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Multidrug resistance protein 4 (MRP4/ABCC4) regulates thrombus formation in vitro and in vivo Li-Ming Lien, Zhih-Cherng Chen, Chi-Li Chung, Ting-Lin Yen, Hou-Chang Chiu, Duen-Suey Chou, Shih-Yi Huang, Joen-Rong Sheu, WanJung Lu, Kuan-Hung Lin www.elsevier.com/locate/ejphar

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S0014-2999(14)00350-1 http://dx.doi.org/10.1016/j.ejphar.2014.05.001 EJP69277

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European Journal of Pharmacology

Received date: 12 August 2013 Revised date: 23 April 2014 Accepted date: 7 May 2014 Cite this article as: Li-Ming Lien, Zhih-Cherng Chen, Chi-Li Chung, Ting-Lin Yen, Hou-Chang Chiu, Duen-Suey Chou, Shih-Yi Huang, Joen-Rong Sheu, WanJung Lu, Kuan-Hung Lin, Multidrug resistance protein 4 (MRP4/ABCC4) regulates thrombus formation in vitro and in vivo, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2014.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

EJP-38619-R3

Multidrug resistance protein 4 (MRP4/ABCC4) regulates thrombus formation in vitro and in vivo

Li-Ming Liena,b,1, Zhih-Cherng Chenc,d,e,1, Chi-Li Chungf,g, Ting-Lin Yene, Hou-Chang Chiub,h, Duen-Suey Choue, Shih-Yi Huangi, Joen-Rong Sheue, Wan-Jung Lue,*, and Kuan-Hung Line,j,*

a

School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan, Department of Neurology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan, c Department of Cardiology, Chi-Mei Medical Center, Tainan City, Taiwan, d Department of Pharmacy, Chia Nan University of Pharmacy & Science, Tainan City, b

Taiwan, eDepartment of Pharmacology, and Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan, fDivision of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei, Taiwan, g School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan, hCollege of Medicine, Fu-Jen Catholic University, Taipei, Taiwan, i School of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan, and jCentral Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan

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These authors contributed equally to this work.

*Correspondence to: Dr. Kuan-Hung Lin, Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd., Taipei 111, Taiwan. Tel: +886-2-28332211 ext. 2436, Fax: +886-2-28383005, e-mail: [email protected]; Dr. Wan-Jung Lu, Graduate Institute of Medical Sciences, Taipei Medical University, 250 Wu-Hsing St., Taipei 110, Taiwan. Tel: +886-2-27361661 ext. 3201, Fax: +886-2-27390450, e-mail: [email protected].

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EJP-38619-R3 ABSTRACT The multidrug resistance protein 4 (MRP4) is a member of the ABCC subfamily of the adenosine triphosphate-binding cassette transporters that remove cyclic nucleotides from platelets and uptake ADP into dense granule in platelets. However, whether MRP4 directly involves platelet activation remains unclear. Thus, the aim of our study was to determine the detailed mechanisms underlying the regulation of MRP4 in platelet activation. Our results revealed that the MRP4 inhibitor MK571 inhibited collagen-induced platelet aggregation which was partially reversed by the PKA inhibitor H89, but not by the adenylyl cyclase (AC) inhibitor SQ22536 and the guanylyl cyclase (GC) inhibitor ODQ, suggesting that MK571 can prevent collagen-induced aggregation via a route independent of cyclic nucleotide production. In the present study, we found that MK571 inhibited collagen-induced ATP release and calcium mobilization. The phosphorylation of protein kinase C, JNK, and Akt was also inhibited by MK571, and electron spin resonance experiment showed that MK571 significantly reduced hydroxyl radical formation. Moreover, MK571 delayed platelet plug formation in vitro by a PFA-100 device, and delayed thrombus formation in mesenteric venules of mice irradiated by fluorescein sodium. However, previous studies have reported that MK571 also blocks MRP1 and leukotriene D4 (LTD4) receptor. Therefore, whether MK571 inhibits platelet activation through MRP1 or LTD4 receptor needs to be considered and further defined. In conclusion, in addition to blocking the transport of cyclic nucleotides, MRP4 inhibition may prevent thrombus formation in vitro and in vivo. Our findings also support the idea that MRP4 may represent a potential target for the development of novel therapeutic interventions for the treatment of thromboembolic disorders.

