25 ~,M).
Figure 4 shows the direct binding of biotinylated-NAT12 (SEQ ID
N0:2) to RPPGF (SEQ ID N0:7), RPPGC (SEQ ID N0:9), RPP MAP-4, LNA7
(SEQ ID N0:8), FSPFR (SEQ ID NO:10), or bovine serum albumin (BSA).
Figure 5 shows a-thrombin-induced calcium mobilization on
fibroblasts grown to confluence in microtiter plates (panel A), and inhibition
of a-
thrombin-induced calcium mobilization by HK, high molecular weight kininogen
(panel B), RPPGF (SEQ ID NO: 7, panel C), and RPP (panel D).
i 5 Figure 6 shows a-thrombin-induced calcium mobilization on human
umbilical vein endothelial cells (HUVEC) grown to confluence in microtiter
plates
(panel A), and inhibition of a-thrombin-induced calcium mobilization by HK.
high
molecular weight kininogen (panel B), RPP MAP-4 (panel C), and RPPGF MAP-4
(panel D).
Detailed Description of the Invention
Definitions
"Natural amino acid" means any of the twenty primary, naturally
occurring amino acids which typically form peptides, polypeptides, and
proteins.
"Synthetic amino acid" means any other amino acid, regardless
of whether it is prepared synthetically or derived from a natural source. As
used
herein, "synthetic amino acid" also encompasses chemically modified amino
acids.
including but not limited to salts, derivatives (such as amides), and
substitutions.
"Human kininogen" means, unless otherwise indicated, both high
and low molecular weight forms of any kininogen molecule, in all its various
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forms derived from human plasma, platelets. endothelial cells, granulocytes,
or
skin or other tissues or organs, regardless of whether it is found in the
fluid or the
tissue phase.
"Light chain", when referring or relating to human kininogen.
means the 56 kDa intermediate plasma kallikrein-cleavage fragment of HK which
has the ability to correct the coagulant defect in total kininogen-deficient
plasma.
"Heavy chain", when referring or relating to human kininogen.
means the 64 kDa kallikrein-cleavage fragment of HK or LK, which is free of
bradykinin and "light chain".
"Domain 3". with respect to the kininogen heavy chain, means the
trypsin-cleavage fragment of the human kininogen heavy chain which is about 21
kDa.
"Bradykinin" (BK) means the nonapeptide having the sequence
SEQ ID NO: 5.
"BK analog" means a compound comprising an amino acid
sequence analogous to all or pan of the sequence of the nonapeptide
bradykinin,
which is capable of inhibiting a-thrombin from cleaving its receptor on
platelets
and other cells, such that the peptide prevents the alteration or loss of the
SPAN12
epitope on the thrombin receptor. and blocks cleavage of a peptide, NAT12 (SEQ
ID N0:2), which spans the a-thrombin cleavage site on the thrombin receptor.
"Heterodimer" means a compound comprising two different
single-chain BK analogs joined by a linker.
"Homodimer" means a compound comprising two identical single
chain BK analogs joined by a linker. "Symmetric homodimer" means a
homodimer in which the linker joins amino acids which occupy the same position
in the single chain BK analogs. "Asymmetric homodimer" means a homodimer
in which the linker joins amino acids which occupy different positions in the
single chain BK analogs.
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Abbreviations
Some of the nomenclature of the subject matter of the present invention
involves lengthy terms. It is customary for those skilled in the art to
abbreviate
these terms in a manner well-known to the art. These general and customary
abbreviations are set forth below and may be utilized in the text of this
specification.
ATAP138: monoclonal antibody specific for an epitope
on the thrombin
receptor, which epitope is preserved following
a-thrombin
cleavage of the receptor
BK: bradykinin
D3: domain 3 of kininogen
EDTA: ethylenediaminetetraacetic acid
FITC: fiuorescein isothiocyanate
HBTU: 2-( 1-H-benzotriazole- I -YL)-1,1,3,3-tetramethyl-
uroniumhexofluorophosphate
HOBt: 1-hydroxybenzotriazole
HK: human high molecular weight kininogen
LK: human low molecular weight kininogen
NAT12: peptide sequence Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-
Leu-Leu-Arg (SEQ ID N0:2), which spans
the a-thrombin
cleavage site on the thrombin receptor
PGE 1: prostaglandin E 1
SPAN 12: monoclonal antibody specific for the sequence
Asn-Ala-Thr-
Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg (SEQ
ID N0:2)
which spans the a-thrombin cleavage site
on the thrombin
receptor
TRAP: thrombin receptor activation peptide. which has the amino
acid sequence Ser-Phe-Leu-Leu-Arg-Asn (SEQ ID N0:4)
The invention is directed to a method for preventing thrombosis by the use
of BK analogs, bradykinin sequence-related analogous peptides that act as
selective
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anti-thrombins. The BK analogs are selective anti-thrombins because they are
able
to inhibit human a-thrombin and y-thrombin from activating platelets and other
cells without interfering with a,-thrombin's ability to proteolyze its various
substrates, e. g. , fibrinogen and factor XIII. Most known thrombin
inhibitors,
hirudin, hirulog and PPACK, interfere with a-thrombin's action by blocking all
of its proteolytic activity. Use of these proteolytic inhibitors to inhibit oc-
thrombin
activation of platelets may result in excessive anticoagulation and
hemorrhage.
The BK analogs utilized in the present method allow for inhibition of thrombin-
induced cell activation (e.g. platelet activation, mitogenesis) without
interfering
with a-thrombin's enzymatic activity on other substrates. such as proteolysis
of
fibrinogen and activation of factor XIII. BK analogs may be used to prevent
arterial occlusions which occur in coronary thrombosis and stroke.
The BK analogs of the present invention block 1 nM y-thrombin from
activating platelets in the presence of I00 mg/dl fibrinogen and in platelet-
rich
plasma.
The BK analogs of the present invention do not appear to inhibit platelet
activation by the same mechanism as intact kininogen and its isolated domain
3,
because the BK analogs do not inhibit ''-5I-a-thrombin binding to platelets,
as does
a molar excess of purified HK. LK, or isolated domain 3
Without wishing to be bound by any theory, the BK analogs of the present
invention are believed to inhibit platelet and other cell activation by
inhibiting a,-
thrombin from cleaving its receptor, which is expressed on platelets and other
cells. The BK analogs thus have the ability to inhibit thrombin-induced
platelet
activation by blocking cleavage of the thrombin receptor and subsequent
activation
of platelets by exposure of the new amino terminus of the cleaved receptor.
Administration of a BK analog as described herein comprises a therapeutic
method for inhibiting thrombin-induced activation of platelets, endothelial
cells,
brain cells, fibroblasts, smooth muscle cells, or other cells that express the
thrombin receptor. This function inhibits platelet thrombus formation and
other
activities mediated by the thrombin receptor.
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The thrombin receptor peptide NAT12 and monoclonal antibodies SPAN12
and ATAP138 are useful for characterizing the compounds, compositions. and
methods of the present invention, as illustrated in Example 1. NAT12 has a
sequence, Asn-Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg (SEQ ID N0:2),
which spans the a-thrombin cleavage site on the thrombin receptor.
Monoclonal antibodies to the thrombin receptor, SPAN12 and ATAP138.
were obtained from Dr. Lawrence F. Brass of the University of Pennsylvania,
and
were prepared according to the method of Brass et al., J. Biol. Chem. 267.
