http://informahealthcare.com/plt ISSN: 0953-7104 (print), 1369-1635 (electronic) Platelets, Early Online: 1–8 ! 2013 Informa UK Ltd. DOI: 10.3109/09537104.2013.863856

ORIGINAL ARTICLE

Hinokitiol is a novel glycoprotein VI antagonist on human platelets Wan-Jung Lu1*, Ming-Ping Wu1,2*, Kuan-Hung Lin1,3, Yu-Chen Lin1, Hsiu-Chu Chou4, & Joen-Rong Sheu1 Department of Pharmacology and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan, 2Department of Obstetrics and Gynecology, Chi-Mei Medical Center, Tainan, Taiwan, 3Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, and 4 Department of Anatomy, Taipei Medical University, Taipei, Taiwan

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Abstract

Keywords

Hinokitiol (4-isopropyl-tropolone) is a bioactive compound with various pharmacological activities that is found in the wood of cupressaceous plants. Platelet activation plays an important role in thrombogenesis. In our previous study, hinokitiol specifically inhibited collagen-induced platelet aggregation ex vivo and prolonged thrombogenesis in vivo. The glycoprotein (GP) VI and integrin a2b1 are major collagen receptors that mediate platelet adhesion and aggregation. In our current study, we investigated which of these collagen receptors is involved in the hinokitiol-mediated inhibition of platelet activation. Treatment with 2–100 mM hinokitiol caused a dose-dependent right, parallel shift in the collagen concentration–response curve (0.5–10 mg/ml), with no change in the maximal responses. Furthermore, hinokitiol inhibited platelet aggregation and relative [Ca2þ]i mobilization stimulated by convulxin, an agonist of GP VI, but not by aggretin, an agonist of integrin a2b1, indicating that hinokitiol mediates the inhibition of platelet activation through GP VI, rather than through integrin a2b1. Hinokitiol also specifically inhibited the convulxin-mediated activation of protein kinase C, phospholipase Cg2, Akt, mitogen-activated protein kinases, and Lyn. Hinokitiol markedly diminished the co-immunoprecipitation of GP VI-bound Lyn after convulxin stimulation. In conclusion, hinokitiol, an antagonist of collagen GP VI may represent a novel antiplatelet drug for the prevention of thrombi associated with coronary and cerebral artery diseases.

Collagen receptors, convulxin, glycoprotein VI, hinokitiol, platelet activation

Introduction Platelets are anuclear blood cells that play a central role in hemostatic processes and wound healing, but are also involved in the pathophysiology of various diseases, such as stroke, atherosclerosis, and arterial thrombosis. When vessels are damaged, collagen promotes the initial adhesion of platelets to the site of injury. Activated platelets then recruit circulating platelets through the binding of fibrinogen to integrin aIIbb3 [1]. Integrin aIIbb3 is important for platelet–platelet interactions [1], and the binding of fibrinogen to integrin aIIbb3 stimulates tyrosine phosphorylation of the b3 subunit, which is critical for clot retraction and the formation of a firm platelet plug [2]. Platelet collagen receptors are grouped on the basis of their interaction with collagen. Glycoprotein VI (GP VI), integrin a2b1, and CD36 bind collagen directly, whereas GP Iba and integrin aIIbb3 interact with collagen-bound von Willebrand factor [3]. Platelet GP VI is the major platelet collagen receptor in the formation of platelet aggregates on collagen surfaces under blood flow [4]. Integrin a2b1 is also a major collagen receptor on both endothelial cells and platelets [3]. Hinokitiol (4-isopropyl-tropolone; b-thujaplicin) is a bioactive tropolone-related compound found in the wood of cupressaceous *These authors contributed equally in this work. Correspondence: Dr Joen-Rong Sheu, 250 Wu-Hsing St., Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 110, Taiwan. Tel: +886-2-27361661-3199. Fax: +886-2-27390450. E-mail: [email protected]

