- Email: [email protected]
Cyclocurcumin from Curcuma longa selectively inhibits shear stress-induced platelet aggregation
Journal of Functional Foods 61 (2019) 103462
Contents lists available at ScienceDirect
Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Cyclocurcumin from Curcuma longa selectively inhibits shear stress-induced platelet aggregation
T
Thien Ngoa, Keunyoung Kima, Yiying Biana,1, Gwang-Jin Ana, Ok-Nam Baeb, Kyung-Min Limc, ⁎ Jin-Ho Chunga, a
College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea College of Pharmacy, Hanyang University, Ansan, Gyeonggido 15588, Republic of Korea c College of Pharmacy, Ewha Womans University, Seoul 03760, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cyclocurcumin Curcumin SIPA Antiplatelet Antithrombotic Cardiovascular disease
Curcuma longa (C. longa) has been the subject of intensive research for pleiotropic therapeutic potential. However, except for curcumin, the biological activities of other active ingredients of C. longa are not well known. Here, we demonstrated that C. longa extract significantly attenuated shear stress-induced platelet aggregation and cyclocurcumin exhibited the most potent activity (IC50 of 6.33 ± 3.29 μM). Cyclocurcumin potently inhibited shear stress-induced platelet activation, primarily through modulating the initial step, vWF – platelets GP Ib interaction. Moreover, cyclocurcumin effectively inhibited thrombus formation, without influencing platelet cell viability or blood clotting time. Collectively, we demonstrated that cyclocurcumin might be a promising therapeutic agent for the treatment or prevention of thrombotic diseases.
1. Introduction The powdered rhizome of Curcuma longa (C. longa), which contains a mixture of curcuminoids, has been used widely for millenniums as a spice, food coloring, and traditional herbal remedies for diverse therapeutic purpose (Ammon & Wahl, 1991; Ghosh, Banerjee, & Sil, 2015). Curcumin, the most enriched ingredient among these curcuminoids, is generally believed as the vital constituent contributing to the diverse pharmacological activities of C. longa (Ammon & Wahl, 1991; Goel, Kunnumakkara, & Aggarwal, 2008). Numerous biological activities of curcumin have been discovered including anti-oxidant, anti-inflammatory, and anti-neuro-degradation, and their potential benefits for various diseases have drawn attention (Ghosh et al., 2015; Goel et al., 2008). Interestingly, recent studies have highlighted that the distinct pharmacological effects of curcuminoids other than curcumin; demethoxycurcumin, bisdemethoxycurcumin, and cyclocurcumin may at least in part contribute to the therapeutic benefits of C. longa (Kim, 2017; Pei et al., 2016; Sheu et al., 2013). C. longa may be beneficial against cardiovascular diseases through various bioactivities such as lowering cholesterol and ameliorating
myocardial damage (Goel et al., 2008; Wongcharoen & Phrommintikul, 2009). In addition, the favorable effects of C. longa on inflammation, an important player in triggering coronary artery diseases and thrombotic complications of atherosclerosis (Libby, 2012; Tsoupras, Lordan, & Zabetakis, 2018), have been highlighted (Lantz, Chen, Solyom, Jolad, & Timmermann, 2005). Due to the crucial role in both hemostasis and thrombosis (Li, Delaney, O'Brien, & Du, 2010; Palomo, Toro, & Alarcon, 2008), the influence of C. longa on platelet activity has also been assessed (Srivastava, 1989). The antiplatelet effects of curcumin, as well as some other constitutes of C. longa, were also known (Prakash et al., 2011; Srivastava, Dikshit, Srimal, & Dhawan, 1985). In these studies, the physiological agonists, such as collagen, ADP, and arachidonic acid (Prakash et al., 2011; Shah et al., 1999) were used to evaluate the antiplatelet effects. However, it was not known whether the C. longa could affect the shear stress-induced platelet aggregation (SIPA), a distinct phenomenon of platelet activity, of which role in pathological thrombosis is gaining attention recently (Kamada, Imai, Nakamura, Ishikawa, & Yamaguchi, 2017; Kim et al., 2012). Blood flow produces shear stress, which can increase in atherosclerotic lesion or vasospasm, thereby directly triggering platelet aggregation regardless of other
Abbreviations: SIPA, shear stress-induced platelet aggregation; CYCL, cyclocurcumin; vWF, von Willebrand factor; GP, glycoprotein; Ab, antibody; AM, acetoxymethyl ester; FITC, fluorescein isothiocyanate; PE, phycoerythrin; LDH, lactate dehydrogenase, PGE1, prostaglandin E1; PRP, platelet-rich plasma; WPs, washed platelets; PT, prothrombin time; aPTT, activated partial thromboplastin time ⁎ Corresponding author at: College of Pharmacy, Seoul National University, Shinrim-dong San 56-1, Gwanak-Gu, Seoul 08826, Republic of Korea. E-mail address: [email protected] (J.-H. Chung). 1 Permanent address: School of Public Health, China Medical University, 110122, PR China. https://doi.org/10.1016/j.jff.2019.103462 Received 28 April 2019; Received in revised form 5 July 2019; Accepted 12 July 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
endogenous stimuli (Kroll, Hellums, McIntire, Schafer, & Moake, 1996). SIPA plays a critical role in thrombo-pathogenesis but is relatively unassociated with normal hemostasis (Jackson, 2007; Kroll et al., 1996). Accordingly, SIPA is considered as a promising target for antiplatelet and antithrombotic agents without bleeding complications, which is the most serious adverse effects of current platelet inhibitors (McFadyen, Schaff, & Peter, 2018; Michelson, 2010). Here, we investigated the effects of C. longa along with its active ingredients on SIPA. Of all, cyclocurcumin showed the most potent effects. The mechanism on anti-SIPA effects and antithrombotic potential of cyclocurcumin was further elucidated.
1: 6, v/v), supplemented with PGE1 1 μM. PRP, prepared as described above was centrifuged for 10 min at 500g to obtain platelet pellets. Platelet pellets were washed with calcium-free Tyrode's buffer (134 mM NaCl, 2.9 mM KCl, 1.0 mM MgCl2, 10.0 mM HEPES, 5.0 mM glucose, 12.0 mM NaHCO3, 0.34 mM Na2HPO4, and 0.3% bovine serum albumin, pH 7.4) containing 10% ACD and PGE1 1 μM. After centrifugation at 400g for 10 min, platelets were finally suspended at 3 × 108 platelets/ml in Tyrode's buffer containing 2 mM CaCl2, as described previously (Kim et al., 2012).
2. Material and methods
Reactions were performed in a programmable cone-plate viscometer (RotoVisco 1; Thermo Fisher Scientific, Waltham, MA) with the shear rate at 10,800 s−1 for 3 min at 37 °C. For WPs, before applying shear stress, 10 µg/ml of vWF was added. 500 μL of platelet suspension treated with cyclocurcumin in DMSO (final, 0.5%) or vehicle (DMSO) for 10 min at 37 °C, was loaded to the cone-plate viscometer. The platelets after exposing to shear stress were collected and fixed with 0.5% glutaraldehyde in Tyrode's buffer. Platelet aggregation was determined based on the number of single platelets per microliter under a phasecontrast light microscope (CX41; Olympus, Tokyo, Japan) as described previously with a minor modification (Cattaneo, Lombardi, Bettega, Lecchi, & Mannucci, 1993). The level of platelet aggregation was calculated depending on the loss of single cell as compared to the unsheared sample. The inhibition of platelet aggregation was compared to that of vehicle treated sample. As the maximal of the inhibition of platelet aggregation due to the shear stress of all the tested compounds was 50%, this level was defined as the maximal inhibition by shear stress.
2.3. Shear stress-induced platelet aggregation (SIPA) assay.
2.1. Reagents and plant materials Reagents and antibodies. Thrombin was purchased from Calbiochem (San Diego, CA). Von Willebrand factor (vWF) was from Molecular Innovations, Inc. (Michigan, USA), and collagen, ADP, and ristocetin were from Chrono-log (Havertown, PA). Fluo-4/AM, pluronic F-127, and alexa fluor 488-conjugated fibrinogen were from Invitrogen (Carlsbad, CA). Phycoerythrin (PE)-labeled monoclonal antibody against human CD42b (antiCD42b-PE Ab), fluorescein isothiocyanate (FITC)-labeled anti-CD62P antibody (anti-CD62P-FITC Ab), and FITClabeled PAC-1 (PAC-1-FITC) were from BD Biosciences (San Jose, CA), and FITC-labeled anti-vWF antibody (anti-vWF-FITC) was from Abcam plc (Cambridge, UK). Cyclocurcumin (95.6% pure as determined by HPLC, Karl Fischer (water content), GC (residual solvent) including mass spectroscopy, and NMR profiles by the manufacturer) was obtained from Chromadex Inc. (Irvine, CA, USA). Curcumin (> 98% pure as determined by HPLC including mass spectroscopy and melting point by the manufacturer) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other ingredients in C. longa were provided from the National Center for Standardization of Herbal Medicines in Korea, with a purity of > 98%. For all the experiments, platelet suspensions were treated with various concentration of tested compounds in DMSO (final, 0.5%). The effects were compared to those of the control, DMSO-treated samples. Plant material. The plant was authenticated, and a voucher specimen (CU2009-06) has been deposited at the herbarium of Chosun University (Gwangju, Korea). Dry powders of C. longa were extracted with 70% ethanol at 70–80 °C for 3 h. The extraction was repeated three times. After filtration and concentration under reduced pressure, extracts were lyophilized, and the resultant powder was stored at −20 °C. For experiments extracts were dissolved in DMSO. Identification and quantification of the constitution of the C. longa extract was described in our previous study (Kim, 2017).
