Inhibition of platelet aggregation by carbon monoxide-releasing molecules (CO-RMs): comparison with NO donors.

Abstract

Carbon monoxide (CO) and CO-releasing molecules (CO-RMs) inhibit platelet aggregation in vitro. Herein, we compare the anti-platelet action of CORM-3, which releases CO rapidly (t (½) 1 min), and CORM-A1, which slowly releases CO (t(½) = 21 min). The anti-platelet effects of NO donors with various kinetics of NO release were studied for comparison. The effects of CO-RMs and NO donors were analyzed in washed human platelets (WP), platelets rich plasma (PRP), or whole blood (WB) using aggregometry technique. CORM-3 and CORM-A1 inhibited platelet aggregation in human PRP, WP, or WB, in a concentration-dependent manner. In all three preparations, CORM-A1 was more potent than CORM-3. Inhibition of platelets aggregation by CORM-A1 was not significantly affected by a guanylate cyclase inhibitor (ODQ) and a phosphodiesterase-5 inhibitor, sildenafil. In contrast, inhibition of platelet aggregation by NO donors was more potent with a fast NO releaser (DEA-NO, t (½) = 2 min) than slow NO releasers such as PAPA-NO (t (½) = 15 min) or other slow NO donors. Predictably, the anti-platelet effect of DEA-NO and other NO donors was reversed by ODQ while potentiated by sildenafil. In contrast to NO donors which inhibit platelets proportionally to the kinetics of NO released via activation of soluble guanylate cyclase (sGC), the slow CO-releaser CORM-A1 is a superior anti-platelet agent as compared to CORM-3 which releases CO instantly. The anti-platelet action of CO-RMs does not involve sGC activation. Importantly, CORM-A1 or its derivatives representing the class of slow CO releasers display promising pharmacological profile as anti-platelet agents.

Full text

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ORIGINAL ARTICLE
Inhibition of platelet aggregation by carbon
monoxide-releasing molecules (CO-RMs):
comparison with NO donors
Stefan Chlopicki & Magdalena Lomnicka &
Andrzej Fedorowicz & Elżbieta Grochal &
Karol Kramkowski & Andrzej Mogielnicki &
Włodzimierz Buczko & Roberto Motterlini
Received: 2 October 2011 /Accepted: 21 January 2012 /Published online: 25 February 2012
#The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract Carbon monoxide (CO) and CO-releasing mole-
cules (CO-RMs) inhibit platelet aggregation in vitro. Herein,
wecomparetheanti-plateletactionofCORM-3,whichreleases
CO rapidly (t½1 min), and CORM-A1, which slowly releases
CO (t½021 min). The anti-platelet effects of NO donors with
various kinetics of NO release were studied for comparison.
The effects of CO-RMs and NO donors were analyzed in
washed human platelets (WP), platelets rich plasma (PRP), or
whole blood (WB) using aggregometry technique. CORM-3
and CORM-A1 inhibited platelet aggregation in human PRP,
WP, or WB, in a concentration-dependent manner. In all three
preparations, CORM-A1 was more potent than CORM-3.
Inhibition of platelets aggregation by CORM-A1 was not
significantly affected by a guanylate cyclase inhibitor (ODQ)
and a phosphodiesterase-5 inhibitor, sildenafil. In contrast,
inhibition of platelet aggregation by NO donors was more
potentwith afastNOreleaser (DEA-NO,t½02min) thanslow
NOreleaserssuchasPAPA-NO(t½015min)orotherslowNO
donors. Predictably, the anti-platelet effect of DEA-NO and
other NO donors was reversed by ODQ while potentiated by
sildenafil. In contrast to NO donors which inhibit platelets
proportionally to the kinetics of NO released via activation of
soluble guanylate cyclase (sGC), the slow CO-releaser
CORM-A1 is a superior anti-platelet agent as compared to
CORM-3 which releases CO instantly. The anti-platelet action
of CO-RMs does not involve sGC activation. Importantly,
CORM-A1 or its derivatives representing the class of slow
CO releasers display promising pharmacological profile as
anti-platelet agents.