History Received 8 July 2013 Revised 16 October 2013 Accepted 5 November 2013 Published online 27 November 2013

plants, and has various pharmacological activities, including antimicrobial, anticancer, and anti-inflammatory properties [5–7]. Hinokitiol has been shown to suppress tumor growth by inhibiting cell proliferation and inducing cell differentiation and apoptosis in various carcinoma cell lines [6, 8, 9]. Hinokitiol has also been shown to regulate immune cell function by inhibiting the production of tumor necrosis factor-a from lipopolysaccharide-stimulated macrophages through the inhibition of nuclear factor-B activity [10]. We demonstrated previously that hinokitiol specifically inhibits collagen-induced platelet activation, as evidenced by the inhibition of phospholipase C (PLC)g2 activation and free radical formation in washed human platelets. It did not, however, inhibit platelet activation mediated by other agonists, including thrombin, arachidonic acid, and adenosine diphosphate (ADP) [11]. Thus, hinokitiol specifically antagonizes collagen-mediated platelet activation. In our current study, we investigated the mechanisms involved in the hinokitiol-mediated inhibition of collagen-induced platelet activation.

Methods Materials Hinokitiol (99%), type I collagen, heparin, and prostaglandin E1 (PGE1) were purchased from Sigma-Aldrich (St. Louis, MO). The Fura 2-AM was purchased from Molecular Probes (Eugene, OR). The protein A/G plus-agarose, the anti-phospho-p38 mitogenactivated protein kinase (MAPK) Ser182 monoclonal antibody (mAb), and the anti-Lyn polyclonal antibody (pAb) were

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purchased from Santa Cruz (Santa Cruz, CA). The anti-p38 MAPK, anti-phospho-c-Jun N-terminal kinase (JNK) (Thr183/ Tyr185), and anti-p44/p42 extracellular signal-regulated kinase (ERK1/2) mAbs and the anti-PLCg2, anti-phospho (Tyr759) PLCg2, anti-phospho (Ser) protein kinase C (PKC) substrate, anti-SPAK/JNK, and anti-phospho-ERK1/2 (Thr202/Tyr204) pAbs were purchased from Cell Signaling (Beverly, MA). The antiphospho Akt (Ser473) and anti-Akt mAbs and the anti-GP VI pAb were purchased from Biovision (Mountain View, CA). The antia-tubulin mAb was purchased from NeoMarkers (Fremont, CA). The Hybond-P polyvinylidene difluoride (PVDF) membrane, the enhanced chemiluminescence (ECL) Western blotting detection reagent, the horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG), and the sheep anti-mouse IgG were purchased from Amersham (Buckinghamshire, UK). The hinokitiol was dissolved in 0.5% dimethyl sulfoxide (DMSO) and stored at 4  C.

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the platelets were immediately solubilized in 200 ml of lysis buffer. Samples containing 80 mg of protein were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% acrylamide gel, and the protein bands were electrotransferred to PVDF membranes using a Bio-Rad semidry transfer unit (Hercules, CA). The blots were blocked with TBST (10 mM Tris-base, 100 mM NaCl, and 0.01% Tween 20) containing 5% BSA for 1 hour, and probed with the various primary antibodies. The membranes were incubated with HRPconjugated anti-mouse IgG or anti-rabbit IgG (diluted 1:3000 in TBST) for 1 hour. Immunoreactive bands were detected using an ELC system. The ratios of the semiquantitative results were obtained by scanning the reactive bands and quantifying the optical density using a videodensitometer and the Bio-profil Biolight software, Version V2000.01 (Vilber Lourmat, Marnela-Valle´e, France). Immunoprecipitation