2.4. Measurement of agonist-induced platelet aggregation Agonist-induced platelet aggregation was determined by the turbidometric method using an aggregometer (Chrono-log). After incubation with cyclocurcumin for 10 min at 37 °C, PRP were loaded on the aggregometer and stimulated with thrombin (0.6–0.8 U/mL), collagen (2–4 µg/mL), ADP (5–10 µM), or ristocetin (1.0–1.25 mg/mL) for 6 min. Platelet aggregation was measured by light transmission, with 100% calibrated as the absorbance of PPP. 2.5. Measurement of intraplatelet activation events induced by shear stress To detect intracellular calcium levels, WPs were loaded with fluo-4/ AM (5 µM) and pluronic F-127 (0.2%) for 45 min at 37 °C in the dark. The cyclocurcumin treated platelets were exposed to shear stress, then diluted by Tyrode’s buffer. The change of intracellular calcium was analyzed on the FACS Calibur cytometer (BD Biosciences) equipped with an argon laser (λex 488 nm). The light scatters, and fluorescence channels were set on a log scale. Data from 10,000 events were collected and analyzed by using CellQuest Pro software (BD Biosciences) (Kim et al., 2012). To detect P-selectin expression, glycoprotein (GP) IIb/IIIa activation, fibrinogen binding, or vWF binding, CD62P-FITC Ab, PAC-1-FITC, alexa fluor 488-conjugated fibrinogen, and anti-vWF-FITC Ab was used as the marker, respectively. WPs were treated with cyclocurcumin before exposing to high shear stress. Anti-CD42b-PE Ab was used as platelet identifier. Platelets were incubated with the mixture of antibodies for 20 min in the dark, at room temperature, and then analyzed on the flow cytometer as described above.
2.2. Human platelet preparation The work was approved by the Ethics Committee of the Health Service Center at Seoul National University. Blood was collected from 35 non-smoking healthy male volunteers (18–25 years old), who were medication free for two weeks, and was used on the day of collection. For PRP preparation, venous whole blood was collected from forearm vein to a vacuum vacutainer (Vacuette; Greiner Bio‐One GmbH, Kremsmünster, Austria) containing sodium citrate 3.2% (1: 9, v/v) by a 21-gauge needle. Whole blood was centrifuged for 15 min at 150g to obtain platelet-rich-plasma (PRP). Platelet-poor-plasma (PPP) was obtained from the precipitated fraction of PRP by centrifugation at 2000g for 20 min. Platelets are counted with a hemocytometer (Marienfeld GmbH, Marienfeld, Germany), and platelet count in PRP was adjusted to 3 × 108 platelets/ml with PPP. For washed platelets (WPs) preparation, venous whole blood was collected from forearm vein by a 16gauge needle to blood collecting bag containing acid citrate-dextrose (ACD, 85 mM trisodium citrate, 71 mM citric acid, and 111 mM glucose,
2.6. Measurement of serotonin secretion Platelets were treated with cyclocurcumin at various concentrations in DMSO (final, 0.5%) or vehicle (DMSO) for 10 min at 37 °C before subjected to shear stress at 10,800 s−1 for 3 min. The sheared platelets 2
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
B 100
% of maximal inhibition
80 60 40 20 0
10 20 50 100 250
80
E
60 40 20 0
C sito yc st l (+ ocu ero )rc l ar um 4 tu -d in eh Bi me s yd ab ron ro xy ola e bi trae sa -h c n yd ro Bi uro xy sa ne c c D em inn uro et am ne ho ic xy a cu cid Bi sd rc um em i et Cu ho rc n xy um cu i rc n um in
% of maximal inhibtion
A
Curcuma longa ( g/mL)
D
100
% of maximal inhibition
% of maximal inhibition
C 80 60 40 20 0 ( M)
0.5
1
5
10
10
Cyclocurcumin
50
100 250
Curcumin
100
WP
80 60 40 20 0
0.5
1
5
10
Cyclocurcumin ( M)
F Cyclocurcumin
Platelet activator
IC50 ( M)
Physical stimuli: Shear stress
6.33 ± 3.29
Physiological stimuli: - Thrombin
69.3 ± 50.5
- Collagen
32.8 ± 14.7
- ADP
65.6 ± 34.1
Fig. 1. Effects of C. longa extract and the active ingredients on shear stress-induced platelet aggregation (SIPA). Effects of C. longa extract (A) or the active ingredients in C. longa (25 μM, B) on platelet aggregation-induced by shear stress in freshly isolated human platelet-rich-plasma (PRP). C, Concentration-dependent inhibitory effects of cyclocurcumin (left) or curcumin (right) on SIPA in human PRP. D, Inhibitory effects of cyclocurcumin on SIPA in human washed platelets (WPs). E, Fluorescent microscopy observation of inhibitory effects of cyclocurcumin on SIPA where platelets were stained by Calcein-green AM for 30 min before exposing to shear. F, Comparison of effects of cyclocurcumin on platelet aggregation-induced by different platelet agonists. Values are mean ± SEM of three to five independent experiments from different blood donors, * significant differences from the control group (p < 0.05). Arrowhead indicated platelet aggregates (E). Scale bar (E): 100 μm.
were collected, centrifuged at 12,000 rpm for 5 min at 4 °C to obtain the supernatant for detecting serotonin content. Serotonin release was measured with a serotonin ELISA kit (Labor Diagnostika Nord GmbH & Co., Nordhorn, Germany) according to manufacturer’s instructions.
calculated the average of the coverage area of three random fields.
2.7. Measurement of platelet adhesion to vWF in high shear stress condition
Leakage of lactate dehydrogenase (LDH) from platelets was measured by spectrophotometric analysis as described previously (Kim et al., 2012) with brief modification. After incubation with cyclocurcumin, the platelet pellets, obtained from centrifugation reaction mixture, were washed by Tyrode’s buffer 2 times before totally lysed by 0.3% Triton X-100. The resulting aliquot was used in the LDH assay. The extent of cell lysis was expressed as the percentage of total enzyme activity compared to that a control incubation lysed with 0.3% Triton X-100.
2.8. Measurement of platelet cytotoxicity
To measure platelet adhesion to vWF in high shear force condition, flow chambers (microslide VI0.1, iBidi) were coated with 50 µg/mL human purified vWF for 60 min at room temperature. After washing with phosphate buffered saline (PBS), the chambers were blocked with 1% bovine serum albumin in PBS. Whole blood, which was loaded with calcein-green AM 5 µM and treated with cyclocurcumin, then was perfused to coated flow chambers at the shear rate of 1500 s−1 for 5 min by a syringe pump. The adhesion of platelets was imaged by confocal microscopy LSM-710 and the data was analyzed by ImageJ as 3
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
* *
50
No shear
100 80
*
40
*
20 0
0 0.5 1 5 10
Cyclocurcumin ( M)
D
*
60
No shear
0 0.5 1 5 10
E Fibrinogen binding (fold of basal)
GP IIb/IIIa activation (fold of basal)
5
*
* *
2
0
0
0.5
1
5
Ristocetin
100
0 1
80
5
10
0 3
6
Time (min)
20
*
2
*
*
1 0
0.5
1
5
* * *
15 10 5 0
No shear
0 0.5 1 5 10
10
10 8
* * *
6 4 2 0
0
0.5
1
5
10
Cyclocurcumin ( M)
H
( M)
50
0
3
Cyclocurcumin ( M)
Aggregation (%)
Aggregation (%)
100
4
0
10
Cyclocurcumin ( M)
G
25
Cyclocurcumin ( M)
F
6
4
30
Cyclocurcumin ( M)
40
*
60
*
40 20 0
0
0.5
1
5
Platelet adhesion (%)
0
P-selectin expression (%)
100
C
Serotonin release (ng/mL)
Intracellular calcium (MFI)
B 150
vWF (binding fold of basal)
A
10
Cyclocurcumin ( M)
30
*
20
*
*
10 0
0
1
5
10
Cyclocurcumin ( M)
Fig. 2. Effects of cyclocurcumin on platelet activation-stimulated by shear stress. A to F, Human WPs were treated with various concentration of cyclocurcumin for 10 min before exposure to high shear stress. A, Intraplatelet calcium levels were determined with Fluo-4AM loaded platelets. B, Serotonin release was measured by ELISA kit. C to F, P-selectin (C), GP IIb/IIIa activation (D), fibrinogen binding (E), and vWF binding to platelets (F) - stimulated by high shear stress were measured by flow cytometry. G, Effects of cyclocurcumin on ristocetin-induced platelet aggregation. H, Effects of cyclocurcumin on platelet adhesion to vWF (50 μg/ml) coated surface in the flow chamber. The adhesion of platelet to vWF coated surface was determined as described in methods. Values are mean ± SEM of four independent experiments from different blood donors, * significant differences from the control group (p < 0.05).
5 µM and incubated with tested compounds for 10 min, then perfused to coated flow chambers at the shear rate of 1500 s−1 for 5 min. The thrombus formation on the coated surface was observed by confocal microscopy LSM-710 and the data was analyzed by ImageJ as calculated for the average of a coverage area of three random fields.