Keywords CO-releasing molecule(CO-RMs).Platelet
aggregation.NOdonors.Humanplatelets
Abbreviations
CORM-3
CORM-A1
DEA-NO
Tricarbonylchloro(glycinato)ruthenium(II)
Sodium boranocarbonate
Diethylammonium (Z)-1-(N,N-diethylamino)
diazen-1ium-1,2-diolate
(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)
amino]diazen-1-ium-1,2-diolate
(Z)-1-[N-(3-aminopropyl)-N-(3-
ammoniopropyl)amino]diazen-1-ium-1,
2-diolate
CORM-A1 deactivated by nitrogen bubbling
in acidified solution
N-nitro-L-arginine methyl ester
1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one
(Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]
diazen-1-ium-1,2-diolate
S-nitroso-N-acetyl penicillamine
DETA-NO
DPTA-NO
iCORM-A1
L-NAME
ODQ
PAPA-NO
SNAP
S. Chlopicki (*):M. Lomnicka:E. Grochal
Department of Experimental Pharmacology, Chair
of Pharmacology, Jagiellonian University Medical College,
Krakow, Poland
e-mail: s.chlopicki@jmrc.org.pl
S. Chlopicki:A. Fedorowicz:E. Grochal
Jagiellonian Centre for Experimental Therapeutics (JCET),
Jagiellonian University,
Krakow, Poland
K. Kramkowski:A. Mogielnicki:W. Buczko
Pharmacodynamics, Medical University in Bialystok,
Bialystok, Poland
R. Motterlini
INSERM U955, Faculté de Médecine, Université Paris Est,
Creteil, France
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:641–650
DOI 10.1007/s00210-012-0732-4

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Introduction
Endogenouscarbon monoxide (CO) formation inmammals is
catalyzed by a family of heme oxygenase enzymes (HO), an
inducible isoform (HO-1) and a constitutive protein (HO-2),
thatdecomposehemeintoCO,ferrousiron,andbiliverdin,the
latter being converted to bilirubin by biliverdin reductase
(Maines and Kappas 1977; Tenhunen et al. 1969). CO is
recognized as a signalling molecule within the cardiovascular
system being involved in the regulation of vascular tone as
well as exerting anti-inflammatory, anti-apoptotic, anti-
atherogenic, anti-proliferative, and cytoprotective activities
(Motterlini and Otterbein 2010). The biological effects of
CO, mainly manifested through vascular smooth muscle
relaxation and mitigation of inflammatory processes, involve
stimulation of soluble guanylate cyclase (sGC) and cyclic
guanosine monophosphate (cGMP) production (Furchgott
and Jothianandan 1991; Utz and Ullrich 1991), activation of
calcium-dependentpotassium channels(Wang and Wu 1997),
stimulation of p38 mitogen-activated protein kinase (MAPK)
(Otterbein et al. 2000; Otterbein et al. 2003), and direct
binding to the heme moiety of structural and functional pro-
teins such as iNOS (Foresti and Motterlini 1999; Sawle et al.
2005).
Carbon monoxide-releasing molecules (CO-RMs), of
which the chemical and biochemical features have been thor-
oughlycharacterizedbyMotterliniandco-workers(Johnsonet
al. 2003; Motterlini 2007; Motterlini et al. 2003), liberate CO
in biological systems providing a useful research tool for
exploring the mechanism by which CO exerts its pharmaco-
logical activities (Motterlini et al. 2002). Two of these com-
pounds, tricarbonylchloro(glycinato)ruthenium(II) (CORM-3)
and sodium boranocarbonate (CORM-A1), have unique fea-
tures as they are fully water-soluble and have been shown to
simulate the bioactivities of gaseous CO including vessel
relaxation (Clark et al. 2003; Foresti et al. 2003), protection
against organ ischemia-reperfusion injury (Clark et al.
2003; Foresti et al. 2004; Guo et al. 2004), prevention of
organ rejection following transplantation (Clark et al.
2003), and inhibition of the inflammatory response (Sawle
et al. 2005).
Importantly, CORM-3 is a metal carbonyl complex that
rapidlyliberatesCOinphysiologicalbuffers(half-life<1min)
(Clark et al. 2003), while CORM-A1 that does not possess a
transition metal but a carboxylic moiety liberates CO at a
much slower rate (half-life 21 min) under physiological con-
ditions (Motterlini et al. 2005). Recently, we have shown that
CORM-3 inhibits platelet aggregation in vitro through a
mechanism that is independent of soluble guanylate cyclase
activation (Chlopicki et al. 2006). The aim of the present
study was to use the two mostly characterized water-soluble
CO-RMs to evaluate how the different rates of CO release
affect platelet aggregation in vitro and finally compare their
anti-platelet profile with the one exerted by NO donors pos-
sessing various kinetics of NO release.
Materials and methods
Detection of CO release by assessing carbonmonoxy
myoglobin formation
ThereleaseofCOfromCORM-3andCORM-A1wasassessed
spectrophotometricallybymeasuringthekineticsofconversion
of deoxymyoglobin (deoxy-Mb) to carbonmonoxy myoglobin
(MbCO) as described previously (Motterlini et al. 2003). Myo-
globin solutions (50 μmol/l final concentration) were prepared
freshly by dissolving the protein in 0.1 mol/l phosphate buffer
(pH 7.4). Sodium dithionite (0.1%) was added to convert
myoglobinto deoxy-Mb prior toeach experiment. CO released
from CORM-3 and CORM-A1 (final concentrations: 10, 30,
40 μM) was quantified by adding aliquots of stock solutions
(10μl)ofthecompounds(inpuredistilledwater)directlytothe
deoxy-Mb.The kinetics ofMbCOformationwasquantifiedby
measuring the change in absorbance at 541 nm (background
wavelength 558 nm) for deoxy-Mb at 37°C using a spectro-
photometer equipped with kinetic software package. The rates
of MbCO formation were calculated and expressed as change
in MbCO concentration (μM).
