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221http://bmbreports.org BMB reports
*Corresponding author. Tel: +82-53-950-8570; Fax: +82-53-950-
8557; E-mail: baejs@knu.ac.kr
#First two authors contributed equally to this work.
http://dx.doi.org/10.5483/BMBRep.2012.45.4.221
Received 9 August 2011, Revised 28 September 2011,
Accepted 11 October 2011
Keywo rds : aPTT, Curcumin, HUVECs, PT
Anticoagulant activities of curcumin and its derivative
Dong-Chan Kim1,#, Sae-Kwang Ku2,# & Jong-Sup Bae3,*
1Laboratory of Microvascular Circulation Research, NEUORNEX Inc. Daegu 711-823, 2Department of Anatomy and Histology, College of
Oriental Medicine, Daegu Haany University, Gyeongsan 712-715, 3College of Pharmacy, Research Institute of Pharmaceutical Sciences,
Kyungpook National University, Daegu 702-701, Korea
Curcumin, a polyphenol responsible for the yellow color of
the curry spice tu rmeric, p os sesses anti inflammatory, anti-
proliferative and antiangiogenic activities. However, anti-
coagulant activities of curcumin have not been studied. Here,
the anticoagulant properties of curcumin and its derivative
(bisdemethoxycurcumin, BDMC) were determined by monitor-
ing activated partial thromboplastin time (aPTT), prothrombin
time (PT) as well as cell- base d thrombi n and activated factor X
(FXa) generation activities. Data showed that curcumin and
BDMC prolonged aPTT and PT significantly and inhibited
thrombin and FXa activities. They inhibited the generation of
thrombin or FXa. In accordance with these anticoagulant activ-
ities, curcumin and BDMC showed anticoagulant effect in
vivo. Surprisingly, these anticoagulant effects of curcumin
were be tter th an those of BDMC indi catin g that methoxy
group in curcumin positively regulated anticoagulant function
of curcumin. Therefore, these results suggest that curcumin
and BDMC pos sess antithrombotic activities and dai ly con-
sumption of the curry spice turmeric might help mai ntain anti-
coagulant status. [BMB reports 2012; 45(4): 221-226]
INTRODUCTION
The key of the blood clotting pathway is the production of
thrombin which is required for the conversion of fibrinogen to
fibrin (1, 2). Thrombin resides in the cell in an inactive form,
called prothrombin, and is activated by the coagulation casca-
devia formation of a complex called the prothrombin activator
complex (1-5). The formation of the prothrombin activator com-
plex occurs by two different pathways: the intrinsic pro-
thrombin activation pathway and the extrinsic prothrombin ac-
tivation pathway. Though the ultimate goal of both the path-
ways is the generation of the prothrombin activator complex,
alternate routes are used, each giving rise to a different form of
the prothrombin activator (1-5). In the extrinsic pathway, pro-
thrombin activator complex consists of activated factor X (FXa),
tissue factor (TF), activated factor VII (FVIIa) and the cofactor
activated factor V (FVa) (1-5). This complex, specifically FXa,
along with the cofactor FVa, then converts prothrombin to ac-
tive thrombin. Fibrin forms a mesh within the platelet aggregate
to stabilize clots (1-5). In contrast, in the intrinsic pathway, pro-
thrombin activator complex consists of FXa, FVa, activated fac-
tor VIII (FVIIIa) and phopspholipid (PL) (1-5). The clotting time
assay measures the lag time of thrombin generation (6) and the
activated partial thromboplastin time (aPTT) is a performance
indicator measuring the efficacy of both the contact activation
pathway and the common coagulation pathways (6). Further,
the prothrombin time (PT) is measure of the extrinsic pathway
of coagulation (7, 8).
The rhizome of Curcuma longa has been used in indigenous
medicine for the treatment of inflammatory disorders and its me-
dicinal activity has been known since ancient times. Turmeric
derived from the rhizome has been widely used by the people in
the Middle East for centuries as a food component (9, 10). The
use of turmeric extract or turmeric oil as a spice and household
remedy has been known to be safe for centuries. Bhide, et al. al-
so revealed the safety and tolerance of turmeric through human
clinical trials (11). In many previous studies, extracts prepared
from Curcuma longa have been used as antiinflammatory agents
to treat gas, colic, toothaches, chest pains, menstrual difficulties,
stomach and liver ailmetns (9, 12, 13). Polyphenolic phytochem -
icals are common in the diet and have been suggested to have a
wide range of beneficial health effects and the polyphenolic
compounds in turmeric are responsible for a number of its bene-
ficial health effects (14, 15). Turmeric contains three major poly-
phenolic analogues. The majority is curcumin and the com-
pounds in smaller amounts are demethoxycurcumin, and bisde-
methoxycurcumin (BDMC) (16, 17). Recent studies indicate that
dietary administration of curcumin may have beneficial effects in
conditions such as cancer (18), Alzheimer’s disease (19) and
cystic fibrosis (20). With regard to mode of action, curcumin ex-
hibits a diverse array of metabolic, cellular and molecular activi-
ties. Although curcumin analogues exhibit activities very similar
to curcumin, their potencies compared to curcumin have not
been clearly established. In most systems, curcumin is found to
be most potent (21, 22) and in some systems, BDMC was found
Anticoagulant activities of curcumin
Dong-Chan Kim, et al.
