Modulating Innate Immunity for Biodefense and Emerging Infectious Diseases: Stimulation of Platelets by a Naturally-occurring

Immunomodulatory Peptide

Introduction

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.

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

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-sn-glycerol (“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 TGF-β, 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.]

Abstract

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.

sPDAG does not cause or inhibit platelet aggregation

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 1-3108 /ml platelets for 30 min. Nonadhered platelets were removed and adhered cells lysed to measure acid phosphatase activity.

sPDAG & peptide do not cause RBC hemolysis

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.

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.

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.

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 Channel-related Protein 1 (TRPC1).

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.

The first step in characterizing these compounds was to test their potential cytotoxicity. In vivo toxicity studies are ongoing.

acALY-18 induces release ofhuman platelet α-granule contents

O O

O

O

OALYDKGYTSKEQKDCVGIac

12

3

PDAG (EC99 3ng/mL)

acALYDKGYTSKEQKDCVGI

acALY-18 (EC99 100 ng/mL)

Role of platelets in innate immunity

ACQUIRED

CONSTITUTIVEACTIVATION-INDUCED

or SECRETED

Endothelial cell effects

Inflammatory effectsCoagulation and

suppressive effects

Effects on neutrophilsImmune costimulation

Effects on endothelial cells

Temporary functional capacities

MHC cl

ass I

Plasm

a

protei

nsCD14

CD80 (B7) CD66M

HC Class II

PAFPF45-HTTGF-βPDGFRANTESIL-1β

CD40L

TLR 9

CD40

GP αIIbβ3

P-selectin

PSGL-1

TLR 1,2,3,4,6,7,9 TREM 1 ligand

GP IbIX

Chemokine receptors

Platelet specific

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).

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

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 PDGF-AB and TGF- in the supernatants was measured by ELISA.

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 Chrono-lume 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.

Indirect activation of monocytes by acALY-18

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 1106 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.

acALY-18 mediates platelet adhesion

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.