Acetoxy drug: Protein transacetylase catalyzed activation of human platelet nitric oxide synthase by polyphenolic peracetates

Article abstract of DOI:10.1016/j.bmc.2005.08.044

An enhanced intracellular level of Nitric oxide (NO) is essential to ameliorate several pathological conditions of heart and vasculature necessitating the activation of NOS. We have projected in this report the acetylation of eNOS by polyphenolic peracetates (PA) catalyzed by the novel enzyme acetoxy drug: protein transacetylase (TAase) discovered in our laboratory as an unambiguous way of activating NOS which results in the manifestation of physiological action. The human platelet was chosen as the experimental system in order to validate the aforementioned proposition. PA caused profound irreversible activation of platelet NADPH cytochrome c reductase mediated by TAase. The convincing biochemical evidences are presented to show that PA could cause acetylation of the reductase domain of NOS leading to the activation of eNOS in tune with their specificities to platelet TAase. As a result, the enhanced level of NO due to activation of platelet eNOS by PA was found to inhibit the ADP-induced platelet aggregation. The present studies highlight for the first time the role of PA as the novel potent agent for enhancing the intracellular NO levels.

Full text of DOI:10.1016/j.bmc.2005.08.044

Bioorganic & Medicinal Chemistry 14 (2006) 575–583
Acetoxy drug: Protein transacetylase catalyzed activation
of human platelet nitric oxide synthase by polyphenolic peracetates
a
Pulkit Khurana, Ranju Kumari, Parag Vohra, Ajit Kumar, Seema, Garima Gupta,
a
a
a
a
a
a,
Hanumantharao G. Raj, Bilikere S. Dwarakanath, Virinder S. Parmar, Daman Saluja,
b
c
d
*
e
Mridula Bose, Anjana Vij, Nabo K. Chaudhary, Jawahar S. Adhikari,
f
b
b
a
Yogesh K. Tyagi and Ekta Kohli
a
a
Department of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi 110 007, India
b
Institute of Nuclear Medicine and Allied Sciences, DRDO, Lucknow Road, Delhi, India
c
Chemistry Department, University of Delhi, Delhi 110 007, India
Dr. B.R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110 007, India
d
e
Department of Microbiology, V.P. Chest Institute, University of Delhi, Delhi 110 007, India
f
Defence Institute of Physiology and Allied Sciences, DRDO, Lucknow Road, Delhi, India
Received 30 June 2005; revised 18 August 2005; accepted 19 August 2005
Available online 7 October 2005
Abstract—An enhanced intracellular level of Nitric oxide (NO) is essential to ameliorate several pathological conditions of heart and
vasculature necessitating the activation of NOS. We have projected in this report the acetylation of eNOS by polyphenolic perac-
etates (PA) catalyzed by the novel enzyme acetoxy drug: protein transacetylase (TAase) discovered in our laboratory as an unam-
biguous way of activating NOS which results in the manifestation of physiological action. The human platelet was chosen as the
experimental system in order to validate the aforementioned proposition. PA caused profound irreversible activation of platelet
NADPH cytochrome c reductase mediated by TAase. The convincing biochemical evidences are presented to show that PA could
cause acetylation of the reductase domain of NOS leading to the activation of eNOS in tune with their specificities to platelet TAase.