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EJP-38619-R3 Keywords MRP4, Platelet activation, Thrombus formation, Cyclic nucleotides

Abbreviations cPLA2, cytosolic phospholipase A2; ESR, electron spin resonance; HO, hydroxyl radical; MRP4, multidrug resistance protein 4; PKA, protein Kinase A; PKC, protein kinase C; PKG, protein kinase G; PRP, platelet-rich plasma; PGE1, prostaglandin E1; ROS, reactive oxygen species; TxA2, thromboxane A2.

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EJP-38619-R3 1. Introduction Blood platelets are known to play important roles in haemostatic processes, wound healing, inflammation, immunity, angiogenesis, atherosclerosis, and tumor progression (Li et al., 2010). The main physiological functions of platelets are to prevent blood loss and maintain vascular integrity. Thrombosis resulting from the dysregulation of platelet functions may contribute to a wide variety of cardiovascular diseases. Upon vascular injury, platelets are activated by von Willebrand factor and collagen, or by soluble platelet agonists, such as ADP, thrombin, and thromboxane A2 (TxA2). These agonists induce signal transduction via their respective receptors, subsequent platelet shape and granule secretion, finally leading to platelet activation. Platelets express several receptors, including collagen, thrombin, and TxA2 receptors. GPVI, a member of the immunoglobulin superfamily, is required for collagen-induced platelet activation (Li et al., 2010). When platelets are exposed to collagen, a signaling complex, including LAT, SLP-76, and Gads, activates PLCJ2, leading to PKC activation and Ca2+ release. Equally important, soluble platelet agonists, such as thrombin and TxA2, activate platelets via G-protein-coupled receptors (GPCRs), a family of 7-transmembrane domain receptors that transmit signals through heterotrimeric G proteins. These agonists activate PLCE-PKC/IP3pathway through Gq proteins, ultimately leading to platelet activation (Li et al., 2010). The multidrug resistance protein 4 (MRP4) is a member of the ABCC subfamily of the adenosine triphosphate (ATP)-binding cassette transporters, which are able to transport a range of endogenous molecules, including cyclic nucleotides (cAMP and cGMP) (Chen et al., 2001; Russel et al., 2008). It has been shown that high MRP4 is associated with poor prognosis in neuroblastoma (Norris et al., 2005), and MRP4 may

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EJP-38619-R3 regulate cancer cell proliferation, differentiation, and migration through cAMP and cGMP (Copsel et al., 2011; Sinha et al., 2013). In addition, the expression of MRP4 increases in pulmonary arteries from patients with idiopathic pulmonary arterial hypertension (PAH), and the inhibition of MRP4 may prevent PAH in a mice model (Hara et al., 2011). In human platelets, MRP4 is highly expressed in dense granules and, to a lesser extent, on the plasma membrane, where it may be involved in the storage of ADP in the granules and the release of ADP from platelets (Jedlitschky et al., 2004). This distribution of MRP4 may be altered under some pathologic conditions. Mattiello et al. (2011) found higher levels of MRP4 proteins in platelets of patients after coronary artery bypass grafting (CABG) surgery, in which the MRP4 preferentially localized at the plasma membrane and exported aspirin, attenuating the inhibitory effect of aspirin on cyclooxygenase-1. This phenomenon may be one of the major causes of aspirin resistance (Mattiello et al., 2011). Borgognone and Pulcinelli (2012) recently reported that the MRP4 inhibitor MK571 potentiated the inhibition of platelet activation by cAMP-elevating agent (forskolin) and cGMP-elevating agent (sodium nitroprusside) via enhancing both cAMP and cGMP concentrations. In addition, Niessen et al. (2010) found that the level of MRP4 mRNA was 465% higher in platelets than in megakaryocytic progenitor cells, and that MRP4 transcript increased during differentiation of the CD34+ cells towards megakaryocyte. These evidences reveal that MRP4 may play a regulatory role on platelet functions. Although the inhibition of MRP4 has been shown to increase the inhibitory effects of forskolin and sodium nitroprusside on platelet aggregation via enhancing the concentrations of cAMP and cGMP, whether MRP4 directly regulates platelet