2.9. Measurement of PT and aPTT To determine effects of cyclocurcumin on prothrombin time (PT) and activated partial thromboplastin time (aPTT), PPP was incubated with cyclocurcumin at various concentrations (in DMSO, final 0.5%) for 10 min at 37 °C. The resulting samples were subjected to measure PT and aPTT in BBL Fibrometer (Becton Dickinson, Cockeysville, Maryland), according the procedure of manufacturer's recommendation.
2.11. Statistical analysis The data are all shown as mean ± SEM and were subjected to oneway analysis of variance (ANOVA) followed by Duncan’s multiple ranged tests or Student’s t-test using SPSS software (Chicago, IL, USA) to determine which means were significantly different from the control. In all cases, p < 0.05 was used to determine significance. IC50 values were determined by nonlinear regression using Sigma Plot (Systat Software, Inc., San Jose, CA, USA).
2.10. Measurement of in vitro thrombus formation To measure in vitro thrombus formation in arterial shear force, flow chambers (microslide VI0.1, iBidi) were coated with 100 µg/mL collagen (type I, equine tendon, Chrono-log) for 60 min at room temperature. After washing with PBS, the chambers were blocked by 1% bovine serum albumin in PBS. Whole blood was loaded with calcein-green AM 4
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
of 6.33 ± 3.29 μM, superior to that of curcumin (> 250 μM), which is generally believed to be responsible for the most of biological activities of C. longa (Fig. 1C). Further investigation on human WPs, which are free from the involvement of plasma proteins, confirmed the inhibition on SIPA by cyclocurcumin in a consistent pattern to that observed in human PRP (Fig. 1C and D). Also, fluorescent microscopic observation indicated the reduction of the platelet aggregate formation both in number and size under high shear stress condition following the treatment with cyclocurcumin (Fig. 1E), supporting the major role of cyclocurcumin for the inhibitory effects of C. longa on SIPA. Platelet aggregation can be stimulated by various physiological agonists which bind to their respective receptors on the platelet
3. Results 3.1. Cyclocurcumin significantly inhibited platelet aggregation-depending on high shear stress The extract of C. longa significantly inhibited SIPA in a concentration-dependent manner in freshly isolated human platelets (Fig. 1A). To identify the bioactive ingredients accountable for the activity of C. longa extract, we further evaluated anti-SIPA effects of 10 major components of C. longa at 25 μM (Fig. 1B). As a result, cyclocurcumin exhibited the strongest potent effects as compared to other components from C. longa extract. Interestingly, the IC50 value of cyclocurcumin against SIPA was
A
60
* PT (sec)
20
*
40
0
0 25 100250
60 40
20
20
0
DIG
0
25 100 250
LB
Cyclocurcumin ( M)
Vehicle
0
25 100 250
5 M
LB
0
Cyclocurcumin ( M)
Curcumin
Cyclocurcumin 0 M
80
30
10
Cyclocurcumin ( M)
C
*
50
60 40
100
aPTT (sec)
LDH leakage (% of total)
B 80
10 M
50 μm
+ Shear stress
No shear
Thrombus formation (%)
40 30
*
20
* 10 0 ( M)
No shear
0
1
5
10
Curcumin
Cyclocurcumin
Fig. 3. Effects of cyclocurcumin on in vitro thrombus formation. A, Nonspecific cytotoxicity of cyclocurcumin on platelets was measured by the leakage of LDH. Effects of cyclocurcumin on blood clotting time, as measured with prothrombin time (PT) and activated partial thromboplastin time (aPTT). C, Whole blood, loaded with calcein-green AM, was treated with cyclocurcumin or curcumin for 10 min before subjected to high shear stress in flow chambers (coated with 100 μg/ml fibrillary collagen type I). The thrombus formation was observed by confocal microscopy LSM-710 at the magnification of 400×, and the size of the thrombi was analyzed by ImageJ as a coverage area. Values are mean ± SEM of three to four independent experiments from different blood donors, * significant differences from the control group (p < 0.05). Scale bar (C): 50 μm. 5
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
preventing pathological thrombosis. Distinct from other pathways dependent on physiological agonists to induce platelet activation, SIPA occurs solely in pathological condition, such as in stenotic vessels or atherosclerotic lesson (Jackson, 2007; Li et al., 2010). Notably, cyclocurcumin, a bioactive ingredient from C. longa, selectively inhibited SIPA (Fig. 1F), that may provide an advantage in modulating pathological thrombosis without interfering with the normal hemostasis (McFadyen et al., 2018). Furthermore, cyclocurcumin did not prolong blood clotting time (Fig. 3B), supporting its therapeutic benefit for the management of pathological thrombosis without bleeding complications, the most serious adverse effects of current therapeutic antiplatelet agents (McFadyen et al., 2018; Michelson, 2010). In high shear stress condition, the binding of vWF to GP Ib leads to signal transduction, thereby activating platelets (Kroll et al., 1996). When platelets are activated, the degranulation and GP IIb/IIIa activation further propagates this process and stabilize platelet aggregates, and subsequently accelerating thrombus formation (Li, 2010; Nieswandt, Pleines, & Bender, 2011). Cyclocurcumin alleviated the earliest event of platelet activation under high shear stress, as disturbing the interaction of vWF and platelet GP Ib (Fig. 2F–H), suggesting that cyclocurcumin might be used to broadly prevent thrombotic events and related cardiovascular disorders. C. longa is a popular food ingredient, of which, curcumin is considered to be the major constituent accountable for its effects on platelet activity (Ammon & Wahl, 1991; Ghosh et al., 2015). Interestingly, in this study, we demonstrated that cyclocurcumin, although present in a low content (Ahmed & Gilani, 2014), exhibited highly potent and broad inhibitory effects on platelet activation/ aggregation induced by high shear stress as well as by other physiological stimuli (Fig. 1C–F). Cyclocurcumin showed the much higher potency than the reported effects of curcumin (Shah et al., 1999). Moreover, in a good line with the inhibitory effects on platelet activity, cyclocurcumin significantly suppressed thrombus formation in arterial shear stress condition, suggesting that it could provide an effectively contribution to the antiplatelet and antithrombotic effects of C. longa.
membrane surface (Li et al., 2010). Cyclocurcumin exhibited the relatively selective effects on platelet function under high shear stress as demonstrated by the IC50 value against SIPA was much lower than that of cyclocurcumin against platelet aggregation-induced by other endogenous agonists, including thrombin, collagen, and ADP (Fig. 1F, Suppl. Fig. 1). Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jff.2019.103462. 3.2. Cyclocurcumin inhibited shear stress-induced platelet activation through modulating vWF-GP Ib interaction High shear stress exerting the elevation of intraplatelet calcium level was significantly inhibited by treatment with cyclocurcumin (Fig. 2A). Moreover, cyclocurcumin remarkably reversed the degranulation of platelets by shear stress from both dense granules and αgranules, which could be determined by the significant reduction in serotonin release (Fig. 2B) and P-selectin expression (Fig. 2C). Furthermore, cyclocurcumin caused a significant attenuation on glycoprotein (GP) IIb/IIa activation and fibrinogen binding to the activated platelet in a concentration-dependent manner (Fig. 2D and E), reflecting the inhibition of platelet activation events under pathological shear stress by cyclocurcumin. Treatment with cyclocurcumin significantly blocked the engagement of von Willebrand factor (vWF) to platelets under high shear (Fig. 2F), which plays a fundamental role as the initial step of SIPA (Ikeda, Murata, & Goto, 1997). This effect was further confirmed when cyclocurcumin remarkably suppressed platelet aggregation stimulated by ristocetin (Fig. 2G), an exogenous platelet activator inducing the binding of vWF and GP Ib in the absence of shear stress (Dong et al., 2001). And cyclocurcumin also inhibited the adhesion of platelet to the vWF-coated surface in the flow chambers in arterial high shear stress (Fig. 2H). 3.3. Cyclocurcumin prevented thrombus formation without inducing cytotoxicity nor prolonging blood clotting time The effects of cyclocurcumin on platelet function was not mediated by non-specific cytotoxicity as no effects on lactate dehydrogenase (LDH) leakage could be observed up to a concentration of 250 µM (Fig. 3A). Cyclocurcumin neither influenced on blood clotting time as determined by prothrombin time (PT) and activated partial thromboplastin time (aPTT) up to a concentration of 250 µM (Fig. 3B). Interestingly, cyclocurcumin significantly inhibited in vitro thrombus formation, as could be determined by the remarkably reduced number and size of the formed thrombi, in a concentration-dependent manner (Fig. 3C). Most importantly, cyclocurcumin exhibited a much stronger prevention effect on thrombus formation than curcumin. At the concentration of 10 µM, cyclocurcumin attenuated formed thrombi by 62.2% while curcumin did not show any significant inhibition (Fig. 3C), highlighting the potent antithrombotic effects of cyclocurcumin.
5. Conclusion In addition to our previous study on the anticontractile effects of cyclocurcumin (Kim, 2017), this study revealed the novel and potent antiplatelet and antithrombotic effects of cyclocurcumin, which is selective to pathological high shear stress condition through modulating vWF - platelet interaction. Further study is necessary to re-evaluate and elucidate the anti-thrombotic effects of cyclocurcumin and its potential applications for the prevention and treatment of cardiovascular diseases. 6. Ethics statements With the approval from the Ethics Committee of Health Service Center at Seoul National University, human blood was obtained from healthy male volunteer donors.