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Platelet aggregation Our study was approved by the Institutional Review Board of Taipei Medical University, and conformed to the directives of the Helsinki Declaration. All participants have provided their written informed consent to participate in this study. Human platelet suspensions were prepared as previously described [12]. The blood was collected from healthy human volunteers who had taken no medication during the preceding 2 weeks, and was mixed with acid-citrate-dextrose (ACD) solution (9:1; v/v). After centrifugation at 120 g for 10 min, the supernatant (platelet-rich plasma (PRP)) was supplemented with PGE1 (0.5 mM) and heparin (6.4 IU/ml), and then incubated for 10 min at 37  C and centrifuged at 500 g for 10 min. The platelet pellets were suspended in 5 ml of Tyrode’s solution, pH 7.3 (containing (mM) NaCl 11.9, KCl 2.7, MgCl2 2.1, NaH2PO4 0.4, NaHCO3 11.9, and glucose 11.1), then apyrase (1.0 U/ml), PGE1 (0.5 mM), and heparin (6.4 IU/ml) were added, and the mixture was incubated for 10 min at 37  C. After centrifugation of the suspensions at 500 g for 10 min, the washing procedure was repeated. The washed platelets were finally suspended in Tyrode’s solution containing bovine serum albumin (BSA) (3.5 mg/ml) and adjusted to about 4.5  108 platelets/ml. A Lumi-Aggregometer (Payton Associates, Scarborough, ON, Canada) was used to measure platelet aggregation, as previously described [12]. Platelet suspensions (3.6  108 cells/ml) were pre-incubated with various concentrations of hinokitiol or an identical volume of solvent control (0.5% DMSO) for 3 min before the addition of the agonists, including collagen, convulxin, and aggretin. The reaction was incubated for 6 min, and the extent of aggregation was expressed in light-transmission units. Measurement of relative intracellular calcium mobilization by Fura 2-AM fluorescence The citrated whole blood was centrifuged at 120  g for 10 min. The supernatant was incubated with 5 mM Fura 2-AM for 1 hour. Human platelets were prepared as described above. The platelet suspensions were adjusted to 1 mM Ca2þ. Relative cytoplasmic calcium ion concentration ([Ca2þ]i) was measured using a Jasco CAF 110 fluorescence spectrophotometer (Tokyo, Japan) with excitation wavelengths of 340 and 380 nm and an emission wavelength of 500 nm [12]. Immunoblotting Washed platelets (1.2  109 cells/ml) were pre-incubated with 1 or 2 mM hinokitiol or 0.5% DMSO for 3 min, and the agonists were added to trigger platelet activation. The reaction was stopped, and

Washed platelets (1  109 cells/ml) were stimulated with 100 ng/ ml convulxin for 0.5 to 1 min, and were lysed in an immunoprecipitation buffer as described previously [13]. An equal amount of protein from each supernatant was pre-cleared with protein A/G-agarose-conjugated beads for 2 hour. Samples were then rotated overnight with 1 mg/ml anti-Lyn primary antibody or control IgG. On the following day, 20 ml of the beads were added, and samples were mixed by rotation overnight. The immunoprecipitates were washed three times, and analyzed by immunoblotting as described. Data analysis The experimental results are expressed as the means  standard error of the mean (SEM) and are accompanied by the number of observations (n). The values of n refer to the number of experiments, each performed with blood from different donors. The results of experiments were evaluated using an analysis of variance (ANOVA). If the ANOVA indicated significant differences among the group means, each group was compared using the Student–Newman–Keuls method. The results of comparisons with a p value less than 0.05 were considered statistically significant. All statistical analyses were performed using the SAS, Version 9.2 software package (SAS Inc., Cary, NC).

Results Hinokitiol on platelet aggregation and relative [Ca2þ]i mobilization stimulated by GP VI and integrin a2b1 agonists Although 0.5–2 mM hinokitiol inhibited platelet aggregation and the ATP-release reaction in platelets stimulated with 1 mg/ml collagen, concentrations of hinokitiol up to 50 mM did not significantly inhibit platelet aggregation stimulated by 1 mM U46619, 20 mM ADP, 0.05 U/ml thrombin, or 60 mM arachidonic acid [11]. As shown in Figure 1(A), we demonstrated that treatment with 2, 10, or 100 mM hinokitiol produced a dosedependent right, parallel shift in the collagen concentration– response curve (0.5–10 mg/ml), with no change in the maximal responses. These results indicate that hinokitiol may competitively antagonize collagen activity in platelets. Integrin a2b1 and GP VI are major collagen receptors that mediate platelet adhesion and aggregation. Treatment with 1 or 2 mM hinokitiol markedly inhibited platelet aggregation and relative [Ca2þ]i mobilization stimulated by 100 ng/ml convulxin, an agonist of GP VI, which is purified from the venom of Crotalusdurissus terrificus [14], but not by aggretin (3.6 mg/ml), an agonist of integrin a2b1, which is purified from the venom of Calloselasma rhodostoma

