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Introduction
Prostaglandins are derived from arachidonic acid liberated from the cell membrane. They are produced in a variety of
tissues and mediate an array of physiologic and pathologic processes, including regulation of respiratory, vascular,
intestinal and inflammatory
activities[1]. Cyclopentenone prostaglandins (cycPG), including the A and J series, are formed from the
enzyme-induced dehydration of prostaglandin E and D. They have some unique biological activities that are different from
those of classic prostaglandins, such as inducing cell
differentiation[2],
anti-tumor[3-5] and anti-virus
activities[6,7]. Prostaglandin A1 (PGA1) has many pharmacological actions, including inhibiting tumor growth, inflammation and viral
replication[8-11]. We have reported previously that PGA1 inhibits excitotoxin-induced apoptosis of striatal neurons
in vivo and rotenone-induced apoptosis of cultured SH-SY5Y
cells[12,13]. In a recent study, we found that PGA1 significantly reduced infarction
volume in rodent models of focal cerebral
ischemia[14,15]. PGA1 influences several cellular signaling pathways, including
activation of peroxisome proliferator-activated receptor
g (PPARg)[16], inhibition of nuclear factor-kappaB
(NF-κB)[11,17], and induction of heat shock
proteins[18,19]. The molecular signaling pathways involved in its neuroprotection, however, remain to
be determined.
The activation and aggregation of platelets play an important role in the pathological process of cerebral ischemia
through interactions with endothelial cells and formation of
thrombosis[20,21]. It has been shown that drugs inhibiting platelet
function, including aspirin, could be beneficial in preventing ischemic attack. The effects of some prostaglandins on platelets
have been reported[22,23]. However, information about the effects of PGA1 on platelets is incomplete and inconsistent;
therefore, the aim of the present study was to determine if PGA1 had an inhibitory effect on the functions of human platelets.
We found that PGA1 inhibited platelet aggregation, release of
TXB2 and platelet adhesion to endothelial cells. These actions
could contribute to its neuroprotective effects in rodent models of stroke.
Materials and methods
Drugs and reagents PGA1, thrombin, Fluo-3 acetoxy-methyl ester (Fluo-3 AM), sulfinpyrazone, and A23187 were
purchased from Sigma (St Louis, MO, USA); adenosine 5¡¯ diphosphate (ADP),
N-(2-hydroxyethylpiperazine)-NV-(2-ethanesulfonic
acid) (HEPES), and ethylene glycol
bi-b-aminoethylether)-N,N,NV-tetraacetic acid (EGTA) were purchased from Shanghai
Sangon Bioengineering and Technology Service (Shanghai, China). Collagen was purchased from the Jiangsu Institute of
Hematology (Suzhou, China).
Platelet preparations Venous blood was obtained from healthy donors with no drug history for at least 14 d, and
immediately mixed with a one-ninth volume of citrate acid
(3.8%). Platelet enriched plasma (PRP) was obtained by
centrifugation at 200×g for 10 min. For experiments with thrombin, platelets were washed with HEPES-buffed solution (140 mmol/L
NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 10 mmol/L glucose and 20 mmol/L HEPES), and then resuspended in
Tyrode¡¯s solution (137 mmol/L NaCl, 2.7 µmol/L KCl, 0.36
µmol/L NaH2PO4, 12 µmol/L
NaHCO3, 1 µmol/L MgCl2, 5 µmol/L HEPES, 5 µmol/L glucose,
2 µmol/L CaCl2).
Platelet aggregation assay Aggregation of platelets was evaluated by using an MPG-3E aggregometer (Shanghai Silong
Medical Instrument Factory, Shanghai, China) according to the manufacturer¡¯s instructions. PRP was first incubated with
ethanol (with a final concentration of 0.8%, as a vehicle control), or different concentrations of PGA1 at 37 °C for 5 min. Platelet
aggregation was triggered by adding 20 µL of ADP, collagen and thrombin to
200 µL of PRP to achieve final concentrations of
40 µmol/L, 40 µg/mL and 0.25 U/mL, respectively.
Platelet adhesion assay Platelets were washed and treated as described earlier. Human umbilical endothelial cells were
cultured in 96-well plates and grown to cover the entire surface of wells. Endothelial cells were pre-treated with Triton X-100
(0.5%) before use. Thrombin-stimulated platelet suspension was added to each well and incubated for 30 min. The adherent
platelets were stained with Rose Bengal (0.25%) at 37
°C and then dissolved in absolute ethanol 30 min later. The number of
adherent platelets was determined by measuring absorption using a wavelength of 570 nm.