4. Discussion Owing to the crucial role of platelets in thrombogenesis progression (Jackson, 2007; Palomo et al., 2008), antiplatelet therapy has become an important pharmacotherapeutic strategy for prevention and treatment cardiovascular disorders, one of the most leading mortality cause globally (Michelson, 2010). Conventionally, physiological agonists, such as thrombin, collagen, ADP, or arachidonic acid, are employed for exploring novel drugs inhibiting platelet activation (McEwen, 2015; Tsoupras, Zabetakis, & Lordan, 2019). However, contribution of physical activator, high shear stress, which is critical in pathological thrombosis (Kamada et al., 2017; Kroll et al., 1996), has not been well studied. Here, we newly demonstrated the anti-SIPA effects of C. longa extracts which might explain the beneficial effects of C. longa in
Author contributions T.N. designed the experiments, analyzed the data and wrote the manuscript; K.K., Y.B., G-J.A., and O-N.B analyzed the data, K-M.L. analyzed the data and edited the manuscript. J-H.C. supervised the study. All authors have approved the final version of the manuscript. Declaration of Competing Interest All the authors declared that there is no conflict of interest. 6
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
Acknowledgements
Libby, P. (2012). Inflammation in atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology, 32(9), 2045–2051. https://doi.org/10.1161/ATVBAHA.108. 179705. McEwen, B. J. (2015). The influence of herbal medicine on platelet function and coagulation: A narrative review. Seminars in Thrombosis and Hemostasis, 41(3), 300–314. https://doi.org/10.1055/s-0035-1549089. McFadyen, J. D., Schaff, M., & Peter, K. (2018). Current and future antiplatelet therapies: Emphasis on preserving haemostasis. Nature Reviews Cardiology, 15(3), 181–191. https://doi.org/10.1038/nrcardio.2017.206. Michelson, A. D. (2010). Antiplatelet therapies for the treatment of cardiovascular disease. Nature Reviews Drug Discovery, 9(2), 154–169. https://doi.org/10.1038/ nrd2957. Nieswandt, B., Pleines, I., & Bender, M. (2011). Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke. Journal of Thrombosis and Haemostasis, 9(Suppl 1), 92–104. https://doi.org/10.1111/j.1538-7836.2011. 04361.x. Palomo, I., Toro, C., & Alarcon, M. (2008). The role of platelets in the pathophysiology of atherosclerosis (Review). Molecular Medicine Reports, 1(2), 179–184. https://doi.org/ 10.3892/mmr.1.2.179. Pei, H., Yang, Y., Cui, L., Yang, J., Li, X., Yang, Y., & Duan, H. (2016). Bisdemethoxycurcumin inhibits ovarian cancer via reducing oxidative stress mediated MMPs expressions. Scientific Report, 6, 28773. https://doi.org/10.1038/ srep28773. Prakash, P., Misra, A., Surin, W. R., Jain, M., Bhatta, R. S., Pal, R., ... Dikshit, M. (2011). Anti-platelet effects of Curcuma oil in experimental models of myocardial ischemiareperfusion and thrombosis. Thrombosis Research, 127(2), 111–118. https://doi.org/ 10.1016/j.thromres.2010.11.007. Shah, B. H., Nawaz, Z., Pertani, S. A., Roomi, A., Mahmood, H., Saeed, S. A., & Gilani, A. H. (1999). Inhibitory effect of curcumin, a food spice from turmeric, on plateletactivating factor- and arachidonic acid-mediated platelet aggregation through inhibition of thromboxane formation and Ca2+ signaling. Biochemical Pharmacology, 58(7), 1167–1172. https://doi.org/10.1016/S0006-2952(99)00206-3. Sheu, M. J., Lin, H. Y., Yang, Y. H., Chou, C. J., Chien, Y. C., Wu, T. S., & Wu, C. H. (2013). Demethoxycurcumin, a major active curcuminoid from Curcuma longa, suppresses balloon injury induced vascular smooth muscle cell migration and neointima formation: An in vitro and in vivo study. Molecular Nutrition & Food Research, 57(9), 1586–1597. https://doi.org/10.1002/mnfr.201200462. Srivastava, K. C. (1989). Extracts from two frequently consumed spices–cumin (Cuminum cyminum) and turmeric (Curcuma longa)–inhibit platelet aggregation and alter eicosanoid biosynthesis in human blood platelets. Prostaglandins, Leukotrienes & Essential Fatty Acids, 37(1), 57–64. https://doi.org/10.1016/0952-3278(89)90187-7. Srivastava, R., Dikshit, M., Srimal, R. C., & Dhawan, B. N. (1985). Anti-thrombotic effect of curcumin. Thrombosis Research, 40(3), 413–417. https://doi.org/10.1016/00493848(85)90276-2. Tsoupras, A., Lordan, R., & Zabetakis, I. (1985). Inflammation, not cholesterol, is a cause of chronic disease. Nutrients, 10(5), https://doi.org/10.3390/nu10050604. Tsoupras, A., Zabetakis, I., & Lordan, R. (2019). Platelet aggregometry assay for evaluating the effects of platelet agonists and antiplatelet compounds on platelet function in vitro. MethodsX, 6, 63–70. https://doi.org/10.1016/j.mex.2018.12.012. Wongcharoen, W., & Phrommintikul, A. (2009). The protective role of curcumin in cardiovascular diseases. International Journal of Cardiology, 133(2), 145–151. https:// doi.org/10.1016/j.ijcard.2009.01.073.
This work was supported by the National Research Foundation of Korea (NRF) grant (MSIT; 2018R1A5A2025286) funded by the Korea Government. References Ahmed, T., & Gilani, A. H. (2014). Therapeutic potential of turmeric in Alzheimer's disease: Curcumin or curcuminoids? Phytotherapy Research, 28(4), 517–525. https://doi. org/10.1002/ptr.5030. Ammon, H. P., & Wahl, M. A. (1991). Pharmacology of Curcuma longa. Planta Medica, 57(1), 1–7. https://doi.org/10.1055/s-2006-960004. Cattaneo, M., Lombardi, R., Bettega, D., Lecchi, A., & Mannucci, P. M. (1993). Shearinduced platelet aggregation is potentiated by desmopressin and inhibited by ticlopidine. Arteriosclerosis and Thrombosis, 13(3), 393–397. Dong, J. F., Berndt, M. C., Schade, A., McIntire, L. V., Andrews, R. K., & Lopez, J. A. (2001). Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with sheardependent interactions. Blood, 97(1), 162–168. Ghosh, S., Banerjee, S., & Sil, P. C. (2015). The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update. Food and Chemical Toxicology, 83, 111–124. https://doi.org/10.1016/j.fct.2015.05.022. Goel, A., Kunnumakkara, A. B., & Aggarwal, B. B. (2008). Curcumin as “Curecumin”: From kitchen to clinic. Biochemical Pharmacology, 75(4), 787–809. https://doi.org/ 10.1016/j.bcp.2007.08.016. Ikeda, Y., Murata, M., & Goto, S. (1997). Von Willebrand factor-dependent shear-induced platelet aggregation: Basic mechanisms and clinical implications. Annals of the New York Academy of Sciences, 811, 325–336. https://doi.org/10.1111/j.1749-6632.1997. tb52012.x. Jackson, S. P. (2007). The growing complexity of platelet aggregation. Blood, 109(12), 5087–5095. https://doi.org/10.1182/blood-2006-12-027698. Kamada, H., Imai, Y., Nakamura, M., Ishikawa, T., & Yamaguchi, T. (2017). Shear-induced platelet aggregation and distribution of thrombogenesis at stenotic vessels. Microcirculation, 24(4), https://doi.org/10.1111/micc.12355. Kim, K., Bae, O. N., Lim, K. M., Noh, J. Y., Kang, S., Chung, K. Y., & Chung, J. H. (2012). Novel antiplatelet activity of protocatechuic acid through the inhibition of high shear stress-induced platelet aggregation. Journal of Pharmacology and Experimental Therapeutics, 343(3), 704–711. https://doi.org/10.1124/jpet.112.198242. Kim, K., et al. (2017). Cyclocurcumin, an antivasoconstrictive constituent of Curcuma longa (Turmeric). Journal of Natural Product, 80(1), 196–200. https://doi.org/10. 1021/acs.jnatprod.6b00331. Kroll, M. H., Hellums, J. D., McIntire, L. V., Schafer, A. I., & Moake, J. L. (1996). Platelets and shear stress. Blood, 88(5), 1525–1541. http://www.bloodjournal.org/content/ 88/5/1525. Lantz, R. C., Chen, G. J., Solyom, A. M., Jolad, S. D., & Timmermann, B. N. (2005). The effect of turmeric extracts on inflammatory mediator production. Phytomedicine, 12(6–7), 445–452. https://doi.org/10.1016/j.phymed.2003.12.011. Li, Z., Delaney, M. K., O'Brien, K. A., & Du, X. (2010). Signaling during platelet adhesion and activation. Arteriosclerosis, Thrombosis, and Vascular Biology, 30(12), 2341–2349. https://doi.org/10.1161/ATVBAHA.110.207522.