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Figure 1. Effects of hinokitiol on platelet aggregation and relative [Ca2þ]i mobilization in washed human platelets. Washed platelets (3.6  108 cells/ ml) were pre-incubated with solvent control (0.5% DMSO, *) or hinokitiol (2 mM, œ; 10 mM, 5; and 100 mM, ^), followed by the addition of 0.5–10 mg/ml collagen, 100 ng/ml convulxin, or 3.6 mg/ml aggretin to trigger (A and B) platelet aggregation or (C) relative [Ca2þ]i mobilization. The data in A are presented as the means  SEM (n ¼ 4). Profiles in B and C are representative of four separate experiments.

(Figure 1B and C) [15], even treatment with 10 mM hinokitiol (data not shown). Hinokitiol (1 mM) also markedly inhibited ATPrelease reaction stimulated by convulxin, but not by aggretin (data not shown). These data indicate that hinokitiol inhibits platelet activation through GP VI but not integrin a2b1. Hinokitiol inhibits convulxin-induced activation of PKC, PLCg2, Akt, and MAPK The stimulation of platelets by various agonists induces PKC activation and the subsequent phosphorylation of p47 proteins

[16]. As shown in Figure 2(A), a protein with an apparent molecular weight similar to p47 (47-kDa, pleckstrin) was predominately phosphorylated stimulated by 1 mg/ml collagen, 100 ng/ml convulxin, or 3.6 mg/ml aggretin, compared with the protein profile of non-activated platelets. Treatment with 2 mM hinokitiol reduced the amount of the phosphorylated 47-kDa protein in both the collagen- and the convulxin-activated platelets, but did not reduce the amount of phosphorylated 47-kDa protein in the aggretin-activated platelets (Figure 2A). Furthermore, hinokitiol (2 mM) did not significantly influence outside-in signaling of PKC activation (p47 phosphorylation) initiated

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by platelet interaction with immobilized fibrinogen (data not shown). The PLCg2 hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate the secondary messengers, inositol 1,4,5trisphosphate (IP3), and diacylglycerol (DAG). The DAG activates PKC, inducing protein phosphorylation and ATP release. Thus, PLCg2 plays an important role as an upstream regulator of PKC activation in activated platelets [16]. As shown in Figure 2(B) and (C), after treatment with 2 mM hinokitiol, both the PLCg2 and Akt phosphorylation were obviously reduced in the convulxin-stimulated platelets. Furthermore, the MAPKs control major cellular responses in eukaryotic organisms, and contribute to cell proliferation, migration, differentiation, and apoptosis. In our current study, we also found that 2 mM hinokitiol significantly inhibited the convulxin-mediated phosphorylation of p38 MAPK, ERK1/2, and JNK1/2 (Figure 3). These results collectively demonstrated that hinokitiol specifically antagonizes convulxin-mediated intracellular signaling events in platelet activation. Inhibition of GP VI binding of Lyn by hinokitiol To further evaluate the inhibitory activity of hinokitiol in Lynmediated association with GP VI in human platelets, the co-immunoprecipitation was performed by using the anti-GP VI Ab. As shown in Figure 4(A), platelets in the resting state or stimulated by 100 ng/ml convulxin were immunoprecipitated with anti-Lyn Ab, and the Lyn could co-immunoprecipitate with GP VI in the resting state, following stimulation with 100 ng/ml convulxin, the GP VI-bound Lyn increased in a time-dependent manner, reaching a maximum at 0.5 min. Treatment with 2 mM hinokitiol significantly diminished the co-immunoprecipitation of GP VI-bound Lyn at 0.5 min after convulxin stimulation (Figure 4B). Furthermore, either bead only or control IgG did not significantly co-immunoprecipitate with GP VI as compared with the anti-Lyn Ab in 100 ng/ml convulxin-stimulated platelets (Figure 4B). Regulation of Lyn phosphorylation by hinokitiol in convulxin-stimulated platelets To further establish the regulatory role of hinokitiol in the GP VI binding of Lyn in platelets, the anti-phospho Lyn Ab was used to assess the kinase activation state induced by convulxin treatment. As shown in Figure 5(A), stimulation with 100 ng/ml convulxin resulted in a time-dependent increase of Lyn phosphorylation within 0.5–2 min following convulxin treatment. In addition, the phosphorylation of Lyn was markedly inhibited in a concentration-dependent manner using 1 and 2 mM hinokitiol (Figure 5B).

