1. Art and Diseño

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Prostaglandin E, Potentiates Platelet Aggregation by Priming Protein Kinase C
By Roberta Vezza, Rita Roberti, Giuseppe Giorgio Nenci, and Paolo Gresele
Prostaglandin E, (PGE,) is produced by activated platelets
and by several other cells, including capillary endothelial
cells. PGE, exerts a dual effect on platelet aggregation:
inhibitory, at high, supraphysiologic concentrations, and
potentiating, at low concentrations. No information exists
on the biochemical mechanisms through which PGE, exerts its proaggregatory effect on human platelets. We have
evaluated the activity of PGE, on human platelets and have
analyzed the second messenger pathways involved. PGE,
(5 t o 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing
calcium transients. At a high concentration (50 pmol/L),
PGE, inhibited both aggregation and calcium movements.
PGE, (5 to 500 nmol/L) significantly enhanced secretion of
&thromboglobulin (j3TG) and adenosine triphosphate from
U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE, also increased the binding of radiolabeled fibrinogen to the platelet surface and
increased the phosphorylation of the 47-kD protein in 32Plabeled platelets stimulated with subthreshold doses of
U46619. Finally, the amplification of U46619-induced aggregation by PGE, (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C,
staurosporine, H7, and TMB8). Our results suggest that
PGE, exerts its facilitating activity on agonist-induced
platelet activation by priming PKC t o activation by other
agonists. PGE, potentiates platelet activation at concentrations produced by activated platelets and may thus be of
pathophysiologic relevance.
0 1993by The American Society of Hematology.
A
function by itself, but it can affect aggregation when platelets have been previously challenged by an agonist. Indeed,
low concentrations of PGE, have a proaggregatory effect,
whereas high doses of this prostanoid inhibit platelet aggregation.'-'' It is quite conceivable that the amounts of PGE,
normally produced by activated platelets contribute to physiologic platelet activation because they are in the range of
those found to potentiate aggregation when exogenously
added (1 to 15 n m ~ l / L ) " "and
~ because platelets appear to
possess specific receptors for PGE,.I4 In addition, when the
enzyme Tx-synthase is pharmacologically blocked, the concentrations of PGE, generated by stimulated platelets increase up to 20 time^,^^^,'^,'^,'^ attaining levels that cause a
more pronounced potentiation of platelet aggregation. Indeed, the failure of Tx-synthase inhibition to suppress
arachidonate-induced aggregation in a subset of normal
subjects (nonresponders) depends largely on PGE, formation.4,9.15
Studies with citrated and heparinized platelet-rich plasma
(PRP) have led to the conclusion that the proaggregatory
effect of PGE, is mediated by an increased influx of extracellular calcium into platelets,' whereas the inhibitory effect
is due to a nonspecific binding to the PGI, receptor with
stimulation of adenylate cyclase.I6No recent investigations
have been performed to ascertain the biochemical mechanisms through which PGE, potentiates aggregation in human platelets. The aim of our study was thus to assess the
effect of PGE, on different excitatory second messenger systems in human platelets. Our results suggest that PGE, is
capable of amplifying the platelet response to stimuli by
facilitating the activation of protein kinase C (PKC).
RACHIDONIC ACID metabolism plays an important
role in the regulation of the functional response of
platelets to stimuli.' Once liberated from membrane phospholipids by the activity of a phospholipase A, or phospholipase
arachidonate undergoes further metabolism
either through the 12-lipoxygenaseor through the cyclooxygenase (prostaglandin G, [PGG,]/PGH,-synthase) pathway. The main products of cyclooxygenase are the PG endoperoxides (PGG, and PGH,) and thromboxane A,
(TxA,) that is produced by their metabolism through the
Tx-synthase enzyme. Minor amounts of the PG endoperoxides are also transformed, by specific isomerases, into
PGD,, PGF,,, and PGEz.',4Whereas the role of PGH, and
TxA, in the regulation of platelet function in health and
disease has been deeply e~plored,'.~
the physiologic functions of PGD,, PGF,,, and PGE, have been much less investigated. PGD, acts on a specific receptor located on the
platelet membrane to stimulate adenylate cyclase and thus
suppresses platelet function, whereas PGF, appears to be
inactive on platelets, if not at high, supraphysiologic, concentration~.'.~
PGE, does not have any effect on platelet
From the Institute of Internal and Vascular Medicine and Institute of Medical Biochemistry, University of Perugia, Perugia, Italy.
Submitted March 3, 1993; accepted June 30, 1993.
Supported in part by a grant to P.G. of the Italian National Research Council (Grant No. 920104104/115).
Presented in part at the 7th International Congress on Prostaglandins and Related Compounds (Florence, May 28-June I , 1990)
(abstr p 26). at the 1lth Congress ofthe Italian Society on Thrombosis and Haemostasis (Bari. September 24-28, 1990) (abstr 85), and
at the 13th Congress ofthe InternationalSocietyon Thrombosis and
Haemostasis (Amsterdam, June 30-July 6 , 1991) (abstr 1447).
Address reprint requests to Paolo Gresele. MD, PhD, Institute of
Internal and Vascular Medicine, University of Perugia, via E. dal
Pozzo, I-06126 Perugia, Italy.
The publication costs ofthis article were defrayed in part by page
charge payment. This article must therefore be herebjJ marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8209-0014$3.00/0
2704
MATERIALS AND METHODS
Preparation of platelets. Blood was collected into 1/ 10 vol/vol
trisodium citrate 3.8% from drug-free, healthy donors who had
been fasting for I 2 hours; PRP and platelet-poor plasma (PPP) were
obtained as previously described"; platelet count in PRP was adjusted to 2.5 X 108/mL with autologous PPP. In some selected experiments, PRP was incubated with aspirin at 1 mmol/L for 15
minutes at 37°C before use. In other experiments, creatine phosphate (CP; 2 mmol/L) and creatine phosphokinase (CPK; 20 U/
mL) were added to PRP to exclude the contribution of endogenous
Hood, Vol82, No 9 (November 1). 1993: pp 2704-2713
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PROSTAGLANDIN E2 AND PLATELET AGGREGATION
adenosine diphosphate (ADP) to the results obtained. In other experiments, EGTA at 2 mmol/L was added to citrated PRP 30 seconds before adding PGE2 to analyze the effects of PGE, in a medium without calcium (external calcium <lo-’ mol/L). Before
starting each experiment, PRP was tested to see whether the blood
donor was a “responder” or a “nonresponder” to in vitro Tx-synthase inhibition? Briefly, PRP was incubated with two different
Tx-synthase inhibitors (either dazoxiben at 10 pmol/L or OKY-046
at 10 pmol/L) for 15 minutes at 37°C and then platelets were stimulated with arachidonic acid at the threshold aggregating concentration (TAC) defined as the minimal amount of the stimulus giving
full, irreversibleplatelet aggregation (more than 60%light transmission) starting within 2 minutes from the addition of the inducer.
