135
KYNURENIC ACID AS AN ADDITIONAL ENDOGENOUS
ANTI-AGGREGATORY FACTOR
Kamal E.H. ELTahir* and Dana M. Bakheet
‫يعتبررحمض رريمورييك (كرر)ممويررر م ر م يضرررحمض ض ر ك موحاتكورررامديتك كررممورردمورروم ر م ضررم ر مداعرريم ضررر م‬
‫مإام رمدوعمإلكح مهره م ضم ةرةمهركموفرردرمإورح م‬. ‫ضطحوكةم ثوم د كةم ضم كيةمد ضكلكتك مد ضقلبمد عر مد ضعك‬
‫هه م ضح يم م د كةم ضم كيةم ضتدموفح مأيضرًم ردودماحدةترةريكلك مدأدوسكمم دحم ض عرحدوتك ماقرم وا رم لر م‬
‫موشفتمهه م ضم ةةمأاموعحييم ضفرفكحرحم ضم كيرةم ض علدضرةم ر مضكرك ام ضركاحمض ررد م‬.‫يعموكمسم ضففكحرحم ضم كية‬
‫م لدم كال مأدحمإض موثبكطموكمسم ضفرفكحرحم ضم كيرةم ض حرمبما ررد مئيررسدموسرفرحم‬2-‫م‬0.1‫موير ماتح وكلموح دضتماك م‬
‫م لردم ركال مدكضر)م ض حرمبماح ريمأ وكرمد(كك)ماجح رةم(فر م‬0.06 ± 1.1‫أدييك ي ماجح ةم(فر موعرضرةم قرم هرم‬
‫م لردم‬0.5‫مد يمم يرد موحوكرلم ررد مورضسرككلم ض حكطرةمارضفرفكحرحم ضم كيرةما قرم م‬.‫م لدم كل‬0.08 ± 0.9‫وعرضةم قم هرم‬
‫موردمضرضرةمض ريم‬%6.3 ± 71.5‫موردمضرضرةمئيررسدموسرفرحمأدييرك ي مدايسربةم‬%4.5 ± 60‫كلموممإاطرلموأئكحممويرر مايسربةم‬
‫مدوشكحمهه م ضم ةةمإضر مإ كر(كرةم ضي رحموردمض ريمورييك (كر)موعر رومد للردم‬.ً‫أ وكمد(كك)مافك ميعتمماارمإضفرسكر‬
‫ م‬.‫إضرودمض يعموكمسم ضففكحرحم ضم كية‬
‫م‬
‫م‬
‫م‬
Kynurenic acid (KNA) is an endogenous metabolite of tryptophan that has been characterized
both in the brain and various peripheral organs that included blood vessels, kidneys, hearts,
intestine and the eye. Its release from the vascular tissues which also release prostacyclin and‫م‬
nitric oxide directed us to investigate its influence on platelets aggregation. Exposure of guinea-‫م‬
pig platelets to KNA in concentrations ranging from 0.1 to 2mM inhibited adenosine diphosphate
(ADP) and arachidonic acid (AA) induced aggregations in a dose-dependent manner.The
inhibitory dose 50 values against ADP and AA were found to be 1.1±0.06 and 0.9±0.08 mM
,respectively. (N=8). The anti-aggregatory effect was significantly reversed in presence of
exogenous Ca2+. Elevation of the level of the plasma by 0.5mM Ca2+ reversed the antiaggregatory effect KNA by 60±4.5 and 71.5±6.3% against ADP and AA, respectively (P<0.01 ,
N=8). It is suggested that KNA may be considered as an additional endogenous anti-aggregatory
factor.
Introduction
Kynurenic acid was identified in 1904 as a
metabolite of tryptophan (1,2). The sequence of its
formation has been outlined by Schwarcz and
Pellicciari (3). Initially, tryptophan is acted on by the
enzyme tryptophan 2,3-dioxygenase to produce Nformylkynurenine which is acted on by the enzyme
formamidase to produce kynurenine. The latter is
subjected to the enzyme kynurenine aminotransDepartment of Pharmacology, College of Pharmacy, King Saud
University, Riyadh 11451, P.O. Box 2457, Kingdom Saudi Arabia
*
To whom correspondence should be addressed.
Saudi Pharmaceutical Journal, Vol. 15, No.2, April 2007
ferase to produce kynurenic acid (KNA). In 1988, it
was identified as a modulatory constituent of the
brain (4,5). It is considered as an endogenous
blocker to the ionotropic glutamate receptors, the NMethyl-D-aspartate (NMDA) receptors (6,7) and as
a non-competitive blocker to the α7-nicotinic
receptor in the brain (8). It is also considered as a
non-neuronal costituent of the brain that is
synthesized in the astrocytes and microglia at the
vicinity of the glutamergic neurones (3,5).
Besides its presence in the brain as a
neuroprotective against degenerations and seizures
(9), it has been shown to be present in several
peripheral organs that included the liver and
136
ELTAHIR & BAKHEET
intestine of rat (10), the rat kidney (2,11), the heart
(12), the umbilical cord and amniotic fluid (13,4),
the vascular endothelium of the rat aortic rings (15),
the bovine aortic endothelial cells (16), the rat retina
(17), and human gingival fibroblasts (18). It is also
detected in the blood (13,14), the synovial fluid (19)
and in the RBCs (20).