Discussion

Figure 2. Inhibitory effects of hinokitiol on protein kinase C (PKC), phospholipase Cg2 (PLCg2), and Akt activation in human platelets. Washed platelets were pre-incubated with 0.5% DMSO or 2 mM hinokitiol, followed by treatment with 1 mg/ml collagen, 100 ng/ml convulxin, or 3.6 mg/ml aggretin to induce platelet activation. Platelets were collected, and subcellular extracts were analyzed for (A) PKC (p47 phosphorylation) and (B) PLCg2 and (C) Akt activation. Profiles in A are representative of five separate experiments. The data in B and C are presented as the means  SEM (n ¼ 4) **p ¼ 0.005 (B) and ***p ¼ 0.001 (C), compared with the control (resting state) platelets; ##p ¼ 0.008 (B) and ##p ¼ 0.002 (C), compared with the platelets receiving the convulxin treatment only.

In our previous study, hinokitiol specifically inhibited collageninduced platelet activation ex vivo and effectively inhibited thrombogenesis in vivo [11]. Our current study showed that hinokitiol specifically blocks convulxin-stimulated platelet activation associated with PLCg2-PKC, MAPK, and AKT activation. The stimulation of platelets by collagen or snake-venom toxin convulxin each results in the PLCg2-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and concomitant formation of IP3 and DAG [17] (Figure 6). The DAG activates PKC, inducing p47 (pleckstrin) phosphorylation. In our current study, hinokitiol inhibited the convulxin-induced phosphorylation of both PLCg2 and p47, but did not inhibit the phosphorylation of these proteins when platelets were stimulated by aggretin, indicating that hinokitiol is a specific antagonist of platelet GP VI on platelet membrane.

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Figure 4. Regulation of glycoprotein (GP) VI-bound Lyn by hinokitiol. Washed platelets (1  109/ml) were pre-incubated (A) without or (B) with 2 mM hinokitiol for 3 min, and stimulated using 100 ng/ml convulxin for (A) 0.5–1 min or (B) 0.5 min, respectively. The platelets were lysed, and proteins were co-immunoprecipitated (IP) with bead only, anti-Lyn antibody or control IgG (B). The immunoprecipitates were analyzed by immunoblotting (WB) using an anti-GP VI primary antibody. The profiles are representative of four separate experiments.

Figure 3. Effects of hinokitiol on p38 MAPK, ERK1/2, and JNK1/2 phosphorylation in convulxin-stimulated human platelets. Washed platelets (1.2  109 cells/ml) were pre-incubated with 0.5% DMSO or 2 mM hinokitiol, followed by treatment with 100 ng/ml convulxin to induce platelet activation. Platelets were collected, and subcellular extracts were analyzed for (A) p38 MAPK, (B) ERK1/2, and (C) JNK1/2 phosphorylation. The data are presented as the means  SEM (n ¼ 4). **p ¼ 0.002 (A) and ***p ¼ 0.001 (B and C), compared with the control (resting state) platelets; ##p ¼ 0.003 (A) and ###p ¼ 0.001 (B and C), compared with the platelets receiving the convulxin treatment only.