Subjects were defined as “responders” when no aggregation was
In the
observed within 4 minutes after arachidonicacid
experiments on PRP, only the platelets from nonresponder donors
were used to assess the proaggregatoryactivity of PGE,. Indeed, in
PRP from responder people, PGEz does not exert any clear proaggregatory effect even at low concentration^.^ In the experiments on
washed platelets (WP), instead, no selection was made between responder and nonresponder donors because PGE, potentiates systematically the aggregation of platelets resuspended in a buffer.
For experiments in which WP were used, blood was collected
either in 1/6 vol/vol of acid citrate dextrose, pH 5 (ACD formula A;
Baxter, Lessines, Belgium), or in EDTA and processed according to
the methods described below.
Platelet aggregation. Platelet aggregation was studied either in
PRP or in WP with the photometric method by using a Chrono-Log
540 dual channel aggregometer (Chrono-Log Corp, Havertown,
PA).
The calibrationwas performed with autologousPPP in studies on
PRP and with HEPES-Tyrode buffer in studies using WP.
The samples (0.25 mL) were incubated under continuous stirring
for 2 minutes with PGE, or its vehicle and were then challenged
with various inducers. Stimuli used were the stable PG endoperoxides analogue U466 19 (9,l I-dideoxy-1l a , 9a-epoxymethano-PG
Fh), ADP, thrombin, and phorbol 12-myristate 13-acetate(PMA).
To assess the proaggregatory activity of PGE,, a dose-response
curve to the aggregatory agent under study was built in every experiment and the subthreshold concentration was defined as the dose
giving an aggregation tracing with an amplitude between 20% and
40% of maximal; for thrombin, the amplitude was between 40%
and 60% of maximal because, due to the extreme steepness of the
dose-responsecurve, it was impossibleto obtain reproducibly a 20%
to 40% maximal amplitude. Percentage increases produced by different concentrations of PGE, were calculated from the increase of
either the maximal amplitude or the slope of the aggregation tracings in relation to the values obtained in the paired solvent experiments. For the aggregation studies, and for all the other experiments, control samples always contained the identical amount of
organic solvents as the treated samples, the final concentration of
which never exceeded 0.04%, even in complex experiments in
which platelets were treated with three reagents.
Aggregations were observed for 3 minutes after the inducer was
added; in the experiments with PMA, the aggregations were recorded for 5 minutes.
Platelet secretion. Adenosine triphosphate (ATP) release was
measured using the luciferin-luciferase method. ATP release and
aggregation were monitored simultaneously in PRP samples (0.45
mL) using a Chrono-Log Platelet Ionized Calcium Aggregometer
(PICA; Chrono-Log). ATP secretion was quantitated by the luminescenceemitted on the addition of a mixture of luciferin-luciferase
(50 pL; Chrono-Lume 395; Chrono-Log) to the sample and expressed as micromoles of ATP per liter. The calibration ofthe signal
2705
of ATP release was performed by adding ATP (2 pmol/L) at the end
of the stimulation period to each sample. The release of 0-thromboglobulin (@E)
was also assessed, essentially as previously de~cribed.~
Briefly, 3 minutes after the inducer was added, the samples
were rapidly transferred into Eppendorf tubes and immediately
centrifuged at 12,OOOg for 2 minutes; the supernatant was frozen at
-20°C for subsequent assay. Total PTG and ATP content were
assessed in the supernatant of samples submitted to three cycles of
freezing in liquid nitrogen and thawing at 37°C; for @TG, a blank
value was obtained by centrifuging unstimulated samples at
12,OOOgfor 2 minutes and was subtracted from the values obtained
in the supernatant of stimulated samples for the measurement of
the actual PTG release. PTG was measured by a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Boehringer
Mannheim, Mannheim, Germany), as described.’
Platelet shape change. Shape change can be evaluated only
when aggregation can not take place, ie, when there is no calcium in
the external medium. Thus, blood was collected into EDTA (0.1%
final concentration) and PRP was obtained as described above.
Platelet shape change was studied with a turbidimetric method” by
using an Elvi 840 aggregometer(Elvi Logos, Milan, Italy). The Calibration of the instrument was performed with PRP (10% light
transmission)and PPP (90% light transmission). The shape change
recording signal was then amplified fivefold to obtain curves of a
larger amplitude. The amplitude of shape change was then measured on the recorded tracings and expressed as a percentage of
control. In experiments using U46619 as a stimulus, aspirin at 1
mmol/L was added to exclude any contribution from endogenous
arachidonic acid metabolites.
Calcium measurements. Blood was collected into ACD and
WP were prepared as described.” If not differently specified, calcium measurements were performed in platelet suspensionsloaded
with aequorin by using small amounts of dimethyl sulfoxide
(DMSO).” Aequorin-loaded platelets were finally diluted at a concentration of 108/mLin HEPES-Tyrode buffer (129 mmol/L NaCl,
9.9 mmol/L NaHCO,, 2.9 mmol/L KCI, 0.8 mmol/L KHzP04,0.8
mmol/L MgCI, X 6H,O, 5.6 mmol/L glucose, 10 mmol/L HEPES,
pH 7.4) containing Ca2* I mmol/L.
Some experimentswere also performed by loadingaequorin with
the hypo-osmotic shock treatment (HOST) technique, as previously d e s ~ r i b e d . ’ Aequorin
~.~~
signals were measured by using a
calibration curve obtained in a cell-free system. A fixed amount of
aequorin (in the order of nanomolar concentrations) was added to a
solution containing 5 mmol/L HEPES, 150 mmol/L KCl, 1 mmol/
L Mg2+and Ca2+between
and IO-, mol/L. The ratio between
the luminescenceobtained for each Ca2+concentration (L) and the
luminescence obtained when aequorin was exposed to a saturating
CaZ+concentration (1 mmol/L) (Lmax) were measured and the
calibration curve was obtained by plotting Log L/Lmax against Log
[Ca’’].