13795
( 1992). Monoclonal antibody SPAN12 was reared to the 12 amino acids, Asn-
Ala-Thr-Leu-Asp-Pro-Arg-Ser-Phe-Leu-Leu-Arg (SEQ ID N0:2), that bridge the
a.-thrombin cleavage site on the thrombin receptor. Monoclonal antibody
ATAP 138 recognizes an epitope, Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe (SEQ ID
N0:3). on the thrombin receptor which is preserved after cleavage by a-
thrombin.
When a,-thrombin cleaves the thrombin receptor, it eliminates the epitope
which
is recognized by the SPAN 12 antibody but not the epitope which is recognized
by
the ATAP138 antibody.
The complete sequence for human kininogen heavy chain is found in
Kellerman et al. , Eur. J. Biochem. 154:471-478 ( 1986), the entire disclosure
of
which is incorporated herein by reference. The amino acid sequence of the
human
kininogen parent segment, which spans kininogen amino acid residues 333 to
396.
is given herein as SEQ ID NO:1. The core peptide sequence Arg-Pro-Pro
corresponds to kininogen amino acid residues 363-65.
According to the present invention, naturally occurring or synthetic amino
acids having the general formula
Co2 _
R-CH
i
NH3'
where R is a hydrogen atom or an organic side chain, are added to either the
carboxyl or amino terminus of a peptide comprising the core sequence Arg-Pro-
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Pro, in order to form chain expansion analogs. Up to thirty amino acids may be
added to either the carboxyl or amino terminus of the core sequence.
Preferably,
from zero to seven amino acids are added to the amino terminus, and zero to
nine
amino acids are added to the carboxyl terminus of the core sequence. An
example of the BK analogs included in this invention is the BK analog SEQ ID
N0:6, which has the sequence Arg-Pro-Pro Ala-Phe. Another example is the
tripeptide Arg-Pro-Pro.
According to one embodiment of the invention, the BK analog has the
sequence X,-Arg-Pro-Pro-X~, wherein X, is from zero to thirty amino acids from
the amino terminal portion of the kininogen heavy chain parent segment (amino
acids 1-30 of SEQ ID NO:1) and X, is from zero to thirty natural or synthetic
amino acids, provided that the N-terminal amino acid of X~ is not glycine.
In another embodiment of the invention, the BK analog has the sequence
X,-Arg-Pro-Pro-X2, wherein X, is from zero to seven amino acids from the amino
terminal portion of the kininogen heavy chain parent segment (amino acids 24-
30
of SEQ ID NO:1) and X~ is from zero to nine natural or synthetic amino acids,
provided that the N-terminal amino acid of X~ is not glycine.
According to another embodiment of the invention, two or more single
chain BK analogs are joined by one or more linkers, L, to form homodimers,
heterodimers, trimers, or other multimers. The linker can be either a covalent
bond or a chemical group. The number of single-chain BK analogs that can be
joined is from two to thirty-two. Preferably, the number of BK analogs joined
is from two to twenty, more preferably from two to eight, and most preferably,
from two to four. The BK analogs to be joined can be identical or they can be
different. A heterodimer is comprised of two different single-chain BK
analogs;
a homodimer is comprised of two identical single-chain BK analogs. The
multimers can be symmetric or asymmetric. A "symmetric homodimer" means
a homodimer in which the linker joins amino acids which occupy the same
position in the single chain BK analogs. An "asymmetric homodimer" means a
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homodimer in which the linker joins amino acids which occupy different
positions
in the single chain BK analogs.
An example of a covalent bond linking two single-chain BK analogs is the
disulfide bond formed by the oxidation of two single chain BK analogs
containing
cysteine amino acids. This may require initially modifying the parent peptide
so
that the peptide includes a Cys residue in the appropriate position. Cysteine
residues on single-chain BK analogs can be oxidized to form BK analog dimers
by dissolving 1 mg of the single-chain peptide in 1.5 ml of 0.1 % (v/v) 17. S
mM
acetic acid, pH 8.4, followed by flushing with nitrogen and then 0.01 M
K,Fe(CN)6. After incubation for one hour at room temperature, the dimer
peptide
is purified by HPLC.
Another example of a suitable covalent bond for linking two single-chain
BK analogs is the amide bond formed by reacting the amino group of a lysine
amino acid residue on one chain with the carboxylic acid group of a glutamic
or
aspartic amino acid residue of another chain.
Alternatively, the linking group can be formed by the covalent bond
between two single-chain BK analogs using a cross-linking reagent. For
example,
homodimers and heterodimers can be prepared by first preparing S-(-N-
hexylsuccinimido)-modified peptide monomers according to the method of
Cheronis et al. , J Med. Chem. 37: 348 (1994). N hexylmaleimide, a precursor
for the modified peptide monomers, is prepared from N-
(methoxycarbonyl)maleimide and N hexylamine by mixing the two compounds in
saturated NaHC03 at 0°C according to the procedure of Bodanszky and
Bodanszky, The Practice of Peptide Synthesis; Springer-Verlag, New York, pp.
29-31 ( 1984). The product of the resulting reaction mixture is isolated by
extraction into ethyl acetate, followed by washing with water, dried over
Na,SOa,
and is then concentrated in vacuo to produce N-hexylmaleimide as a light
yellow
oil. S-(N Hexylsuccinimido)-modified peptide monomers are then prepared from
a cysteine-containing peptide (monomer) and N-hexylmaleimide by mixing one
part peptide with 1.5 parts N hexylmaleimide in dimethylformamide (3.3 ml/mM
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peptide) followed by addition to 30 volumes of 0.1 M ammonium bicarbonate, pH
7.5. The S-alkylation reaction carried out in this manner is complete in 30
min.
The resulting S-(N hexylsuccinimido)-modified peptide monomer is purified by
preparative reverse-phase HPLC, followed by lyophilization as a fluffy, white
powder.
Bissuccinimidohexane peptide dimers, either as homodimers or
heterodimers, may be prepared according to the method of Cheronis et al. ,
supra
from cysteine-substituted peptides in the same or different positions,
respectively.
A mixture of one part bismaleimidohexane is made with two parts peptide
monomer in dimethyIformamide (3.3 ml/mM peptide) followed by addition to 0.1
ammonium bicarbonate, pH 7.5. The reaction mixture is stirred at room
temperature and is usually completed within 30 min. The resulting
bissuccinimidohexane peptide dimer is purified by preparative reverse-phase
HPLC. After lyophilization the material is a fluffy, white powder.
Covalently cross-linked BK analog dimers of the present invention may be
prepared by utilizing homobifunctional cross-linking reagents, e. g. ,
disuccinimidyi
tartrate, disuccinimidyl suberate, ethylene glycolbis(succinimidyl succinate),
1,5-
difluoro-2,4-dinitrobenzene ("DFNB"), 4,4'-diisothiocyano-2,2'-disuifonic acid
stilbene ("DIDS"), and bismaleimidohexane ("BMH"). The cross-linking reaction
occurs randomly between the single-chain BK analogs.
Alternatively, heterobifunctional cross-linking reagents may be employed.