Measurement of CO release from CORMs using a CO
electrode
Blood specimens (20 ml) were collected on the day of exper-
iment fromhealthy volunteers. Specimenswereadded in5-ml
volumes to TEKLAB™ blood collection tubes containing
8.75 mg of the anti-coagulant tripotassium EDTA (1.75 per
ml of blood). These specimens were then gently mixed by
inversion for 10 min to guarantee complete solubilizing of the
tripotassium EDTA.
Each blood sample was centrifuged for 15 min at 100×g at
4°C resulting in the three following layers: the inferior layer
composed of red cells, the intermediate layer composed of
white cells, and the superior layer made up of plasma. The
plasma layer was examined for red cells. If red cells were
present,thesamplewasre-centrifugedforanadditional5min.
The plasma layer was removed and retained as platelet rich
plasma(PRP).Theremainingbloodspecimenwasthencentri-
fugedat1,500×gfor20mintocollecttheplateletpoorplasma
layer.
The release ofCO fromCORM-A1and CORM-3 inbuffer
or PRP was detected using a prototype electrode purchased
from World Precision Instrument (WPI; Stevenage, Herts,
UK) and used as previously described (Motterlini et al.
2005).ThisCOelectrodeisamembrane-coveredamperometric
sensor which has been designed on a basic operating principle
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similar to the nitric oxide (NO) sensor. The CO sensor can be
connectedtotheWPIISO-NOMarkIImeterfordetectionofthe
current signals providing that the poise potential is set to a
different value (900 mV for CO as opposed to 860 mV for
NO). The electrode was immersed into either 1 ml of buffer
solutionor1mlofPRPandwasequilibratedat37°Cfor30min
prior to addition of CO-RMs. Once equilibrated, the electrode
was zeroed, and CO-RM (0.3 or 3 mM) was added. Readings
were taken until the chart reached the maximal CO release, and
data are expressed as an average of three independent
experiments.
Measurement of carbonmonoxy hemoglobin
in the whole blood upon exposure to CO-RMs
CORM-3 or CORM-A1 was added to the whole blood, and
after 2 or 10 min of incubation, carbonmonoxy hemoglobin
(COHb) content in the blood was analyzed using automatic
blood gases analyzer (NovoMedica). After 2 and 10 min of
incubation at 36°C, blood samples (100 μl) were placed in a
CO-oximeter (Ciba-Corning 270, Siemens), where the
blood was hemolyzed with UV and COHb level was deter-
mined. Before first and after last run of blood with CO-RM,
three runs with control blood were performed. The zero
point was set before first run and after automatically, every
30 min with Wash/Zero solution (Bayer). The calibration
was performed once in a month with CO-OX Slope solution
(Bayer), according to producer instruction.
Platelet aggregation assay
Isolation of human platelets
Venous blood was obtained from human volunteers at the
University Hospital Blood Bank Centre. Volunteer donors
had not taken any medicines for the preceding 2 weeks. Blood
wascollectedintovialscontainingsodiumcitrate(3.2%,9:1v/v
or 3.8%, 10:1v/v) as an anti-coagulant agent.
For the platelet aggregation in full blood, venous blood
samples were diluted with 0.9% NaCl (1:1v/v). To obtain
PRP, blood was centrifuged at 250×g for 20 min. Washed
platelets (WP) were obtained from PRP which were washed
twice in PGI2-containing phosphate buffered saline (PBS)
according to the method of Radomski et al. (1988) and finally
suspended (2×108platelets/ml) in Ca2+-free PBS containing
0.1% albumin and 0.1% glucose. Contamination of neutro-
phils in WP was less than 1/108(Chlopicki et al. 2004).
Platelet aggregation assay in humans
Aggregation of blood platelets in the whole blood was
assessed using Chronolog aggregometer (Chrono-log Corp.,
USA) by measurements of electrical impedance according to
the methoddescribed byCardinal and Flower(1980),while in
PRP and WP, it was assessed by measurements of optic
transmittance according to the method described by Born
(1967).
Whole blood was equilibrated for 13 min, and then,
CORM-A1, CORM-3, or VEH (0.9% NaCl) were added.
Collagen was added after further 2 min of incubation. The
dose of collagen was chosen to cause 50% of full aggregation
(EC50). The stirrer speed was set at 800 rpm. Changes in
impedance were registered 6 min after stimulation with colla-
gen. The maximal extension of the aggregation curve at sixth
minute was expressed as a percentage of control value.