222 BMB reports http://bmbreports.org
In vitro coagulant assay
Sample Dose aPTT (s) PT (s)
Control
Curcumin
Heparin
Saline
0.1 μM
0.5 μM
1 μM
5 μM
10 μM
20 μM
50 μM
36.2 ± 1.2
37.2 ± 1.3
48.5 ± 1.4b
52.6 ± 1.8c
65.3 ± 1.5c
77.5 ± 2.1c
91.8 ± 1.5c
119.8 ± 0.9c
1.5 (μg/ml)
＞300c
17.5 ± 0.4
17.4 ± 0.3
18.2 ± 0.7
19.8 ± 0.5b
21.6 ± 0.6b
27.5 ± 0.5c
31.8 ± 0.4c
35.2 ± 0.4c
15 (μg/ml)
61.5 ± 0.5c
In vivo bleeding time
Sample Dose Tail Bleeding time (s) n
Control
Curcumin
Heparin
Saline
100 mg/kg
50 mg/kg
54.2 ± 8
102 ± 2c
158.6 ± 4c
3
3
3
aEach value represents the means ± SD (n = 5). bP ＜ 0.05 as com-
pared to control. cP ＜ 0.01 as compared to control
Table 1. Anticoagulant activity of curcumina
In vitro coagulant assay
Sample Dose aPTT (s) PT (s)
Control
BDMC
Heparin
Saline
0.1 μM
0.5 μM
1 μM
5 μM
10 μM
20 μM
50 μM
35.8 ± 1.3
38.9 ± 0.8
41.6 ± 1.5b
48.5 ± 2.14b
68.5 ± 1.2c
70.5 ± 1.8c
87.6 ± 1.5c
98.6 ± 1.4c
1.5 (μg/ml)
＞300c
17.5 ± 0.4
17.5 ± 0.5
17.9 ± 0.6
18.2 ± 0.5b
19.2 ± 0.7c
20.9 ± 0.4c
25.4 ± 0.3c
29.8 ± 0.5c
15 (μg/ml)
61.5 ± 0.5c
In vivo bleeding time
Sample Dose Tail Bleeding time (s) n
Control
BDMC
Heparin
Saline
100 mg/kg
50 mg/kg
54.2 ± 8
82 ± 2b
158.6 ± 4c
3
3
3
aEach value represents the means ± SD (n = 5). bP ＜ 0.05 as
compared to control. cP ＜ 0.01 as compared to control
Table 2. Anticoagulant activity of BDMCa
to exhibit different (in some cases, more potent) activities (23-25).
There is an increasing demand for comparison study between
curcumin and BDMC, due to the discovery of their new bio-
logical activities (21, 26, 27). Identification of novel biological
activities of curcumin and its analogues is of interest both pre-
clinically and clinically. Additionally, anticoagulant activities of
curcumin have not been well studied. Herein, the anticoagulant
properties of curcumin and its derivative, BDMC on the gen-
eration of FXa and thrombin as well as the regulation of clotting
time (PT and aPTT) were determined.
RESULTS
Effects of curcumin and BDMC on aPTT and PT
The anticoagulant properties of curcumin and BDMC were
tested in aPTT and PT assays using human plasma and are
summarized in Table 1 and 2. Although the anticoagulant ac-
tivities of curcumin and BDMC were weaker than those of
heparin, aPTT and PT were significantly prolonged by curcu-
min or BDMC at concentrations at or greater than 5 μM.
Prolongation of aPTT suggests inhibition of the intrinsic and/or
the common pathway while prolonged PT indicates that curcu-
min and BDMC could also inhibit the extrinsic pathway of
coagulation. To confirm these in vitro data, in vivo tail bleed-
ing time was determined. As shown in Table 1 and 2, tail
bleeding time was significantly prolonged by curcumin or
BDMC with respect to the control. Surprisingly, effects of cur-
cumin on the clotting time were better than that of BDMC sug-
gesting that methoxy group in curcumin positively regulates
the anticoagulant function of curcumin.