Such agents include, for example, N-succinimidyl-3-(2-pyridyldithio)propionate
("SPDP"), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1-3'-dithiopropionate
("SASD", Pierce Chemical Company, Rockford, IL), N-maleimidobenzoyl-N-
hydroxy-succinimidyl ester ("MBS "), m-maleimidobenzoylsulfosuccinimide ester
("suifo-MBS"), N-succinimidyl(4-iodoacetyl)aminobenzoate ("SIAB"),
succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate ("SMCC"),
succinimidyl-4-(p-maleimidophenyl)butyrate ("SMPB"), sulfosuccinimidyl(4-
iodoacetyl)aminobenzoate ("sulfo-SIAB"), sulfosuccinimidyl 4-(N-
maleimidomethyl)cyclohexane-1-carboxylate ("sulfo-SMCC"), sulfosuccinimidyl
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4-(p-maleimidophenyl)-butyrate("sulfo-SMPB"),bromoacetyl-p-aminobenzoyl-N-
hydroxy-succinimidyl ester, iodoacetyl-N-hydroxysuccinimidyl ester, and the
like.
For heterobifunctional cross-linking, a first single-chain BK analog is
derivatized with, e. g. , the N-hydroxysuccinimidyl portion of the
bifunctional
reagent, and the derivatized BK analog is purified by gel filtration. Next, a
second single-chain BK analog (which may or may not be the same or different
from the first BK analog) is reacted with the second functional group of the
bifunctional reagent, assuring a directed sequence of binding between
components
of the BK dimer.
Typical heterobifunctional cross-linking agents for forming protein-protein
conjugates have an amino-reactive N-hydroxysuccinimide ester (NHS-ester) as
one
functional group and a sulfhydryl reactive group as the other functional
group.
First, epsilon-amino groups of surface lysine residues of the first single
chain BK
analog are acylated with the NHS-ester group of the cross-linking agent. The
second single chain BK analog, possessing free sulfhydryl groups, is reacted
with
the sulfl~ydryl reactive group of the cross-linking agent to form a covalently
cross-
linked dimer. Common thioi reactive groups include maleimides, pyridyl
disulfides, and active halogens. For example, MBS contains a NHS-ester as the
amino reactive group, and a maleimide moiety as the sulthydryl reactive group.
Photoactive heterobifunctional cross-linking reagents, e. g. , photoreactive
phenyl azides, may also be employed. One such reagent, SASD, may be linked
to a single-chain BK analog via its NHS-ester group. The conjugation reaction
is carried out at pH 7 at room temperature for about 10 minutes. Molar ratios
between about 1 and about 20 of the cross-linking agent to the BK analog may
be
used.
The purified, derivatized BK analog is collected by affinity
chromatography using a matrix having affinity for BK analogs, e. g. , a
polyclonal
antibody reared to the BK analog. Antibody for this purpose may be prepared by
coupling the BK analog to key hole limpet hemocyanin using 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide-HCL (Goodfriend et al. , Science 144, 1344
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(1964)). The resulting conjugate is used to immunize rabbits by the procedure
of Miiller-Esterl et al., Methods Enzymol 163, 240 (1988) to produce anti-BK
analog antibodies. The purified antibody is coupled to AFFIGEL 10 (Bio-Rad,
Richmond, CA) to form an affinity column. Immobilized anti-BK analog
antibody, with the derivatized BK analog bound thereto, is then removed from
the
column by 0.2 M glycine elution and suspended in a solution of a second single
chain BK analog. An ultraviolet light source (e.g., Mineralight UVSL-25, Ultra
Violet Products, Inc., San Gabriel, CA) is positioned 1 cm from the gently
stirred
suspension and irradiated in a long-wavelength range for about 10 minutes. The
suspension is put back on the anti-BK analog antibody affinity column and
washed
with a buffer containing 0.15 M NaCI, 0.1 % bovine serum albumin, O.OI %
polysorbate 80 and 2S KIU/ml of aprotinin to remove reaction byproducts. The
covalently cross-linked dimer is eluted with the same buffer system containing
0.2 M glycine or 5 M guanidine. The eluted dimer is dialyzed against buffer to
remove the chaotropic agent.
Following reaction with the BK analog under ultraviolet irradiation, and
chromatography of the reaction mixture as above, the covalently cross-linked
dimer is eluted with either 0.2 M glycine or 5 M guanidine.
While the above-described procedure utilizes SASD, a cleavable cross
linker, non-cleavable cross-linking reagents may be utilized which contain, e.
g. ,
alpha-hexanoate, rather than beta-ethyl-1,3-dithiopropopionate moieties. MSB
is
one example of a non-cleavable cross-linking reagent.
The single-chain BK analogs may be prepared by conventional solid phase
peptide synthesis techniques using automated synthesis. Alternatively, BK
analogs
may be prepared by recombinant DNA techniques. A gene which encodes a BK
analog may be constructed and introduced into an appropriate host by use of an
appropriate cloning vector. Thus, it should be understood that the present
invention is not merely limited to the use of BK analogs as prepared by
peptide
synthetic methods, but also includes polypeptides prepared by recombinant
techniques.
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Moreover, by utilization of such recombinant techniques, one skilled in the
art may prepare additional BK analogs using methods such as site-directed
mutagenesis of the relevant DNA, wherein an amino acid sequence may be
modified to contain single or multiple amino acid changes, additions, or
deletions.
Such sequence modifications are included within the scope of the inven-
tion, provided that the resulting molecule substantially retains the ability
to inhibit
thrombin-induced cell activation.
Additional BK analogs may be identified by screening a combinatorial
library which displays a plurality of peptide analogs. As an example,
degenerate
oligonucleotides may be used to direct the display of heterologous peptides on
the
surface of microorganisms (for example, bacteriophage, bacteria, yeast). One
such method uses a display library expressing the DNA sequence (NNK)". This
DNA encodes the amino acid sequence (Xaa)n, which includes all peptides having
n amino acids. The number of possible peptides (using the 20 natural amino
acids) is 20°. Libraries of such microorganisms are initially screened
for the
ability to bind the cloned thrombin receptor or the NAT12 peptide, as
described
in Example 2. Peptides having activity in a binding assay are screened using
one
of the in vitro functional assays described in Example 1. Activity in the in
vitro
functional assays is predictive of activity in vivo, as shown in Example 3.
New
BK analogs which show activity in the in vitro functional assays are tested
for
safety and efficacy in animals and in human clinical trials.
Combinatorial libraries can also be prepared on a solid phase matrix
support, and screened for the ability to bind to the thrombin cleavage site on
the
thrombin receptor. It is particularly advantageous to screen combinatorial
libraries for compounds which (1) inhibit thrombin-induced platelet
aggregation,
(2) bind to the thrombin cleavage site on the thrombin receptor, and (3)
inhibit
thrombin-induced calcium mobilization in fibroblasts. This combination
screening
method identifies compounds which are selective inhibitors of thrombin-induced
platelet and other cell activation. Additional methods of preparing and
screening
combinatorial libraries are known to those of skill in the art.
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Purified BK analogs may be administered in any circumstance where
inhibition of thrombin-induced or ADP-induced platelet activation or platelet
aggregation is sought. BK analogs are administered to subjects experiencing
platelet thrombosis from any cause, and may also be used prophylactically in
subjects undergoing surgery or catherization for insertion of artificial
dacron
grafts and stems to prevent reocclusion events due to platelet thrombi. BK
analogs may be infused into individuals to prevent strokes and cerebral edema.