Aggregation and calcium release were monitored simultaneously
by the use of a Chrono-Log PICA (Chrono-Log). One-milliliteraliquots of aequorin-loaded platelets were incubated with microliter
amounts of PGE, or its vehiclefor 2 minutes at 37°C under continuous stirring and then challenged with the inducer. Finally, a separate series of experiments was performed using the fura-2 technique.” Briefly, human blood was collected into disodium EDTA
(15 mg/lO mL of blood) and centrifuged at 7008 for 5 minutes, and
PRP was then removed. PRP was incubated for 30 minutes at 37°C
with I pmol/L fura-2-AM (CalbiochemCorporation, La Jolla, CA)
and then centrifuged at 3508 for 20 minutes to obtain a platelet
pellet. After careful removal of the supernatant plasma, fura-2loaded platelets were resuspended in HEPES buffer containing 145
mmol/L NaC1, 10 mmol/L glucose, 10 mmol/L HEPES, and 1
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VEZZA ET AL
2706
mmol/L MgCl,, pH 7.4, at a count of 4 X 107/mL. Each test was
performed by adding to I part of this platelet suspension 1 part of
the same buffer as above, but containing 2 mmol/L CaC1,. A doseresponse curve to U466 19 was built and the experiment was performed with the dose of the inducer giving the half-maximal calcium peak. PGE, or its vehicle (saline) was incubated for 2 minutes
at 37°C before adding the inducer. Fluorescence was measured,
under gentle stimng, at 37°C using an Aminco-Bowman spectrofluorimeter (Silver Spring, MD), adjusting the excitation wavelength at 335 nm and the emission wavelength at 505 nm. The
calibration of the signal was performed in each sample by adding
0.2% Triton X-100 to obtain the maximal fluorescence (Fmax) and
then 1 mmol/L MnC1, to remove all the calcium bound to fura-2 to
get the minimal fluorescence (Fmin). Cytosolic free calcium concentration was calculated by the following equation: [CaZ+]= kd[F
- Fmin]/[Fmax - F], where kd is the apparent dissociation constant (224 nmol/L) of fura-2 and F is the fluorescence of nonstimulated platelets (basal level of calcium) or the peak of fluorescence
obtained in each sample after the stimulation.z2
Fibrinogen binding. Human
fibrinogen (specificradioactivity, 122 pCi/mg) was dissolved in distilled water at a concentration
of 0.8 mg/mL. The preparation of iodinated fibrinogen was found
to be 9790 k 4% (n = 4) clottable. WP were prepared according to
Mustard” with slight modifications as previously described3 and
diluted to 3 X 108/mL in Tyrode’s buffer (140 mmol/L NaCI, 2.7
mmol/L KCI, 12 mmol/L NaHCO,, 0.4 mmol/L NaH,PO,, 2
mmol/L CaCl,, 1 mmol/L MgCl,) containing 0.35% bovine serum
albumin and 0. I % glucose, pH 7.4. The binding of fibrinogen to
WP was measured as rep0rted.2~Briefly, 200-pL aliquots of WP
were placed into Eppendorf tubes at 37°C without stirring and 5 FL
oflabeled fibrinogen was added. A time- and dose-response curve to
U466 19 were built by adding the stimulus 30 seconds after labeled
fibrinogen. The reactions were stopped by removing three 50+L
aliquots from each sample. These aliquots were immediately
layered onto 300 pL of a sucrose solution (Tyrode’s buffer containing 2% bovine serum albumin and 20% sucrose, pH 7.4).
Free and platelet-bound ligand were separated by centrifugation
for 2 minutes at 12,OOOgin an Eppendorf centrifuge. Platelet pellets
were counted for the radioactivity in a gammacounter (Packard
Instrument CO, Meriden, CT). Nonspecific binding was measured
in the presence of a large excess of unlabeled fibrinogen and was
always less than 10% of the total binding. Results are expressed as
molecules of fibrinogen bound per latel let.'^ In the experiments in
which the effects of PGE, were assessed, the PG or its vehicle were
added to the samples 30 seconds after labeled fibrinogen; after 2
minutes, the samples were stimulated with increasing concentrations of U466 19 and the reactions were stopped 10 minutes later.
Protein phosphorylation. Blood was collected into ACD plus
aspirin (1 mmol/L) and platelets were washed twice in a phosphateand calcium-free Tyrode’s buffer (136 mmol/L NaCl, 2.7 mmol/L
KCl, 12 mmol/L NaHCO,, 2 mmol/L MgCI2, 5 mmol/L glucose,
pH 6.8), resuspended at 2 X lo9in a volume of I mL, and incubated
with 500 pCi of carrier-free ”P, neutralized with 1 mol/L Tris-HCI
(pH 7.4), for 1 hour at room temperature. After this period, platelets were centrifuged (1,0008 for 10 minutes) and resuspended in
Tyrode’s buffer containing phosphate and calcium (1 36 mmol/L
NaCl, 2.7 mmol/L KCI, 12 mmol/L NaHCO,, 0.4 mmol/L
Na2HP0,, 1 mmol/L MgCl,, 2 mmol/L CaCl,, 5 mmol/L glucose,
pH 7.4) at a count of 2 x 108/mL;each test was performed with 250
pL of the ”P-labeled platelet suspension. An aliquot of platelets,
prepared as above, was studied by awegometry to establish the
TAC to U46619. Platelets were then incubated with PGE2 for 2
minutes and challenged with U466 19 for 90 seconds. The reactions
were stopped by the addition of 15% trichloroacetic acid (TCA) and
energetic vortexing for 2 minutes. Protein pellets were washed twice
with water and then solubilized in 2% sodium dodecyl sulfate
(SDS). Samples were subjected to 10%SDS-polyacrylamide gel electrophoresis according to Laemmli.2’ Gels stained with Coomassie
blue were dried and autoradiographed through a 17-hour exposure
to Kodak X-Omat-AR film (Eastman Kodak, Rochester, NY) at
-80°C. The extent ofphosphorylation ofthe 47-kD protein (molecular mass was determined by comparison with concomitantly run
molecular mass standards) was assessed by scanning densitometry
(Scanning Densitometer SG300; Hoefer Scientific Instruments,
San Francisco, CA). In addition, bands containing the 47-kD protein were cut out from the dried gels and radioactivity was determined by liquid scintillation counting.