Effects of curcumin and BDMC on inactivation of thrombin or
FXa
To elucidate the inhibitory mechanism of curcumin and
BDMC on coagulation time, their inhibitory effect on thrombin
and FXa activities was measured using chromogenic substrates
in the absence or presence of antithrombin III (AT III). In the
absence of AT III, the amidolytic activity of thrombin was in-
hibited by curcumin and BDMC in a dose-dependent manner,
showing that the anticoagulant directly inhibited thrombin
activity. However, in the presence of AT III, thrombin activity
was essentially unchanged (Fig. 1A, B). Thus, AT III was un-
able to potentiate the activity of curcumin or BDMC. Further,
the effects of curcumin and BDMC on FXa activity in the ab-
sence or presence of AT III were also investigated. The anti-
coagulant showed direct inhibitory effects on FXa activities at
high concentrations, and the inhibitory effect of AT III was not
changed by curcumin or BDMC (Fig. 1C, D). These results
were consistent with the antithrombin assay. Therefore, these
results suggested that the antithrombotic mechanism of curcu-
min and BDMC appears to be due to inhibition of fibrin poly-
merization and/or the intrinsic/extrinsic pathway without po-
tentiation by AT III. Furthermore, the methoxy group in curcu-
min positively regulates the anticoagulant effects on the in-
hibition of thrombin or FXa activity because the anticoagulant
effects of curcumin were better than those of BDMC.
Anticoagulant activities of curcumin
Dong-Chan Kim, et al.
223http://bmbreports.org BMB reports
Fig. 1. Effect of curcumin and BDMC on the inactivation o
f
thrombin and factor Xa. Inhibition of thrombin (Th) in the ab-
sence of antithrombin III (○) or in the presence of antithrombin
III (●) by curcumin (A) or DBMC (B) was monitored by a chro-
mogenic assay as described in “Materials and Methods”. Inhibition
of factor Xa (FXa) in the absence of antithrombin III (○) or in
the presence of antithrombin III (●) by curcumin (C) or DBMC
(D) was monitored by chromogenic assay as described in
“Materials and Methods”. *P ＜ 0.01 as compared to 0.
Fig. 2. Inhibition of thrombin and FXa generation by curcumin
and BDMC in HUVECs. (A) FVa (100 pM) and FXa (1 nM) were
preincubated on HUVECs monolayers for 10 min with indicated
concentrations of curcumin (□) or BCMC (■). Prothrombin was
added to a final concentration of 1 μM and the amounts of pro -
thrombin activated were determined at 30 min as described in
“Materials and Methods”. (B) HUVECs were preincubated with in-
dicated concentrations of curcumin (□) or BCMC (■) for 10 min.
TNF-α (10 ng/ml for 6 h) stimulated HUVECs were incubated with
FVIIa (10 nM) and FX (175 nM) in the absence or presence o
f
anti-TF IgG (25 μg/ml) and FXa generation was then determined
as described in “Materials and Methods”. (C) HUVECs were cul-
tured with curcumin (□) or BCMC (■) in the absence or pres -
ence of TNF-α (10 ng/ml) for 18 h and PAI-1 concentration in the
culture mediums was examined as described in “Materials and
Methods”. (D) the same as C except that cells were preincubated
with curcumin (□) or BCMC (■) and cultured with SP600125 (2
μM), emodin (2 μg/ml), and PD98059 (10 μM) in presence o
f
TNF-α (10 ng/ml) for 18 h. *P ＜ 0.05 as compared to 0 (A) or
TNF-α alone (B, C) or to (+) control (D).
Effects of curcumin and BDMC on the generation of thrombin
and FXa
Sugo et al. reported that endothelial cells are able to support
prothrombin activation by FXa (28). Preincubation of FVa and
FXa in the presence of CaCl2 with HUVECs before addition of
prothrombin resulted in thrombin generation (Fig. 2A). The ef-
fect of curcumin and BDMC on thrombin generation showed
that curcumin and BDMC inhibited thrombin activation of pro-
thrombin dose- dependently (Fig. 2A). Rao et al. showed that
the endothelium provides the functional equivalent of procoa-
gulant phospholipids and supports FX activation (29) and that
in TNF-α stimulated HUVECs, FVIIa could activate FX, which
was completely dependent on TF expression (30). Thus, it is
likely that the endothelium can provide support for FVIIa acti-
vation of FX. If so, it would be of interest to investigate the ef-
fect of curcumin and BDMC on FVIIa activation of FX.