PRP (500 μl) was equilibrated for 2 min at 37°C with
continuous stirring at 1,100 rev/min and then stimulated with
collagen to cause aggregation. At the beginning of each exper-
iment, concentrations of collagen that induced sub-maximum
aggregationresponseweredetermined.Thesewereintherange
of 0.5–1.5 μg/ml. CORM-3, CORM-A1, RuCl3, inactive form
of CORM-A1, DEA-NO, PAPA-NO, DETA-NO, DPTA-NO,
and SNAP were added usually 2 min before stimulation of
platelets with collagen unless longer incubation was indicated.
Insomeexperiments,ODQ(10μM)orsildenafil(100nM)was
added 1 min prior to the addition of CORMs or NO donors.
WP (500 μl) was equilibrated for 2 min at 37°C with
continuous stirring at 1,100 rev/min in PBS containing CaCl2
and MgCl2both at a concentration of 1 mM and then stimu-
lated with thrombin to cause aggregation. At the beginning of
each experiment, concentrations of thrombin that induced
sub-maximum aggregation response were determined. These
wereintherange15–20mU/ml.CORM-3,CORM-A1,DEA-
NO,PAPA-NO,DETA-NO,andDPTA-NOwereadded2min
before stimulation of platelets unless longer incubation time
was indicated. In some experiments, ODQ or sildenafil was
added 1 min prior to the addition of CORMs or NO donors.
Reagents and drugs
CollagenwasobtainedfromChrono-Par(USA),thrombinfrom
Biomed (Poland),DEA-NO(half-life ~2min at 37°C andpH0
7.4), PAPA-NO (half-life ~15 min), DPTA-NO (half-life ~3 h),
DETA-NO (half-life ~20 h), and ODQ were purchased from
Cayman(USA).Ru(CO)3Cl(glycinate)(CORM-3)andsodium
boranocarbonate (Na[H3BCO2H] or CORM-A1) were synthe-
sized as previously described (Clark et al. 2003; Foresti et al.
2003) (Motterlini et al. 2005). The inactive form of CORM-A1
which does not release CO (iCORM-A1) was prepared as
described previously (Motterlini et al. 2005). Ruthenium chlo-
ride (RuCl3) was used as a negative control for CORM-3.
Statistical analysis
AllcalculationswereperformedwithGraphPadPrismsoftware.
Values of (lg) IC50were calculated by sigmoidal dose–response
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:641–650643

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curve fitting (variable slope, and fixed bottom and top values
to 0 and 100, respectively). Least squares method was used
unless the residuals did not follow normal distribution; in this
case, robust fit was used. However, this last method did not
reportconfidence intervals for estimated parameters. Compar-
ison of active form of CORMs with inactive counterparts and
influence of ODQ or sildenafil on action of CORMs or NO
donorswereassessedbyMann–WhitneytestorwithKruskal–
Wallis test followed by Dunn’s multiple comparisons. Results
of whole blood aggregation were compared by unpaired t test.
Results
Characteristics of CO release from CORM-3
and CORM-A1
As detected by the myoglobin assay (Fig. 1a), CORM-3
released CO almost instantly in buffer (half-life <1 min at
37°C, pH 7.4), while CORM-A1 released CO at much
slower rate (half-life≈21 min at 37°C, pH 7.4). Although
the CO electrode detected the release of CO from CORM-A1
in PRP, which occurred in a slow fashion as previously
described in buffer, this device was not sensitive enough to
detect measurable amounts of CO from CORM-3 (Fig. 1b).
Scavenging of CO by plasma constituents interacting with
CORM-3 could represent one possible reason for the failure
of the CO electrode to detect any CO in PRP. Alternatively,
CORM-3 releases CO effectively only in the presence of an
avid acceptor, as in the case of reduced myoglobin. In fact, as
exemplified in Fig. 1c, there was a striking difference in CO
detection also from CORM-A1 between PRP and buffer sol-
utions showing much less CO being measured in PRP
(Fig. 1c). When CO-RMs were incubated in the whole
blood for few minutes, we found that COHb increased from
1.5±0.17% (control) to 2.6±0.15% with 1 mM CORM-3,
while 300 μM CORM-A1 did not significantly changed this
parameter (1.76±0.17%).