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EJP-38619-R3 activation remains unclear. Therefore, we systemically investigate the role of MRP4 in platelet activation.

2. Materials and methods 2.1. Materials MK571, collagen (type I), luciferin-luciferase, 9,11-dideoxy-11,9-epoxymethanoprostaglandin (U46619), phorbol-12,13-dibutyrate (PDBu), 5,5-dimethyl-1 pyrroline N-oxide (DMPO), SQ22536, 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), H89, KT5823, and thrombin were purchased from Sigma (St. Louis, MO). Arachidonic acid (AA) was purchased from Chrono-Log (Havertown, PA). The Dade Behring PFA collagen/epinephrine (CEPI) and collagen/ADP (CADP) test cartridges were obtained from Siemens Healthcare (Marburg, Germany). The Fura 2-AM and fluorescein isothiocyanate (FITC) were purchased from Molecular Probes (Eugene, OR). The rabbit anti-phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182), rabbit anti-phospho-p44/p42 extracellular signal-regulated kinase (Erk) (Thr202/Tyr204), rabbit anti-c-Jun N-terminal kinase (JNK), rabbit anti-phospho-Akt (Ser473), and rabbit anti-phospho-PKC substrate (Ser) polyoclonal antibodies (pAbs) and the mouse anti-p38 MAPK, rabbit anti-Erk, rabbit anti-phospho JNK, and mouse anti-Akt monoclonal antibodies (mAbs) were purchased from Cell Signaling (Beverly, MA). The goat anti-pleckstrin pAb was purchased from GeneTex (Irvine, CA). The Hybond-P polyvinylidene difluoride (PVDF) membrane, the enhanced chemiluminescence 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

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EJP-38619-R3 MK571 was dissolved in dd-H2O, and stored at 4°C.

2.2. Platelet aggregation Washed human platelet suspensions were prepared as previously described (Sheu et al., 2000). Our study was approved by the Institutional Review Board of Shin Kong Wu Ho-Su Memorial Hospital (No. 20120710R), and conformed to the guidelines of the Helsinki Declaration. Participants provided informed consent before participating in our study. Blood was collected from healthy human volunteers who had taken no medication during the preceding 2 weeks, and was mixed with acid-citrate-dextrose solution. After centrifugation, the supernatant (platelet-rich plasma; PRP) was supplemented with 0.5 M prostaglandin E1 (PGE1) and 6.4 IU/ml heparin. Washed platelets were finally suspended in Tyrode’s solution containing 3.5 mg/ml bovine serum albumin (BSA). The final concentration of Ca2+ in Tyrode’s solution was 1 mM. A Lumi-Aggregometer (Payton Associates, Scarborough, ON, Canada) was used to measure platelet aggregation, as previously described (Sheu et al., 2000). Platelet suspensions (3.6 × 108 cells/ml) were pre-incubated with the various concentrations of MK571 (a MRP4 inhibitor) for 3 min before the addition of agonists. The reaction was allowed to proceed for 6 min, and the extent of aggregation was expressed in light-transmission units. For measuring ATP release, 20 l of a luciferin-luciferase mixture was added 1 min before the addition of agonists, and the amount of ATP released was compared to the control.