The vascular endothelium is known to release
the anti-aggregatory substance nitric oxide (21)
while the vascular smooth muscles are known to
release the anti-aggregatory substance prostacyclin
(22 - 24) and kynurenic acid (15, 16). This coincidence raised the question, could kynurenic acid also
affect the platelet aggregability and hence help in the
maintenance of the non-coagulability of the circulating blood. Thus, this study was designed to
investigate the effect of various doses of kynurenic
acid on the guinea-pig platelets and to investigate its
mechanism of action.
Methods
1. Preparation of guinea-pig platelet-rich plasma:
Guinea-pig platelet-rich plasma (PRP) was
prepared as described for rat and rabbit platelets [25,
26]. In brief : guinea-pigs were anaethetized with
ether and from each animal 9 ml blood were
collected in a plastic syringe containing 1ml of 3.6%
trisodium citrate in water using cardiac puncture.
The blood was centrifuged using an aggregometer
(Profiler Model P A P-4) at 1600 rpm for 10
minutes. The upper (PRP) layers were aspirated and
pooled together and mixed. The PRP was then
divided into 0.5 ml aliquots into siliconized glass
cuvettes (3ml capacity). To obtain platelet poor
plasma, an aliquot of PRP was centrifuged at
3000rpm for 10minutes and the supernatant was
collected.
2. Induction of Platelets aggregation and quantification of the Anti-aggregatory effect:
For induction of platelets aggregation, the
cuvettes containing PRP were initially stored in
various chambers in the aggregometer (Profiler
Model PAP-4) heated at 37ºC. To initiate aggregation, the limit of 100% aggregation was determined
using an aliquot (0.5ml) of the platelet-poor plasma.
The cuvette was removed and another cuvette
containing 0.5ml of PRP was placed in the aggregometer chamber and a stainless steel stirrer was
Saudi Pharmaceutical Journal, Vol. 15, No. 2, April 2007
added to mix the platelets at a rate of 1100 rpm.
After 2 minutes of stirring and obtaining of a base
line, aggregation was induced by addition of 10 µl of
a solution of either ADP (adenosine diphosphate) to
produce a final concentration of 1.5µM or arachidonic acid (AA) to produce a final concentration of
0.65 mM. The aggregation response was automatically displayed in the chart coupled to the
aggregometer and allowed to continue for its
maximum response (after 3 – 4 minutes). The
concentrations of ADP and AA used were the
minimums that produced irreversible aggregation.
The% magnitude of the aggregation induced by each
aggregating agent was then recorded.
To quantify the anti-aggregatory effect of
kynurenic acid (KNA), different doses in a fixed
volume of 10µl each were used. These doses were
those that produced final concentrations of 0.1, 0.5,
1 and 2mM in the PRP aliquot under test. In the
corresponding control aliquots PRP, 10µl volumes
of water were added as a control in all experiments.
The aliquot of PRP undertest (containing KNA)
in the aggregonmeter chamber was stirred for 5
minutes and then the selected dose of the
aggregating agent was added. Aggregation was
allowed to continue for its maximum. The %
inhibition of aggregation induced by each dose of
KNA compared with the control was then calculated. Each dose was tested 8 times in PRP obtained
in 4 different days. The inhibitory dose 50 value
(ID50) i.e the dose of KNA that inhibited the control
aggregation by 50% against each aggregating agent
was then calculated using linear regression
To examine the influence of exogenous Ca2+ on
KNA-induced anti-aggregatory activity, the following procedure described previously was used (27).
To an aliquot of PRP, the ID50 of KNA in a
volume of 10µl was added followed by 10µl of an
aqueous CaCl2 solution to obtain a final
concentration of Ca2+ in the PRP of 0.5 or 1mM. To
the control aliquots of PRP, the same procedure was
used except that no KNA was added and the doses
of CaCl2 added were contained in 20µl volumes.
Each cuvette was then stirred for 5 minutes and the
aggregatory dose of the aggregating agent under test
was added. Aggregation was allowed to continue for
its maximum. The percentage aggregation induced
in each case was calculated and compared with the
control aggregation. The percentage effectiveness of
Ca2+ in reversing the anti-aggergatory effect of KNA
was then calculated as follows:
KYNURENIC ACID AS AN ANTI-AGGREGATORY FACTOR
A – B x 100 %
A
where A & B are the % anti – aggregatory effects of
the ID50 of KNA in absence and presence of the
specific dose of Ca+2, respectively.
Statistical analysis:
All values reported in this study were mean ±
s.e. mean with N= number of experiments performed
for each dose tested. Significant differences between
the antiaggregatory effects of KNA in presence and
absence of Ca2+ were calculated using student's 't'
test for paired samples.
Drugs used:
Kynurenic acid, ADP and arachidonic acid were
obtained from (Sigma, USA). Trisodium citrate
dihydrate and CaCl2 dihydrate (BDH. U.K).
