Lysophosphatidylcholine Acts as an Anti-hemostatic Molecule in the Saliva of the Blood-sucking Bug Rhodnius prolixus*
Daniel M. Golodne,
Robson Q. Monteiro,
Aurélio V. Graça-Souza,
Mário A. C. Silva-Neto and
Georgia C. Atella
From the
Departamento de Bioquímica Médica, Instituto de
Ciências Biomédicas, Centro de Ciências da Saúde,
Universidade Federal do Rio de Janeiro, P.O. Box 68041, Cidade
Universitária, Ilha do Fundão, Avenida Bauhínia 400, Rio
de Janeiro, CEP-21941-590, Rio de Janeiro, Brazil
Received for publication, December 6, 2002
, and in revised form, May 9, 2003.
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ABSTRACT
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Blood-sucking arthropods possess a variety of anti-hemostatic factors in
their salivary glands to maintain blood fluidity during feeding. In this work
we demonstrate the anti-hemostatic properties of lysophosphatidylcholine
(lysoPC) isolated from the salivary glands of Rhodnius prolixus.
First, we examined salivary glands of fourth and fifth instar nymphs for their
phospholipid composition. The lumen displayed an accumulation of its
phospholipid content, mainly phosphatidylcholine and lysoPC, with a 6-fold
increase for the latter. To determine the presence of phospholipids in the
saliva, fourth instar nymphs were fed with a32P-enriched blood
meal. After 28 days their saliva was collected and subjected to lipid
extraction, thin-layer chromatography, and autoradiography. The results showed
the presence in the saliva of the same phospholipids present in the lumen. We
then examined possible biological roles of these phospholipids when compared
with other known effects of lysoPC. The luminal lipid extract and purified
lysoPC from the lumen and saliva were tested for inhibition of washed rabbit
platelets' aggregation induced by
-thrombin and platelet-activating
factor. Both the luminal lipid extract and salivary lysoPC showed an
increasing inhibition of aggregation, which correlated with the response of
the platelets to standard lysoPC (up to 13 µg/ml). Next, salivary lysoPC
was incubated with porcine arterial endothelial cells for 24 h. After
incubation, culture medium was assayed for nitric oxide and showed increased
nitric oxide production, similar to control cells exposed to standard lysoPC
(up to 20 µg/ml). Together these data demonstrate the presence of lysoPC in
the saliva of Rhodnius prolixus and its potential anti-hemostatic
activities.
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INTRODUCTION
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Hematophagous arthropods rely on a wide array of anti-hemostatic substances
to counteract vertebrate responses to blood loss and to obtain their blood
meal successfully (1). These
salivary compounds show distinct properties and are generally involved with
inhibition of coagulation, platelet aggregation, and vasoconstriction
(2). These molecules include
peptides such as tachykinins
(3), maxadilan
(4), nitric oxide
(NO)1-binding proteins
such as nitrophorins (5), and
prostaglandins (6). The small
lesions elicited by the mouthparts of the arthropod most likely evoke platelet
aggregation and vasoconstriction by the vertebrate host. Therefore, the
formation of the platelet plug is specifically inhibited by collagen
inhibitors, apyrases, catechol oxidases, thrombin inhibitors, NO-releasing
proteins, fibrinogen receptor agonists, and a specific platelet aggregation
inhibitor (7,
8). The complexity of
anti-hemostatic mechanisms has recently been addressed with the use of
proteomic techniques (9,
10). Hundreds of gene
sequences were obtained, and most of them await future tests concerning the
anti-hemostatic properties of the proteins they code for.
Lysophosphatidylcholine (lysoPC) is a component of oxidized low-density
lipoprotein, which is involved in the pathogenesis of atherosclerosis and
inflammation (11,
12). Recently, great research
efforts have been directed to understand and characterize lysoPC effects on
cell biology (13). The list is
continuously growing and is quite diverse, including the induction of
endothelial genes involved in early atherosclerosis, such as adhesion
molecules and growth factors
(14,
15), and secretion of matrix
metalloproteinase (16).
Indeed, the lysoPC content of atherosclerotic arteries is higher than in
normal vessels, and oxidized low-density lipoprotein displays a great
proportion of lysoPC (17).