HUVECs were stimulated with TNF-α to induce TF expression.
As shown in Fig. 2B, the rate of FX activation by FVIIa was
100-fold higher in stimulated HUVECs (53.3 ± 5 nM) com-
pared with non-stimulated HUVECs (0.54 ± 0.2 nM), which
was completely attenuated by anti-TF IgG (4.8 ± 0.4 nM).
Moreover, preincubation with curcumin or BDMC dose-de-
pendently inhibited FVIIa activation of FX (Fig. 2B). Therefore,
these results suggested that curcumin could inhibit the gen-
eration of thrombin or FXa and the methoxy group in curcu-
min positively regulated these functions of curcumin.
Because plasminogen activator inhibitor type 1 (PAI-1) de-
termines fibrinolytic activity (1), the effect of TNF-α and curcu-
min on PAI-1 secretion from HUVECs were investigated. As
shown in Fig. 2C, curcumin dose dependently inhibited TNF-α
induced PAI-1 secretion from HUVECs. To define the molec-
ular targets of curcumin in the signal transduction pathways
leading to TNF-α induced PAI-1 expression, we investigated
the effects of three signal transduction inhibitors, emodin (a
NF-κB inhibitor), PD98059 (an extracellular signal regulated
kinase, ERK, inhibitor), and SP600125 (a c-Jun N-terminal kin-
ase, JNK, inhibitor) on TNF-α induced PAI-1 expression in the
presence or absence of curcumin. Experiments performed
showed that neither SP600125 nor emodin showed any addi-
tional inhibitory effects in the presence of curcumin (Fig. 2D).
However, the inhibitory effects of PD98059 were essentially
additive with those of curcumin (Fig. 2D).
DISCUSSION
The vascular endothelium provides a number of important
functions in order to maintain adequate blood supply to vital
organs. These functions include prevention of coagulation,
regulation of vascular tonus, orchestration of the migration of
blood cells by the expression of adhesion molecules and regu-
Anticoagulant activities of curcumin
Dong-Chan Kim, et al.
224 BMB reports http://bmbreports.org
lation of vasopermeability (31). Among these, regulation of he-
mostatic activity was regulated through a balance of pro- and
anticoagulant properties (1). Impaired endothelial function
causes thrombus-related complications including myocardial
infarction, stroke and thromboembolism (32). In this study, we
presented curcumin as a potent anticoagulant by inhibitiing
thrombin or FXa. The anticoagulant activity of curcumin was
evidenced by the prolongation of the clotting time in plas-
ma-based PT and APTT assays. Additionally, the inhibitory ef-
fects by curcumin on FXa generation and further thrombin
generation support the anticoagulant activities of curcumin.
It is well known that FXa has no effect on platelet activation,
however, once it is assembled into the prothrombinase com-
plex, it triggers enormous amounts of thrombin (2, 5). Throm-
bin is the final enzyme in the blood clotting cascade respon-
sible for clot formation and platelet activation (2, 5). Based on
the results that curcumin could inhibit generation of FXa and
thrombin, the anticoagulant activity of curcumin was initiated
from the inhibition of the penultimate and final enzyme in the
blood clotting cascade.
TNF-α has been known to activate JNK, NF-κB, and ERK in
human endothelial cells (33-35). Here, we used a JNK
(SP600125), NF-κB (emodin), and ERK (PD98059) inhibitors to
define the molecular target of curcumin. We observed that
PD98059, but not emodin or SP600125, was additive to the in-
hibitory effects of curcumin on TNF-α induced PAI-1 secretion.
These results suggest that the NF-κB and JNK pathway are in-
volved in curcumin mediated inhibition of TNF-α induced
PAI-1 expression in HUVECs. Thus, these results seem to in-
dicate that curcumin decreases PAI-1 levels via inhibition of the
NF-κB and JNK pathways.
Noting that the effects of curcumin on the anticoagulant ac-
tivity was better than BDMC, it suggests that the ortho-methoxy
group in curcumin positively regulates anticoagulant functions
of curcumin. In a previous report, curcumin and BDMC had
different redox properties due to the presence of the ortho-me-
thoxy group in position 3 of the phenyl moiety in curcumin.