The BK analogs may be administered by any convenient means which will
result in substantial delivery into the bloodstream, including intravenous,
intranasal, and oral administration, as well as administration via a dermal
patch
or rectal suppositories. Intravenous administration is presently contemplated
as
the preferred administration route, although intranasal administration may
also be
utilized. Since BK analogs are soluble in water, they may be effectively
administered in solution. The actual dosage administered may take into account
the size and weight of the patient, whether the nature of the treatment is
prophylactic or therapeutic in nature, the age, health and sex of the patient,
the
route of administration, and other factors. Those skilled in the art can
derive
appropriate dosages and schedules of administration to suit the specific
circumstances and needs of the patient. An effective daily dosage of active
ingredients, based upon in vivo clearance studies using BK analogs related to
those of the present invention, is from about 0.1 to about 10 grams per day
per
70 Kg of body weight. The effective daily dosage is preferably from about 1 to
about 5 grams per day per 70 Kg of body weight. In a preferred embodiment,
a dosage of about 3 grams per day per 70 kg of body weight is given in a
single
bolus infusion of 2.4 grams followed by a continuous infusion of 0.025 g/hour.
The amount of BK analog administered will also depend upon the degree
of platelet activation or aggregation inhibition that is desired. While
infusion of
a BK analog to achieve 3 grams per day dosage may be advantageously utilized,
more or less of the peptide may be administered as needed. The theranem;~ P"r~
point may be determined by monitoring platelet function by aggregation and
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secretion, vessel patency, and bleeding. The actual amount of the BK analog
administered and the length of the therapy regime to achieve the desired
intravas-
cular concentration is readily determinable by those skilled in the art by
routine
methods.
The BK analogs may be administered in a pharmaceutical composition in
a mixture with a pharmaceutically acceptable carrier. The pharmaceutical
composition may be compounded according to conventional pharmaceutical
formulation techniques. The carrier may take a wide variety of forms depending
on the form of preparation desired for administration. For a composition to be
administered parenterally, the carrier will usually comprise sterile water,
although
other ingredients to aid solubility or for preservation purposes may be
included.
Injectable suspension may also be prepared, in which case appropriate liquid
car-
riers, suspending agents and the like may be employed. The preferred
parenteral
route of administration is intravenous administration.
For intravenous administration, the BK analogs may be dissolved in any
appropriate intravenous delivery vehicle containing physiologically compatible
substances, such as sterile sodium chloride having a buffered pH compatible
with
physiologic conditions. Such intravenous delivery vehicles are known to those
skilled in the art.
Examples
The following examples illustrate the practice of the invention. These
examples are illustrative only, and do not limit the scope of the invention.
Example 1
In Vitro Functional Assays
Functional assays are used to evaluate the relative inhibitory efficacies of
the methods and compounds according to the invention. In these assays, native
BK (SEQ ID NO:S), the BK fragment Arg-Pro-Pro-Gly-Phe (SEQ ID N0:7), or
another BK analog, may be used as a positive control peptide.
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A. Inhibition of Platelet Aggregation
For human platelet aggregation studies, 50 ml human blood was collected
into a syringe containing 5 ml of 0.013 M sodium citrate. The anticoagulated
blood was centrifuged at 180 xg for 10 minutes at room temperature and the
platelet-rich plasma was the supernatant. Platelet aggregation studies in
platelet-
rich plasma were performed in the cuvette of an aggregometer using y-thrombin
(Haematologic Technologies, Essex Junction, VT). After standardization of the
aggregometer, the threshold dose of y-thrombin, defined as the minimal
concentration that will induce full platelet aggregation, was ascertained for
each
preparation of platelet-rich plasma. Typically, the threshold dose of y-
thrombin
was between 5 and 30 nM.
Various concentrations of the peptide being tested were added to the
platelet-rich plasma in the cuvette of the aggregometer. Platelet aggregation
was
induced by the addition of ~y-thrombin. The degree of platelet aggregation was
' 15 determined by measuring (in arbitrary units) the increase in light
transmission
through the stirred suspension of platelet-rich plasma.
In a control experiment, the BK fragment SEQ ID N0:7 inhibited ~y-
thrombin-induced (20 nM) platelet aggregation at a concentration of 1 mM
peptide.
B. Inhibition of Platelet Aggregation and Secretion
For simultaneous platelet aggregation and secretion studies, fresh whole
blood was collected and mixed with sodium citrate (final concentration 0.013
M).
Platelet-rich plasma was prepared according to the method of Meloni et al. ,
J.
Biol. Chem. 266, 6786, 1991. Washed platelets were prepared by gel filtration
over Sepharose 2B columns in Hepes-Tyrode's buffer (0.137 M NaCI, 3 mM
KCI, 0.4 mM Na HZP04, 12 mM NaHC03, 1 mM MgCh, 14.7 mM Hepes
containing 20 mM glucose and 0.2% bovine serum albumin, pH 7.35). Platelets
were incubated according to the method of Schmaier et al. , Blood 56, 1013,
1980
with 5-['4C]hydroxytryptamine for 30 min at 37°C. The washed platelets
(2 X
108/ml, final concentration radiolabeled with S-['4C]hydraxytryptamine) were
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added to a cuvette of an aggregometer (Chronolog Corp., Havertown, PA),
standardized using the protocol of Meloni et al. , supra. After the addition
of
ZnCh, final concentration 50 ~.M, purified HK ( 1 ~,M) or various
concentrations
of the peptides (0.1 to 3 mM) or buffer alone was added to the cuvette. Once
the
baseline stabilized, a-thrombin [0.125 U/ml ( 1 nM) final concentration] was
then
added to initiate platelet activation. Stirred platelets were allowed to
incubate
with a-thrombin and additions for 1 min. In other experiments, platelets were
stimulated with TRAP (0.625 to 2.5 ~.M), ADP (1-5 ~cM)(Sigma), collagen (1.25
~g/mI) {Horm, Munich, Germany), or U-46619 (1 ~cM)(Calbiochem Behring, San
Diego, CA). Additional experiments were performed with washed platelets
stimulated with ~y-thrombin ( 1 nM) in the presence of human fibrinogen ( 100
mg/dl). Both y-thrombin and human fibrinogen were purchased from Enzyme
Research Laboratories, South Bend, IN. At the conclusion of the incubation,
the
entire platelet sample was centrifuged at 10,900 xg (Model E, Beckman
Instruments, Palo Alto, CA) over a 0.135 mM formaldehyde, 5 mM EDTA
solution (1 part of formaldehyde-EDTA to 4 parts of platelet suspension) and
stored on ice until an aliquot of the supernatant was assayed for 5-
['4C]hydroxytryptamine secretion. Percent secretion was determined by the
ratio
of the loss of 5-['4C]hydroxytryptamine in the supernatant of the agonist-
treated
specimen to the total 5-['4C]hydroxytryptamine in a platelet suspension of
unstimulated platelets after the level of 5-['4C]hydroxytryptamine in the
supernatant of the unstimulated sample was subtracted from both. Platelet
aggregation was measured in arbitrary units as the initial rate of change in
light
transmittance in the first minute after introduction of agonist.