LysoPC is able to increase the production of NO by endothelial cells by
enhancing endothelial NO synthase (e-NOS) transcription
(18,
19), besides acting as an
inhibitor of platelet aggregation
(20). This evidence shows that
lysoPC is potentially pro-atherogenic by inducing the production of several
growth factors and the expression of chemoattractant genes
(21). On the other hand,
lysoPC also can display an important anti-atherogenic function because it
increases the local synthesis of NO in the initial phases of the formation of
atherosclerotic plaques, leading to vasodilation
(22,
23).
To date, anti-hemostatic activities found in salivary secretions from
blood-feeding arthropods are mainly of proteic nature or are gases such as NO
(24). In the present study we
demonstrate, for the first time, the presence of phospholipids in the saliva
of a blood-sucking insect, Rhodnius prolixus. The dynamics of the
accumulation of phospholipids in the growing salivary glands, as well as
salivary lysoPC anti-hemostatic properties, are also described.
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EXPERIMENTAL PROCEDURES
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Obtainment of Salivary Glands and Phospholipid Profile
Analysis Insects were reared and handled as described previously
(25). Fourth and fifth instar
nymphs of R. prolixus kept in our departmental colony at 28 °C,
70% humidity and fed at 28-day intervals were used in this study. Salivary
glands were isolated in a drop of saline, cleaned of any adhering tissue,
rinsed twice in saline, and gently punctured under a stereo-microscope by
means of a fine syringe needle, while holding the gland with forceps. The
emptied epithelium was removed from the drop and further rinsed, and the drop
containing the luminal contents was recovered. Both samples were subjected to
lipid extraction (26). The
phospholipid profile of the salivary glands was examined after TLC on silica
gel plates using a mixture of acetone:methanol:acetic acid:chloroform:water
(15:13:12:40:8, v/v). After evaporation of the solvents the plate was immersed
for 15 s in a charring solution consisting of 10% CuSO4, 8%
H3PO4 and heated to 170 °C for 510 min
(27). The charred TLC plate
was then subjected to densitometric analysis. Each phospholipid spot was
identified by comparing to phospholipid standards (Sigma) run in parallel.
Salivary Gland Labeling32P-labeled salivary
glands were obtained following a 32P-enriched blood meal offered to
fourth instar nymphs, using established procedures
(28). After 2830 days,
the molted fifth instar nymphs were dissected and their salivary glands
removed and analyzed as above. Following TLC, the plate was subjected to
autoradiography, and the corresponding 32P-phospholipids were
scraped from the plate after coating with Strip-Mix (Alltech, Deerfield, IL)
and subjected to liquid scintillation counting.
Saliva ObtainmentFifth instar nymphs were allowed to
repeatedly probe a Parafilm membrane of an artificial feeder filled with
deionized water for 2 min. After several collection cycles the contents of the
artificial feeder were collected, and the Parafilm was cut and rinsed with
methanol. Samples were combined and subjected to lipid extraction. To obtain
32P-labeled saliva, fourth instar nymphs were fed with a
32P-enriched blood meal as described above, and saliva was
collected after 2830 days.
Lipid Extraction from TLC PlatePurification of
TLC-separated lipids was performed essentially as described by Yuan et
al. (20), with
modifications. Briefly, after staining with iodine, the target phospholipid
spot was scraped into glass tubes and vortexed with 4 ml of an extraction
solution consisting of methanol:iso-butanol:H2O (45.8:11.5: 42.7,
v/v). Tubes were heated to 55 °C in a dry bath incubator for 20 min,
centrifuged at 3000 rpm for 2 min, and the supernatant was collected. This
procedure was repeated two more times, and to the combined supernatants
(
12 ml) 3 ml of chloroform were added. After intense vortexing and
centrifugation (3000 rpm, 8 min), the lower organic phase was recovered and
dried under a stream of N2. Purified lipids were then resuspended
in phosphate-buffered saline and used in the assays.