Effects of CORM-3 and CORM-A1 on human
platelet aggregation in PRP, WP, and whole blood
As shown in Fig. 2a, when added to human PRP, CORM-A1
or CORM-3 inhibited platelet aggregation induced by colla-
gen in a concentration-dependent manner. However,
CORM-A1 was effective in the micromolar concentrations
range, while CORM-3 was effective only at millimolar con-
centrations (IC500160.3 μM and 3170 μM, 95% CI from
2900 to 3464 μM, for CORM-A1 and CORM-3, respective-
ly). Similarly, the inhibitory effect of CORM-A1 on
thrombin-induced platelet aggregation in WP exceeded that
of CORM-3 (Fig. 2b). Even though approximately ten times
lower concentrations of both CORM-3 and CORM-A1 were
0 10 20 30 405060
0
5
10
15
20
25
30
35
40
45
50
40 µM CORM-A1in buffer
40 µM CORM-3 in buffer
Time (min)
MbCO [µM]
a
b
c
Fig. 1 Time course of MbCO formation from CORM-3 and CORM-
A1 in buffer (a) and comparison of CO-releasing capacity from
CORM-3 in PRP and CORM-A1 in PRP and Krebs buffer (b). The
time course of releasing CO by CORM-A1 in buffer and PRP (c)
644Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:641–650

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needed to inhibit washed human platelets as compared to
PRP, CORM-3 was more than a tenfold weaker anti-platelet
agent than CORM-A1 in WP preparation (IC50013.82 μM
and 171.9 μM for CORM-A1 and CORM-3, respectively,
Table 1). Also, the inhibitory effect of CORM-A1 on
collagen-induced platelet aggregation in vitro human whole
blood exceeded that of CORM-3 (Fig. 2c), though the dif-
ference in potency was approximately by twofold (IC500
467.45 μM and 1004.16 μM for CORM-A1 and CORM-3,
respectively; Table 1). Thus, even though the potency of
CORM-A1andCORM-3differed inPRP,WP,andthewhole
blood, CORM-A1 was a more potent anti-platelet agent than
CORM-3 in human PRP, WP, as well as in the whole human
blood.
Forthecomparisonoftheanti-plateletactivitiesofCORM-3
and CORM-A1 in PRP and WP, both compounds were incu-
bated for 2 min prior to the addition of collagen (PRP) or
thrombin (WP) (Fig. 2a, b). If the incubation time for
CORM-3 was prolonged to 5or 10min prior to the stimulation
of platelet aggregation either in PRP (Fig. 3a) or WP (data not
shown), the anti-platelet activity of CORM-3 was not at all
potentiated. In contrast, if CORM-A1 was pre-incubated for 5
or10minpriortothestimulationofplateletaggregationinPRP
(Fig. 3a), the anti-platelet effect of CORM-A1 was more
accentuated.Similarly, prolongation ofCORM-A1incubation
in WP accentuated its anti-aggregatory effects (8.167±1.01%
and 25.17±1.96% after2 and 5 min ofincubationwith30μM
ofCORM-A1,respectively;p<0.001,n05–6).Thedifference
in activity of CORM-A1 but not CORM-3 with the prolonged
pre-incubation is fully compatible with the instant release of
CO from CORM-3 (half-life <1 min at 37°C, pH 7.4) and the
slower rate of CO release from CORM-A1 (half-life≈21 min
at 37°C, pH 7.4). The respective negative controls used for
CORM-3 (RuCl3) and CORM-A1 (iCORM-A1) that do not
possess CO-dependent activity were inactive as anti-platelet
agents (Fig. 3b).
Effects of various NO donors on human platelet aggregation
in PRP and WP
The comparison of anti-platelet activity of various NO donors
(DEA-NO, PAPA-NO, DETA-NO, and DPTA-NO) in PRP is
shown in Fig. 4. Both in PRP and WP, the NO donor with the
shortest half-life was a more potent inhibitor of platelet aggre-
gation than NO donors possessing a slower rate of NO release.
In PRP, DEA-NO, (t½02 min at 37°C and pH07.4), PAPA-
NO (t½015 min), DPTA-NO (t½03 h), and DETA-NO (t½0
20 h) inhibited platelets with an IC50of 0.24 μM, IC500
0.48 μM, IC508.33 μM, and IC50038.9 μM, respectively.
Platelet rich plasma (PRP)
10100100010000
0
20
40
60
80
100
120
CORM-A1
CORM-3
[µM]
Platelet aggregation
(% of control response)
Washed platelets (WP)
1101001000
0
20
40
60
80
100
CORM-A1
CORM-3
[µM]
Platelet aggregation
(% of control response)
Whole blood
10100 1000 10000
0
20
40
60
80
100
120
CORM-A1
CORM-3
[µM]
Platelet aggregation
(% of control response)
b
a
c
„
Fig. 2 Concentration-dependent anti-aggregatory effects of CORM-3
and CORM-A1 in human PRP (a) (n04–52), human WP (b) (n04–39),
and human whole blood in vitro (c) (n03–9)
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:641–650645

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A similar order of potency for these four NO donors was
observed in WP (Table 1).
Differential involvement of soluble guanylate cyclase
in the anti-platelet action of CO-RMs and NO donors
As shown in Fig. 5a, an inhibitor of soluble guanylate
cyclase activity (ODQ) reversed the inhibition of platelet
aggregation induced by DEA-NO, while sildenafil pro-
foundly enhanced the inhibition of platelet aggregation in-
duced by DEA-NO, both in PRP (Fig. 5a) and in WP (data
not shown). ODQ and sildenafil displayed similar effects on
platelet response to other NO donors (e.g., SNAP; data not
shown).