7
Contents lists available at ScienceDirect
Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Cyclocurcumin from Curcuma longa selectively inhibits shear stress-induced platelet aggregation
T
Thien Ngoa, Keunyoung Kima, Yiying Biana,1, Gwang-Jin Ana, Ok-Nam Baeb, Kyung-Min Limc, ⁎ Jin-Ho Chunga, a
College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea College of Pharmacy, Hanyang University, Ansan, Gyeonggido 15588, Republic of Korea c College of Pharmacy, Ewha Womans University, Seoul 03760, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Cyclocurcumin Curcumin SIPA Antiplatelet Antithrombotic Cardiovascular disease
Curcuma longa (C. longa) has been the subject of intensive research for pleiotropic therapeutic potential. However, except for curcumin, the biological activities of other active ingredients of C. longa are not well known. Here, we demonstrated that C. longa extract significantly attenuated shear stress-induced platelet aggregation and cyclocurcumin exhibited the most potent activity (IC50 of 6.33 ± 3.29 μM). Cyclocurcumin potently inhibited shear stress-induced platelet activation, primarily through modulating the initial step, vWF – platelets GP Ib interaction. Moreover, cyclocurcumin effectively inhibited thrombus formation, without influencing platelet cell viability or blood clotting time. Collectively, we demonstrated that cyclocurcumin might be a promising therapeutic agent for the treatment or prevention of thrombotic diseases.
1. Introduction The powdered rhizome of Curcuma longa (C. longa), which contains a mixture of curcuminoids, has been used widely for millenniums as a spice, food coloring, and traditional herbal remedies for diverse therapeutic purpose (Ammon & Wahl, 1991; Ghosh, Banerjee, & Sil, 2015). Curcumin, the most enriched ingredient among these curcuminoids, is generally believed as the vital constituent contributing to the diverse pharmacological activities of C. longa (Ammon & Wahl, 1991; Goel, Kunnumakkara, & Aggarwal, 2008). Numerous biological activities of curcumin have been discovered including anti-oxidant, anti-inflammatory, and anti-neuro-degradation, and their potential benefits for various diseases have drawn attention (Ghosh et al., 2015; Goel et al., 2008). Interestingly, recent studies have highlighted that the distinct pharmacological effects of curcuminoids other than curcumin; demethoxycurcumin, bisdemethoxycurcumin, and cyclocurcumin may at least in part contribute to the therapeutic benefits of C. longa (Kim, 2017; Pei et al., 2016; Sheu et al., 2013). C. longa may be beneficial against cardiovascular diseases through various bioactivities such as lowering cholesterol and ameliorating
myocardial damage (Goel et al., 2008; Wongcharoen & Phrommintikul, 2009). In addition, the favorable effects of C. longa on inflammation, an important player in triggering coronary artery diseases and thrombotic complications of atherosclerosis (Libby, 2012; Tsoupras, Lordan, & Zabetakis, 2018), have been highlighted (Lantz, Chen, Solyom, Jolad, & Timmermann, 2005). Due to the crucial role in both hemostasis and thrombosis (Li, Delaney, O'Brien, & Du, 2010; Palomo, Toro, & Alarcon, 2008), the influence of C. longa on platelet activity has also been assessed (Srivastava, 1989). The antiplatelet effects of curcumin, as well as some other constitutes of C. longa, were also known (Prakash et al., 2011; Srivastava, Dikshit, Srimal, & Dhawan, 1985). In these studies, the physiological agonists, such as collagen, ADP, and arachidonic acid (Prakash et al., 2011; Shah et al., 1999) were used to evaluate the antiplatelet effects. However, it was not known whether the C. longa could affect the shear stress-induced platelet aggregation (SIPA), a distinct phenomenon of platelet activity, of which role in pathological thrombosis is gaining attention recently (Kamada, Imai, Nakamura, Ishikawa, & Yamaguchi, 2017; Kim et al., 2012). Blood flow produces shear stress, which can increase in atherosclerotic lesion or vasospasm, thereby directly triggering platelet aggregation regardless of other
Abbreviations: SIPA, shear stress-induced platelet aggregation; CYCL, cyclocurcumin; vWF, von Willebrand factor; GP, glycoprotein; Ab, antibody; AM, acetoxymethyl ester; FITC, fluorescein isothiocyanate; PE, phycoerythrin; LDH, lactate dehydrogenase, PGE1, prostaglandin E1; PRP, platelet-rich plasma; WPs, washed platelets; PT, prothrombin time; aPTT, activated partial thromboplastin time ⁎ Corresponding author at: College of Pharmacy, Seoul National University, Shinrim-dong San 56-1, Gwanak-Gu, Seoul 08826, Republic of Korea. E-mail address: [email protected] (J.-H. Chung). 1 Permanent address: School of Public Health, China Medical University, 110122, PR China. https://doi.org/10.1016/j.jff.2019.103462 Received 28 April 2019; Received in revised form 5 July 2019; Accepted 12 July 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
endogenous stimuli (Kroll, Hellums, McIntire, Schafer, & Moake, 1996). SIPA plays a critical role in thrombo-pathogenesis but is relatively unassociated with normal hemostasis (Jackson, 2007; Kroll et al., 1996). Accordingly, SIPA is considered as a promising target for antiplatelet and antithrombotic agents without bleeding complications, which is the most serious adverse effects of current platelet inhibitors (McFadyen, Schaff, & Peter, 2018; Michelson, 2010). Here, we investigated the effects of C. longa along with its active ingredients on SIPA. Of all, cyclocurcumin showed the most potent effects. The mechanism on anti-SIPA effects and antithrombotic potential of cyclocurcumin was further elucidated.
1: 6, v/v), supplemented with PGE1 1 μM. PRP, prepared as described above was centrifuged for 10 min at 500g to obtain platelet pellets. Platelet pellets were washed with calcium-free Tyrode's buffer (134 mM NaCl, 2.9 mM KCl, 1.0 mM MgCl2, 10.0 mM HEPES, 5.0 mM glucose, 12.0 mM NaHCO3, 0.34 mM Na2HPO4, and 0.3% bovine serum albumin, pH 7.4) containing 10% ACD and PGE1 1 μM. After centrifugation at 400g for 10 min, platelets were finally suspended at 3 × 108 platelets/ml in Tyrode's buffer containing 2 mM CaCl2, as described previously (Kim et al., 2012).
2. Material and methods
Reactions were performed in a programmable cone-plate viscometer (RotoVisco 1; Thermo Fisher Scientific, Waltham, MA) with the shear rate at 10,800 s−1 for 3 min at 37 °C. For WPs, before applying shear stress, 10 µg/ml of vWF was added. 500 μL of platelet suspension treated with cyclocurcumin in DMSO (final, 0.5%) or vehicle (DMSO) for 10 min at 37 °C, was loaded to the cone-plate viscometer. The platelets after exposing to shear stress were collected and fixed with 0.5% glutaraldehyde in Tyrode's buffer. Platelet aggregation was determined based on the number of single platelets per microliter under a phasecontrast light microscope (CX41; Olympus, Tokyo, Japan) as described previously with a minor modification (Cattaneo, Lombardi, Bettega, Lecchi, & Mannucci, 1993). The level of platelet aggregation was calculated depending on the loss of single cell as compared to the unsheared sample. The inhibition of platelet aggregation was compared to that of vehicle treated sample. As the maximal of the inhibition of platelet aggregation due to the shear stress of all the tested compounds was 50%, this level was defined as the maximal inhibition by shear stress.
2.3. Shear stress-induced platelet aggregation (SIPA) assay.
2.1. Reagents and plant materials Reagents and antibodies. Thrombin was purchased from Calbiochem (San Diego, CA). Von Willebrand factor (vWF) was from Molecular Innovations, Inc. (Michigan, USA), and collagen, ADP, and ristocetin were from Chrono-log (Havertown, PA). Fluo-4/AM, pluronic F-127, and alexa fluor 488-conjugated fibrinogen were from Invitrogen (Carlsbad, CA). Phycoerythrin (PE)-labeled monoclonal antibody against human CD42b (antiCD42b-PE Ab), fluorescein isothiocyanate (FITC)-labeled anti-CD62P antibody (anti-CD62P-FITC Ab), and FITClabeled PAC-1 (PAC-1-FITC) were from BD Biosciences (San Jose, CA), and FITC-labeled anti-vWF antibody (anti-vWF-FITC) was from Abcam plc (Cambridge, UK). Cyclocurcumin (95.6% pure as determined by HPLC, Karl Fischer (water content), GC (residual solvent) including mass spectroscopy, and NMR profiles by the manufacturer) was obtained from Chromadex Inc. (Irvine, CA, USA). Curcumin (> 98% pure as determined by HPLC including mass spectroscopy and melting point by the manufacturer) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other ingredients in C. longa were provided from the National Center for Standardization of Herbal Medicines in Korea, with a purity of > 98%. For all the experiments, platelet suspensions were treated with various concentration of tested compounds in DMSO (final, 0.5%). The effects were compared to those of the control, DMSO-treated samples. Plant material. The plant was authenticated, and a voucher specimen (CU2009-06) has been deposited at the herbarium of Chosun University (Gwangju, Korea). Dry powders of C. longa were extracted with 70% ethanol at 70–80 °C for 3 h. The extraction was repeated three times. After filtration and concentration under reduced pressure, extracts were lyophilized, and the resultant powder was stored at −20 °C. For experiments extracts were dissolved in DMSO. Identification and quantification of the constitution of the C. longa extract was described in our previous study (Kim, 2017).