Platelet Aggregation AssaysWashed rabbit platelets were
obtained from blood anticoagulated with 5 mM EDTA. Platelets were
isolated by centrifugation and washed twice according to Zingali et
al. (29) with
calcium-free Tyrode's buffer, pH 6.5, containing 0.1% glucose, 0.2% gelatin,
0.14 M NaCl, 0.3 M NaHCO3, 0.4 mM
NaH2PO4, 0.4 mM MgCl2, 2.7
mM KCl, and 0.2 mM EGTA. Washed platelets were
resuspended in a modified Tyrode's buffer, pH 7.4, containing 2 mM
CaCl2 at 300,000400,000 cells/µl. Assays were performed
at 37 °C using a Chronolog Aggregometer (Havertown, PA). Aggregation in
the volume of 300 µl was induced either by
-thrombin (5 µl of a
60-nM stock solution, 1 nM final concentration) or
platelet-activating factor (PAF) (5 µl of a 100-nM stock
solution, 1.67-nM final concentration). Molecules to be tested for
inhibition of platelet aggregation were added 1 min before induction. The
inhibition was calculated using the maximum peak height of each tracing, which
was compared with control incubation values.
Cell Culture and NO ProductionPorcine aortas were obtained
in a local slaughterhouse, and endothelial cells (PAEC) were isolated after
dissection of the aortic artery. Arteries were cut open longitudinally,
exposing the endothelium. After being rinsed several times with ice-cold
phosphate-buffered saline, a small amount of Dulbecco's modified Eagle's
medium was added, the surface was gently scrubbed with a cell scraper, and
cells were recovered. Cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum (for 34 passages) and
seeded in 6-well plates. Twenty-four hours before each experiment, serum-free
fresh medium was added. Incubations were carried out for 24 h with serum-free
medium (1 ml), after which the culture medium was recovered. To determine the
nitrite content, 50-µl aliquots were incubated with 10 µl of a solution
containing 2,3-diaminonaphtalene (DAN, Calbiochem) for 10 min in a final
volume of 100 µl according to the manufacturer's instructions. Reactions
were terminated with 20 µl of 2.8 M NaOH, diluted to 1 ml with
deionized water, and the fluorescent nitrite adduct was measured at 365 nm
excitation and 450 nm emission.
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RESULTS
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LysoPC Is Accumulated in Salivary GlandsAfter blood
feeding, insects have to refill the contents of their salivary glands while
preparing for a next blood meal. To characterize the phospholipid profile of
R. prolixus salivary glands, we used fourth instar nymphs from day 7
post blood meal and nymphs from day 28 post blood meal (already molted to
fifth instars). The luminal contents and the epithelium were separated and
subjected to TLC. The results confirmed the presence of phospholipids in both
compartments with an overall increase from day 7 to 28
(Fig. 1A). LysoPC
content is specifically increased by
6-fold in the lumen of the gland
after insect molting, which represents the greatest increase among all lipid
classes analyzed. The epithelium displayed great amounts of
phosphatidylcholine (PC) and phosphatidylethanolamine, and to a lesser extent,
sphingomyelin, phosphatidylinositol, phosphatidic acid, and a non-determined
lipid, with trace amounts of lysoPC (Fig.
1C). Most importantly, the profile of luminal
phospholipids was quite different from that observed for the epithelium,
consisting mainly of PC and lysoPC with minor amounts of sphingomyelin,
phosphatidylinositol, and a non-determined lipid
(Fig. 1B). The
percentage of each phospholipid remained roughly constant in both compartments
throughout development, with the exception of luminal lysoPC (increasing from
5% on day 7 to 13% on day 28) and PC (decreasing from 73% on day 7 to 60% on
day 28). Therefore, on day 28 when the glands have already been refilled and
the nymphs are ready for a new blood meal, the concentration of luminal lysoPC
of a single salivary gland pair would be 75 µg/ml, as estimated with a
standard lysoPC curve.

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FIG. 1. Phospholipid composition of the lumen and epithelium of salivary
glands. Fourth and fifth instar insects (days 7 and 28 post blood meal,
respectively; n = 15) were dissected and the salivary glands removed.
Glands were punctured in a drop of saline, releasing the contents of the
lumen, and the epithelium was recovered. Samples were subjected to lipid
extraction and TLC. A, TLC plate, salivary glands from fourth instar
nymphs (day 7), and fifth instar nymphs (day 28),
O, origin; lysoPC, lysophosphatidylcholine; SM,
sphingomyelin; PI, phosphatidylinositol; PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PA,
phosphatidic acid; nd, not determined; h, heme pigment. The
following phospholipid standards were also applied: PI (30 µg) and PA(10
µg), LysoPC (30 µg), SM (15 µg), and PC (20 µg). B and
C, TLC was scanned and subjected to densitometric analysis (gray
bars, day 7; black bars, day 28; AU, arbitrary
units).