In contrast, in the presence of an inhibitor of soluble
guanylate cyclase activity (ODQ), the effect of CORM-A1
was fully preserved either in collagen-stimulated platelets
aggregation in PRP (Fig. 5b) or in thrombin-induced platelet
aggregation in WP (data not shown). In the presence of a
selective inhibitor of phosphodiesterase-5 (sildenafil), the
anti-platelet effect of CORM-A1 in PRP (Fig. 5b) and in
WP (data not shown) was not potentiated.
Table 1 IC50values for inhibition of platelets aggregation by CO-RMs
andNOdonorsinwholeblood,plateletrichplasma,andwashedplatelets.
All values are interpolated from concentration-dependent curves with the
exception ofIC50for DETA-NOinPRP whichwas extrapolatedfrom the
concentration-dependent curve since even at the concentration of 30 mM
DETA-NO inhibited platelet aggregation by approximately 40%
Inhibition of platelet aggregation (IC50)
CO-RMs
CORM-3
CORM-A1
NO donors
DEA-NO
PAPA-NO
DPTA-NO
DETA-NO
t½
<1 min
21 min
Whole blood (μM)
1004.2
467.4
Platelet rich plasma (μM)
3170.0
160.3
Washed platelets (μM)
171.9
13.8
2 min
15 min
3 h
20 h
N.D.
N.D.
N.D.
N.D.
0.24
0.48
8.33
38.9
0.022
0.051
0.37
6.5
N.D. not determined
024681012
0
20
40
60
80
100
*
Time of incubation [min]
Platelet aggregation
(% of control response)
CORM-A1
CORM-3
CORM-A1 (500 µM)
iCORM-A1 (500 µM)
CORM-A1 (1000 µM)
iCORM-A1 (1000 µM)
CORM-3 (5 mM)
(5 mM)
3
RuCl
0
20
40
60
80
100
120
**
****
**
Platelet aggregation
(% of control response)
ba
Fig. 3 a Influence of
incubation time on the anti-
aggregatory effect of CORM-3
and CORM-A1 in PRP (a)
(n07–14), 2 mM CORM-3 and
100 μM CORM-A1 were used,
respectively). b Lack of the
anti-aggregatory effect of
inactive forms of CO-RMs in
PRP (n05–46). One asterisk
and three asterisks denote
statistical significance vs.
standard incubation of 2 min
646 Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:641–650

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Discussion
In the present work, we evaluated the time- and concentration-
dependent anti-platelet effects of two water-soluble CO-
releasing agents (Motterlini et al. 2002; Motterlini et al.
2003; Motterlini et al 2005). These two compounds are (1) a
metal carbonyl complex that rapidly liberates CO (CORM-3)
and(2)anovelreleaserofCOthatdoesnotcontainatransition
metal (sodium boranocarbonate, CORM-A1) and generates
CO at a much slower rate under physiological condi-
tions. Using three different in vitro systems for platelet aggre-
gation (PRP, WP, and whole blood), we demonstrated a
superior anti-platelet profile of activity for CORM-A1 com-
pared to CORM-3. Indeed, the anti-aggregatory effects of
CORM-A1 in PRP and whole blood were achieved in a con-
centration range that does not significantly affect the formation
of COHb. Furthermore, prolongation of pre-incubation of pla-
telets with CORM-A1 but not CORM-3 potentiated the
observed effect suggesting that effective anti-aggregatory
action by micromolar concentration of CORM-A1 may be
achieved through a gradual liberation of CO over time.
Takinginto account that CORM-3elicited prompt andrapid
vasodilatory effects in vitro andinvivo whereas CORM-A1
promoted prolonged vasodilation and milder hypotensive
effect in vivo (Motterlini et al. 2005), one can speculate that
CORM-A1 could afford an anti-aggregatory effect in vivo
without a hypotensive effect and possibly without a toxic
effect related to increased COHb formation which might com-
promise oxygen delivery to tissues. Predictably, exogenous
0.010.1
concentration [µM]
1 10100
0
20
40
60
80
100
120
DEA-NO
PAPA-NO
DETA-NO
DPTA-NO
Platelet aggregation
(% of control response)
Fig. 4 Concentration-dependent anti-aggregatory effect of NO donors
in PRP
DEANO (0.5 µM)
DEANO (0.5 µM) + ODQ (10 µM)
DEANO (0.3 µM)
DEANO (0.3 µM) + Sild (100 nM)
0
20
40
60
80
100
*
**
Platelet aggregation
(% of control response)
CORM-A1 (300 µM)
CORM-A1 (300 µM) + ODQ (10 µM)
CORM-A1 (300 µM)
CORM-A1 (300µM) + Sild (100nM)
0
20
40
60
80
100
Platelet aggregation
(% of control response)
ab
Fig. 5 Effect of ODQ (10 μM)
(n011–14) and sildenafil (100
nM) (n07–12) on the anti-
aggregatory action of DEA-NO
(a) (n03–16) and CORM-A1
(b) (n07–14) in PRP
Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:641–650647

Page 8

NO affords simultaneously hypotensive and anti-aggregatory
effects on platelets, which are both mediated by activation of
sGC. In the present work, the pharmacological effects of both
CO-RMs on platelets do not appear to involve activation of
soluble sGC supporting our previous work on CORM-3 and
pointingoutthatthe identificationofaspecifictargetforCOin
platelets remains a challenge (Chlopicki et al. 2006).