(36) While curcumin has two symmetric ortho-methoxy phe-
nols linked through the a,b-unsaturated b-diketone moiety,
BDMC, which is also symmetric, is deficient in the two or-
tho-methoxy substitutions. Although curcumin and bisdeme-
thoxycurcumin differ in their chemical structures only with re-
gard to the ortho-methoxy substitution, they exhibit signifi-
cantly different antioxidant, antitumor, and antiinflammatory
activities. The hydrogen bonding interaction between the phe-
nolic OH and the ortho-methoxy groups in curcumin markedly
influences the O-H bond energy and H-atom abstraction by
free radicals, thus making it a better free radical scavenger than
BDMC (37). In another investigation, the ortho-methoxy- defi-
cient BDMC was a more potent ROS inducer and the ortho-me-
thoxy substituted curcumin was a more potent suppressor of
NF-kB activation (38). According to our results, the ortho-me-
thoxy group in curcumin is important for the anticoagulant
effect. Thus, we can postulate that the anticoagulant activities
of curcumin could be mainly caused by interaction of the target
molecules with the ortho-methoxy group.
The significant progress made in understanding the role of
FXa and thrombin in various thrombotic disease states has
clearly demonstrated potential therapeutic benefits of blocking
these key enzymes in the blood coagulation cascade (39). A
potent and selective small molecule FXa or thrombin inhibitor
has the potential to offer substantial therapeutic benefits (39).
Curcumin exhibits the potency and selectivity required for such
a candidate and is currently undergoing additional evaluations.
In conclusion, this study showed that curcumin inhibited the
extrinsic and intrinsic pathways of blood coagulation by in-
hibiting FXa and thrombin generation in HUVECs. These re-
sults adds to previous work and may be helpful for the rational
design of pharmacological strategies for treating or preventing
vascular diseases via regulation of thrombin generation.
MATERIALS AND METHODS
Reagents
Curcumin (product catalog #: C2302) and bisdemethoxycurcu-
min (product catalog #: B3347) were purchased from TCI
Korea (Tokyo Chemical Industry Co., Ltd. Seoul, South Korea).
TNF-α, JNK inhibitor (SP600125), NF-κB inhibitor (emodin),
and ERK inhibitor (PD98059) were purchased from R&D
Systems (Minneapolis, MN). Anti-tissue factor antibody was
purchased from Santa Cruz Biologics (Santa Cruz, CA). Factor
V, Vll, Vlla, FX, FXa, antithrombin III (AT III), prothrombin and
thrombin were obtained from Haematologic Technologies
(Essex Junction, VT, USA). aPTT assay reagent and PT reagents
were purchased from Fisher Diagnostics (Middletown, Virgi-
nia, USA). Chromogenic substrates S-2222, and S-2238 were
from Chromogenix AB (Sweden).
Anticoagulation assay
Determination of aPTT and PT were performed according to
the manufacture’s specifications using Thrombotimer (Behnk
Elektronik, Germany). In brief, citrated normal human plasma
(90 μl) was mixed with 10 μl of curcumin or BDMC and in-
cubated for 1 min at 37°C. Then, aPTT assay reagent (100 μl)
was added to the mixture and incubated for 1 min at 37°C.
Thereafter, 20 mM CaCl2 (100 μl) was added and the clotting
time was recorded. For the PT assay, citrated normal human
plasma (90 μl) was mixed with 10 μl of a curcumin or BDMC
stock and incubated for 1 min at 37oC. Then, PT assay reagent
(200 μl), preincubated for 10 min at 37oC, was added and the
clotting time was recorded.
Cell culture
Primary HUVECs were obtained from Cambrex Bio Science
(Charles City, IA) and maintained as described before (40).
Briefly, cells were cultured to confluency at 37oC at 5% CO2
in EBM-2 basal media supplemented with growth supplements
(Cambrex Bio Science).
Anticoagulant activities of curcumin
Dong-Chan Kim, et al.
225http://bmbreports.org BMB reports
Factor Xa generation on the surface of HUVECs
HUVECs were preincubated with indicated concentrations of
curcumin or BDMC for 10 min. TNF-α (10 ng/ml for 6 h in
serum-free medium) stimulated confluent monolayers of
HUVECs in a 96-well culture plate were incubated with FVIIa
(10 nM) in buffer B for 5 min at 37°C in presence or absence
of anti-TF IgG (25 μg/ml). FX (175 nM) was then added to the
cells (final reaction mixture volume, 100 μl) and incubated for
15 min. The reaction was stopped by adding buffer A contain-
ing 10 mM EDTA and the amount of FXa generated in the re-
action period was measured by using a chromogenic substrate,
and the change in absorbance at 405 nm was monitored in a
microplate reader for 2 min. The initial rate of color develop-
ment was converted into FXa concentrations from a standard
curve prepared with known dilutions of purified human FXa.