In a control experiment, native BK (SEQ ID NO:S) inhibited a-thrombin-
induced ( 1 nM) platelet activation with an ICSO of 0.25 mM and 1.0 mM for 100
percent aggregation and secretion inhibition, respectively.
C. Inhibition of a-thrombin-induced Calcium Mobilization
For calcium mobilization studies, the cytoplasmic free Ca2+ concentration
([Ca2+];) was measured using the fluorescent Ca2+ indicator fura-2
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acetyloxymethylester (fura-2 AM, Molecular Probes, Inc., Eugene, OR). Gel
filtered platelets in HEPES-Tyrode's buffer were loaded with fura-2 by
incubation
at 37°C with 1 ~cM fura-2 AM for 45 min according to the method of
Rasmussen
et al. , J. Biol. Chem. 268, 14322 (1993). The labeled platelets were then re-
gel
filtered to remove any excess probe. Aliquots of the labeled platelet
suspension
were transferred into a quartz cuvette with a magnetic stirrer, which was then
placed in a thermostatically controlled chamber at 37 °C in a
fluorescence
spectrophotometer (Dual Wave Length Shimazdu SP5000 Spectrofluorometer,
Shimazdu USA, Pittsburgh, PA). Reagents were directly added to the cuvette.
The excitation wave lengths varied between 340 and 380 nm. The fluorescence
was measured by recording emitted light at 510 nm as reported by Fisher et al.
,
Mol. Pharm. 35, 195 (1989). The minimum emission was determined on a 20
mM digitonin, 10 mM EGTA solubilized platelet sample; maximum emission was
determined on the same sample with 10 mM Ca2+ added. The intraplatelet free
Ca'+ concentration was calculated by the method of Grykiewicz et al. , J.
Biol.
Chem. 260, 3440 (1985). The ratio of the fluorescence readings was calculated
as R = 340/380 nm and processed according to the equation [Ca2+]; = KD((R-
R",;~)/Rmax-R))(Sa/Sbz) to determine the intraplatelet free Ca2+
concentration. The
Ko for fura-2 was assumed to be 224 nM. R",aX and Rm;" are the maximum and
minimum fluorescence ratios measured at the end of the experiment,
respectively;
S~, and Sb~ are the fluorescence values at 380 nm in the absence and presence
of
saturating [Caz+], respectively.
In a control experiment, the BK fragment SEQ ID N0:7 was able to
inhibit a-thrombin-induced calcium mobilization in a concentration dependent
manner. One mM SEQ ID N0:7 produced 80 % inhibition of a-thrombin-induced
calcium mobilization. These data indicate that BK analogs can interfere with a-
thrombin activation of platelets at the level of the stimulus-response
coupling
mechanism.
A variation of the calcium mobilization assay uses fibroblasts (which
express a single receptor for thrombin, PAR 1), umbilical vein endothelial
cells,
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or other cells which express the cloned thrombin receptor, and which grow in
monolayers on the bottom of plastic wells. Examples of other kinds of cells
which express the thrombin receptor, and which may have their calcium
mobilization abilities blocked, include smooth muscle cells, astrocytes and
neuronal cells.
Fibroblasts or endothelial cells were grown in 24-96 well plates and loaded
with the fluorescent Ca'+ indicator furs-2 acetyloxymethylester (S~,M,
Molecular
Probes Inc.) by incubation for 60 min. in HEPES-Tyrode's buffer at
37°C. After
washing the cells, the fluorescence of the cells was monitored on a
thermostatically controlled heated pad at 37°C for 1 min, then the
cells were
incubated with a-thrombin (1 to 5 nM) in the absence or presence of a BK
analog. Fluorescence was detected by a Perkin-Elmer LS-SOB Luminescence
Spectrofluorometer. Excitation was measured at 340 and 380 nm wavelength and
emission was assessed at 510 nm. The cytosolic Ca'+ levels were determined as
described above. The RmaX value was determined using 10 mM Ca'-+ in the
presence of 20 ~M Ionophor A23187; R",;~ was determined in the presence of 20
mM EDTA.
D. Inhibition of the Elimination of the Epitope Recognized by SPAN12
Flow cytometry studies were performed to determine whether BK analogs
prevent a-thrombin from eliminating an epitope on the thrombin receptor which
is lost following a-thrombin cleavage of the receptor. SPAN12 is an antibody
to
the thrombin receptor on platelets, which is specific for such an epitope.
Platelets for flow cytometry studies were prepared from 53.3 ml fresh
blood anticoagulated with 8.7 ml acid citrate dextrose (10 mM trisodium
citrate,
66 mM citric acid, 111 mM glucose, pH 4.6). Washed platelets from platelet
rich plasma were prepared by centrifugation at 180 x g for 15 min. at room
temperature. The platelet-rich plasma was brought to a final concentration of
2.8
~cM with PGE, (Sigma) and 1:25 (vol:vol) with 1 M sodium citrate. After a 5
min. incubation at room temperature, the platelet-rich plasma was centrifuged
at
1200 x g for 10 min. at room temperature. The platelet pellet was then re-
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suspended in 10 ml of platelet wash buffer (128 mM NaCI, 4.26 mM NaH,P04,
7.46 mM Na~HP04, 4.77 mM sodium citrate, 2.35 mM citric acid, 5.5 mM
glucose, 3.5 mg/ml bovine serum albumin, pH 6.5) followed by centrifugation at
1200 x g for 5 min. at room temperature. After re-suspension in 5 ml of
platelet
suspension buffer ( 137 mM NaCI, 2.6 mM KCI, 13.8 mM NaHC03, 5.5 mM
glucose, 1 mM MgCh, 0.36 mM NaH2P04, 10 mM Hepes, 3.5 mg/ml bovine
serum albumin, pH 7.35), the platelet count was adjusted to 400,000/p,l. One
hundred ~,l of washed platelets were then placed in a 5 ml round bottom
polystyrene tube in the absence or presence of the BK analogs before treatment
with a-thrombin (0.125 U/ml or 1 nM). Primary antibodies were added at a final
concentration of 2 ~,g/ml and the antibodies were incubated with the platelets
for
30 min at 4°C. After incubation, the platelets were diluted with 500
~.l of platelet
suspension buffer and again centrifuged at 1200 x g for 5 min. at room
temperature. The platelet pellets were then re-suspended in 100 ~cl of
platelet
suspension buffer and incubated with a 1:40 dilution of an anti-mouse IgG
conjugated with FITC. After an additional incubation for 30 min. at
4°C, the
platelets were again centrifuged at 1200 x g for 5 min. followed by re-
suspension
in 500 ~,1 of platelet suspension buffer.
Mouse IgG and an antibody to the epitope CD62 were used as controls.
Mouse IgG (Code #4350) was purchased from BioSource, Camarillo. CA. The
fluorescence of bound FITC-anti-IgG to platelets was monitored on an Epics-C
flow cytometer (Coulter Electronics, Hialeah, FL). Light scatter and
fluorescence
channels were set at logarithmic gain. Laser excitation was at 488 nm. Green
fluorescence was observed through a 525 nm band pass filter. The relative
fluorescence intensity of at least 15,000 platelets was analyzed in each
sample.
An antibody to CD62 (P-selectin) was purchased from Becton-Dickinson
(Catalogue # 550014), San Jose, CA.