Assay of 5-hydroxytryptamine release from platelets
Levels of 5-hydroxytryptamine (5-HT) released from platelets were
determined by using O-phthaldialdehyde (OPT) fluorospectrophotometry as described
elsewhere[2]. Briefly, platelets were obtained and washed as described earlier, and different concentrations of PGA1 (20-80 µmol/L) or vehicle (final
concentration=0.8% ethanol) were incubated with washed human platelets for 5 min. 5-HT was extracted after stimulation with thrombin (0.25
µmol/L) for 5 min and reacted with OPT in 10 mol/L HCl for 15 min. Fluorescence was then measured with a 960 CRT
fluorospectrophotometer (Shanghai Jinmi Scientific Instrument Company, Shanghai, China) using excitation and emission
wavelengths of 365 nm and 480 nm, respectively. The concentration of 5-HT was calculated as follows:
[5-HT]=(Fsample-Fblank
)/(Fstandard-Fblank
)×0.5 (µg/mL)
Evaluation of ultrastructure of
platelets The effect of PGA1 on ultrastructural changes of platelets induced by thrombin
was examined with an electron microscope. Washed platelets were pretreated with PGA1 (20-80 µmol/L), then thrombin
(0.25 U/mL) was added to activate platelets 5 min later. Platelets were then fixed with 4%
(w/v) glutaraldehyde after a 5 min incubation with thrombin, stained with osmium tetroxide, dehydrated, and embedded in Araldite. Ultrathin sections were
examined with a Philips CM-120 electron microscope.
TXB2 assay Platelets were obtained as described earlier. Washed platelets were prepared and pre-incubated with PGA1
(20-80 µmol/L) or vehicle (final concentration=0.8% ethanol) for 5 min. Then thrombin was added and the reaction was
terminated (500 µL of ice-cooled stop solution
containing 50 mmol/L EDTA; 2 mmol/L indomethacin, 130 mmol/L
NaCl) 5 min later. Samples were then centrifuged at
500×g for 10 min at 4 °C. Supernatants were used for determination of the levels of
TXB2 with radioimmunoassay (TXB2
Radioimmunoassay kit, Jiangsu Institute of Hematology).
Determination of cytosolic free calcium The intracellular
[Ca2+] was determined using Fluo-3 AM, essentially as
described elsewhere[1]. Briefly, PRP was incubated with 8 µmol/L Fluo-3 AM for 30 min at 37 °C; the dyed platelets were spun
down and gently re-suspended at a concentration of approximately
1×108 cells/mL in the HEPES-buffered
solution containing 140 mmol/L NaCl, 5 mmol/L KCl, 1
mmol/L MgCl2, 10 mmol/L glucose and 20 mmol/L HEPES, supplemented with 100 mmol/L
sulfinpyrazone to prevent the cellular efflux of Fluo-3 acid. The external
[Ca2+] was adjusted to 1 mmol/L and the fluorescence
was measured with flow cytometer using excitation and emission wavelengths of 488 nm and 526 nm, respectively.
Calibration of the ratio of fluorescence signals into pseudo fluorescence was performed using 1 µmol/L A23187 to obtain the maximal
ratio, followed by 5 mmol/L EGTA to obtain the minimal ratio. Platelets were incubated with PGA1 (20-80 µmol/L) at 37 °C for
5 min in HEPES-buffered solutions before the addition of thrombin. The full response was obtained in solutions containing
1 mmol/L CaCl2. Calcium concentration was estimated as follows:
Fp=(F-Fmin
)/(Fmax-Fmax). To clarify whether the rise in
intracellular calcium is derived from intracellular reservoirs or the entry of extracellular
Ca2+, platelets were stimulated in a
Ca2+-free solution, and calcium concentration was estimated as described earlier.
Statistical analysis Statistical analyses of the differences between vehicle control and PGA1-treated samples were
carried out with an unpaired, two-tailed Student¡¯s
t-test, and P<0.05 was considered significant.
Results
PGA1 inhibited platelet activation Adding ADP (40
mmol/L), collagen (40 mg/mL) or thrombin (0.25 U/mL) to human
platelets triggered robust aggregation. Pretreatment with PGA1 (20-80 µmol/L) dose-dependently inhibited the platelet
aggregation induced by collagen, ADP and thrombin. The maximal inhibition of PGA1 (20, 40, and 80 µmol/L) on platelet
aggregation induced by collagen was 46.63%,
48.70%, and 64.11% (P<0.05); that by ADP was 33.13%,
42.45%, and 49.43% (P<0.05); and that by thrombin was
41.63%, 90.29%, and 96.39% (P<0.05), respectively (Figure
1). The IC50 values of PGA1 on platelet aggregation induced by
collagen, ADP and thrombin were 31.02, 80.56, and 20.67
mmol/L, respectively. Thus, PGA1 had greater effects on the
thrombin-induced aggregation of platelets. Thrombin-induced adhesion of platelets to endothelial cells was inhibited by pretreatment
with PGA1 5 min before thrombin. The inhibitory rates of PGA1 at concentrations of 20 µmol/L, 40
µmol/L, and 80 µmol/L on platelet adhesion were 38.7%, 34.1%, and
40.8%, respectively (P<0.05 vs vehicle; Figure 2). PGA1 significantly decreased the release of 5-HT from the dense granules
of platelets induced by thrombin (P<0.05; Figure 3). The lactate dehydrogenase (LDH) assay showed that there was no
difference in LDH leakage among groups with and without PGA1 treatment using the same samples for 5-HT assay, excluding
the possibility that the increase in 5-HT in supernatant was caused by damage of platelets during preparation (data not
shown).