Reagents. PGE, (Sigma Chemical CO, St Louis, MO) was dissolved in ethanol. EGTA (Sigma) was dissolved in NaOH at the
concentration of 0.25 mol/L. Acetylsalicylic acid, lysine salt (Lyrca
Synthelabo, Milan, Italy); dazoxiben (4-[2-( 1H-imidazol- I -yl)ethoxylbenzoic acid hydrochloride; Pfizer, Sandwich, UK), kindly
given by Dr H. Tyler (Pfizer); OKY-046 sodium salt (sodium (E)-3ONO Pharmaceuti[4-( 1-imidazolylmethyl)phenyl]-2-propanoate;
cal CO,Osaka, Japan), kindly given by Dr M. Tsuboshima (ONO);
creatine phosphate, creatine phosphokinase, and apyrase (all
Sigma) were dissolved in distilled water.
PGE, (Sigma) was dissolved in ethanol at a concentration of 50
mmol/L and further diluted in 0. I mol/L phosphate buffer. For the
experiments in which calcium was measured with the fura-2 technique, PGE, was diluted in saline. U466 19 (9,l I-dideoxy-1 1q 9 a epoxymethano-PGF,,; Sigma) was dissolved in ethanol and further
diluted with saline. Thrombin (bovine thrombin; Behring, Marburg, Germany) was dissolved in distilled water. PMA (Sigma) was
dissolved in DMSO at a stock concentration of I O mmol/L and
further diluted in distilled water. Arachidonic acid, sodium salt
(>99% pure; Sigma) were dissolved in 0.05 mol/L phosphate buffer,
pH 7.4. ADP (Sigma) was dissolved in ice-cold saline. Adrenaline
bitartrate, a 5 mmol/L solution in Tris buffer, was from Mascia
Brunelli (Milan, Italy). PGI, (Sigma) was dissolved in glycine buffer
at a concentration of 20 pmol/L. Human ’2SI-fibrinogenwas purchased from Amersham (Amersham, UK) and human cold fibrinogen from Kabi (Stockholm, Sweden). Phosphorus-32, as orthophosphoric acid in 1 mL of HC1-free water, was from New England
Nuclear (Boston, MA). Reagents for electrophoresis and molecular
mass standards were from Bio-Rad (Richmond, CA). Staurosporine (Streptomyces species),Z6calphostin C (Cladosporium cladosporioides),” and 4a-phorbol 12, 13 didecanoate (4aPDD)” (Calbiochem, San Diego, CA) were dissolved in DMSO and further
diluted in water (staurosporine and 4aPDD) or saline. H-7 (1-[5isoquinoline sulfonyl]-2 methylpiperazine dihydrochloride; RBI,
Natick, USA)29and TMBI ([S-(N,N-diethylamino)octil-3,4,5-trimethoxybenzoate]; Sigma)” were dissolved in water. Sulprostone,
kindly given by Dr K.H. Thierauch (Schering AG, Berlin, Germany), was dissolved in saline.
Statistical analysis. Data are expressed as mean k SEM. Oneway analysis of variance followed by Tukey’s multiple comparison
test between all pairs was applied. A probability level less than .05
was considered to be significant. When data are presented as a percentage of control, statistical analysis was performed on absolute
values.
RESULTS
Efects of PGE, on platelet aggregation, calcium movements, and secretion. PGE, ( 5 to 500 nmol/L) caused an
increase of the maximal amplitude of aggregation of aequorin-loaded WP stimulated with subthreshold concentrations of U466 19 (0.1 to 0.4 Kmol/L). The increase was sta-
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2707
PROSTAGLANDIN E, AND PLATELET AGGREGATION
crease of aggregation (+33%) observed only with PGE, at 5
pmol/L; PGE, at 50 pmol/L decreased thrombin-induced
aggregation significantly (-43.5%) (Fig 2).
Low doses of thrombin induced a calcium increase of
I .44 2 0.2 pmol/L; PGE, ( 5 nmol/L to 5 pmol/L) did not
increase calcium transients induced by thrombin, whereas
high concentrations of the prostaglandin (50 pmol/L) decreased calcium increase by 41% (Fig 2).
We also studied the effects of PGE, on aggregation in WP
challenged with low doses (10 to 50 nmol/L) of PMA, a
phorbol ester that can directly stimulate PKC. The maximal
amplitude of PMA-induced aggregation was increased by
PGE, at 50 nmol/L (+15.7% k lo%, n = 5) and at 500
nmol/L (+71.7% k 4.4%, n = 5, P < .05), whereas it was
depressed by PGE, at 50 pmol/L (-52.6%
18.6%,n = 4).
On the other hand, PGE, was not able to increase calcium
movements in platelets stimulated by low doses of PMA
(from 0.95 k 0.14 pmol/L without PGE, to 0.87 k 0.19
pmol/L with PGE, at 500 nmol/L, n = 5 ) despite its proaggregatory effect (data not shown). When 4aPDD (40 nmol/
L to 1 pmol/L), a phorbol ester lacking the ability to stimulate PKC?' was used as a stimulus, no platelet aggregation
was seen either in the absence or in the presence of PGE,
(500 nmol/L; data not shown).
In washed platelets stimulated with fully aggregatory
doses of U466 19 (0.2 to 0.5 pmol/L), thrombin (0.05 to 0.1
U/mL), or PMA (40 to 500 nmol/L), PGE, ( 5 to 500 nmol/
L) caused only minor, not significant increases of platelet
aggregation (maximum +20%), with no effects on the calcium transients.
In platelets loaded with aequorin by either the DMSO or
the HOST technique, PGE, alone, in the range of concentrations used (5 nmol/L to 50 pmol/L), never caused an
increase ofcalcium levelsabove the basal in any ofthe preparations tested.