The three major subgroups of MAPKs that have been identified in platelets consist of ERK2, JNK1, and p38 MAPK [18]. The pathophysiological roles of ERK1/2 and JNK1/2 are unclear, but previous studies have shown that they may suppress integrin aIIbb3 activation or negatively regulate platelet activation [17]. In contrast, p38 MAPK provides a crucial signal for collagen-induced platelet aggregation. The stimulation of platelets by agonists results in Akt activation, and Akt is one of several downstream effectors of PI3-kinase [19]. However, the molecular mechanism involved in Akt activation in platelets has not been well established. When tissues are damaged, platelets adhere to exposed collagen, resulting in a change in platelet shape and the release of granules. Adhesion is partly dependent on the release of ADP and thromboxane A2 (TxA2), whereas aggregation is entirely dependent on the release of ADP and TxA2 [20]. The intercellular matrix protein collagen is present in the vascular subendothelium and vessel wall, and acts as a substrate for platelet adhesion. Collagen is also an endogenous platelet activator. Among the platelet receptors known to interact directly with collagen, integrin a2b1 (GP Ia/IIa) and GP VI [15] appear to play key roles. The GP VI is a 60 - to 65-kDa type I transmembrane GP of the immunoglobulin superfamily [21]. The GP VI ligands, such as collagen and convulxin, induce receptor clustering that facilitates the phosphorylation of the tandem tyrosines found in the immunoreceptor tyrosine-based activation motifs (ITAM) of the noncovalently associated Fcg-chain receptor (FcRg) adaptor by Src-family tyrosine kinases, such as Fyn and Lyn [22, 23] (Figure 6). The Src was the first proto-oncogenic non-receptor tyrosine kinase characterized in humans. Tyrosine kinases, such as Fyn and Lyn, are involved in GP VI-dependent activation, and may phosphorylate the FcRg [24]. The Fyn and Lyn FcRg adaptors were shown to bind to the proline-rich domain of the GP VI cytoplasmic tail in platelets [25], suggesting that the GP VI-dependent activation mechanism may be similar to that

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Figure 5. The influence of Lyn phosphorylation by hinokitiol in convulxin-stimulated human platelets. Washed platelets (1  109/ml) were pre-incubated (A) without or (B) with 1 or 2 mM hinokitiol for 3 min, and stimulated using 100 ng/ml convulxin for (A) 0.5–5 min and (B) 2 min, respectively. The platelets were collected, and subcellular extracts were analyzed for Lyn phosphorylation. The data in A are presented as the means  SEM (n ¼ 4), *p ¼ 0.01, **p ¼ 0.001, and ***p ¼ 0.001, compared with the control (resting state) platelets; the data in B are presented as the means  SEM (n ¼ 4), ***p ¼ 0.001, compared with the control (resting state) platelets; #p ¼ 0.035 and ###p ¼ 0.001, compared with the platelets receiving the convulxin treatment only.

Figure 6. Hypothesis of inhibitory mechanisms of hinokitiol in platelet activation. Hinokitiol binds to glycoprotein (GP) VI and activates collagenmediated signal events, such as Lyn and phospholipase Cg2 (PLCg2)-diacylglycerol (DAG)-protein kinase C (PKC), and MAPKs or Akt pathway to ultimately trigger platelet activation. DTS, dense tubular system; IP3, inositol 1,4,5-trisphosphate. ITAM, immunoreceptor tyrosine-based activation motifs.

of cytokine receptors. Receptor-bound tyrosine kinases, such as Src, phosphorylate the cytoplasmic tail of receptors upon binding. This phosphorylation initiates the signal transduction pathway through Syk kinases, linker activation of T-cells (LAT), SPL-76, and PLC [24]. The cross-linking of the GP VI/FcRg complex in platelets places GP VI-bound Fyn or Lyn in close proximity to FcRg, facilitating the phosphorylation of FcRg ITAM. This triggers the

phosphorylation of downstream signals, including LAT, leading to the activation of a kinase cascade, such as the PLCg2PKC-mediated pathways. In addition, integrin a2b1 is also a major collagen receptor on endothelial cells and platelets. Many intracellular signaling cascades, including tyrosine phosphorylation and matrix remodeling pathways, are activated following integrin a2b1-mediated cell adhesion to collagen [26]. Recent findings suggest that integrin a2b1 and GP VI may contribute