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EJP-38619-R3 2.3. Flow cytometry analysis Triflavin, an IIb3 disintegrin, was conjugated to FITC as previously described (Sheu et al., 1999). Platelet suspensions (1 × 106 cells/ml) were pre-incubated with 5 mM EDTA, 20 M MK571, or 50 M MK571 for 3 min, followed by the addition of 2 g/ml FITC-triflavin. Suspensions were assayed for FITC-labeled platelets by using a Beckman Coulter flow cytometer (Brea, CA). The data were collected from 50 000 platelets for each experimental group, and platelets were identified on the basis of their forward and orthogonal light-scattering profiles. All experiments were repeated at least 3 times.

2.4. Measurement of 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 M fura 2-AM for 1 h. Human platelets were prepared as described above. The platelet suspensions were adjusted to 1 mM Ca2+. The relative cytoplasmic calcium ion concentration ([Ca2+]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 (Sheu et al., 2000).

2.5. Immunoblotting Washed platelets (1.2 × 109 cells/ml) were pre-incubated with 20 M or 50 M MK571 for 3 min, followed by the addition of 1 g/ml collagen to trigger platelet activation. The reaction was stopped, and the platelets were immediately resuspended in 200 l of lysis buffer. Samples containing 80 g of protein were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide gel, and the proteins were electrotransferred to the PVDF membranes

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EJP-38619-R3 using a Bio-Rad semidry transfer unit (Hercules, CA). Blots were blocked with TBST (10 mM Tris-base, 100 mM NaCl, and 0.01% Tween 20) containing 5% BSA for 1 h, and probed with the various primary antibodies (1:1000 dilution). The membranes were incubated with a 1:3000 dilution of HRP-linked anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG in TBST for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence system. Ratios of the semiquantitative results were obtained by scanning the reactive bands and quantifying the optical density using a videodensitometer and Bio-profil Biolight software, version V2000.01 (Vilber Lourmat, Marne-la-Vallée, France).

2.6. Measurement of hydroxyl radicals by electron spin resonance spectrometry The electron spin resonance (ESR) spectrometry method was performed by using a Bruker EMX ESR spectrometer (Billerica, MA), as described previously (Chou et al., 2005). The platelet suspensions (3.6 × 108 cells/ml) were pre-incubated with MK571 (20 M or 50 M) for 3 min before the treatment of 1 g/ml collagen for 5 min. Thereafter, 100 M DMPO was added for the ESR analysis.

2.7. Platelet function assay (PFA-100) The PFA-100 System (Dade Behring, Marburg, Germany) was used to measure the platelet functions under high-shear conditions that mimic in vivo blood vessel injury (Jilma, 2001). A small volume (0.8 ml/cartridge) of blood samples collected in 3.8% sodium citrate was aspirated from the sample reservoir through the capillary, which exposed platelets to high shear flow condition (5000 to 6000s-1), and began to seal a small central aperture (147 Pm) within a collagen/epinephrine (CEPI) or collagen/ADP (CADP)-coated membrane. The closure time (CT) was defined as the

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EJP-38619-R3 time required to occlude the aperture by platelet plugs. The measurements are stopped at a maximum of 5 min, and the instrument will give a result > 300 s, if the CT is exceeded.

2.8. Fluorescein-induced thrombus formation in mesenteric microvessels of mice ICR mice (aged 5 wk) were purchased from BioLASCO (Hsinchu, Taiwan). All procedures were approved by Affidavit of Approval of Animal Use Protocol-Taipei Medical University (No. LAC-99-0175) and are in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, 1996). As described previously (Hsiao et al., 2005), mice were anesthetized (a mixture of 75% air and 25% O2 gases containing 3% isoflurane) and an external jugular vein was cannulated with a PE-10 for intravenous (IV) administration of the dye and drugs. An IV bolus of 15 Pg/kg fluorescein sodium was administered, followed 1 min later by a second IV bolus of 2.5 or 5 mg/kg MK571. Venules (30-40 Pm) were selected for irradiation at wavelengths below 520 nm to produce a microthrombus. The time lapse between the induction of thrombus formation and the cessation of blood flow (occlusion time) was recorded. At the end of experimental sections, animals were euthanized by carbon dioxide.