When using the fura-2 technique, low doses (0.1 to 0.2
pmol/L) of U466 19 gave an increase of calcium above the
basal level of 58% k 7%; with PGE, (5 to 500 nmol/L), no
further increases of calcium were observed. On the other
tistically significant with PGE, at 50 nmol/L (+61%), 250
nmol/L (+ 1 13%), and 500 nmol/L (+ 150%);the highest
dose of PGE, tested (50 pmol/L) caused a strong inhibition
of platelet aggregation (-88%) (Fig 1). The slope of aggregation was also increased significantlyby PGE, at 250 nmol/L
and 500 nmol/L (+61% and +79%, respectively), whereas
PGE, at 50 pmol/L had a significant depressing effectalso
on this parameter (-83.5%) (Fig 1). Subthreshold doses of
U46619 induced a calcium rise of 1.37 k 0.17 pmol/L;
PGE, (50 to 500 nmol/L), despite its clear potentiating effect on aggregation, did not enhance calcium increases significantly as compared with the control. The maximum,
not significant, increase was obtained with PGE, at 500
nmol/L (+38%), a concentration that enhanced aggregation
by 150%.On the other hand, PGE, at 50 pmol/L caused a
significant decrease of calcium transients (-78%) (Fig I).
When the same experiments were repeated in platelets
loaded with aequorin by the HOST te~hnique,'',~~
identical
results were obtained (namely, subthreshold U466 19 = calcium increase of l. 17 k 0.23 pmol/L [n = 41; +PGE, at 500
nmol/L = 1.29 k 0.22 pmol/L [+IO%, n = 4, P = NS]).
Similar data (potentiation of aggregation by PGE, with
no increase of calcium movements) were obtained with
aspirin-treated ( 1 mmol/L) platelets stimulated with
subthreshold U466 19 (data not shown). Experiments were
also performed to assess the effects of sulprostone (a PGE,
analogue)I0on platelets challenged with subthreshold doses
of U466 19. Sulprostone (5 nmol/L to 100 pmol/L) caused a
significant increase of the maximal amplitude of aggregation at all the doses tested (maximum increase, 106%with
sulprostone at 50 pmol/L; n = 5, P < .0001) without a
parallel increase of calcium increases as detected with the
aequorin method (maximum increase, +36% with sulprostone at 500 nmol/L; n = 5, P = NS). At none of the doses
tested did sulprostone induce any inhibition of platelet aggregation or calcium movements.
When WP were stimulated with low doses of thrombin
(0.02 to 0.03 U/mL), the proaggregatory effects of PGE,
were less evident than with U46619, with a significant in-
*
+
75
*
h.
K
v
Fig 1. Maximal amplitude of
aggregation (0). and slope of
aggregation (m) and calcium
movements (E) in aequorinloaded WP stimulated by
subthreshold doses of U46619
(0.1 to 0.4 pmol/L) and preincubated with increasing concentrations of PGE, or with its vehicle (C).*A significant difference
as compared with the control (P
< .05, at least). With threshold
doses of U46619 (0.2 to 0.5
pmol/L), calcium increase was
2.56 f 0.23pmol/L(n = 7).
F
50
*
*
T
h.
1
T
dw
3
4
25
0
0
50nM
C
5nM
500nM
25pM
250nM
5pM
50pM
+ PGE2
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VEZZA ET AL
270%
100
h
x
v
T
++
a
7 l
*I
1 -V
1
0
C
50nM 250nM 500nM 5pM
50pM
+ PGE2
hand, PGE, at 5 and 50 pmol/L significantly inhibited calcium increases by 65% and loo%, respectively (Fig 3).
The effects of PGE, were studied also in PRP stimulated
with subthreshold doses of U46619 (0.4 to 0.7 pmol/L) or
ADP (0.6 to 1 pmol/L). Aggregation induced by U46619
was increased by PGE, up to a maximum of +465% (PGE,
at 500 nmol/L); PGE2 at 50 pmol/L totally suppressed aggregation. ADP-induced aggregation was also potentiated
with a maximal increase of + 179%with PGE, at 500 nmol/
L. PGE, at 50 pmol/L exerted an inhibitory effect (-55%)
(Fig 4).
Another set of experiments was performed in PRP supplemented by EGTA, ie, in a low extracellular calcium environment. Without external calcium, the dose-response curve to
U46619 was shifted to the right, but PGE, at 500 nmol/L
was still able to give an increase of aggregation (Fig 5). The
Fig 2. Maximal amplitude of aggregation (U)
and calcium increases (E!) in aequorin-loaded WP
stimulated by subthreshold doses (0.02 to 0.03 U/
mL) of thrombin and preincubated with PGE, or its
vehicle (C). *A significant difference as compared
with the control (P< .05, at least). With threshold
doses of thrombin (0.05 to 0.1 U/mL), calcium
transients attained 3.6 -C 0.8 pmol/L (n = 7).
stimulatory effect of PGE, on U466 19-induced aggregation
was not diminished by the preincubation of PRP with
aspirin and CP/CPK (data not shown).
In the range of concentrations tested (5 nmol/L to 50
pmol/L), PGE, alone did not induce platelet aggregation in
either WP or in PRP in any of the volunteers tested.
From these data, it appears that PGEz potentiates aggregation both in WP and in PRP. We then studied the effects
of PGE, on secretion of a- and dense-bodies in PRP.
With subthreshold concentrations of U466 19 (0.4 to 0.7
pmol/L), the release of ATP was negligible. PGE, dose-dependently increased ATP release up to a maximum of 1.2 f
0.13 pmol/L at 500 nmol/L (Fig 6). In aspirin-treated platelets, the release of ATP induced by U46619, although
blunted, was still significantly enhanced by PGE, at 500
nmol/L (from 0 to 0.74 k 0.27 pmol/L, n = 5, P < .05, data
not shown). ATP release induced by subthreshold doses of
70 1
*
C
5nM 50nM 500nM 5pM
50pM
+ PGEz
Fig 3. Effects of PGE, on calcium movements as measured by
the fura-2 technique in platelets stimulated with low doses of
U46619 (0.1 to 0.2 pmol/L) (n = 5). Results are expressed as the
percentageof increase above the basal level (111.5 -C 6.2 nmol/L,
n = 25); maximal doses of the inducer (0.2 to 1 pmol/L) increased
calcium by 137% 18%above basal (n = 7). *A significant difference as compared with the control (P < .05, at least).