As a result, the enhanced level of NO due to activation of platelet eNOS by PA was found to inhibit the ADP-induced platelet aggre-
gation. The present studies highlight for the first time the role of PA as the novel potent agent for enhancing the intracellular NO
levels.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1. Introduction
ing various protein kinase(s), has received much atten-
2
tion. Furthermore, the phosphorylation of NOS at
Accumulated evidences strongly suggest the presence of
non-adrenergic, non-cholinergic (i-NANC) inhibitory
nerves in many organ system and blood vessels. Nitric
oxide (NO) is an established candidate for the mediation
different sites mediated by different protein kinases
may evoke diverse responses. Phosphorylation of
NOS, suggested to be one of the pathways for its activa-
tion, is beset with limitations. For example, phosphory-
lation of eNOS at Ser-1179 leads to activation, while
1
of i-NANC related physiological effects. Therefore,
3
,4
there is considerable amount of interest in understand-
ing the mechanisms involved in the activation of Nitric
oxide synthase (NOS) leading to the alterations in the
intracellular NO levels. Posttranslational modification
of NOS, especially the phosphorylation of NOS involv-
phosphorylation of tyrosine leads to inhibition. In or-
der to overcome these complexities, we propose the acet-
ylation of NOS by polyphenolic acetates (PA) as an
effective approach to activate NOS thereby enhancing
NO levels in human platelet. We have in this report
demonstrated the role of acetoxy drug: protein transa-
cetylase (TAase), a unique membrane-bound enzyme
5
,6
identified by us, in mediating the possible transfer of
acetyl groups from PA to platelet NOS resulting in the
enhancement of NO levels in platelet. A positive corre-
lation was found between the specificities of various
Keywords: Nitric oxide; Nitric oxide synthase; Transacetylase; Platelet
aggregation; Polyphenolic peracetate; Protein acetylation.
*
Corresponding author. Tel.: +91 011 27667497; e-mail: rajhg@
yahoo.com
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.044

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P. Khurana et al. / Bioorg. Med. Chem. 14 (2006) 575–583
PA for platelet TAase and the efficacy of the former to
activate platelet eNOS leading to NO-mediated inhibi-
tion of platelet aggregation. The enhancement of intra-
cellular NO level by this novel mechanism further
strengthens the concept evolved by us of acetyl CoA
activated by various PA in a manner similar to their
specificities to TAase (Table 1). Accordingly, the PA
caused the activation of platelet reductase in the follow-
ing order: compound (1) > (3) > (5) > (4) ꢀ (2) (Fig. 4).
7
,8
independent enzymatic protein acetylation.
2.2. RT-PCR
An attempt was made to examine the expression of NOS
in human blood platelet. For this purpose, RNA was iso-
latedfrom platelet (Fig. 5A) andRT-PCRwasperformed.
The results (Fig. 5B) indicated the preponderance of
eNOS and no iNOS under the present conditions.
2
. Results
In the present investigations, the characterization of
TAase catalyzed reaction (Fig. 1) in platelet was carried
out by examining the specificity of various PA (Fig. 2) to
platelet TAase. Further, the TAase mediated activation
of NADPH cytochrome c reductase and NOS by PA,
and the effects of PA on ADP-induced aggregation of
platelet were also studied with specific reference to the
role of TAase. The kinetic parameters of TAase present-
ed in Table 1 demonstrate the specificities of various PA
to TAase. The specificity of monoacetoxychromone (4)
was found to be equivalent to that of monoacetoxy-
coumarin (2). It is evident from the data (Table 1) that
for TAase, compound (1) exhibited the least Km value
followed in order by compound (3) < (5) < (4) ꢀ (2). Re-
sults depicted in (Fig. 3) demonstrated that the TAase
mediated inhibition of GST by compound (1) was nearly
abolished when PHMB was included in the preincuba-
tion mixture.
2.3. TAase mediated activation of platelet NOS by PA
Platelets were incubated with L-arginine in order to mea-
sure the activity of NOS and a sensitive flow cytometric
method was employed. The data presented are fluores-
cence intensities and the values are means of four mea-
surements with an error less than 5%. It is evident that
platelet when incubated with increasing concentrations
of L-arginine led to enhanced activity of NOS (Fig. 6)
as seen by DCFH oxidation resulting in the fluorescence
due to DCF which is a measure of the NO levels. The
platelet preincubated with PA and L-arginine exhibited
a significantly higher level of NO as compared to that
Table 1. Substrate specificity for acetoxy drug protein: transacetylase
PA
K
m
(lM)
Vmax (units)
2
.1. TAase mediated activation of platelet NADPH
cytochrome c reductase by PA
DAMC
1050
1422
1818
1588
1612
400
222
188
200
190
7
1
7
7
-AMTC
-AX
Platelet was preincubated with PA, followed by the
addition of substrates of cytochrome c reductase, name-
ly NADPH and cytochrome c, in order to assay the
reductase activity. The results documented in (Fig. 4)
indicated that the platelet reductase was remarkably
-AAMC
-AMC
The details are outlined under Section 4.1. Values are means of four
experiments with variations less than 2%.