Solubilization of the Drugs:
KNA, ADP, trisodium citrate dihydrate and
CaCl2. 2H2O were dissolved in distilled water.
Arachidonic acid was purchased as an oil to which
an equivalent weight of NaHCO3 was initially added
and then diluted with water to obtain the required
concentration.
Results
1.Effect of KNA on ADP-and AA-induced platelets
aggregation:
Pretreatment of the aliquots of PRP for 5
minutes with KNA in doses of 0.1 , 0.5 , 1 and 2mM
final concentrations, inhibited ADP-and AA-induced
aggregations in a dose-dependent manner. Table 1
depicts the % inhibitions of aggregations induced by
the various doses of KNA against the used
aggregatory agents. The ID50 values of KNA
against AA and ADP were 0.9±0.08 and 1.1±0.06
mM, respectively (N=8).
2. Effect of Ca2+ on KNA antiaggregatory activity
against ADP and AA:
The co-presence of 0.5 & 1 mM Ca2+ with the
ID50s of KNA either that against ADP or AA
decreased the anti-aggregatory effect of KNA in a
dose-dependent manner. The presence of 1mM Ca2+
(final concentration in the PRP) completely
Saudi Pharmaceutical Journal, Vol. 15, No. 2, April 2007
137
abolished the anti-aggregatory effect of KNA
against both ADP & AA. The % effectiveness of
Ca2+ 0.5 mM (final concentration) in reversing the
antiaggregatory effects KNA were 60±4.5 and
71.5±6.3% against ADP and AA, respectively (N=8;
P<0.01).
Table 1: The percentage anti-aggregatory effects of
KNA against ADP – and AA- induced Platelets
aggregation (N = 8).
Aggregatory Concentration of Kynurenic Acid (mM)
Agent
0.1
0.5
1
2
ADP
11.2±2.1
26.1±3.2
46.3±5.9
100
AA
21.3±1.9
39.6±4.1
60.1±5.3
100
Discussion
The results of this study clearly demonstrated the
ability of Kynurenic acid (KNA) in inhibiting
platelets aggregation induced by two different
aggregatory agents: arachidonic acid that induces its
activity via the enzymes cyclo-oxygenase type -1
(COX-1) and thromboxane synthase (28) and ADP
(adenosine diphosphate) via activation of P2T
receptors (29). Both agents require Ca2+ to induce
their aggregatory activity (30,31) . The ability of
exogenous Ca2+ addition to the platelets – rich
plasma (PRP) to reverse the anti-aggregatory effect
of KNA points to the possible mechanism of action
of this acid as inhibition of influx of extracellular
Ca2+ mediated by any of the two aggregatory agents
used in this study. At the same time, one can not rule
out any direct partial effect of KNA on COX-1,
thromboxane synthase or even the partial blockade
of ADP or TXA2 receptors located on the platelets
membranes.
Confirmation or support to the ability of KNA to
inhibit influx of Ca2+ can be drawn from the ability
of KNA to inhibit presynaptic influx of Ca2+ in the
rat retinal neurones (32) resulting in inhibition of
excitatory post synaptic currents and inhibition of
the Ca2+ dependent glutamate release (33). Further
support to the suggested mechanism can be derived
from the ability of KNA to block actions mediated
via the NMDA (6,7) and the α7-nicotinic receptors
(8) because both these two receptors mediate an
increase in influx of Ca2+ [8,34].
138
ELTAHIR & BAKHEET
With regard to the milli molar doses of KNA
used in this study, one has to admit that they are
higher than those that are expected to be in the
circulating human blood, since Urbanska et al., (35)
reported that the human plasma level is 3.9±1.8 n
mole /litre. However, it should also be noted that
there are no reports regarding the plasma levels of
KNA in guinea-pigs or other rodents. Further more,
the doses used in this study are in the same range to
that reported previously regarding the bactericidal
action of KNA against oral cavity Staphylococous
aureus and Escherichia coli (18) and its concentration that suppressed the respiratory function of the
rat heart mitochondria (36).
On a broad basis, it can be suggested that albeit
the high doses of KNA required to inhibit platelets
aggregation, yet it can be considered as a new
additional factor that can operate in concert with
other antiaggregatory factors known to exist in the
circulating blood such as PGI2 and Nitric oxide [NO
(21). This is strengthened by the continuous release
of this acid from the vascular endothelium (15,16).
Furthermore, an increase in KNA synthesis within
the cardiovascular system may benefit those who
show platelets hyperaggregability and low prostacyclin production as seen in patients with diabetes
mellitus (37), hypertension or thrombosis (35). A
suggested mechanism for the increase in blood level
of KNA is via decrease in the methionine metabolite
homocysteine that is shown to affect the endothelial
production of KNA in a biphasic way : stimulating
its synthesis and release in low concentrations and
decreasing its synthesis and release in high doses
(15). Alternatively, a direct way of increasing KNA
blood level is via drugs such as (RS)-3-4- dichlorobenzoylalanine (39).
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Acknowledgement
We thank the Research Centre, College of
Pharmacy, King Saud University for the financial
grant No.195 to support this research.
16.
17.
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