C
50nM
250nM 500nbi 50pM
f
PGEz
Fig 4. Effects of PGE2on aggregation induced by subthreshold
doses of ADP (E!) or of U46619 (0)in PRP from nonresponder donors. Data represent mean -C SEM of four experiments for U46619
and of five for ADP. *A significant difference as compared with the
control (P < .05, at least). The inset shows representative aggregation tracings in response to a subthreshold dose of U46619 (0.4
pmol/L) or of ADP (1 pmol/L) in the absence (C) or in the presence of
PGE, at 500 nmol/L ( +PGE,).
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PROSTAGLANDIN E2 AND PLATELET AGGREGATION
2709
n
60 -
3
h
be
v
3
W
40-
2W
F:
4
0
4
W
k
200
4
0-
0.4
i
2
B
[U466191 (pM)
-T
Fig 5. Effects of PGE, (500 nmol/L) on the platelet aggregation
dose-response curve to U46619 in PRPfrom nonresponder donors.
Thirty seconds after the addition of EGTA at 2 mmol/L (0, 0) or
water (A,A), PGE, or itsvehicle was added and PRP was incubated
for additional 2 minutes. Platelets were then stimulated with increasing concentrations of U46619 and the maximal amplitude of
aggregation was recorded. One experiment representative of three
others is shown. Open symbols represent controls (vehicle-pretreated); solid symbols represent PGEz (500 nmol/L)-pretreated
samples.
C
ADP (0.12 & 0.04, n = 5) was also strikingly enhanced by
PGE, at 500 nmol/L (1.85 & 0.72 pmol/L, n = 5 , P < .05),
whereas PGE, at 50 pmol/L totally suppressed it (Fig 6).
PGE, was also able to increase U466 19-induced /3TG secretion from 145.5 k 145.5 ng/108 platelets to 967 k 145 ng/
10' platelets with PGE, at 500 nmol/L; with PGE, at 50
pmol/L, PTG release was 142 k 50 ng/108 platelets (Fig 6).
Eflects of PGE, on shape change. In our experimental
conditions, all the platelet agonists tested induced shape
change (U466 19, thrombin, ADP, and collagen) except
adrenaline (10 to 100 pmol/L) and PMA (40 to 200 nmol/
L). From a dose-response curve to U466 I9 or thrombin an
average concentration of the inducer giving half-maximal
amplitude of shape change was identified (U46619, 400
nmol/L; thrombin, 0.1 U/mL) and used throughout the experiments. At proaggregatory concentrations (50 to 500
nmol/L), PGE, did not affect the shape change induced
either by U466 19 or by thrombin. High concentrations of
PGE, (50 pmol/L) significantly reduced shape change.
Effects ofPGE, onfibrinogen binding. U466 19 caused a
time- and dose-dependent binding of labeled fibrinogen to
human platelets. In time-course experiments of fibrinogen
binding induced with U466 19 at 2.5 pmol/L, a plateau was
reached in 5 minutes and the values remained stable up to
20 minutes (data not shown). For this reason, a 10-minute
stimulation period was chosen for the experiments with
PGEl. A dose-response curve for fibrinogen binding induced by U466 19 was performed in the absence and in the
presence of PGE,; PGE, at 500 nmol/L shifted the dose-response curve to the left. Indeed, whereas the maximal number of fibrinogen molecules bound in the absence or in the
presence of PGE, did not differ, the concentration of
U466 I9 giving half-maximal binding was lowered by PGE,
from 0.88 to 0.26 pmol/L (Fig 7).
PGE, at 50 pmol/L inhibited fibrinogen binding induced
50nM 250nM500nM 50pM
+ PGE2
Fig 6. Effects of PGE, on platelet secretion induced by subthreshold doses of U46619 or ADP. (A) ATP release; (B) j3TG release. (A) ATP levels inthe supernatant of lysed platelets (totalATP
content) were 7.1 1 2 0.3 pmol/L (n = 3). ATP release in platelets
or ADP
stimulated with maximal aggregatory doses of U46619 (0)
(H) was 1.9 2 0.1 25 pmol/L (n = 4) and 2.9 2 1 pmol/L (n = 4).
respectively. (B) j3TG levels in the supernatant of resting platelets
(blank value) were 754 2 142 ng/108 platelets (n = 4); total platelet j3TG content was 2,850 f 137 ng/108 platelets (n = 4). *A
significant difference as compared with the control (P C .05, at
least).
by U466 19 (1 pmol/L) by 76% f 7% (P< .05, n = 5 ) (data
not shown).
Eflect ofPGE, on protein phosphorylation. Platelet stimulation with U466 19 at subthreshold concentrations in-
1
12
I
T
'
8 .
4
0 -
10-6
10-7
10-6
[U466191 (M)
Fig 7. Dose-response curve of fibrinogen binding induced by
U46619 in the presence of PGE, (500 nmol/L) (A)or its vehicle
(control, 8) (n = 4). The EC,, for U46619 was shifted from 0.88
pmol/L (control) to 0.26 pmol/L ( + PGE, at 500 nmol/L).
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2710
P47
VEZZA ET AL
4
100 189 259 294 298 107 256 249 409 %
U46 SdaM UdDM SOODM PGE.2 U46 +%Ea PMA
STIM mb -----SbOllM
TAC %hf
NOT
TAC
+PG&
Fig 8. Protein phosphorylation was assessed as described in
Materials and Methods by stimulating platelets with U46619
(U46)at 6 pmol/L (subTAC), in the presence of increasing concentrations of PGE, or its vehicle, with U46619 at 12 pmol/L (TAC) in
the presence of PGE, at 50 pmol/L or its vehicle, or with PMA (2.5
pmol/L). The radioactivity of the 47-kD protein, expressed as a percentage of the nonstimulated sample (not stim), is reported below
each lane.