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Following a 32P-enriched blood meal, we verified the
incorporation of label in the phospholipids present both in the lumen and
epithelium of the salivary glands (Fig.
2) with a pattern similar to that observed with non-labeled
phospholipids from the 28th day post blood meal. Next, we sought to determine
whether the saliva also contained these phospholipids. After collection of
saliva from 32P-labeled nymphs
(Fig. 3) and lipid extraction
followed by TLC (Fig.
3A), the autoradiograph confirmed the presence of labeled
PC and lysoPC (Fig.
3B). The amount of label in each phospholipid present in
the saliva was determined (Fig.
3C) and found to be similar to the profile previously
observed for the luminal phospholipids. Therefore, these experiments linked
blood feeding with the refilling of phospholipids (and other anti-hemostatic
components) in the salivary glands: nymphs are able to use precursor molecules
obtained from their meal to accumulate luminal phospholipids that could be
used during the next feeding. The experiments also show that phospholipids,
including lysoPC, are a component of R. prolixus saliva.

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FIG. 2. In vivo 32P-labeling of phospholipids of the
salivary glands. Fourth instar insects were fed with a
32P-enriched blood meal (<1µCi of 32P per insect).
After 28 days, molted insects were dissected (2 groups of 20 insects each),
and the lumen and epithelium were isolated. Samples were subjected to lipid
extraction followed by TLC and autoradiography. The radioactive spots were
scraped from the plate and subjected to liquid scintillation counting.
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FIG. 3. Phospholipid composition of the saliva. Fourth instar insects
(n = 200) were fed with a 32P-enriched blood meal
(<1µCi of 32P per insect). After 2832 days, molted
insects were separated in small groups (20 each) and the saliva collected by
means of an artificial feeder filled with deionized water. Samples were
subjected to lipid extraction, followed by TLC (panel A,
iodine-stained) and autoradiography (panel B). Radioactive spots were
scraped and subjected to liquid scintillation counting (C).
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Salivary LysoPC Shows Anti-hemostatic PropertiesTo clarify
the possible roles of this lipid component present in the saliva we focused on
lysoPC, a molecule with well documented effects in important aspects of
hemostasis. The first possibility analyzed was that the presence of lysoPC in
saliva injected into the vessel could inhibit platelet aggregation. When
washed rabbit platelets were induced to aggregate by
-thrombin and PAF,
addition of increasing amounts of standard lysoPC resulted in a dose-dependent
inhibition (Fig. 4A).
This inhibition was also verified when the assay was promoted in the presence
of the luminal lipid extract; when using extract from 20 pairs of salivary
glands, the final concentration of lysoPC in the assay corresponds to 10
µg/ml (Fig. 4B).
This effect suggested lysoPC as an effective component of the luminal lipids
in platelet aggregation inhibition. When lysoPC was purified from the luminal
extract (Fig. 4C),
addition of increasing amounts of this purified lysoPC to the platelet
suspension led to a similar inhibition response
(Fig. 4D). Control
assays showed that either PC or lysophosphatidylethanolamine, respectively a
lysoPC parent molecule and a related lysophospholipid, could not inhibit
platelet aggregation (Fig.
4D, inset). PC purified from the lumen showed no
inhibition of aggregation, similar to standard PC (data not shown). By adding
excess inducers (9-fold for PAF and 140-fold for
-thrombin) in the
presence of lysoPC, aggregation was readily restored, confirming platelet
viability (data not shown). Moreover, lysoPC purified from the saliva elicited
a marked inhibition on platelet aggregation induced by
-thrombin
(Fig. 4E). Hence the
lysoPC found in the lumen of the salivary glands and secreted into the
vertebrate host as saliva acts as an inhibitor of platelet aggregation.

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FIG. 4. Lysophosphatidylcholine from the lumen and saliva of salivary glands
inhibits platelet aggregation. Washed rabbit platelets were isolated, and
aggregation in the volume of 300 µl was induced by 1 nM
-thrombin ( ) or 1.67 nM PAF ( ) in the presence or
absence of the inhibitors. A, standard lysoPC. B, lipids
extracted from the lumen of salivary glands. C, TLC showing the
luminal lipid extract (1) and purified luminal lysoPC (2).