Thrombin generation on the surface of HUVECs
HUVECs were preincubated in 300 μl containing curcumin or
BDMC in 50 mM Tris–HCl buffer, 100 pM FVa and 1 nM FXa
for 10 min and prothrombin was added to a final concen-
tration of 1 μM. After 10 min, duplicate samples (10 μl each)
were transferred to a 96-well plate containing 40 μl of 0.5 M
EDTA in Tris-buffered saline in each well to terminate pro-
thrombin activation. Activated prothrombin was determined
by measuring the rate of hydrolysis of S2238 measured at 405
nm. Dilutions with known amounts of purified thrombin were
used for standard curves.
Thrombin activity assay
Curcumin or BDMC in 50 mM Tris–HCl buffer, pH 7.4 con-
taining 7.5 mM EDTA and 150 mM NaCl was mixed in the ab-
sence or presence with 150 μl of AT III (200 nM). After the
mixture was incubated at 37°C for 2 min, thrombin solution
(150 μl; 10 U/ml) was added and incubated at 37°C for 1 min.
Then, substrate for thrombin (S-2238, 150 μl; 1.5 mM) sol-
ution was added and absorbance at 405 nm was monitored for
120 s with a spectrophotometer (TECAN, Switzerland).
Factor Xa (FXa) activity assay
These assays were performed similar to the thrombin activity
assay. Instead of thrombin and S-2238, factor Xa (1 U ml/1)
and substrate S-2222 were used.
ELISA for PAI- 1
The concentrations of PAI-1 in HUVEC cultured supernatants
were determined by ELISA methods, according to the manu-
facturer’s recommended protocol (American Diagnostica Inc.,
Stamford, CT, USA).
Effect on bleeding time
The tail transection bleeding time was determined according
to the method of Dejana et al. (41) Male C57BL/6 mice were
fasted overnight and curcumin or BDMC was administered or-
ally to mice. One hour after administration, the mouse tail was
transected at 2 mm from the tip. Bleeding time was measured
as time elapsed until bleeding stopped. When bleeding time
lasted longer than 15 min, measurement was stopped and
bleeding time was recorded as 15 min for statistical analyses.
Statistical analysis
Data are expressed as the means ± standard deviation of at
least three independent experiments. Statistical significance
between two groups was determined by a Student’s t-test. The
significance level was set at P ＜ 0.05.
Acknowledgements
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government [MEST]
(No. 2011-003410, 2011-0026695, 2011-0030124).
REFERENCES
1. Davie, E. W. (1995) Biochemical and molecular aspects of the
coagulation cascade. Thromb. Haemost. 74, 1-6.
2. Davie, E. W., Fujikawa, K. and Kisiel, W. (1991) The coagu-
lation cascade: initiation, maintenance, and regulation.
Biochemistry 30, 10363-10370.
3. Hoffman, M. M. and Monroe, D. M. (2005) Rethinking the
coagulation cascade. Curr. Hematol. Rep. 4, 391-396.
4. Hoffmann, J. N., Vollmar, B., Romisch, J., Inthorn, D.,
Schildberg, F. W. and Menger, M. D. (2002) Antithrombin ef-
fects on endotoxin-induced microcirculatory disorders are
mediated mainly by its interaction with microvascular
endothelium. Crit. Care. Med. 30, 218-225.
5. Monroe, D. M., Hoffman, M. and Roberts, H. R. (2002)
Platelets and thrombin generation. Arterioscler. Thromb.
Vasc. Biol. 22, 1381-1389.
6. Quinn, C., Hill, J. and Hassouna, H. (2000) A guide for diag-
nosis of patients with arterial and venous thrombosis. Clin.
Lab. Sci. 13, 229-238.
7. Della Valle, P., Crippa, L., Garlando, A. M., Pattarini, E., Safa,
O., Vigano D'Angelo, S. and D'Angelo, A. (1999)
Interference of lupus anticoagulants in prothrombin time as-
says: implications for selection of adequate methods to opti-
mize the management of thrombosis in the antiphos-
pholipid-antibody syndrome. Haematologica 84, 1065-
1074.
8. Furie, B. and Furie, B. C. (2005) Thrombus formation in vivo.
J. Clin. Invest. 115, 3355-3362.
9. Jurenka, J. S. (2009) Anti-inflammatory properties of curcu-
min, a major constituent of Curcuma longa: a review of pre-
clinical and clinical research. Altern. Med. Rev. 14, 141-153.
10. Bengmark, S., Mesa, M. D. and Gil, A. (2009) Plant-derived
health: the effects of turmeric and curcuminoids. Nutr. Hosp.
24, 273-281.