OH
OCOCH3
Acetoxy Drug:Protein
Transacetylase ( TAase)
HO
O
O
H COCO
O
O
3
Protein-OAc
Acetylated
+
Protein
CH3
CH3
7
,8-Diacetoxy-4-methylcoumarin
7,8-Dihydroxy-4-methylcoumarin Protein
Figure 1. TAase catalyzed reaction.
OCOCH3
H COCO
O
S
H COCO
O
O
H COCO
O
O
3
3
3
CH3
CH3
CH3
(
1)
(2)
(3)
OH
H COCO
O
CH3
O
3
O
HO
O
COCH3
O
O
OCOCH
3
CH3
(
4)
(
5)
(6)
Figure 2. Structures of test compounds.

P. Khurana et al. / Bioorg. Med. Chem. 14 (2006) 575–583
577
DAMC+PHMB
DAMC
with the primary antibody, showing that eNOS was
not acetylated. These observations clearly document
the TAase catalyzed acetylation of eNOS by DAMC.
1
00
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
2.5. TAase mediated inhibition of ADP-induced platelet
aggregation by PA
ADP-induced platelet aggregation was significantly re-
duced by compound (1) at 100 lM, while (6) had very
little effect under similar conditions (Fig. 9). Another
acetoxycoumarin, 7-AMTC, was also found to be effec-
tive albeit to a lesser extent as compared to DAMC
(
Fig. 10). These results have clearly pointed out that
PA is very effective in causing the inhibition of platelet
aggregation in tune with their specificities to TAase.
0
5
10
15
20
25
Preincubation time (min)
Figure 3. TAase activity in platelet: effect of PHMB. Platelet
lysate(50 lg protein) was preincubated with DAMC (100 lM) along
with cytosol (10–12 lg protein) for different periods and TAase was
assayed as described under Section 4.1. The concentration of PHMB
2.6. Demonstration by confocal microscopy of enhanced
NO production in platelet due to PA
The remarkable activation of NOS by compound (1)
leading to an enhancement in the NO levels in the plate-
let is clearly shown in Figure 11. Under these conditions,
platelet incubated with arginine and compound (1)
exhibited green fluorescence significantly higher than
the cells incubated with arginine alone (Figs. 12 and 13).
ꢁ2
(where added) was 10 M. The values are means of three observations
with variation less than 5%.
of L-arginine alone signifying the activation of platelet
NOS (Fig. 6). It is clear from the data presented in Figure
7
that the preincubation of platelets with polyphenol like
compound (6) other than PA itself failed to enhance the
levels of NOS. Preincubation of platelets with L-arginine
and L-NAME (an inhibitor of NOS) resulted in drastic
reduction in the levels of NO compared to cells incubated
with L-arginine alone, implying that the reactive species
oxidizing DCFH to DCF under the conditions was indeed
NO (Fig. 6). Similarly, the enhancement of platelet NO
levels due to DAMC was also profoundly reduced when
L-NAME was included in the reaction mixture (Fig. 6).
Several classes of PA were also found to be effective in
activating NOS (Fig. 8) in the following order:
3. Discussion
The previous studies carried out in our laboratory dem-
5
,19
onstrated
the irreversible inhibition of several liver
microsomal cytochrome P-450-linked mixed function
oxidases (MFO) in vitro by a model PA, compound
(1). DAMC-mediated irreversible inhibition of rat liver
MFO activity was distinct from the action of the classi-
cal suicidal inhibitors of P-450 such as chloramphenicol,
secobarbital, 21-chlorinated steroids, N-acetyl amin-
obenzotriazoles in that compound (1) needed no prior
(
1) > (3) > (2) ꢀ (4) ꢀ (5).
2
0–23
oxidative metabolism unlike the other inhibitors.