PGA1 inhibited the ultrastructural changes of platelets
Stimulated platelets transform from a discoid form to a spiny
spherical shape with numerous pseudopodia (shape change); simultaneously, the fibrinogen receptors that mediate
aggregation are exposed. We investigated the effects of PGA1 on platelet morphology after thrombin stimulation with an electron
microscope. We found that thrombin elicited robust changes in platelet morphology, including formation of numerous
pseudopodia and loss of dense granules. Pretreatment with PGA1 almost completely blocked the thrombin-induced shape
changes (formation of pseudopodia) and aggregation of human platelets (Figure 4).
PGA1 inhibited TXA2 synthesis and calcium influx in
platelets TXA2 is a potent activator of platelets. Addition of
thrombin to platelets stimulated synthesis of
TXB2, a stable metabolite of
TXA2. PGA1 dose-dependently inhibited the
production of TXB2 (P<0.05). The
IC50 of PGA1 on TXB2 production was 34.1 µmol/L
(P<0.05; Figure 5). Calcium is a key player in activation of platelets. PGA1 (20-80 µmol/L downregulated the increase in calcium concentration inside the
platelets induced by thrombin (P<0.05; Figure 6A). When calcium was absent in the extracellular solution, we failed to
observe an inhibition of calcium increase by PGA1 (Figure 6B), suggesting that PGA1 inhibited calcium influx from the
extracellular space.
Discussion
Most actions of the cyclopentenone prostaglandins, including PGA2, PGA1, and PGJ2, are not mediated by binding to
G-protein-coupled prostanoid receptors, but result from their direct interaction with other cellular target
proteins[8]. The pharmacological actions of PGA1 with respect to its anti-inflammatory, anti-neoplastic, and anti-viral activities have been
well documented. Recently, our in vivo
and in vitro studies suggested that PGA1 has neuroprotective
actions[12,14,15], but the mechanisms underlying its neuropro-tective effects have not been fully understood.
Platelet aggregation, adhesion to endothelial cells and release of
TXA2 play important roles in the formation of thrombosis.
TXA2 causes constriction of blood vessels, which could further reduce blood flow in the brain. Inhibitors of platelet function
are commonly used in therapeutic approaches in the treatment of brain
ischemia[24,25]. Previous studies have found inconsistent
results with respect to the effect of PGA1 on platelet aggregation. PGA1 has been reported to
have no effect on aggregation of rabbit platelets induced by
ADP, or formation of thrombus in
rats[26,27], or weakly inhibited platelet aggregation at high
concentrations[28]. Since platelets from different species commonly respond differently to platelet inhibitors, we thought that human platelets might be
sensitive to PGA1. The present studies thus examined the effects of PGA1 on the functions of human platelets. The results
showed that PGA1 significantly inhibited thrombin-induced human platelet aggregation, the release of 5-HT, and production
of TXA2. Similar inhibitory results were obtained with respect to platelet aggregation induced by collagen and ADP with less
potency. PGA1 inhibited the thrombin-induced increase in cytosolic
[Ca2+]. These actions could be part of mechanisms by
which PGA1 inhibits platelet activation. It has been well documented that in almost every step of platelet activation, calcium
is required. We found that the inhibitory effect of PGA1 on rises in calcium concentration was not seen in the absence of
extracellular calcium, suggesting that PGA1 may inhibit calcium influx. However, additional studies are required to confirm
whether PGA1 blocks calcium entry into platelets. In our study of the effect of PGA1 on thrombin-induced 5-HT release and
increases in intraplatelet calcium concentration, the medium dose of PGA1 (40
mmol/L) was less effective. The reason for this is unknown at present. Also, the effects of PGA1 on calcium entry into platelets need to be further investigated under other
experimental conditions.
In addition to the above-discussed pharmacological actions, PGA1 inhibits
IkB-a degradation and NF-κB activation, induces expression of heat shock proteins and activates
PPAR-g. The significance of these pharmacological actions of PGA1
with respect to the ability of PGA1 to inhibit platelets and neuronal injury in ischemia need to be further investigated.
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