SPAN 12 detected an antigen on the thrombin receptor on unstimulated
platelets. When the washed platelets were treated with 1 nM a-thrombin, there
was a decrease in the antigenic expression of the epitope of the monoclonal
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antibody SPAN 12. The forward scatter of the SPAN 12 epitope seen on
unstimulated platelets was shifted towards the origin on a-thrombin activated
platelets, giving an absent antigen detection pattern similar to that for
mouse IgG
used as a control.
S In a control experiment, I mM native BK (SEQ ID NO:S) prevented the
loss of the epitope of the thrombin receptor on a-thrombin activated
platelets.
E. Inhibition of a-thrombin Cleavage of NAT12
The peptide NAT12 (SEQ ID N0:2), which spans amino acids 35-46 of
the a-thrombin cleavage site on the thrombin receptor (Vu et al., Cell 64,
1057
(1991), was also used to determine whether BK analogs blocked a-thrombin
cleavage of the cloned receptor.
The cleavage studies were performed according to the method of Molino
et al., J. Biol. Chem. 270, 11168 (1995). Briefly, NAT12 (SEQ ID N0:2) was
dissolved in a solution of 0.01 M NaHzP04 and 0.15 M NaCI, pH 7.4. The
mixture was then incubated with 8 nM a-thrombin for one hour at 37° C
either
in the absence (control) or presence of 1 mM of a BK analog. Following
incubation, the mixture was applied to a Vyadec C-18 HPLC column in 0.1 %
trifluoroacetic acid and eluted with a gradient from 0 % to 100 % of 80 % MeCN
and 0.1 % trifluoroacetic acid. The sizes of the separated products were
confirmed by mass spectrometry .
NAT12 (SEQ ID N0:2) produced a single peak (peak 1) when analyzed
by HPLC. When NAT12 was treated with a-thrombin, its peak area was reduced
by 81% and two new peaks appeared, the new peaks constituting 44%o (peak 3)
and 37 % (peak 2), respectively, of the original peak area. The additional
peaks,
peaks 3 and 2, represent the cleavage products of NAT12.
In a control experiment, in the presence of the BK fragment SEQ ID
N0:7, peak 1 of NAT12 was reduced by only 57% after treatment with a-
thrombin, and the cleavage products (peaks 3 and 2) of NAT12 constituted only
31 % and 26 % , respectively, of the original peak area.
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Example 2
Binding Assays
Binding assays are used to show protein-protein interactions, and can be
used to determine which domains of proteins participate in the binding and to
ascertain the relative binding affinity of various domains. Binding assays are
also
used to screen large numbers of peptides, such as those in a combinatorial
library.
It is particularly useful to screen peptides for the ability to bind to the
NAT12
peptide or to the thrombin receptor. Peptides that show activity in one or
more
of the binding assays are also tested in one or more of the in vitro
functional
assays, such as those described in Example 1. A combination of binding and
functional assays can be used to identify compounds that selectively inhibit
thrombin-induced platelet and other cell activation.
A. Assav for Binding to the NATI2 Peptide
Peptides from a combinatorial library are linked to microtiter plates and
the wells are blocked with 1 % BSA. Biotinylated-NAT12 is incubated with the
microtiter plates. After washing, the presence of biotin-NAT12 attached to the
matrix support expressing peptides is detected by incubation with ImmunoPure
streptavidin horseradish peroxidase conjugate (Pierce Chemical Co. , Rockville
IL)
followed by peroxidase-specific fast reacting substrate, turbo-3,3',5,5'
tetramethylbenzidine (turbo-TMB, Pierce Chemical Co., Rockville IL). After
incubation for 5 min at room temperature, the color reaction of the turbo-TMB
is stopped by the addition of 1 M phosphoric acid. The bound peptide is
quantified by measuring the absorbance of the reaction mixture at 450 nm using
a Microplate auto reader EL311 (BioTek Instrument, Winooski VT).
B. Assav for Binding to the Thrombin Receptor
Peptides from a combinatorial library on a solid phase matrix support are
incubated with biotinylated thrombin receptor. The thrombin receptor may be
expressed in bacterial, baculovirus, or other recombinant system, using
methods
known to those skilled in the art. Isolated receptor is biotinylated by
procedures
known to those skilled in the art. The labeled receptor is incubated with the
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peptides on the matrix support and detected as described for the NAT12 binding
assay, above.
Example 3
In Vivo Clearance and Function:
Correlation With In Vitro Results
Clearance studies were performed in New Zealand white rabbits weighing
between 2.0 and 2.5 kg. Rabbits were premedicated according to the method of
Michelson et al. , J. Mol. Cell Cardiol. 20, 547 ( 1988) with 10 mg/kg 1 M
xylazine and 10 mglkg 1 M ketamine. After tracheostomy, intubation, and
positive pressure ventilation done with room air (Harvard instruments), stage
III
surgical anesthesia was maintained with 20 mg/ml of intravenous pentobarbital.
A carotid artery and a jugular vein were then exposed. A catheter was inserted
into the exposed carotid artery for withdrawal of blood samples and monitoring
the animal's blood pressure (Gould, Inc., Cardiovascular Products, Oxnard,
CA).
In a similar manner, a catheter was inserted into the exposed jugular vein for
administering the anesthetic and BK analog.
A single intravenous infusion of BK analog was injected. The amount of
BK analog injected was calculated from the weight of the animal such that the
blood concentration was 1 mM peptide. For example: for a 2.5 kg rabbit, 7%
of its weight gives an estimated blood volume of 175 ml. Accordingly, 89 mg
of the control BK fragment SEQ ID N0:7 was injected (giving a 1 mM
concentration in the 175 ml plasma). Depending upon the size of the animal, 75
to 90 mg peptide was injected. Blood samples were collected at 2, 4, 6, 8, 10,
20, 30, 40, 60, 90, and 120 minute intervals after infusion into a 0.013 M
sodium
citrate anticoagulant solution. Plasma was prepared from each of the blood
samples collected over time by centrifugation of the blood samples at 10,000
xg
for two minutes. Aliquots of the plasmas were assayed for the presence of the
BK analog antigen by the ELISA technique using a MARKIT-M [1-5] BK assay
from Dainippon Pharmaceutical Co., Ltd., Osaka, Japan.
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For the function inhibition study, New Zealand white rabbits weighing
between 2.0 and 2.5 kg were surgically prepared as described above. After a
single bolus infusion of BK analog calculated as described above. 5 mi blood
samples were collected at 2, 6, 10, 30, 60, 90, 120, 150, I80, 210, and 240
minute intervals following infusion into a 0.013 M sodium citrate
anticoagulant
solution. The collected blood samples were centrifuged at 180 xg ( 1000 rpms)
for 15 minutes at room temperature. The platelet-rich plasma (PRP) portion of
the blood was contained in the supernatant. The platelet count of the PRP,
obtained with an H-10 Cell counter (Texas International Laboratories, Inc.,
Houston, TX), was adjusted with rabbit platelet-poor plasma to 200,000-250,000
platelets/~,1.
Platelet aggregation studies on the PRP were conducted on a 4-channel
aggregometer (BioData-PAP-4, Bio Data Corp., Hatboro, PA). The degree of
platelet aggregation was determined by measuring the increase in light
IS transmission through a stirred suspension of PRP maintained at 37°C.