2.4. Measurement of agonist-induced platelet aggregation Agonist-induced platelet aggregation was determined by the turbidometric method using an aggregometer (Chrono-log). After incubation with cyclocurcumin for 10 min at 37 °C, PRP were loaded on the aggregometer and stimulated with thrombin (0.6–0.8 U/mL), collagen (2–4 µg/mL), ADP (5–10 µM), or ristocetin (1.0–1.25 mg/mL) for 6 min. Platelet aggregation was measured by light transmission, with 100% calibrated as the absorbance of PPP. 2.5. Measurement of intraplatelet activation events induced by shear stress To detect intracellular calcium levels, WPs were loaded with fluo-4/ AM (5 µM) and pluronic F-127 (0.2%) for 45 min at 37 °C in the dark. The cyclocurcumin treated platelets were exposed to shear stress, then diluted by Tyrode’s buffer. The change of intracellular calcium was analyzed on the FACS Calibur cytometer (BD Biosciences) equipped with an argon laser (λex 488 nm). The light scatters, and fluorescence channels were set on a log scale. Data from 10,000 events were collected and analyzed by using CellQuest Pro software (BD Biosciences) (Kim et al., 2012). To detect P-selectin expression, glycoprotein (GP) IIb/IIIa activation, fibrinogen binding, or vWF binding, CD62P-FITC Ab, PAC-1-FITC, alexa fluor 488-conjugated fibrinogen, and anti-vWF-FITC Ab was used as the marker, respectively. WPs were treated with cyclocurcumin before exposing to high shear stress. Anti-CD42b-PE Ab was used as platelet identifier. Platelets were incubated with the mixture of antibodies for 20 min in the dark, at room temperature, and then analyzed on the flow cytometer as described above.
2.2. Human platelet preparation The work was approved by the Ethics Committee of the Health Service Center at Seoul National University. Blood was collected from 35 non-smoking healthy male volunteers (18–25 years old), who were medication free for two weeks, and was used on the day of collection. For PRP preparation, venous whole blood was collected from forearm vein to a vacuum vacutainer (Vacuette; Greiner Bio‐One GmbH, Kremsmünster, Austria) containing sodium citrate 3.2% (1: 9, v/v) by a 21-gauge needle. Whole blood was centrifuged for 15 min at 150g to obtain platelet-rich-plasma (PRP). Platelet-poor-plasma (PPP) was obtained from the precipitated fraction of PRP by centrifugation at 2000g for 20 min. Platelets are counted with a hemocytometer (Marienfeld GmbH, Marienfeld, Germany), and platelet count in PRP was adjusted to 3 × 108 platelets/ml with PPP. For washed platelets (WPs) preparation, venous whole blood was collected from forearm vein by a 16gauge needle to blood collecting bag containing acid citrate-dextrose (ACD, 85 mM trisodium citrate, 71 mM citric acid, and 111 mM glucose,
2.6. Measurement of serotonin secretion Platelets were treated with cyclocurcumin at various concentrations in DMSO (final, 0.5%) or vehicle (DMSO) for 10 min at 37 °C before subjected to shear stress at 10,800 s−1 for 3 min. The sheared platelets 2
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
B 100
% of maximal inhibition
80 60 40 20 0
10 20 50 100 250
80
E
60 40 20 0
C sito yc st l (+ ocu ero )rc l ar um 4 tu -d in eh Bi me s yd ab ron ro xy ola e bi trae sa -h c n yd ro Bi uro xy sa ne c c D em inn uro et am ne ho ic xy a cu cid Bi sd rc um em i et Cu ho rc n xy um cu i rc n um in
% of maximal inhibtion
A
Curcuma longa ( g/mL)
D
100
% of maximal inhibition
% of maximal inhibition
C 80 60 40 20 0 ( M)
0.5
1
5
10
10
Cyclocurcumin
50
100 250
Curcumin
100
WP
80 60 40 20 0
0.5
1
5
10
Cyclocurcumin ( M)
F Cyclocurcumin
Platelet activator
IC50 ( M)
Physical stimuli: Shear stress
6.33 ± 3.29
Physiological stimuli: - Thrombin
69.3 ± 50.5
- Collagen
32.8 ± 14.7
- ADP
65.6 ± 34.1
Fig. 1. Effects of C. longa extract and the active ingredients on shear stress-induced platelet aggregation (SIPA). Effects of C. longa extract (A) or the active ingredients in C. longa (25 μM, B) on platelet aggregation-induced by shear stress in freshly isolated human platelet-rich-plasma (PRP). C, Concentration-dependent inhibitory effects of cyclocurcumin (left) or curcumin (right) on SIPA in human PRP. D, Inhibitory effects of cyclocurcumin on SIPA in human washed platelets (WPs). E, Fluorescent microscopy observation of inhibitory effects of cyclocurcumin on SIPA where platelets were stained by Calcein-green AM for 30 min before exposing to shear. F, Comparison of effects of cyclocurcumin on platelet aggregation-induced by different platelet agonists. Values are mean ± SEM of three to five independent experiments from different blood donors, * significant differences from the control group (p < 0.05). Arrowhead indicated platelet aggregates (E). Scale bar (E): 100 μm.
were collected, centrifuged at 12,000 rpm for 5 min at 4 °C to obtain the supernatant for detecting serotonin content. Serotonin release was measured with a serotonin ELISA kit (Labor Diagnostika Nord GmbH & Co., Nordhorn, Germany) according to manufacturer’s instructions.
calculated the average of the coverage area of three random fields.
2.7. Measurement of platelet adhesion to vWF in high shear stress condition
Leakage of lactate dehydrogenase (LDH) from platelets was measured by spectrophotometric analysis as described previously (Kim et al., 2012) with brief modification. After incubation with cyclocurcumin, the platelet pellets, obtained from centrifugation reaction mixture, were washed by Tyrode’s buffer 2 times before totally lysed by 0.3% Triton X-100. The resulting aliquot was used in the LDH assay. The extent of cell lysis was expressed as the percentage of total enzyme activity compared to that a control incubation lysed with 0.3% Triton X-100.
2.8. Measurement of platelet cytotoxicity
To measure platelet adhesion to vWF in high shear force condition, flow chambers (microslide VI0.1, iBidi) were coated with 50 µg/mL human purified vWF for 60 min at room temperature. After washing with phosphate buffered saline (PBS), the chambers were blocked with 1% bovine serum albumin in PBS. Whole blood, which was loaded with calcein-green AM 5 µM and treated with cyclocurcumin, then was perfused to coated flow chambers at the shear rate of 1500 s−1 for 5 min by a syringe pump. The adhesion of platelets was imaged by confocal microscopy LSM-710 and the data was analyzed by ImageJ as 3
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
* *
50
No shear
100 80
*
40
*
20 0
0 0.5 1 5 10
Cyclocurcumin ( M)
D
*
60
No shear
0 0.5 1 5 10
E Fibrinogen binding (fold of basal)
GP IIb/IIIa activation (fold of basal)
5
*
* *
2
0
0
0.5
1
5
Ristocetin
100
0 1
80
5
10
0 3
6
Time (min)
20
*
2
*
*
1 0
0.5
1
5
* * *
15 10 5 0
No shear
0 0.5 1 5 10
10
10 8
* * *
6 4 2 0
0
0.5
1
5
10
Cyclocurcumin ( M)
H
( M)
50
0
3
Cyclocurcumin ( M)
Aggregation (%)
Aggregation (%)
100
4
0
10
Cyclocurcumin ( M)
G
25
Cyclocurcumin ( M)
F
6
4
30
Cyclocurcumin ( M)
40
*
60
*
40 20 0
0
0.5
1
5
Platelet adhesion (%)
0
P-selectin expression (%)
100
C
Serotonin release (ng/mL)
Intracellular calcium (MFI)
B 150
vWF (binding fold of basal)
A
10
Cyclocurcumin ( M)
30
*
20
*
*
10 0
0
1
5
10
Cyclocurcumin ( M)
Fig. 2. Effects of cyclocurcumin on platelet activation-stimulated by shear stress. A to F, Human WPs were treated with various concentration of cyclocurcumin for 10 min before exposure to high shear stress. A, Intraplatelet calcium levels were determined with Fluo-4AM loaded platelets. B, Serotonin release was measured by ELISA kit. C to F, P-selectin (C), GP IIb/IIIa activation (D), fibrinogen binding (E), and vWF binding to platelets (F) - stimulated by high shear stress were measured by flow cytometry. G, Effects of cyclocurcumin on ristocetin-induced platelet aggregation. H, Effects of cyclocurcumin on platelet adhesion to vWF (50 μg/ml) coated surface in the flow chamber. The adhesion of platelet to vWF coated surface was determined as described in methods. Values are mean ± SEM of four independent experiments from different blood donors, * significant differences from the control group (p < 0.05).
5 µM and incubated with tested compounds for 10 min, then perfused to coated flow chambers at the shear rate of 1500 s−1 for 5 min. The thrombus formation on the coated surface was observed by confocal microscopy LSM-710 and the data was analyzed by ImageJ as calculated for the average of a coverage area of three random fields.