2.9. Data analysis The experimental results are expressed as the mean ± S.E.M. and are accompanied by the number of observations (n). Paired Student’s t-test was used to determine significant differences of the closure time by PFA-100 and the occlusion time in mice. Other experiments were assessed by the method of analysis of variance (ANOVA). If this analysis indicated significant differences among group means, then

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EJP-38619-R3 each group was compared using the Student-Newman-Keuls method. P < 0.05 was considered statistically significant. Statistical analyses were performed using the SAS software, version 9.2 (SAS institute Inc., Cary, NC).

3. Results 3.1. Effects of MK571 on platelet aggregation and intracellular calcium mobilization As shown in Fig. 1A, MK571 (10~50 M) inhibited collagen-induced platelet aggregation and the ATP-release reaction in a concentration-dependent manner. The 50% inhibitory concentration (IC50) value of MK571 for platelet aggregation induced by collagen was approximately 20 PM. We also found that MK571 inhibited AA (60 M)-induced platelet aggregation at a higher concentration of MK571 (100 M) (Fig. 1B). However, MK571 even at the concentration of 100 M did not affect platelet aggregation induced by thrombin (0.05 U/ml) or U46619 (1 M) (Fig. 1C). In addition, MK571 could concentration (20~50 M)-dependently inhibit calcium mobilization in collagen (1 g/ml)-stimulated human platelets (Fig. 1D).

3.2. Effects of MK571 on platelet aggregation independent of cyclic nucleotide production As shown in Fig. 2A, prostagladin E1 (PGE1) and nitroglycerin (NTG) completely inhibited collagen-induced platelet aggregation, which was reversed by the adenylyl cyclase (AC) inhibitor SQ22536 and the guanylyl cyclase (GC) inhibitor ODQ, respectively. However, neither SQ22536 nor ODQ reversed the MK571mediated inhibition of collagen-induced platelet aggregation (Fig. 2B), indicating that neither AC nor GC are involved in the MK571-mediated inhibition of platelet activation. Fig. 2C showed that the cAMP-dependent protein kinase (PKA) inhibitor

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EJP-38619-R3 H89 significantly reversed the PGE1-mediated inhibition of platelet aggregation. However, H89 only reversed the effect of MK571 to a small extent (Fig. 2C). These data suggest that MRP4 can prevent collagen-induced platelet aggregation via a route independent of cyclic nucleotide production. The inhibitory effects of MK571 in platelet activation were determined in the following experiments.

3.3. Effects of MK571 on IIb3 integrin binding To investigate whether MK571 inhibits platelet aggregation by interfering with the conformation of the IIb3 integrin of platelets, thereby blocking the interaction of platelets, the IIb3 integrin antagonist FITC-triflavin (Sheu et al., 1999) was used in the flow cytometry study to determine whether MK571 competes with triflavin to bind to the IIb3 integrin. As shown in Fig. 3A, the FITC-triflavin fluorescence was significantly reduced in the presence of 5 mM EDTA (negative control), which chelates Ca2+ and disrupts the confirmation of the IIb3 integrin, resulting in reduced FITC-triflavin binding. However, treatment with MK571 (20 and 50 M) did not affect the intensity of FITC-triflavin fluorescence, indicating that MK571 did not interfere with the conformation of the IIb3 integrin.

3.4. Effects of MK571 on the phosphorylation of p47 protein, Akt, p38, ERK and JNK The phosphorylation of the p47 kDa protein (pleckstrin), the substrate of protein kinase C (PKC) (Singer et al., 1997), was used to determine the activity of PKC. As shown in Fig. 3B, treatment with MK571 (20 and 50 M) significantly inhibited the collagen-induced phosphorylation of p47. Platelet aggregation analysis showed that MK571 did not inhibit platelet aggregation induced by 150 nM PDBu (a PKC activator), indicating that the inhibitory effect of MK571 on PKC is indirect (Fig. 3C).