2.4. Demonstration of TAase catalyzed acetylation of
eNOS by DAMC (Western blot)
Compound (6), the deacetylated product of (1), failed
to inhibit MFO and also the 7,8-diacetoxy-4-methyl-
coumarin mediated mechanism based inhibition of
MFO was effectively abolished by thiol blockers such
as PHMB and iodoacetamide. These observations
strongly suggested the existence of an enzyme in liver
microsomes, which possibly transferred acetyl groups
from (1) to cytochrome P-450 apoprotein leading to
The immunoblot shown in (Fig. 9) highlights the inter-
action of polyclonal anti-lysine with eNOS that was
incubated with DAMC and purified rat liver TAase.
In the controls, eNOS, when incubated in the absence
of DAMC and TAase, failed to show any interaction
1
9
70
60
50
40
30
20
10
0
5
10 15 20
5
10 15 20
5
10 15 20
7-AMC
5 10 15 20
7-AAMC
5
10 15 20
7
,8-DAMC
7-AMTC
1-AX
Figure 4. TAase catalyzed irreversible activation of NADPH cytochrome c reductase by PA. Preincubation time (min) indicated inside the bar. The
control received DMSO in place of PA. The increment in the activity of NADPH cytochrome c reductase due to preincubation with PA over the
control is expressed as percent activation. Concentration of the PA was 25 lM. Values are means of three observations with variation less than 2%.

578
P. Khurana et al. / Bioorg. Med. Chem. 14 (2006) 575–583
1
01
102
DCF Fluorescence
Figure 7. Activation of platelet eNOS by DAMC: effect of L-NAME.
Details of NOS assay procedure given under Section 4.1. Solid light
line, control; solid dark line, arginine; dotted and line, Arg + DAMC;
dotted only, Arg + DAMC + L-NAME.
Figure 5. (A) Total RNA isolated from human platelet. (B) RT-PCR
of human platelet RNA using NOS primers. Lane 1, RT-PCR using
iNOS primers. Lane 2, DNA ladder. Lane 3, RT-PCR using eNOS
primers.
2
4
3
5
1
1
01
102
DCF Fluorescence
Figure 8. Effects of DHMC and DAMC on platelet eNOS. Solid light
line, control; solid dark line, arginine; dot and line, Arg + DAMC;
dotted only, Arg + HMC.
01
102
103
1
DCF Fluorescence
Figure 6. Effect of increasing concentration of L-Arg on human
platelet NOS. Platelets were incubated with DCFH-DA for 30 min
followed by the measurement of fluorescence due to intracellular.
DCFH oxidation. Several concentrations of L-Arg were incubated
separately with DCFH-DA and fluorescence was measured. The
increment in fluorescence due to L-Arg represents the formation of
NO. 1, control; 2, L-Arg (50 lM); 3, L-Arg (100 lM); 4, L-Arg
other protein in the P-450 cycle was modified by (1) cat-
alyzed by microsomal TAase in a manner similar to P-
450-linked MFO. Compound (1) contrary to its inhibi-
tory action on hepatic P-450 caused kinetically discern-
ible hyperbolic activation of liver microsomal NADPH
16
cytochrome c reductase. Compound (1) was also
(150 lM); 5, L-Arg (200 lM).
found to be competent in causing substantial activation
of reductase of rats in vivo. Since the aforementioned
work carried out in our laboratory firmly established
the TAase catalyzed profound activation of NADPH
cytochrome c reductase by PA, it was considered worth-
while to examine whether NOS which bears a domain of
the reductase could similarly be activated leading to en-
2
4,25
inhibition of P-450-catalyzed MFO reaction.
The
enzyme was termed TAase in general, since apart from
DAMC a large number of PA and related compounds
were found to be acted upon by this enzyme. In the ear-
lier study, an effort was made to examine whether any

P. Khurana et al. / Bioorg. Med. Chem. 14 (2006) 575–583
579
250
200
150
100
50
0
Figure 9. Platelet was incubated separately with several classes of PA (100 lM) along with arginine, the DCF fluorescence due to the formation of
NO was measured. The details and abbreviations are given under Section 4.1.
