Transcript acALY-18 stimulates release of - TherimuneX Pharmaceuticals, Inc

Modulating Innate Immunity for Biodefense and Emerging Infectious
Diseases: Stimulation of Platelets by a Naturally-occurring
Immunomodulatory Peptide
Mitali Purohit*, Gerald Soslau1, James D. Thacker2*, Richard F. Rest*
*Department of Microbiology and Immunology, Drexel University College of Medicine, 1Department of Biochemistry and Molecular Biology,
Drexel University College of Medicine, 2TherimuneX Pharmaceuticals, Inc., Doylestown, PA
acALY-18 (EC99 100 ng/mL)
A new approach to characterize active peptides is to determine their
effect on platelet function.
Mammalian platelets are unique and
multipurpose inflammatory cells exhibiting archetypal features. We sought
to determine the effects of acALY-18 on various aspects of platelet
function. Our current research is focused on uncovering data that will help
develop acALY-18 for clinical use.
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acALY-18
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pDAG
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Figure 3B: Neither pDAG nor acALY-18 inhibit platelet aggregation.
Various concentrations of pDAG or acALY-18 were added to washed
human platelets along with thrombin (0.2 units) and incubated in an
aggregometer. Neither pDAG nor acALY-18 suppressed the aggregation
process
---------------concentration (ng /ml) ---------------------Figure 1: Neither pDAG nor acALY-18 cause RBC hemolysis. Human
erythrocytes were washed three times with PBS, centrifuged at 500g for 10
min, and resuspended in PBS-BSA to a concentration of 5% (v/v) and
peptides added. After incubation for 1 h at 37°C, mixtures were centrifuged at
800g for 1 min and hemolysis recorded at OD535.
Several lines of evidence suggest that granule secretion is not
dependent upon platelet aggregation or shape change. Platelet
granule exocytosis plays a critical role in inflammation, thrombosis
and wound healing. Platelets have three major types of secretory
granules that are defined by their unique contents, kinetics of
exocytosis and morphologies. We determined the effect of acALY-18
on platelet secretion from - and dense granules.
Adherence of platelets to acALY-18
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----------------------acALY-18 (ng)-----------------Figure 5: acALY-18 induces platelet adhesion. 96 well plates
were coated with various concentrations of acALY-18 O/N;
blocked, washed and incubated with 1-3108 /ml platelets for 30
min. Nonadhered platelets were removed and adhered cells lysed
to measure acid phosphatase activity.
Indirect activation of
monocytes by acALY-18
acALY-18 induces release of
human platelet α-granule contents
Temporary
functional capacities
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CD40
Effects on neutrophils
Immune costimulation
Effects on endothelial cells
PAF
PF4
5-HT
TGF-β
PDGF
RANTES
IL-1β
ACTIVATION-INDUCED PSGL-1
or SECRETED
concentration (pg)
Endothelial cell effects P-selectin
CD40L
Inflammatory effects
Coagulation and
TLR 9
suppressive effects
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PDGF-AB
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TGF-β
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CONSTITUTIVE
Figure 2: Platelets store or express and secrete many immunomodulatory
molecules that significantly affect innate immune mechanisms. Some are
constitutively expressed while others are expressed or secreted upon platelet
activation (Cell Mol Life Sci, 2010, 67:499).
-------------acALY-18 (ng /ml)----------Figure 4A: acALY-18 stimulates release of -granule PDGF-AB and
TGF- from human platelets. Washed platelets were incubated with
acALY-18 for 3 min. Platelets were then centrifuged and release of PDGFAB and TGF- in the supernatants was measured by ELISA.
sPDAG does not cause or
inhibit platelet aggregation
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ATP conc. (nM)
0.3
% aggregation
acALYDKGYTSKEQKDCVGI
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untreated thrombin Thrombin Thrombin Thrombin Thrombin
+ pDAG + pDAG 10 + ac-ALY18 + ac-ALY18
100 ng
ng
100 ng
10 ng
Introduction
PDAG (EC99 3ng/mL)
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ACQUIRED
Recent years have yielded a plethora of discoveries of new bioactive
peptides. Small peptide segments, often parts of larger proteins are
increasingly found to have critical roles in biological processes especially
in initiating innate immune responses. We have identified an
immunomodulatory lipopeptide, pDAG and its 18 amino acid peptide
acALY-18. acALY-18 has full sequence homology to the internal sequence
of amino acids 558-574 in the Transient Receptor Potential Channelrelated Protein 1 (TRPC1).
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Role of platelets in innate immunity
Developing new medical countermeasures for biodefense and
emerging infectious diseases is associated with numerous challenges.