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to the overall processes of platelet adhesion and activation [14, 27, 28]. The stimulation of GP VI induces platelet activation, leading to secretion and inside-out signaling through integrins a2b1 and aIIbb3, which stabilize platelet interactions with the wall of damaged blood vessels and mediate platelet aggregation [28]. We also found that GP VI binding of collagen directly activates Lyn, and that GP VI-bound Lyn in resting platelets is in an active state before receptor ligation (Figure 5). Platelets deficient in GP VI have been shown to be unresponsive to collagen-induced aggregation and adhesion in vitro [29]. Massberg et al. [30] also demonstrated in mice that GP VI is a major determinant of arterial thrombus formation. In addition, Stephens et al. [31] also found that direct activation of GP VI by collagen or convulxin induced a marked cleavage of metalloproteinase-dependent GP VI, which may cause down-regulation of platelet aggregation stimulated by collagen. Thus, platelet collagen receptors, especially GP VI, have emerged as highly interesting targets for novel antiplatelet drugs. At present, platelet activation is mainly employed as a valuable research tool, but it appears to have a number of potential clinical implications. Measurement of the markers of platelet activation could be useful in identifying patients with a variety of cardiovascular disorders at high risk of thromboembolism [32]. An essential role of platelets in cardiovascular diseases and cardiac mortality has been demonstrated in several landmark clinical trials in which inhibition of platelet activation and/or aggregation, improved cardiovascular outcomes [33]. Thus, in the present study, the inhibitory effects of hinokitiol, as an antagonist of collagen GP VI in platelet activation may have a potential to be clinically informative in cardiovascular diseases.

Conclusions In conclusion, hinokitiol acts as an antagonist of the collagen receptor GP VI on human platelets. However, our experiments did not rule out the possibility that other as-yet-unidentified mechanisms might be involved in hinokitiol-mediated inhibition of platelet activation. Our findings provide insight into the inhibitory mechanism of hinokitiol in specific signaling events in platelet activation. Hinokitiol may have therapeutic potential for the prevention of the pathologic thrombi associated with coronary and cerebral artery diseases.

Acknowledgements The authors thank Prof. Tur-Fu Huang (Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan) for his kindness to provide the convulxin and aggretin in this study. This work was supported by grants from the National Science Council, Taiwan (NSC100-2320-B-038-021-MY3, NSC101-2811-B-038-002, NSC1012811-B-038-006 and NSC102-2320-B-341-001-MY3), and the Chi-Mei Medical Center of Taipei Medical University (100CM-TMU-08); and Shin Kong Wu Ho-Su Memorial Hospital (SKH-8302-102-NDR-04). The funders had no role in study, data collection and analysis, decision to publish, or preparation of the manuscript.

Declaration of interest The authors declare that they have no conflicts of interest.

References 1. Jackson SP, Nesbitt WS, Kulkarni S. Signaling events underlying thrombus formation. J Thromb Haemost 2003;1:1602–1612. 2. Phillips DR, Nannizzi-Alaimo L, Prasad KS. Beta3 tyrosine phosphorylation in alphaIIbbeta3 (platelet membrane GP IIb-IIIa) outside-in integrin signaling. Thromb Haemost 2001;86:246–258. 3. Clemetson KJ, Clemetson JM. Platelet collagen receptors. Thromb Haemost 2001;86:189–197.

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Hinokitiol is a novel glycoprotein VI antagonist on human platelets.

Hinokitiol (4-isopropyl-tropolone) is a bioactive compound with various pharmacological activities that is found in the wood of cupressaceous plants. ...
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