1
33 KDa
Lane1
Lane 2
Lane 3
Lane1: Control (eNOS+7,8-Diacetoxy-4-methyl coumarin)
Lane2: Control (TAase+eNOS+Dimethylsulfoxide)
Lane3: Acetylated eNOS (TAase+ eNOS+7,8-Diacetoxy-4-methylcoumarin)
Figure 10. Western blot using polyclonal anti-acetyl lysines. Lane 1, control (eNOS + 7,8-diacetoxy-4-methylcoumarin). Lane 2, control
(TAase + eNOS + dimethylsulfoxide). Lane 3, acetylated eNOS (TAase + eNOS + 7,8-diacetoxy-4-methylcoumarin).
Control
7
-AMTC (100µM)
DAMC (75µM)
Figure 12. Inhibition of ADP-induced platelet aggregation by 7-
AMTC and DAMC. (Details and abbreviations described under
Section 4.1.)
Figure 11. Comparison of inhibition of ADP-induced platelet aggre-
gation by DAMC and DHMC. Details and abbreviations described
under Section 4.1.
2
6
to NO pathway. As a first step, the presence of TAase
in platelet was confirmed by the inhibition of cytosolic
glutathione S-transferase (GST) activity by PA. Hu-
man platelets were found to have considerable TAase
activity and the specificities for various PA were in the
hanced intracellular production of NO. In order to
investigate this proposition, we chose human platelet
as the suitable experimental system. Human platelet is
recognized as a suitable model for investigation related
5

580
P. Khurana et al. / Bioorg. Med. Chem. 14 (2006) 575–583
2
8
the activation of reductase activity. Hence, it is plausi-
ble to infer that the TAase catalyzed acetylation of the
reductase domain of platelet NOS could similarly ac-
count for the observed significant activation of NOS.
Protein acetylation is often seen as a phenomenon,
either independent or as adjunct to protein phosphory-
2
9
lation, that leads to the control of physiological events.
The multiple regulation of p53 tumor suppressor protein
by phosphorylation and acetylation can be viewed in
30,31
Control
7,8-Diacetoxy-4-methyl coumarin
this context.
The fact of the matter is that PA cause
significant activation of platelet eNOS (Fig. 3) catalyzed
by TAase. The enhanced formation of NO in platelet
leads to the increased production of cAMP, which caus-
es degranulation resulting in the platelet aggregation.
These studies purport for the first time a possible mod-
ification of NOS by way of enzymatic acetylation cata-
lyzed by TAase resulting in the activation of platelet
NOS. Such a conclusion was ratified by the observation
that the incubation of purified eNOS with compound (1)
and purified liver TAase resulted in the formation of the
acetylated eNOS, which was found to avidly interact
with the antibody against protein bearing acetylated ly-
sines. These investigations have also projected for the
first time PA as the novel potent activators of NOS that
can lead to substantial release of NO as visualized by
confocal microscopy (Fig. 11) leading to the inhibition
of platelet aggregation. To our knowledge, no such
xenobiotic acetates (PA) other than aspirin related com-
pounds have been reported in the causation of signifi-
cant physiological effects by way of acetylation of the
target protein. Another remarkable distinction is that
the action of PA is enzyme (TAase) mediated, while
the effect of aspirin is solely non-enzymatic in nature.
We have presented in this paper a unique mechanism
for the enhancement of intracellular NO levels by PA,
which can culminate in the expression of physiological
effect, viz., inhibition of platelet aggregation.
Figure 13. Demonstration by confocal microscopy of enhanced
production of NO in platelets by compound (1) in comparison with
compound (6), the deacetylated product which served as a control.