11. Joshi, J., Ghaisas, S., Vaidya, A., Vaidya, R., Kamat, D. V.,
Bhagwat, A. N. and Bhide, S. (2003) Early human safety study
of turmeric oil (Curcuma longa oil) administered orally in
healthy volunteers. J. Assoc. Physicians. India. 51, 1055-1060.
12. Ammon, H. P. and Wahl, M. A. (1991) Pharmacology of
Curcuma longa. Planta. Med. 57, 1-7.
13. Bao, W., Li, K., Rong, S., Yao, P., Hao, L., Ying, C., Zhang,
Anticoagulant activities of curcumin
Dong-Chan Kim, et al.
226 BMB reports http://bmbreports.org
X., Nussler, A. and Liu, L. (2010) Curcumin alleviates etha-
nol-induced hepatocytes oxidative damage involving heme
oxygenase-1 induction. J. Ethnopharmacol. 128, 549-553.
14. Shankar, R., de la Motte, C. A., Poptic, E. J. and DiCorleto,
P. E. (1994) Thrombin receptor-activating peptides differ-
entially stimulate platelet-derived growth factor production,
monocytic cell adhesion, and E-selectin expression in human
umbilical vein endothelial cells. J. Biol. Chem. 269,
13936-13941.
15. Bachmeier, B., Nerlich, A. G., Iancu, C. M., Cilli, M.,
Schleicher, E., Vene, R., Dell'Eva, R., Jochum, M., Albini, A.
and Pfeffer, U. (2007) The chemopreventive polyphenol
Curcumin prevents hematogenous breast cancer metastases
in immunodeficient mice. Cell. Physiol. Biochem. 19,
137-152.
16. Aggarwal, B. B., Sundaram, C., Malani, N. and Ichikawa, H.
(2007) Curcumin: the Indian solid gold. Adv. Exp. Med. Biol.
595, 1-75.
17. Inoue, K., Nomura, C., Ito, S., Nagatsu, A., Hino, T. and Oka,
H. (2008) Purification of curcumin, demethoxycurcumin,
and bisdemethoxycurcumin by high-speed countercurrent
chromatography. J. Agric. Food. Chem. 56, 9328-9336.
18. Greenwald, P., Milner, J. A., Anderson, D. E. and McDonald,
S. S. (2002) Micronutrients in cancer chemoprevention.
Cancer. Metastasis. Rev. 21, 217-230.
19. Lim, G. P., Chu, T., Yang, F., Beech, W., Frautschy, S. A. and
Cole, G. M. (2001) The curry spice curcumin reduces oxida-
tive damage and amyloid pathology in an Alzheimer trans-
genic mouse. J. Neurosci. 21, 8370-8377.
20. Zeitlin, P. (2004) Can curcumin cure cystic fibrosis? N. Engl.
J. Med. 351, 606-608.
21. Ahsan, H., Parveen, N., Khan, N. U. and Hadi, S. M. (1999)
Pro-oxidant, anti-oxidant and cleavage activities on DNA of
curcumin and its derivatives demethoxycurcumin and
bisdemethoxycurcumin. Chem. Biol. Interact. 121, 161-175.
22. Sreejayan, N. and Rao, M. N. (1996) Free radical scavenging
activity of curcuminoids. Arzneimittelforschung 46, 169-171.
23. Thapliyal, R., Deshpande, S. S. and Maru, G. B. (2001) Effects
of turmeric on the activities of benzo(a)pyrene-induced cyto-
chrome P-450 isozymes. J. Environ. Pathol. Toxicol. Oncol.
20, 59-63.
24. Syu, W. J., Shen, C. C., Don, M. J., Ou, J. C., Lee, G. H. and
Sun, C. M. (1998) Cytotoxicity of curcuminoids and some nov-
el compounds from Curcuma zedoaria. J. Nat. Prod. 61,
1531-1534.
25. Guo, L. Y., Cai, X. F., Lee, J. J., Kang, S. S., Shin, E. M., Zhou,
H. Y., Jung, J. W. and Kim, Y. S. (2008) Comparison of sup-
pressive effects of demethoxycurcumin and bisdemethox-
ycurcumin on expressions of inflammatory mediators in vitro
and in vivo. Arch. Pharm. Res. 31, 490-496.
26. Li, Y. B., Gao, J. L., Lee, S. M., Zhang, Q. W., Hoi, P. M. and
Wang, Y. T. (2011) Bisdemethoxycurcumin protects endothe-
lial cells against t-BHP-induced cell damage by regulating the
phosphorylation level of ERK1/2 and Akt. Int. J. Mol. Med. 27,
205-211.