duced only a slight incorporation of radioactivity into the
47-kD protein, whereas with fully aggregatory concentrations of the inducer the radioactivity of the 47-kD band was
higher (Fig 8). When PGE, (50 to 500 nmol/L)-preincubated platelets were stimulated with subthreshold U466 19,
a higher degree of phosphorylation of the 47-kD protein was
found. In basal conditions. the radioactivity in the 47-kD
band was 108 cpm/108 platelets: in the presence of low
doses of U466 I9 alone. the count was 204 cpm/ IO8 platelets, whereas with PGE, the radioactivity increased up to
321 cpm/lO* platelets (+l98% as compared with the
nonstimulated sample). In staurosporine (200 nmol/L)preincubated platelets, the increase of radioactivity of the
47-kD protein given by PGE, at 500 nmol/L was suppressed
(+26% over the basal) (data not shown). PGE, alone (500
nmol/L) did not induce any phosphorylation of the 47-kD
protein. PMA (2.5 pmol/L) induced a marked increase of
the radioactivity of the 47-kD band (+309% over the basal)
(Fig 8). The analysis of the autoradiography of the gels with
scanning densitometry gave results similar to those obtained with scintillation counting concerning the 47-kD
band and showed that the stimulation of platelets with
PMA, as well as with U46619 plus PGE,, enhances the
phosphorylation of other proteins, including a band of a p
proximate 20 kD that may represent myosin light chain
(data not shown). The latter result is compatible with PKC
activation because a 20-kD protein is also a substrate of this
enzyme.”
E / ~ Y qf
. YPKC inliihitors. In view of the ability of PGE,
to enhance the phosphorylation of the 47-kD protein, we
undertook a series of experiments to assess the effects of
different PKC inhibitors on the proaggregatory activity of
this prostaglandin.
Experiments were performed with staurosporine.a powerful PKC inhibito?6 that also interferes with some tyrosine
kinase activities’,: H7, an inhibitor of PKC’’ also active on
cyclic-nucleotide-dependent protein kinase and on calmodulindependent protein kinase 11”; TMBR, an intracellular
calcium antagonist’’ also able to interfere with the PKC
path~ay’~;
and, finally, calphostin C, a recently described,
highly selective PKC inhibit~r.~’
Aspirin-treated ( I mmol/L) PRP was preincubated with
staurosporine (100 and 200 nmol/L), H7 (100 and 500
pmol/L), TMB8 (50 and 100 pmol/L), calphostin C (200
nmol/L). or their vehicle, for 2 minutes before adding
PGE,. Subthreshold aggregatory concentrations of U466 19
(0.2 to 0.6 pmol/L) were then added and maximal amplitude of aggregation at 3 minutes was measured. PGE, at 500
nmol/L increased significantly the maximal amplitude of
aggregation from 1 1 % f 7% to 36.5% f 9.5% (+228%, n =
5. P < .Ol): with staurosporine at 100 and 200 nmol/L, the
increase induced by PGE, was reduced to 63% and 4% respectively, and with calphostin C at 200 nmol/L to +44%:
H7 and TMB8 also blunted the amplifying effect of PGE,
on U466 19-induced aggregation (Fig 9). The increase of aggregation induced by PGE, in the presence of the four PKC
inhibitors was no longer significant as compared with control.
The inhibitory effect of the PKC inhibitors on PGE,-induced potentiation of aggregation was present also in nonaspirinated platelets (data not shown). At the concentra-
/46+g;
U46
Fig 9. Effect of calphostin C (calph C; 200 nmol/L), staurosporine (stauro; 200 nmol/L), H7 (100 pmol/L), or T M B I (100 pmol/L)
on the ampliwing activity of PGE, (500 nmol/L) on aggregation induced by subthreshold U46619 (U46). In the last tracing, the effects of calphostin C, staurosporine, and T M B I on aggregation induced by fully aggregatory doses of U46619 (0.4 to 1 pmol/L) are
shown. Aggregation tracings of one representative experiment
from a total of five are reported.
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271 1
PROSTAGLANDIN E, AND PLATELET AGGREGATION
calcium in the activity of PGE, .37 In addition, PGE, potentiates platelet aggregation even in the presence of EGTA
added to chelate external Ca", which is also against a preeminent role of calcium influx in the proaggregatory activity of this prostaglandin, in contrast to what was previously
~uggested.~
PGE, did not potentiate agonist-induced platelet shape
change, whereas it strongly amplified both dense- and agranule secretion; shape change is strongly dependent on
calcium and is not significantly affected by PKC,38,39
whereas PKC activation is essential for platelet secretion, a
phenomenon in which calcium increases are less important.36340341
The activation of PKC is also involved in the
expression of fibrinogen receptors on platelet^^^^^^ and we
found that FGE, strongly increases fibrinogen binding to platelets stimulated with the endoperoxide analogue U466 19.
Potentiation by PGE, of platelet aggregation and secretion induced by U466 19 was accompanied by an enhanced
phosphorylation of the 47-kD protein that is the main substrate of PKC in platelets,& although PGE, itself did not
induce any phosphorylation. In addition, three substances
(staurosporine, H7, and TMB8) able to suppress nonselectively PKC but with a pattern of accessory activities different from one another:6.29,32-34and a highly selective PKC
DISCUSSION
inhibitor (calphostin C),27all abolished the amplifying effect
Our results confirm that PGE, exerts a proaggregatory
of PGE, on U466 19-induced platelet aggregation. Interesteffect at concentrations generated by activated platelets. A
ingly, staurosporine only marginally affected, instead, the
potentiation of agonist-induced platelet aggregation is obamplifying effect of adrenaline on U466 19-induced aggreserved both in PRP and in WP. PGE, amplifies the platelet
gation. Similar to PGE,, adrenaline is able to potentiate the
response to a range of agonists acting on different receptors,
platelet aggregatory response to other stimuli35but with a
such as U466 19 (TxA,/PGH, receptor agonist), thrombin,
mechanism largely independent from PKC a ~ t i v a t i o n . ~ ~
or ADP, as well as the response to PMA, a PKC activator,
Thus, our results indicate that low concentrations of
implying that the proaggregatoryeffect is exerted at the level
PGE, amplify the platelet response to various agonists by
of a second messenger system common to different agonists.