D, lysoPC purified from the lumen of salivary glands. Inset,
controls. 1, PC; 2, lysophosphatidylethanolamine; and
samples subjected to the lipid purification procedure (3, blank
silica; 4, lysoPC; and 5, PC). Black bars,
-thrombin; white bars, PAF. E, inhibition of
-thrombin-induced platelet aggregation by lysoPC purified from the
saliva. The standard lysoPC concentration required in all assays to achieve
100% inhibition of -thrombin-induced platelet aggregation ranged from
813 µg/ml.
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Another important effect of lysoPC is increased NO production by
endothelial cells. Using cultured PAEC we were able to test the effect of
standard lysoPC or salivary lysoPC on the amount of NO end products released
to the culture medium (Fig. 5).
Cells incubated with increasing amounts of standard lysoPC showed an increase
in NO production when compared with control cells, whereas standard PC was
ineffective (Fig. 5,
inset). When cells were exposed to the purified salivary lysoPC there
was a correspondent increase in NO production similar to that observed for the
standard lysoPC response; salivary PC did not elicit NO production in these
cells. These data confirm that the lysoPC present in the saliva is able to
increase NO production by the endothelial cells in culture.

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FIG. 5. Salivary lysoPC elicits increased NO production by PAECs. Confluent
PAECs were incubated for 24 h in the presence or absence of either standard
lipids (inset: lysoPC, gray bars; PC, white bar),
purified salivary lysoPC (050 µg/ml), or salivary PC (50 µg/ml).
The incubation medium was collected and assayed for NO2 produced as
described under "Experimental Procedures."
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DISCUSSION
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Hematophagous arthropods are able to effectively block the host's response
that would lead to reduced blood ingestion. In R. prolixus the main
anti-hemostatic activities can be assigned to a series of NO-releasing
proteins denoted nitrophorins, which release NO when injected into the
bloodstream of the host, leading to increased vasodilation and inhibition of
platelet aggregation; to an apyrase, which upon cleavage of ADP also reduces
platelet aggregation; and finally to an ADP-binding lipocalin termed RPAI-1
(R. prolixus aggregation inhibitor-1)
(8). Hence the formation of the
platelet plug is the target for several molecules described in R.
prolixus saliva, as well as other arthropods.
The lipid profile of the salivary glands of blood-sucking arthropods has
been examined in detail for the lone-star tick, Amblyomma americanum
(30). In this case careful
work has been undertaken in analyzing the arachidonic acid (AA) profile
associated with each lipid present in the salivary glands of the tick,
especially phospholipids, and in relating AA levels to the occurrence of
salivary prostaglandins. One of the main aspects concerning the presence of
these prostaglandins, besides immunosuppressive and vasodilatory activities,
is platelet aggregation inhibition. Thus, ticks are able to produce
prostaglandins from AA derived from dietary lipids, which is mobilized upon
hydrolysis of phospholipids present in the epithelium of the salivary glands
by phospholipase A2
(31). However, these studies
focus on the neutral lipid and phospholipid profiles of whole salivary glands,
using homogenized glands as the starting material. Also, the phospholipids
present in the epithelium are considered mainly as a source of AA, which is
subsequently converted to prostaglandins via the cyclooxygenase pathway and
secreted into the lumen. The presence of salivary prostaglandins is, to date,
the only demonstration of lipid-derived anti-hemostatic molecules in
arthropods. The present work is the first to consider the presence of
phospholipids in the salivary glands, besides the epithelium of the glands,
where the presence of phospholipids is expected because of its membranous
structure. We have examined the lumen of glands for their phospholipid content
because it contains the salivary secretion to be injected into the host. The
method used throughout this work, consisting of extrusion of the luminal
contents followed by recovery and analysis of the separated
"compartments," unequivocally showed the presence of phospholipids
in the lumen of the glands with a distinct composition when compared with the
epithelium. This luminal phospholipid profile suggested to us that
phospholipids are possibly being injected into the vertebrate bloodstream
during feeding. To further demonstrate this and confirm their occurrence, we
collected saliva from insects, and the analysis showed the main presence of PC
and lysoPC.
The presence of lysoPC in the saliva would suggest the possibility of
increasing blood-feeding effectiveness by reducing anti-hemostatic responses.