2.9. Measurement of PT and aPTT To determine effects of cyclocurcumin on prothrombin time (PT) and activated partial thromboplastin time (aPTT), PPP was incubated with cyclocurcumin at various concentrations (in DMSO, final 0.5%) for 10 min at 37 °C. The resulting samples were subjected to measure PT and aPTT in BBL Fibrometer (Becton Dickinson, Cockeysville, Maryland), according the procedure of manufacturer's recommendation.
2.11. Statistical analysis The data are all shown as mean ± SEM and were subjected to oneway analysis of variance (ANOVA) followed by Duncan’s multiple ranged tests or Student’s t-test using SPSS software (Chicago, IL, USA) to determine which means were significantly different from the control. In all cases, p < 0.05 was used to determine significance. IC50 values were determined by nonlinear regression using Sigma Plot (Systat Software, Inc., San Jose, CA, USA).
2.10. Measurement of in vitro thrombus formation To measure in vitro thrombus formation in arterial shear force, flow chambers (microslide VI0.1, iBidi) were coated with 100 µg/mL collagen (type I, equine tendon, Chrono-log) for 60 min at room temperature. After washing with PBS, the chambers were blocked by 1% bovine serum albumin in PBS. Whole blood was loaded with calcein-green AM 4
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
of 6.33 ± 3.29 μM, superior to that of curcumin (> 250 μM), which is generally believed to be responsible for the most of biological activities of C. longa (Fig. 1C). Further investigation on human WPs, which are free from the involvement of plasma proteins, confirmed the inhibition on SIPA by cyclocurcumin in a consistent pattern to that observed in human PRP (Fig. 1C and D). Also, fluorescent microscopic observation indicated the reduction of the platelet aggregate formation both in number and size under high shear stress condition following the treatment with cyclocurcumin (Fig. 1E), supporting the major role of cyclocurcumin for the inhibitory effects of C. longa on SIPA. Platelet aggregation can be stimulated by various physiological agonists which bind to their respective receptors on the platelet
3. Results 3.1. Cyclocurcumin significantly inhibited platelet aggregation-depending on high shear stress The extract of C. longa significantly inhibited SIPA in a concentration-dependent manner in freshly isolated human platelets (Fig. 1A). To identify the bioactive ingredients accountable for the activity of C. longa extract, we further evaluated anti-SIPA effects of 10 major components of C. longa at 25 μM (Fig. 1B). As a result, cyclocurcumin exhibited the strongest potent effects as compared to other components from C. longa extract. Interestingly, the IC50 value of cyclocurcumin against SIPA was
A
60
* PT (sec)
20
*
40
0
0 25 100250
60 40
20
20
0
DIG
0
25 100 250
LB
Cyclocurcumin ( M)
Vehicle
0
25 100 250
5 M
LB
0
Cyclocurcumin ( M)
Curcumin
Cyclocurcumin 0 M
80
30
10
Cyclocurcumin ( M)
C
*
50
60 40
100
aPTT (sec)
LDH leakage (% of total)
B 80
10 M
50 μm
+ Shear stress
No shear
Thrombus formation (%)
40 30
*
20
* 10 0 ( M)
No shear
0
1
5
10
Curcumin
Cyclocurcumin
Fig. 3. Effects of cyclocurcumin on in vitro thrombus formation. A, Nonspecific cytotoxicity of cyclocurcumin on platelets was measured by the leakage of LDH. Effects of cyclocurcumin on blood clotting time, as measured with prothrombin time (PT) and activated partial thromboplastin time (aPTT). C, Whole blood, loaded with calcein-green AM, was treated with cyclocurcumin or curcumin for 10 min before subjected to high shear stress in flow chambers (coated with 100 μg/ml fibrillary collagen type I). The thrombus formation was observed by confocal microscopy LSM-710 at the magnification of 400×, and the size of the thrombi was analyzed by ImageJ as a coverage area. Values are mean ± SEM of three to four independent experiments from different blood donors, * significant differences from the control group (p < 0.05). Scale bar (C): 50 μm. 5
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
preventing pathological thrombosis. Distinct from other pathways dependent on physiological agonists to induce platelet activation, SIPA occurs solely in pathological condition, such as in stenotic vessels or atherosclerotic lesson (Jackson, 2007; Li et al., 2010). Notably, cyclocurcumin, a bioactive ingredient from C. longa, selectively inhibited SIPA (Fig. 1F), that may provide an advantage in modulating pathological thrombosis without interfering with the normal hemostasis (McFadyen et al., 2018). Furthermore, cyclocurcumin did not prolong blood clotting time (Fig. 3B), supporting its therapeutic benefit for the management of pathological thrombosis without bleeding complications, the most serious adverse effects of current therapeutic antiplatelet agents (McFadyen et al., 2018; Michelson, 2010). In high shear stress condition, the binding of vWF to GP Ib leads to signal transduction, thereby activating platelets (Kroll et al., 1996). When platelets are activated, the degranulation and GP IIb/IIIa activation further propagates this process and stabilize platelet aggregates, and subsequently accelerating thrombus formation (Li, 2010; Nieswandt, Pleines, & Bender, 2011). Cyclocurcumin alleviated the earliest event of platelet activation under high shear stress, as disturbing the interaction of vWF and platelet GP Ib (Fig. 2F–H), suggesting that cyclocurcumin might be used to broadly prevent thrombotic events and related cardiovascular disorders. C. longa is a popular food ingredient, of which, curcumin is considered to be the major constituent accountable for its effects on platelet activity (Ammon & Wahl, 1991; Ghosh et al., 2015). Interestingly, in this study, we demonstrated that cyclocurcumin, although present in a low content (Ahmed & Gilani, 2014), exhibited highly potent and broad inhibitory effects on platelet activation/ aggregation induced by high shear stress as well as by other physiological stimuli (Fig. 1C–F). Cyclocurcumin showed the much higher potency than the reported effects of curcumin (Shah et al., 1999). Moreover, in a good line with the inhibitory effects on platelet activity, cyclocurcumin significantly suppressed thrombus formation in arterial shear stress condition, suggesting that it could provide an effectively contribution to the antiplatelet and antithrombotic effects of C. longa.
membrane surface (Li et al., 2010). Cyclocurcumin exhibited the relatively selective effects on platelet function under high shear stress as demonstrated by the IC50 value against SIPA was much lower than that of cyclocurcumin against platelet aggregation-induced by other endogenous agonists, including thrombin, collagen, and ADP (Fig. 1F, Suppl. Fig. 1). Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jff.2019.103462. 3.2. Cyclocurcumin inhibited shear stress-induced platelet activation through modulating vWF-GP Ib interaction High shear stress exerting the elevation of intraplatelet calcium level was significantly inhibited by treatment with cyclocurcumin (Fig. 2A). Moreover, cyclocurcumin remarkably reversed the degranulation of platelets by shear stress from both dense granules and αgranules, which could be determined by the significant reduction in serotonin release (Fig. 2B) and P-selectin expression (Fig. 2C). Furthermore, cyclocurcumin caused a significant attenuation on glycoprotein (GP) IIb/IIa activation and fibrinogen binding to the activated platelet in a concentration-dependent manner (Fig. 2D and E), reflecting the inhibition of platelet activation events under pathological shear stress by cyclocurcumin. Treatment with cyclocurcumin significantly blocked the engagement of von Willebrand factor (vWF) to platelets under high shear (Fig. 2F), which plays a fundamental role as the initial step of SIPA (Ikeda, Murata, & Goto, 1997). This effect was further confirmed when cyclocurcumin remarkably suppressed platelet aggregation stimulated by ristocetin (Fig. 2G), an exogenous platelet activator inducing the binding of vWF and GP Ib in the absence of shear stress (Dong et al., 2001). And cyclocurcumin also inhibited the adhesion of platelet to the vWF-coated surface in the flow chambers in arterial high shear stress (Fig. 2H). 3.3. Cyclocurcumin prevented thrombus formation without inducing cytotoxicity nor prolonging blood clotting time The effects of cyclocurcumin on platelet function was not mediated by non-specific cytotoxicity as no effects on lactate dehydrogenase (LDH) leakage could be observed up to a concentration of 250 µM (Fig. 3A). Cyclocurcumin neither influenced on blood clotting time as determined by prothrombin time (PT) and activated partial thromboplastin time (aPTT) up to a concentration of 250 µM (Fig. 3B). Interestingly, cyclocurcumin significantly inhibited in vitro thrombus formation, as could be determined by the remarkably reduced number and size of the formed thrombi, in a concentration-dependent manner (Fig. 3C). Most importantly, cyclocurcumin exhibited a much stronger prevention effect on thrombus formation than curcumin. At the concentration of 10 µM, cyclocurcumin attenuated formed thrombi by 62.2% while curcumin did not show any significant inhibition (Fig. 3C), highlighting the potent antithrombotic effects of cyclocurcumin.
5. Conclusion In addition to our previous study on the anticontractile effects of cyclocurcumin (Kim, 2017), this study revealed the novel and potent antiplatelet and antithrombotic effects of cyclocurcumin, which is selective to pathological high shear stress condition through modulating vWF - platelet interaction. Further study is necessary to re-evaluate and elucidate the anti-thrombotic effects of cyclocurcumin and its potential applications for the prevention and treatment of cardiovascular diseases. 6. Ethics statements With the approval from the Ethics Committee of Health Service Center at Seoul National University, human blood was obtained from healthy male volunteer donors.