order (1) > (3) > (5) > (4) ꢀ (2). The fact that the thiol-
blocking agent PHMB blocked the TAase catalyzed
inhibition of GST confirmed that the platelet TAase
was a thiol sensitive enzyme as seen in the case of hepat-
1
9
ic microsomal enzyme. After having established the
presence of TAase in platelet, an effort was made to
examine the influence of Compound (1) and other PA
on NADPH cytochrome c reductase. The results clearly
indicated the time dependent activation of platelet
reductase upon incubation with the test compounds. It
was observed that the substrate (PA) that yielded high
activity of platelet TAase (as measured by inhibition
of GST) was equally effective in causing the activation
of reductase (Fig. 4). After having convinced ourselves
that PA activates the platelet reductase in the manner
described above, we then proceeded to investigate the
influence of PA on NOS of platelet. The result of the
present study has very clearly established that platelet
NOS is activated by the test compounds (PA) in tune
with their specificities to platelet TAase. Accordingly,
Compound (1) having the highest specificity for TAase
activated NOS to the maximum extent. While the other
PA showed varying degrees of capability to activate
NOS in platelet. (6), the deacetylated product of (1),
when incubated with platelet in the presence of L-argi-
nine, failed to activate NOS, pointing the finger toward
the acetyl groups of (1) in causing the profound activa-
tion of NOS. Our earlier work documented the time
dependent inhibition of cytosolic GST by (1), catalyzed
by partially purified TAase from buffalo liver. GST 3-3,
when incubated with purified buffalo liver TAase and
DAMC, was found to undergo acetylation at multiple
sites as demonstrated by MALDI-TOF and LC/MS/
4. Experimental
4.1. Materials and methods
4.1.1. Synthesis and characterization of polyphenolic
peracetates (PA). 7,8-Dihydroxy-4-methylcoumarin (6),
7,8-diacetoxy-4-methylcoumarin (1), 7-acetoxy-4-meth-
ylcoumarin (2), 7-acetoxy-4-methylthiocoumarin (3),
1-acetoxyxanthone (5), and 7-acetoxy-3-acetyl-2-meth-
ylchromone (4) were synthesized as per the published
7
,8
MS studies. Accordingly, N-terminal proline and 6-ly-
sines: Lys-51, -82, -124, -181, -191, and -210 were found
to be acetylated. It is thus logical to postulate, based on
the aforementioned observations that PA catalyzed by
TAase can as well acetylate the platelet eNOS. NADPH
cytochrome P-450 reductase domain of platelet NOS is
possibly the target for TAase catalyzed acetylation, as
the activation of the former is now firmly established
9
–11
procedures.
the compound (1) as the enhancer of intracellular NO
The Indian Patent has been filed for
levels.
4.1.2. Chemicals. Reduced glutathione (GSH), 1-Chloro-
2, 4-dinitrobenzene (CDNB), NADPH, cytochrome c,
dichlorofluorescin diacetate (DCFH-DA), N-nitro-L-ar-
ginine methyl ester (L-NAME), L-arginine and adenosine
diphosphate (ADP), and anti-rabbit HRP conjugated sec-
ondary antibody were procured from Sigma Chemical, St.
Louis, MO (USA). Sodium p-hydroxy mercuribenzoate
(PHMB) was obtained from E. Merck (Germany). Iodo-
acetamide was a product of Lancaster Synthesis. Access
Quick RT-PCR system was procured from Quigen. TRI-
1
6,24,25
by our investigations.
the modification of NOS. The phosphorylation of
There are many reports on
2
7
3
eNOS catalyzed by protein kinase Akt is known to re-
sult in the activation of NOS activity. The enhanced
electron flux owing to the phosphorylation of the reduc-
tase domain is ascribed to be the reason for enhanced
activity of NOS. Nadler observed that the acetylation
of purified reductase by acetic anhydride resulted in

P. Khurana et al. / Bioorg. Med. Chem. 14 (2006) 575–583
581
zol was obtained from Invitrogen. DAB system was pro-
cured from Bangalore Genei, India. Anti-acetyl lysine pri-
mary antibody was obtained from Cell Signalling, USA.
All other chemicals used were of high purity and were ob-
tained from local suppliers.
ucts were analyzed by electrophoresis using 1% agarose
gel and visualized by ethidium bromide staining.
4.1.6. Glutathione S-transferase (GST) assay. The meth-
1
4
od of Habig et al was followed using GSH and CDNB
as the substrates and the assay was carried out in 1.0 ml
spectrophotometric cuvette (1 cm light path). Briefly,
the reaction mixture consisting of 0.25 M phosphate
buffer (pH 6.5), rat liver cytosol (10–12 lg protein),
1 mM CDNB (added in 50 ll ethanol), and 1 mM
GSH was mixed and progress of the reaction was fol-
lowed by monitoring the absorbance at 340 nm using a
Cary Spectrophotometer (Cary Bio 100). The kinetic
software was used to ensure that the analysis was
restricted to the linear range with respect to incubation
time and enzyme concentration.