The biological activities of CO and NO are frequently
compared. Here, we underscore clear differences on the anti-
aggregatory properties of CO-RMs and NO donors that could
reflect the biological activity of endogenous NO and CO on
platelet aggregation. Endogenous NO instantly produced by
the vascular endothelium can be stimulated by increased
blood flow, shear stress, or an agonist, and the rapid kinetics
of endogenous NO release has been detected in biological
systems (Kalinowski etal. 2003).On the other hand, our body
is continuously exposed to small quantities of CO produced
endogenously during the degradation of heme by heme oxy-
genaseenzymes (HO-1and HO-2),while a greater quantityof
COisproduced duringstressconditionsupon the induction of
inducible HO-1. In view of the data presented here, we can
speculate that to mimic an effective anti-aggregatory action of
CO on platelets, slow but sustained kinetics of CO release is
preferable as indicated by the significantly more potent anti-
aggregatory effect obtained with CORM-A1.
It has been consistently shown that sGC is strongly acti-
vated by NO and to a lesser extent by CO (Friebe et al. 1998;
Hoenicka et al. 1999; Stone and Marletta 1998). Here, we
found that in contrast to NO donors, the anti-aggregatory
activity of CORM-A1 was not mediated by sGC. Indeed, in
ourexperiments,ODQ,apotentandselectiveinhibitorofsGC
(Garthwaite et al. 1995), completely reversed the anti-platelet
effectofDEA-NO,whileitwasineffectiveagainstCORM-A1.
Furthermore,sildenafil,aphosphodiesterase(PDE)-5inhibitor,
failed to potentiate the effect of CO liberated by CORM-A1 in
humanplatelets.Atthesametime,sildenafilamplifiedtheanti-
aggregatoryeffectofNOreleasedbyDEA-NO.Previously,we
demonstrated that CORM-3 inhibited platelets by a sGC-
independent mechanism, while others had shown that high
concentrations of gaseous CO (100%) inhibited aggregation
via activation of sGC (Brune et al. 1990). Here, using
ODQ, a selective inhibitor of sGC, and using sildenafil, a
PDE-5inhibitorthatinhibitsdegradationofcGMP,weprovide
evidence that inhibition of platelet aggregation by physiolog-
ically relevant concentrations of CO liberated by CORM-A1
was mediated by a sGC-independent mechanism. Apparently,
inplateletsaswellasothertissues,sGCdoesnotappeartobea
primary target for the bioactivity of micromolar concentration
of CO. Apart from sGC (Christodoulides et al. 1995; Furchgott
and Jothianandan 1991; Hussain et al. 1997), there are a num-
ber of other possible targets for CO such as calcium-activated
potassium channels (Wang and Wu 1997), cytochrome P450
(Coceani et al. 1997), mitochondrial respiratory chain (Lo
Iacono et al. 2011), or p38 MAPK (Amersi et al. 2002; Zhang
et al. 2003). The role of each of these potential targets
responsive to CO in mediating the anti-aggregatory effects of
CO-RMs remains to be fully investigated.
Despite the fact that the specific target(s) for the anti-
aggregatory action of low concentrations of CO in platelets
remain(s) unknown, our data point out that the use of CO-RM
may represent a novel approach for selective and effective
inhibition of platelets in vivo. We previously demonstrated
that in contrast to NO and PGI2, CO effectively inhibited
platelet aggregation even when platelets were excessively ac-
tivated (Chlopicki et al. 2006). These results suggest that CO
may play a role of a retaliatory mediator that comes into play
when NO and PGI2are insufficient to overcome excessive
platelet activation, and this can be mimicked by CO-releasing
agents.