27. Liu, Y. L., Yang, H. P., Gong, L., Tang, C. L. and Wang, H. J.
(2011) Hypomethylation effects of curcumin, demethox-
ycurcumin and bisdemethoxycurcumin on WIF-1 promoter
in non-small cell lung cancer cell lines. Mol. Med. Report. 4,
675-679.
28. Sugo, T., Nakamikawa, C., Tanabe, S. and Matsuda, M. (1995)
Activation of prothrombin by factor Xa bound to the membrane
surface of human umbilical vein endothelial cells: its catalytic
efficiency is similar to that of prothrombinase complex on
platelets. J. Biochem. 117, 244-250.
29. Rao, L. V., Rapaport, S. I. and Lorenzi, M. (1988) Enhance-
ment by human umbilical vein endothelial cells of factor
Xa-catalyzed activation of factor VII. Blood. 71, 791-796.
30. Ghosh, S., Ezban, M., Persson, E., Pendurthi, U., Hedner, U.
and Rao, L. V. (2007) Activity and regulation of factor VIIa ana-
logs with increased potency at the endothelial cell surface.
J. Thromb. Haemost. 5, 336-346.
31. Kaiser, L. and Sparks, H. V. Jr. (1987) Endothelial cells. Not
just a cellophane wrapper. Arch. Intern. Med. 147, 569-573.
32. Chong, A. Y., Blann, A. D. and Lip, G. Y. (2003) Assessment
of endothelial damage and dysfunction: observations in rela-
tion to heart failure. QJM 96, 253-267.
33. Surapisitchat, J., Hoefen, R. J., Pi, X., Yoshizumi, M., Yan, C.
and Berk, B. C. (2001) Fluid shear stress inhibits TNF- alpha
activation of JNK but not ERK1/2 or p38 in human umbilical
vein endothelial cells: Inhibitory crosstalk among MAPK fam-
ily members. Proc. Natl. Acad. Sci. U.S.A. 98, 6476-6481.
34. Berk, B. C., Abe, J. I., Min, W., Surapisitchat, J. and Yan, C.
(2001) Endothelial atheroprotective and anti-inflammatory
mechanisms. Ann. N. Y. Acad. Sci. 947, 93-109; discussion
109-111.
35. Yoshizumi, M., Fujita, Y., Izawa, Y., Suzaki, Y., Kyaw, M., Ali,
N., Tsuchiya, K., Kagami, S., Yano, S., Sone, S. and Tamaki,
T. (2004) Ebselen inhibits tumor necrosis factor-alpha-in-
duced c-Jun N-terminal kinase activation and adhesion mole-
cule expression in endothelial cells. Exp. Cell. Res. 292, 1-10.
36. Anand, P., Thomas, S. G., Kunnumakkara, A. B., Sundaram,
C., Harikumar, K. B., Sung, B., Tharakan, S. T., Misra, K.,
Priyadarsini, I. K., Rajasekharan, K. N. and Aggarwal, B. B.
(2008) Biological activities of curcumin and its analogues
(Congeners) made by man and Mother Nature. Biochem.
Pharmacol. 76, 1590-1611.
37. Somparn, P., Phisalaphong, C., Nakornchai, S., Unchern, S.
and Morales, N. P. (2007) Comparative antioxidant activities
of curcumin and its demethoxy and hydrogenated deriva-
tives. Biol. Pharm. Bull. 30, 74-78.
38. Sandur, S. K., Pandey, M. K., Sung, B., Ahn, K. S., Murakami,
A., Sethi, G., Limtrakul, P., Badmaev, V. and Aggarwal, B. B.
(2007) Curcumin, demethoxycurcumin, bisdemethox-
ycurcumin, tetrahydrocurcumin and turmerones differ-
entially regulate anti-inflammatory and anti-proliferative re-
sponses through a ROS-independent mechanism. Carcino-
genesis. 28, 1765-1773.
39. Fischer, K. G. (2007) Essentials of anticoagulation in
hemodialysis. Hemodial. Int. 11, 178-189.
40. Bae, J. S. and Rezaie, A. R. (2008) Protease activated receptor
1 (PAR-1) activation by thrombin is protective in human pul-
monary artery endothelial cells if endothelial protein C re-
ceptor is occupied by its natural ligand. Thromb. Haemost.
100, 101-109.
41. Dejana, E., Callioni, A., Quintana, A. and de Gaetano, G.
(1979) Bleeding time in laboratory animals. II - A comparison
of different assay conditions in rats. Thromb. Res. 15, 191-197.