4. Discussion Owing to the crucial role of platelets in thrombogenesis progression (Jackson, 2007; Palomo et al., 2008), antiplatelet therapy has become an important pharmacotherapeutic strategy for prevention and treatment cardiovascular disorders, one of the most leading mortality cause globally (Michelson, 2010). Conventionally, physiological agonists, such as thrombin, collagen, ADP, or arachidonic acid, are employed for exploring novel drugs inhibiting platelet activation (McEwen, 2015; Tsoupras, Zabetakis, & Lordan, 2019). However, contribution of physical activator, high shear stress, which is critical in pathological thrombosis (Kamada et al., 2017; Kroll et al., 1996), has not been well studied. Here, we newly demonstrated the anti-SIPA effects of C. longa extracts which might explain the beneficial effects of C. longa in
Author contributions T.N. designed the experiments, analyzed the data and wrote the manuscript; K.K., Y.B., G-J.A., and O-N.B analyzed the data, K-M.L. analyzed the data and edited the manuscript. J-H.C. supervised the study. All authors have approved the final version of the manuscript. Declaration of Competing Interest All the authors declared that there is no conflict of interest. 6
Journal of Functional Foods 61 (2019) 103462
T. Ngo, et al.
Acknowledgements
Libby, P. (2012). Inflammation in atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology, 32(9), 2045–2051. https://doi.org/10.1161/ATVBAHA.108. 179705. McEwen, B. J. (2015). The influence of herbal medicine on platelet function and coagulation: A narrative review. Seminars in Thrombosis and Hemostasis, 41(3), 300–314. https://doi.org/10.1055/s-0035-1549089. McFadyen, J. D., Schaff, M., & Peter, K. (2018). Current and future antiplatelet therapies: Emphasis on preserving haemostasis. Nature Reviews Cardiology, 15(3), 181–191. https://doi.org/10.1038/nrcardio.2017.206. Michelson, A. D. (2010). Antiplatelet therapies for the treatment of cardiovascular disease. Nature Reviews Drug Discovery, 9(2), 154–169. https://doi.org/10.1038/ nrd2957. Nieswandt, B., Pleines, I., & Bender, M. (2011). Platelet adhesion and activation mechanisms in arterial thrombosis and ischaemic stroke. Journal of Thrombosis and Haemostasis, 9(Suppl 1), 92–104. https://doi.org/10.1111/j.1538-7836.2011. 04361.x. Palomo, I., Toro, C., & Alarcon, M. (2008). The role of platelets in the pathophysiology of atherosclerosis (Review). Molecular Medicine Reports, 1(2), 179–184. https://doi.org/ 10.3892/mmr.1.2.179. Pei, H., Yang, Y., Cui, L., Yang, J., Li, X., Yang, Y., & Duan, H. (2016). Bisdemethoxycurcumin inhibits ovarian cancer via reducing oxidative stress mediated MMPs expressions. Scientific Report, 6, 28773. https://doi.org/10.1038/ srep28773. Prakash, P., Misra, A., Surin, W. R., Jain, M., Bhatta, R. S., Pal, R., ... Dikshit, M. (2011). Anti-platelet effects of Curcuma oil in experimental models of myocardial ischemiareperfusion and thrombosis. Thrombosis Research, 127(2), 111–118. https://doi.org/ 10.1016/j.thromres.2010.11.007. Shah, B. H., Nawaz, Z., Pertani, S. A., Roomi, A., Mahmood, H., Saeed, S. A., & Gilani, A. H. (1999). Inhibitory effect of curcumin, a food spice from turmeric, on plateletactivating factor- and arachidonic acid-mediated platelet aggregation through inhibition of thromboxane formation and Ca2+ signaling. Biochemical Pharmacology, 58(7), 1167–1172. https://doi.org/10.1016/S0006-2952(99)00206-3. Sheu, M. J., Lin, H. Y., Yang, Y. H., Chou, C. J., Chien, Y. C., Wu, T. S., & Wu, C. H. (2013). Demethoxycurcumin, a major active curcuminoid from Curcuma longa, suppresses balloon injury induced vascular smooth muscle cell migration and neointima formation: An in vitro and in vivo study. Molecular Nutrition & Food Research, 57(9), 1586–1597. https://doi.org/10.1002/mnfr.201200462. Srivastava, K. C. (1989). Extracts from two frequently consumed spices–cumin (Cuminum cyminum) and turmeric (Curcuma longa)–inhibit platelet aggregation and alter eicosanoid biosynthesis in human blood platelets. Prostaglandins, Leukotrienes & Essential Fatty Acids, 37(1), 57–64. https://doi.org/10.1016/0952-3278(89)90187-7. Srivastava, R., Dikshit, M., Srimal, R. C., & Dhawan, B. N. (1985). Anti-thrombotic effect of curcumin. Thrombosis Research, 40(3), 413–417. https://doi.org/10.1016/00493848(85)90276-2. Tsoupras, A., Lordan, R., & Zabetakis, I. (1985). Inflammation, not cholesterol, is a cause of chronic disease. Nutrients, 10(5), https://doi.org/10.3390/nu10050604. Tsoupras, A., Zabetakis, I., & Lordan, R. (2019). Platelet aggregometry assay for evaluating the effects of platelet agonists and antiplatelet compounds on platelet function in vitro. MethodsX, 6, 63–70. https://doi.org/10.1016/j.mex.2018.12.012. Wongcharoen, W., & Phrommintikul, A. (2009). The protective role of curcumin in cardiovascular diseases. International Journal of Cardiology, 133(2), 145–151. https:// doi.org/10.1016/j.ijcard.2009.01.073.
This work was supported by the National Research Foundation of Korea (NRF) grant (MSIT; 2018R1A5A2025286) funded by the Korea Government. References Ahmed, T., & Gilani, A. H. (2014). Therapeutic potential of turmeric in Alzheimer's disease: Curcumin or curcuminoids? Phytotherapy Research, 28(4), 517–525. https://doi. org/10.1002/ptr.5030. Ammon, H. P., & Wahl, M. A. (1991). Pharmacology of Curcuma longa. Planta Medica, 57(1), 1–7. https://doi.org/10.1055/s-2006-960004. Cattaneo, M., Lombardi, R., Bettega, D., Lecchi, A., & Mannucci, P. M. (1993). Shearinduced platelet aggregation is potentiated by desmopressin and inhibited by ticlopidine. Arteriosclerosis and Thrombosis, 13(3), 393–397. Dong, J. F., Berndt, M. C., Schade, A., McIntire, L. V., Andrews, R. K., & Lopez, J. A. (2001). Ristocetin-dependent, but not botrocetin-dependent, binding of von Willebrand factor to the platelet glycoprotein Ib-IX-V complex correlates with sheardependent interactions. Blood, 97(1), 162–168. Ghosh, S., Banerjee, S., & Sil, P. C. (2015). The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update. Food and Chemical Toxicology, 83, 111–124. https://doi.org/10.1016/j.fct.2015.05.022. Goel, A., Kunnumakkara, A. B., & Aggarwal, B. B. (2008). Curcumin as “Curecumin”: From kitchen to clinic. Biochemical Pharmacology, 75(4), 787–809. https://doi.org/ 10.1016/j.bcp.2007.08.016. Ikeda, Y., Murata, M., & Goto, S. (1997). Von Willebrand factor-dependent shear-induced platelet aggregation: Basic mechanisms and clinical implications. Annals of the New York Academy of Sciences, 811, 325–336. https://doi.org/10.1111/j.1749-6632.1997. tb52012.x. Jackson, S. P. (2007). The growing complexity of platelet aggregation. Blood, 109(12), 5087–5095. https://doi.org/10.1182/blood-2006-12-027698. Kamada, H., Imai, Y., Nakamura, M., Ishikawa, T., & Yamaguchi, T. (2017). Shear-induced platelet aggregation and distribution of thrombogenesis at stenotic vessels. Microcirculation, 24(4), https://doi.org/10.1111/micc.12355. Kim, K., Bae, O. N., Lim, K. M., Noh, J. Y., Kang, S., Chung, K. Y., & Chung, J. H. (2012). Novel antiplatelet activity of protocatechuic acid through the inhibition of high shear stress-induced platelet aggregation. Journal of Pharmacology and Experimental Therapeutics, 343(3), 704–711. https://doi.org/10.1124/jpet.112.198242. Kim, K., et al. (2017). Cyclocurcumin, an antivasoconstrictive constituent of Curcuma longa (Turmeric). Journal of Natural Product, 80(1), 196–200. https://doi.org/10. 1021/acs.jnatprod.6b00331. Kroll, M. H., Hellums, J. D., McIntire, L. V., Schafer, A. I., & Moake, J. L. (1996). Platelets and shear stress. Blood, 88(5), 1525–1541. http://www.bloodjournal.org/content/ 88/5/1525. Lantz, R. C., Chen, G. J., Solyom, A. M., Jolad, S. D., & Timmermann, B. N. (2005). The effect of turmeric extracts on inflammatory mediator production. Phytomedicine, 12(6–7), 445–452. https://doi.org/10.1016/j.phymed.2003.12.011. Li, Z., Delaney, M. K., O'Brien, K. A., & Du, X. (2010). Signaling during platelet adhesion and activation. Arteriosclerosis, Thrombosis, and Vascular Biology, 30(12), 2341–2349. https://doi.org/10.1161/ATVBAHA.110.207522.
7