potentiating PKC activity. Considering that direct activaThe amplifying effect of PGE, on platelet aggregation is not
tion of PKC by PGE, seems unlikely, because this PG does
paralleled by a similar activity on calcium transients. Innot phosphorylate the 47-kD protein, a priming effect on
deed, in conditions in which aggregation is almost triplithe enzyme appears to be the most likely explanation. PGE,
cated, the intraplatelet calcium increase is enhanced only by
might prime PKC to other stimuli by favoring the translocaone-third. Although no linear relation exists between the
tion of the enzyme to the membrane,36by lowering the calmagnitude of calcium increase and the amplitude of the
or by
cium threshold necessary for enzyme a~tivation,~'
platelet aggregation response in conditions of synergistic
other, as yet undefined mechanisms. Alternatively, PGE,
platelet activation by pairs of agonist^,^' an initial priming
might favor the generation of diacylglycerol, a known acticalcium increase must be provoked by the first agonist to
vator of PKC,& as a consequence of either phospholipase C
enhance the aggregation response to the ~ e c o n d .In
~ ~our
. ~ ~ or phospholipase D a ~ t i v a t i o n .Indeed,
~ ~ , ~ ~a stimulatory efexperiments, PGE, alone never induced any calcium tranfect of PGE, on PKC activation has already been described
sients in human platelets; thus, a priming effect exerted
in other cell types, such as the bovine adrenal chromaffin
through calcium movements appears to be unlikely. The
cells,"8 and both phospholipase
and phospholipase DS0
lack of effect of PGE, on calcium transients in platelets was
activation have been reported. Further studies are
detected with fura-2 and with aequorin, the latter loaded
warranted to clarify the exact mechanism of PKC priming
into platelets by two different techniques, the DMSO" and
by PGE, in platelets.
the HOST method^.'^,^" Different aequorin-loading techThe effects of PGE, on platelet activation are likely to be
niques were used because some agonists that induce a calreceptor-mediated because sulprostone, a PGE, analogue
cium increase when platelets are loaded by the HOST techthat binds to the platelet surface, could reproduce the proagnique do not cause such an increase when the cells are
gregatory activity of PGE,. It has been suggested that plateloaded by the DMSO technique, presumably because of an
lets possess at least two different PGE, receptor subtypes,
inhibitory effect of DMSO.I9 The observation that similar
one with high and the other with low affinity.I4One ofthese
results were obtained with various Ca" measurement techmight mediate the priming effect of low concentrations of
niques excludes the possible insensitivity of the method
PGE, on platelet PKC, and the other might be responsible
used as the cause of the apparent lack of involvement of
for the stimulation of adenylate cyclase and platelet inhibitions suppressing the proaggregatory effect of PGE,, the
four PKC inhibitors did not affect aggregation induced by
fully stimulatory doses (0.4 to 1 pmol/L) of U466 19 (Fig 9)
except for the highest concentration of H7 (500 pmol/L),
whichreduceditby81.4%(n = 5,P<.Ol)(datanotshown).
Additional experiments were performed to assess the synergistic effect of adrenaline on U466 19-induced platelet agg r e g a t i ~ n .Adrenaline,
~~.~~
at a dose not provoking aggregation (0.2 to 2 pmol/L), potentiates platelet aggregation
induced by subthreshold doses of U46619 (from 12.9% f
1.8% to 45.4% f 6.8%; +252%, n = 4, P < .05) in 4 of 6
blood donors tested. Staurosporine (200 to 400 nmol/L) did
not suppressthe proaggregatoryeffect ofadrenaline on platelets stimulated with subthreshold doses of U466 19 (adrenaline + U466 19 = 45.4% f 6.8%;staurosporine + adrenaline
U46619 = 38.8% f 12.6%,n = 4, P = NS). In aspirintreated platelets, adrenaline potentiated platelet aggregation
in5 of6blooddonorstested(from 14.6%+ 1.6%to41.3% k
6.6%; + 18370, n = 5, P < .01). Even in these conditions,
staurosporine (200 to 400 nmol/L) did not significantly inhibit the proaggregatory effect of adrenaline (adrenaline
U46619 = 41.3% 6.6%; staurosporine adrenaline
U46619 = 26.4% f 5.7%, n = 5, P = NS).
+
*
+
+
+
c9
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2712
VEZZA ET AL
tion provoked by high concentrations of this prostaglandinI6 and could actually be the PGI, receptor. Sulprostone is
likely t o interact selectively with only one PGE, receptor
subtype as it only potentiates platelet aggregation.
The previously described ability of low concentrations of
PGE, to antagonize the intraplatelet CAMP increase induced by PGE, , PGI,, and PGD; could also be partly explained by its effect on PKC activation. Indeed, platelet adenylate cyclase is negatively regulated by PKC.”
The platelet aggregation facilitating activity of PGE, is
probably of pathophysiologic relevance because PGE, exerts its priming effects already at concentrations normally
produced by activated platelets (1 to 15 nmol/L). A stronger
potentiation is observed with higher concentrations of this
prostanoid (50 t o 500 nmol/L) such as those that may be
produced by platelets under pathologic conditions, eg, in
the nephrotic syndrome, or when the enzyme Tx-synthase is
pharmacologically blo~ked.4,’,’~,’~
Interestingly, nephrotic
syndrome is characterizated by a high incidence of thrombotic events,52 whereas the neutralization of PGE, by specific antibodies enhances the in vitro effectiveness of Txsynthase inhibitors.’ Finally, PGE, is the main product of
endothelial cells in the microcirculations3 where this prostanoid, due to the high endothelial cell/plasma ratio, may
well reach elevated concentration^.^^
The development of specific platelet-type (EP,) PGE, receptor antagonists and purification of the PGE, receptor
will help to clarify the role of PGE, in pathophysiology.
ACKNOWLEDGMENT
We thank Prof E. Boschetti for the assistance given with the statistical analysis of data; and Prof L. Binaglia, Prof C. Riccardi, Prof
R. Donato, and Dr G. Sorci (Department of Experimental Medicine and Biochemical Sciences, University of Perugia) for the help
they gave us for the phosphorylation experiments. We are grateful
to Prof G. Goracci (Institute of Medical Biochemistry) for critical
reading of the manuscript and to Dr A. Sturk, M. Schaap, and Prof
J.W. ten Cate (Department of Haematology, University of Amsterdam) for the suggestionsgiven in the setting up of the fura-2 technique for intraplateletcalcium levels. The technical assistanceof G.
Cipiciani is gratefully acknowledged.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1993 82: 2704-2713
Prostaglandin E2 potentiates platelet aggregation by priming protein
kinase C
R Vezza, R Roberti, GG Nenci and P Gresele
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