Accordingly, some of the diverse cellular responses to lysoPC are related to
counteracting hemostasis, mainly by inhibiting platelet aggregation
(20) and increasing the amount
of e-NOS in endothelial cells
(32). The salivary lysoPC
elicited an increased production of NO in PAEC. This effect is similar to the
described response for lysoPC in other cultured endothelial cells. The role of
lysoPC in the increased synthesis of e-NOS has been clarified in human
umbilical vein endothelial cells and bovine arterial endothelial cells
(18,
19). Treatment of endothelial
cells with lysoPC resulted in an increased transcription of the e-NOS mRNA in
these cells, followed by an increased amount of protein and the corresponding
activity. The time span needed for this response
(33,
34,
18) in the case of injected
saliva would preclude a significant role of lysoPC regarding the increase in
local NO levels, because the feeding of R. prolixus is successfully
accomplished in a few minutes
(35). Perhaps in ticks, which
remain attached to the host on the same site for a greater period, this role
of lysoPC would be more noticeable.
Among the effects evoked by lysoPC, evidence shows that it has potent
platelet inhibitory effects
(20) and is essential for the
inhibition of aggregation by secretory phospholipase A2
(20), which uses circulating
lipoproteins as a lipid substrate source. Inhibition was verified with a
number of different agonists, involving inhibition of fibrinogen binding and
platelet shape change (20).
The molecular mechanism of this inhibition involves a stimulatory G-protein
linked to the activation of adenylyl cyclase, which results in the
accumulation of cytosolic cAMP, an event that down-regulates all signaling
events required for activation
(36). The potent inhibition of
platelet aggregation promoted by lysoPC purified from both the lumen and
saliva confirms its role as an anti-hemostatic molecule. In this case, as
opposed to the long-term response leading to increased NO, the inhibitory
effect occurs in a more appropriate time span and seems to be a more important
role for salivary lysoPC in R. prolixus. The actual concentration of
lysoPC in the feeding site would amount to
15 µg/ml, which is close to
the range capable of full inhibition of platelet aggregation in our
assays.
LysoPC has long been thought of as a pro-atherogenic molecule
(17). Only recently its
anti-atherogenic profile has emerged, specifically by eliciting increased NO
production by endothelial cells
(32), increased thrombomodulin
expression (36), and reduced
tissue-factor expression in human monocytes
(37). Therefore, lysoPC would
have a dual role in atherogenesis, with both pro- and anti-atherogenic
effects. The finding of lysoPC in the saliva of R. prolixus further
suggests an anti-hemostatic/anti-atherogenic character for this molecule,
which would account for several controversies regarding the initial stages of
atherosclerosis (38,
12). Finally, the data
reported in the present work demonstrate the dual anti-hemostatic role of
lysoPC (platelet-inhibitory and NO-inducer) in R. prolixus salivary
secretions, including this lipid molecule as a novel anti-hemostatic compound
found in the saliva of blood-sucking insects.
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FOOTNOTES
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* This work was supported by grants from the Brazilian agencies Conselho
Nacional de Desenvolvimento Científico e Tecnológico
(CNPq-PADCT, Universal), Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES), and Fundação de
Amparo à Pesquisa Carlos Chagas Filho do Estado do Rio de Janeiro
(FAPERJ) and by Grant F/2887-1 from the International Foundation for Science,
Stockholm, Sweden (to M. A. C. S.-N.). The costs of publication of this
article were defrayed in part by the payment of page charges. This article
must therefore be hereby marked "advertisement" in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 51-21-2562-6785; Fax:
55-21-2270-8647; E-mail:
atella{at}bioqmed.ufrj.br.
1 The abbreviations used are: NO, nitric oxide; e-NOS, endothelial nitric
oxide synthase; lysoPC, lysophosphatidylcholine; PAEC, porcine arterial
endothelial cells; PAF, platelet-activating factor; PC, phosphatidylcholine;
TLC, thin-layer chromatography. 
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ACKNOWLEDGMENTS
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We thank Dr. Eliezer J. Barreiro from Faculdade de Farmácia of
Universidade Federal do Rio de Janeiro and we thank José S. Lima, Jr.
and Litiane M. Rodrigues for insect care. We also thank Dr. Tereza Marques de
Oliveira Lima for English revision of the manuscript.
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