4
.1.3. Isolation of platelet-rich plasma (PRP). Nine millili-
ters of venous blood was collected from healthy human
volunteers and was mixed with 1.0 ml of 3.8% trisodium
citrate (anticoagulant). The citrated blood was centri-
fuged at 180g for 10 min at room temperature. The upper
two-third fraction of plasma (PRP) was transferred to
another centrifuge tube leaving behind lower one-third
layer to avoid contamination with WBCs and RBCs.
PRP was then further centrifuged at 4000g for 5 min to
produce a platelet button. The platelet button was lysed
using a hand homogenizer and protein content of the
1
2
homogenate was determined by LowryÕs method. This
homogenate was then accordingly used for the various
spectrophotometric assays mentioned below.
4.1.7. Assay of platelet TAase. TAase in platelet was as-
sayed using PA as the first substrate and cytosolic GST
5
as the second substrate as described earlier. The assay
mixture in a total volume of 0.8 ml consisted of
0.25 M phosphate buffer (pH 6.5), platelet lysate
(50 lg protein), cytosol (10–12 lg protein), and PA
(50 lM in 50 ll DMSO), and preincubated for 10 min,
followed by the addition of GSH and CDNB for the as-
say of GST as described above. The unit of TAase cat-
alytic activity was expressed in terms of % inhibition of
GST under the conditions of the assay.
4
from rat liverfollowing the published procedure for dem-
onstrating TAase catalyzed acetylation of eNOS by (1).
.1.4. Purification of rat liver TAase. TAase was purified
8
4.1.5. RNA isolation and RT-PCR of human platelet
NOS. 4.1.5.1. (a) RNA isolation. Total RNA was isolat-
ed from human platelet by the TRIzol method of Zhang
1
3
et al. Total RNA was purified using chloroform and
precipitated by the addition of isopropanol. Pellet ob-
tained was washed twice with 75% chilled ethanol and
stored at ꢁ80 ꢁC. The RNA was redissolved in 30 ll
RNAase free water and used for RT-PCR.
4.1.8. Measurement of kinetic parameters of platelet
TAase. Platelet lysate (50 lg protein) was mixed separate-
ly with varying concentrations of DAMC (25–400 lM, in
50 ll DMSO), 0.25 M phosphate buffer (pH 6.5), and
cytosol (10–12 lg) in a volume of 0.8 ml, and preincubat-
ed for 10 min followed by the addition of GSH and
CDNB in order to assay GST as described above. The
% inhibition of GST is plotted against the DAMC con-
centration in order to calculate the kinetic constants.
4
.1.5.2. (b) RT-PCR. RT-PCR was carried out using
total RNA extracted from blood platelet using Gradient
PCR Bio-Rad I cycler thermal cycler.
For eNOS the primers used were:
4
.1.9. Effect of PHMB on platelet TAase activity. PHMB
0
ꢁ2
FP: 5 CAG TGT CCA ACA TGC TGC TGG AAA
0
at a concentration of 10 M was included in the prein-
cubation reaction mixture and the assay of TAase was
carried out as described above.
TTG-3
0
0
RP: 5 TAA AGG TCT TCT TCC TGG TGA TGC C 3
RT-PCR was carried out in a total volume of 50 ll con-
taining 1· Access quick master mix, 1 lM forward prim-
er, 1 lM reverse primer, 2 lg RNA, and 5 U AMV
reverse transcriptase. The reaction mixture was incubat-
ed at 48 ꢁC for 45 min followed by PCR cycling. The
conditions for amplification were: an initial denatur-
ation step at 94 ꢁC for 5 min followed by 36 cycles of
4.1.10. TAase catalyzed modulation of NADPH cyto-
1
5
chrome c reductase by PA. For the reductase assay, the
platelet lysate (50 lg protein) was mixed with PA
(25 lM), 0.05 M phosphate buffer (pH 7.7), and water
to make 0.5 ml volume. The contents were preincubated
at 37 ꢁC for 10 min. The aliquots were removed period-
ically into a spectrophotometer cuvette containing
0.1 mM EDTA, 36 mM cytochrome c, and 1 mM
NADPH in a total volume of 1 ml. The progress of
the reaction was monitored by following the absorption
at 550 nm. In the control, PA was replaced by DMSO.