The comparison between the effect of CO-RMs and NO
donors on inhibition of platelet aggregation offers some addi-
tional points of discussion that should be taken into consider-
ation. First of all, it is clear that all NO donors tested are more
potent inhibitors of platelet aggregation than either CORM-3
or CORM-A1. Secondly, the slow CO releaser (CORM-A1,
t½021 min) is in average 15 times more potent than the fast
CO carrier (CORM-3, t½<1 min) in inhibiting aggregation,
and this is true both in PRP and WP. In the whole blood,
CORM-A1 is still more potent than CORM-3 by approxi-
mately twofold. Interestingly, an opposite profile is observed
for the NO donors since DETA-NO, which has a half-life of
approximately 25 min, is 150 times less potent than the fast
NO donor DEA-NO (t½01 min). Notably, the intriguing
observation of our experiments is that the anti-aggregation
effect of both NO donors and CO-RMs is ten times more
pronounced in washed platelets than in PRP. Although the
reduced effect on platelet aggregation in plasma may be
predicted in the case of NO donors, this is not so intuitive
and is rather unexpected for CO-releasing compounds. In
fact, it is well established that NO, apart from interacting
strongly with ferrous (Fe2+) and ferric (Fe3+) metal cen-
ters, has a preferential reactivity with thiols and cysteine
moieties present in proteins (Stamler et al. 2001; Stamler
et al. 1992). Although this post-translational modification
is illustrated in human red blood cells by S-nitrosylation
of hemoglobin which has profound physiological implica-
tions on oxygen transport and delivery (Jia et al. 1996),
the interaction of NO with cysteines in plasma is best
exemplified by the rapid formation of S-nitrosoalbumin and
possibly other stable circulating S-nitrosoproteins (Stamler et
al.2001;Stamleretal.1992).Thefactthatinordertohavethe
same degree of inhibition of aggregation, a higher concentra-
tion of NO donors is required in platelets present in plasma
(PRP) compared to washed platelets which suggests that NO
liberated from these agents is partially scavenged by the
plasma components, possibly proteins rich in cysteines.
648Naunyn-Schmiedeberg's Arch Pharmacol (2012) 385:641–650

Page 9

In the case of CO, it is more difficult to explain why CO-
RMs elicit a reduced inhibition of platelet aggregation in
plasma compared to washed platelets since only the pres-
ence of proteins containing ferrous iron (Fe2+) or other
transition metals with a specific redox state could act as
effective scavengers of CO. From the analysis of our results
on the differential effects of CORM-3 and CORM-A1 in
PRP, washed platelets, and in whole blood, it appears that
the kinetics of CO release is an important factor in deter-
mining the potency of CO-RMs as anti-aggregatory agents.
Our data using a sensitive CO electrode also reveal that
indeed the total amount of CO liberated from CORM-A1
in PRP is much higher than the amount released from
CORM-3; however, both the amount and the rate of CO
release from CORM-A1 are markedly reduced in plasma
compared to PBS especially when very high concentrations
of CORM-A1 are used (3 mM). These data do not exclude
the possibility that transition metal-containing proteins or
enzymes present in plasma could act as potential scavengers
of CO thus diminishing the potency of CORM-A1 in PRP.
The general perception is that once present in the blood
stream, CO would bind irreversibly to hemoglobin and
eliminated through the lung by respiration, but a revision
of this concept may be required as plasma components (i.e,.
metal-containing proteins) may act either as potential targets
of CO or carriers of CO to be transported either in circulat-
ing cells or tissues (Boczkowski et al. 2006; Foresti and
Motterlini 2010). On the other hand, in the case of CORM-
3, the increase in COHb is not substantial if one considers
that heme concentration in human blood hemoglobin is in
the order of 10 mM, and thus, 1 mM CORM-3 should
potentially generate 10% COHb. The fact that much less is
detected with 1 mM CORM-3 indicates that the total trans-
fer of CO from the metal carbonyl to the heme in hemoglo-
bin is not occurring or is somehow prevented by other
factors. In the case of CORM-A1, the lower formation of
COHb suggests that micromolar concentrations of CORM-
A1 may achieve effective anti-platelet effect without a sig-
nificant COHb formation.
In conclusion, CORM-A1 that releases CO at a slower
rateisasuperiorinhibitor ofplateletaggregation thanthefast
CO carriers such as CORM-3. Furthermore, our results un-
derscore the importance of both the chemical features of
compounds releasing gaseous molecules and the kinetics of
thegaseous mediatorreleasethatdeterminetheiranti-platelet
efficacy. There is a clear difference between NO donors and
CO-releasing molecules as regard to their mechanisms and
pharmacodynamic characteristics of anti-platelet action that
might help to determine an intracellular target for anti-
platelet activity of low micromolar concentration of CO that
is not shared by NO. Finally, our work points out that
CORM-A1 displays promising anti-aggregatory activities,
andthus,thiscompoundorsimilarslow-releasersofCOshould
be exploited therapeutically further as an anti-thrombotic drug
in vivo.
Acknowledgments
forsynthesizingCORM-3.Dr.RobertoMotterliniwasrecipientofaVisiting
Professorship (2009–2011) from the Faculty of Medicine, University Paris
Est, Creteil, France. This work was supported by the European Union from
the resources of the European Regional Development Fund under the
Innovative Economy Programme (grant coordinated by JCET-UJ, No.
POIG.01.01.02-00-069/09). Supplementary funding was provided by the
Polish Ministry of Science and Higher Education (N N405 260437).
WethankProf.BrianMann(UniversityofSheffield)
Open Access
tive Commons Attribution License which permits any use, distribution,
and reproduction in any medium, provided the original author(s) and
the source are credited.
This article is distributed under the terms of the Crea-
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