Broad spectrum technologies that improve product potency and ease of
use will benefit many classes of new diagnostics, vaccines, and
therapeutics, regardless of the disease target. Recent therapeutic
advances suggest that innate immunomodulation holds the potential for
improved survival after a bioterror attack with an infectious agent. Thus
products targeting various processes in the innate immune response are
in the initial stages of development.
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Acid phosphatase
(OD 45 0nm)
sPDAG & peptide do not cause
RBC hemolysis
acALY-18 mediates
platelet adhesion
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% aggregation
To limit disease caused by emerging antibiotic-resistant pathogens and
select agents there is a constant quest to develop novel therapeutic
agents and adjuncts to antimicrobials. There has recently been a greater
appreciation of the role of natural host defense peptides in
immunomodulation. A screen of the low-molecular weight (< 10 kDa)
inflammatory proteome for immunoactive agents uncovered a new
immunoregulatory lipopeptide: 1-peptidyl-2-arachidonoyl-3-stearoyl-snglycerol (“pDAG”). The amino acid sequence of the peptide moiety
(acALY-18) is: acetyl-ALYDKGYTSKEQKDCVGI. We studied the
interaction of synthetic pDAG and acALY-18 with various cells involved in
innate immunity. This poster reports the effect of these peptides on
selective modulation of human platelets. Neither pDAG nor acALY-18
were toxic to mammalian cells, nor did they induce or inhibit platelet
aggregation. acALY-18 did, however, induce increased secretion of TGFβ, PDGF-AB and adhesion molecules from platelet -granules. acALY-18
did not stimulate release of dense granule contents (measured as release
of ATP) from platelets, making acALY-18 unique among platelet
activators. Peripheral blood monocytes – another innate immune cell type
– responded poorly to acALY-18. However, monocytes treated with
supernatants of acALY-18-activated platelets exhibited increased
expression of pro-inflammatory cytokines including IL-6, IL-8 and IL-18.
acALY-18 is an exciting new drug-like molecule that has many possible
clinical uses. Since it acts on the host, there is less concern of bacteria
developing resistance to it, indicating that it may be a valuable addition to
current anti-infective therapy. [This work was funded by Drexel University
College of Medicine, and the Institute for Hepatitis and Virus Research.]
% hemolysis
Abstract
The first step in characterizing these compounds was to test their
potential cytotoxicity. In vivo toxicity studies are ongoing.
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ATP release
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-----pDAG----- -----------------acALY-18-----------------Figure 3A: pDAG and acALY-18 induce minimal platelet aggregation.
Different concentrations of pDAG or acALY-18 were added to washed human
platelets and incubated in an aggregometer. Neither pDAG nor acALY-18
caused significant aggregation.
Figure 4B: acALY-18 does not induce ATP release from platelet dense
granules. Platelets were incubated with
acALY-18 (different
concentrations), thrombin (0.2 units) and collagen along with the Chronolume agent (containing the luciferin-luciferase system, Chrono-log) in an
aggregometer for 5 min. AGRO/LINK software measured release of ATP
into platelet supernatants. ATP standards from Chrono-log were used to
construct a standard curve from which released ATP concentrations were
determined.
Figure 6: Sterile, cell-free supernatants from acALY-18-treated
platelets induce expression of inflammatory molecules by
monocytes. Platelets were treated with 500 ng acALY-18 for 3
min. Their supernatants were then used to treat 1106 THP-1
monocytes for 24 hr. After 24 hr treatment, RNA was purified from
the cells. Expression of cytokines or signaling molecules was
determined by quantitative RT-PCR. Values are normalized to actin expression.
Future directions
 Platelets liberate an arsenal of inflammatory mediators after
being activated by acALY-18. It would be interesting to see if
acALY-18 stimulates release of platelet microbicidal proteins.
 Interactions with leukocytes and endothelial cells provide an
additional mechanism by which platelets and acALY-18 may
contribute to innate defense. A detailed study of these interactions in
vitro and in vivo is warranted.
 Bioactive molecules released from platelets can serve as
chemoattractants for monocytes and neutrophils. Chemotaxis
assays are being done to determine if these cells would chemotax
towards supernatants from acALY-18 treated platelets.
 The selective release of platelet granule contents undoubtedly
requires precise packaging of the granules and a highly
orchestrated release reaction. It is thus important to study the
structure-activity relationship inherent to this effect on platelet
activation.
FUNDING: This research was funded in part by Drexel
University College of Medicine in a cooperative agreement with
TherimuneX Pharmaceuticals, Inc., and the Institute for
Hepatitis and Virus Research.