The increment in reductase activity due to PA over the
1
min denaturation at 94 ꢁC, 1 min annealing at 60 ꢁC,
and extension of 1 min at 72 ꢁC. The final cycle was fol-
lowed by strand extension at 72 ꢁC for 5 min.
For iNOS primers used were:
1
6
control was expressed as percent activation.
0
0
0
FP: 5 TGG AAT TCA CTC AGC TGT GC 3
0
RP: 5 GAT GTA GCG CTG GAG G 3
4.1.11. Assay of NOS by flow cytometry. The method
1
7
outlined by Imrich and Koobzik was followed for
the assay of NOS by flow cytometry. Measurements
were made with a 488 nm laser based flow cytometer
(FACScalibur, Becton Dickinson, USA) and data (light
All conditions for iNOS amplification were similar to
those for eNOS (as mentioned above) except that the
annealing temperature was 57 ꢁC. The amplified prod-

582
P. Khurana et al. / Bioorg. Med. Chem. 14 (2006) 575–583
scatter and green fluorescence) were acquired using the
Cell Quest software (Becton Dickinson, USA). Analysis
was performed by applying appropriate gates with refer-
ence to the autofluorescence measured under similar
conditions. Platelet isolated as described earlier was sus-
4.1.14. Confocal microscopy. To 1 ml of PRP, 20 ll com-
pound (1) (100 lM), 20 ll arginine (50 lM), and 10 ll
DCFH-DA (20 lM) were added. The contents were
incubated for 30 min at 37 ꢁC and centrifuged at 1000g
for 15 min at 4 ꢁC. The pellet obtained was washed
and finally suspended in 0.5 ml PBS. Hundred microli-
ters of the cell suspension was placed on microscopic
slide with coverslip for observation under the confocal
microscope (Model LSM 510, Carl, Zeis, Germany).
The images were visualized using 40· oil objective and
the green DCF fluorescence was observed following
excitation at 488 nm from argon laser with the help of
long pass filter LP 505. The fluorescence was confirmed
from intensity profile measurement with the built in soft-
ware provided by the manufacturer.
6
pended in PBS and platelet counts were adjusted to 10 /
ml using electronic particle counter (SYSMEX, Model
No. FA20). DCFH-DA (20 lM) and L-arginine at vari-
ous concentrations were then added to the cell suspen-
sion and preincubated for 37 ꢁC for 30 min while
rotating (10 rpm) to prevent adherence during assay.
To stop the assay, samples were placed on ice for
1
0 min in dark. Relative green DCF fluorescence was
measured. Similarly, various PA (1–5) were preincubat-
ed separately with L-arginine and their relative fluores-
cence was measured thereafter.
4
.1.12. Demonstration of TAase catalyzed acetylation of
Acknowledgments
NOS by 7,8-diacetoxy-4-methylcoumarin. Anti-acetyl ly-
sine as primary antibody was used to show the acetylation
of NOS. For this purpose purified TAase (50 lg) was
incubated with eNOS (50 lg), compound (1) (200 lM),
and 10 mM phosphate buffer (pH 7.2), and incubated
for 30 min at 37 ꢁC. After completion of the reaction,
samplebuffer(loadingdye) was added tothe reaction mix-
ture to stop the reaction. This reaction mixture was used
to detect the acetylated NOS by Western blot.
The financial assistance from the Department of Bio-
technology, Government of India, is gratefully acknowl-
edged. R.K. is a recipient of Senior Research Fellowship
from C.S.I.R, Government of India. Purified eNOS was
generously provided by Prof. Bettie Masters, University
of Texas, Health science Center, Galveston, TX, USA.
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6
7
4.1.13. Aggregation studies. PRP was prepared by the
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8
. Kohli, E.; Gaspari, M.; Raj, H. G.; Parmar, V. S.;
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(
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