Platelet
aggregation was induced in the PRP sample by addition of 20 ~,M ADP and ~y-
thrombin according to the method of Harfenist et al. , Thromb. Haemost. 53,
I83
( 1985).
Like human platelets, rabbit platelets displayed a variable response to y-
thrombin. Each rabbit's platelets were evaluated before BK analog infusion for
their threshold response to ~y-thrombin. The rabbit platelets were typically
responsive to 10 nM to 40 nM y-thrombin. Simultaneous ~y-thrombin-induced
platelet aggregation studies were performed with 10, 20, and 40 nM y-thrombin
and 20 ~,M ADP.
Control experiments were performed using the BK fragment SEQ ID
N0:7. The peak plasma concentration of BK fragment SEQ ID N0:7 after
infusion was 60 ~cg/ml (0.120 mM) for two of three rabbits, as determined by
ELISA. No unfavorable effects were observed in the animals following the bolus
injection of the BK fragment. The rabbits' blood pressure, pulse, and platelet
count remained stable and there was no abnormal bleeding at the surgical sites
of
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cutdowns and intubations. The half-life of BK fragment SEQ ID N0:7 antigen
clearance in plasma has two phases: one at 2.8 minutes and a second at 20
minutes after infusion.
The BK fragment SEQ ID N0:7 had a prolonged biologic clearance,
however. After a single bolus infusion, 10 nM ~y-thrombin-induced platelet
aggregation was inhibited 100 %o for over 4 hours, 20 nM y-thrombin-induced
platelet aggregation was inhibited ~ 50% for 2.75 hours, and 40 nM y-thrombin-
induced platelet aggregation was inhibited >_50% for one hour. There was
>_50% inhibition of ADP-induced platelet aggregation for roughly 45 minutes.
These data demonstrate that after a single bolus infusion of BK analog,
having a peak peptide concentration of only 0.120 mM two minutes after
infusion,
there was a prolonged, selective inhibitory effect on thrombin-induced and ADP-
induced platelet activation in vivo.
When the peptide RPPGF (SEQ ID N0:7) was infused in vivo in rabbits,
there was a prolonged inhibition of y-thrombin-induced platelet aggregation in
rabbit platelet-rich plasma ex vivo, as shown in Figure 1.
A human platelet study was similar to the functional study using white
rabbits described above. Briefly, blood samples were obtained from normal
human volunteers. Platelet counts were measured with a Coulter counter, Mode!
2F (Coulter, Hialeah, FL) and adjusted to a platelet count of 200,000
platelets/~1.
Each individual's platelets at baseline were measured for their threshold
response
to y-thrombin. Typical threshold levels were between 10 nM to 40 nM.
Human platelets in PRP were treated with 20 nM y-thrombin. When 1 mM
BK fragment SEQ ID N0:7 was reacted with 20 nM y-thrombin, the aggregation
tracing was abolished. The specificity of this reaction was demonstrated by
comparing the results to those for a non-BK analog peptide (SEQ ID N0:8). SEQ
ID N0:8 ( 1 mM) was unable to inhibit the ability of y-thrombin to induce
platelet
actmation.
Human platelets were treated with RPPGF (SEQ ID N0:7) in vitro.
RPPGF (SEQ ID N0:7) appears to have a prolonged inhibitory effect. Human
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platelets in platelet-rich plasma were made 1 mM RPPGF (SEQ ID N0:7). After
incubation for 1 h at 37"C, the platelet-rich plasma was centrifuged into a
pellet.
the plasma removed. and the pellet was resuspended in platelet-poor plasma
without RPPGF (SEQ ID N0:7). After resuspension of the RPPGF-treated
platelets in platelet-rich plasma. their ability to respond to y-thrombin was
compared with untreated platelet-rich plasma. In ail instances examined, the
concentration of y-thrombin that served as the threshold to induce full
platelet
aggregation of RPPGF-treated platelet-rich plasma was greater than that seen
with
simultaneous control platelet-rich plasma. Further at the concentration of y-
thrombin that induced full aggregation of control platelet-rich plasma, each
of the
RPPGF-treated platelets was inhibited 62% or more. These data indicate that
RPPGF (SEQ ID N0:7) bound to platelets actually increased their threshold for
platelet aggregation induced by y-thrombin.
Example 4
Preparation of BK Analogs
A. Preparation of Peptides
Peptides analogs of BK were synthesized on an Applied Biosystems Model
431 peptide synthesizer. The carboxy-terminal amino acid was covalently
attached
to a solid phase support, and succeeding amino acids were coupled sequentially
to
the amino terminus unless otherwise stated. The carboxyl group on the amino
acid
to be attached was activated with 2-( 1-H-benzotriazole-1-YL)-1,1,3,3-
tetramethyluroniumhexofluorophosphate (HBTU) and 1-hydroxybenzotriazole
(HOBt). The fluorenyl-methyloxycarbonyl moiety was then attached at the amino-
terminal end as a blocking group. Peptides were purified by preparative
reverse-
phase HPLC.
Synthesis of the peptide Arg-Pro-Pro-Amide was initiated on 4-(2',4'
dimethoxyphenyl)-fmoc-aminomethylphenoxy resin. Once the fmoc group was
removed, synthesis proceeded with attachment of the proline.
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The peptides (D-Arg)-Pro-Pro and Arg-(D-Pro)-Pro were synthesized using
standard methods known in the art.
B. Preparation of RPP MAP-4
"MAP" is an acronym for "multiple antigenic peptide". A four-branch
MAP of RPP, hereinafter called "RPP MAP-4", was prepared. The structure of
RPP MAP-4 was as follows:
Arg-Pro-Pro
Lys
Arg-Pro-Pro
~ L s-
y ~3Ala
Arg-Pro-Pro
Lys
Arg-Pro-Pro
To prepare RPP MAP-4, a resin core, having a ~3-alanine attached through
its carboxyl group, was joined to a free carboxyl of lysine through the free
amine
of ~3-alanine ((3Ala) to form a lysine-~3-alanine complex. Two additional
lysine
residues were then attached by their free carboxyl groups to the two free
amines
of the first lysine. Four molecules of RPP were then attached through their
proline residues to the free amino groups of the two lysine residues,
following
activation with HBTU and HOBt as described above. The RPP MAP-4 was
purified by reverse phase HPLC and then characterized by mass spectroscopy.
Using similar methods, a "PP MAP-4 peptide" (a four-branch MAP of Pro-
Pro) and a "R.PPGF MAP-4 peptide" (a four-branch MAP of Arg-Pro-Pro-Gly-
Phe) were also synthesized.
C. Preparation of RPP Heterodimer
A heterodimer of RPP was prepared. Lysine was attached to the amide
resin, 4-(2',4' dimethoxyphenyl)-fmoc-aminomethylphenoxy resin. After its fmoc
group was removed, RPP was synthesized as described above with the lysine
attached to its carboxy terminus to yield RPPK. Treatment of the C-terminal
lysine with hydrazine and dimethylformamide results in a free amino group to
which an aspartic acid was attached. RPP was then synthesized from the C-
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terminal aspartic acid by standard techniques, resulting in tile heterodimer
shown
below.
Arg-Pro-Pro-Lys
Arg-Pro-Pro-Asp
Example 5
BK Analogs Inhibit Thrombin-Induced Platelet Activation
In Human Platelets In Vitro
The peptides synthesized in Example 4 were assayed for the ability to
inhibit ~y-thrombin-induced platelet aggregation in platelet-rich plasma.
A. Inhibitory Activity of RPPGF and RPPAF
The BK analogs Arg-Pro-Pro-Ala-Phe ("RPPAF", SEQ ID NO: 6) and
Arg-Pro-Pro-Gly-Phe ("RPPGF", a fragment of native BK, SEQ ID N0:7)
both showed inhibition of y-thrombin-induced platelet aggregation in an
aggregation assay like the one described in Example lA.
B. Inhibitory Activity of RPP RPP MAP-4 and RPP Heterodimer
The BK analogs Arg-Pro-Pro ("RPP"), RPP MAP-4, and RPP
heterodimer all showed inhibition of y-thrombin-induced platelet aggregation
in
an aggregation assay like the one described in Example lA.
As shown in Figure 2, RPP at 50 ~cM was able to abolish 27.5 nM 'y-
thrombin-induced platelet aggregation. In three experiments, RPP inhibited y-
thrombin-induced platelet aggregation 100 % at a concentration of 0.089 mM
~ 0.04 (mean ~ SD). RPP was greater than 2.5-fold better as an inhibitor
than RPPGF, which under the same conditions inhibited y-thrombin-induced
platelet aggregation 100% at a concentration of 0.23 mM ~ 0.12.
The peptides RPP-Amide, (D-Arg)-Pro-Pro, and Arg-(D-Pro)-Pro had
reduced ability to inhibit y-thrombin-induced platelet aggregation when
compared to RPP itself. The dipeptide Arg-Pro had no inhibitory activity on
y-thrombin-induced platelet aggregation.
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As shown in Figure 3, RPP MAP-4 inhibited y-thrombin-induced
platelet aggregation 100% at a concentration of 47 ~M peptide (0.047 mM ~
0.019, mean ~ SD of 5 experiments). A PP MAP-4 peptide had no inhibitory
activity on y-thrombin-induced platelet activation.
As shown in Figure 3, the heterodimer of RPP (HETERODIMER)
inhibited y-thrombin-induced platelet aggregation 100% at 75 p,M peptide
(0.079 mM ~ 0.032, a mean ~ SD of 5 experiments). The inhibitory activity
of the heterodimer was better than that of RPPGF but equal to that of RPP.
Example 6
BK Analogs Bind to the Thrombin Receptor Cleavage Site (NAT 12)
The specific binding of biotinylated NAT12 to BK analogs and other
peptides was assayed as described in Example 2A. The results are shown in
Figure 4. Biotinylated NAT12 specifically bound to RPPGF (SEQ ID N0:7),
RPPGC (SEQ ID N0:9), and RPP MAP-4, but not peptides LNA7 (SEQ ID
N0:8) or FSPFR (SEQ ID NO:10).
Example 7
BK Analogs Inhibit a-thrombin-induced Calcium Mobilization in
Fibroblasts and HUVEC Cells
The ability of BK analogs to inhibit a,-thrombin-induced calcium
mobilization in fibroblasts and human umbilical vein endothelial cells
(HUVEC) was assayed as described in Example 1 C. As shown in Figures ~
and 6, HK, RPPGF (SEQ ID NO: 7), and RPP inhibited calcium mobilization
in fibroblasts and HK, RPP MAP-4, and RPPGF MAP-4 inhibited calcium
mobilization in endothelial cells.
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Example 8
Screening a Combinatorial Library to Identify BK Analogs
A. Screening for Ability to Bind the NAT12 Peptide
Peptides from a combinatorial library are linked to a microtiter plate and
blocked with 1% BSA. Biotinylated-NAT12 is incubated with the microtiter
plate. After washing, the presence of biotin-NAT I 2 attached to the matrix
support expressing peptides is detected by incubation with ImmunoPure
streptavidin horseradish peroxidase conjugate (Pierce Chemical Co., Rockville
IL) followed by peroxidase-specific fast reacting substrate, turbo-3,3',5,5'-
tetramethylbenzidine (turbo-TMB, Pierce Chemical Co., Rockville IL). After
incubation for ~ min at room temperature, the color reaction of the turbo-TMB
is stopped by the addition of I M phosphoric acid. The bound peptide is
quantified by measuring the absorbance of the reaction mixture at 450 nm using
a Microplate auto reader EL311 (BioTek Instrument, Winooski VT).
Compounds which bind the NAT12 peptide are also assayed for the ability to
inhibit platelet aggregation and the ability to inhibit calcium mobilization,
using
the assays described in Example 1.
B. Screening for Ability to Bind Thrombin Receptor
Recombinant thrombin receptor is biotinylated, then incubated with
peptides from a combinational library on a solid phase matrix support. Binding
of the labeled receptor is detected as described above.
All references discussed herein are incorporated by reference. One
skilled in the art will readily appreciate that the present invention is well
adapted to carry out the objects and obtain the ends and advantages mentioned,
as well as those inherent therein. The present invention may be embodied in
other specific forms without departing from the spirit or essential attributes
thereof and, accordingly, reference should be made to the appended claims,
rather than to the foregoing specification, as indicating the scope of the
inven-
tion.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: THE REGENTS OF THE UNIVERSITY OF MICHIGAN
APPLICANT AND INVENTOR: Schmaier, Alvin H.
Hasan, Ahmed A.K.
(ii) TITLE OF INVENTION: BRADYKININ ANALOGS AS SELECTIVE
INHIBITORS OF CELL ACTIVATION
(iii) NUMBER OF SEQUENCES: 10
(iv) CORRESPONDENCE ADDRESS:
{A) ADDRESSEE: SEIDEL, GONDA, LAVORGNA & MONACO, P.C.
(B) STREET: Suite 1800 Two Penn Center Plaza
(C) CITY: Philadelphia
(D) STATE: PA
(E) COUNTRY: U.S.A.
(F) ZIP: 19102
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(VII) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: U.S. 60/046,085
(B) FILING DATE: 23-APR-97
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Monaco, Daniel A.
(B) REGISTRATION NUMBER: 30,480
(C) REFERENCE/DOCKET NUMBER: 8820-3PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 568-8383
(B) TELEFAX: (215) 568-5549
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Cys Asn Ala Glu Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro
1 5 10 15
Thr Val Asn Cys Gln Pro Leu Gly Met Ile Ser Leu Met Lys Arg Pro
20 25 30
Pro Gly Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly Glu Ile Lys Glu
35 40 45
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Glu Thr Thr Val Ser Pro Pro His Thr Ser Met Ala Pro Ala Gln Asp
50 55 60
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linexample Bar
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Asn Ala Thr Leu Asp Pro Arg Ser Phe Leu Leu Arg
1 5 10
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Asn Pro Asn Asp Lys Tyr Glu Pro Phe
1 5
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Ser Phe Leu Leu Arg Asn
1 5
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Arg Pro Pro Gly Phe Ser Pro Phe Arg
1 5
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Arg Pro Pro Ala Phe
1 5
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(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Arg Pro Pro Gly Phe
1 5
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Leu Asn Ala Glu Asn Asn Ala
1 5
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:9:
Arg Pro Pro Gly Cys
1 5
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Phe Ser Pro Phe Arg
1 5