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EJP-38619-R3 We also found that the collagen-induced phosphorylation of Akt and JNK was inhibited in the presence of 20 to 50 M MK571 (Fig. 4A and B), and that MK571 did not affect the phosphorylation of ERK and p38 in collagen-activated human platelets (Fig. 4C and D). These data indicate that MK571 prevents collagen-induced platelet activation by inhibiting the phosphorylation of Akt and JNK.

3.5. Effects of MK571 on hydroxyl radical formation in human platelets and thrombus formation in vitro and in mice An ESR signal indicative of hydroxyl radical (HO) formation was recorded for the collagen-activated platelets (Fig. 5A, b), compared with resting platelets (Fig. 5A, a), and treatment with MK571 (20 and 50 M) reduced collagen-induced hydroxyl radical ESR signal by 25.0% and 68.5%, respectively (Fig. 5A, c and d). The results of the PFA-100 system showed that, in the presence of 50 PM MK571, the CADP-CT was longer (99.5 r 8.5 s), compared with the control (77.0 r 9.6 s) (Fig. 5B). However, treatment with 50 PM MK571 did not affect the CEPI-CT (105.8 r 10.7 s), compared with the control (106.3 r 10.2 s) (Fig. 5B). In addition, treatment with 5 mg/kg MK571 prolonged the occlusion time (104.4 r 5.2 s) of thrombus formation in the irradiated venules of fluorescein sodium-pretreated mice,

compared with the

control (69.1 r 2.3 s) (Fig. 5C). 4. Discussion Our findings demonstrate for the first time that MRP4 inhibition prevents thrombus formation in vitro and in vivo (Fig. 6). The MRP4 has been reported to serve as a drug transporter, including antiviral, antibiotic, cardiovascular, and cytotoxic agents. However, no conclusive, direct evidence of the clinical relevance of MRP4 transporter activity regarding drug resistance has been reported (Russel et al., 13

EJP-38619-R3 2008). In fact, an in vitro study has showed that MRP4 is associated with drug resistance in HEK293/MRP4 cells (Norris et al., 2005). Moreover, increased levels of the MRP4 protein on the plasma membrane of platelets of CABG patients has been reported to be one of the major causes of aspirin resistance (Mattiello et al., 2011). This report also indicates that MRP4 may remove aspirin from platelets, thereby reducing the inhibitory effects of aspirin on COX-1 activity (Mattiello et al., 2011). In this study, we found that MK571 blocked collagen-induced platelet aggregation in a concentration-dependent manner (Fig. 1A), but did not affect that induced by thrombin or U46619 (Fig. 1C). Platelet aggregation induced by AA was also blocked by higher concentrations of MK571 (Fig. 1B).The hypothesis that MRP4 may export lipid mediators, such as TxA2 and LTs in platelets was proposed (Jedlitschky et al., 2012). Moreover, TxA2 activates platelet activation mainly through extracellular interaction with the membrane receptors (Jedlitschky et al., 2012). Therefore, MK571 may inhibit the AA-induced platelet activation, at least in part, through the inhibition of MRP4, thereby blocking the export of TxA2, a product of AA metabolism. Although the primary focus of our current study was on the effects of MK571 in collagen-induced platelet activation, future investigations of MK571-mediated inhibition of AA-induced platelet activation are warranted. However, thrombin and U46619 act on their individual receptors, PARs and TxA2 receptor, respectively, which are G-protein couple receptors (GPCRs). In contrast, collagen mainly acts on its receptor glycoprotein (GP) VI, which is a member of the immunoglobulin superfamily and is coupled to the Fc receptor gamma chain (FcRJ) (Li et al., 2010). These discrepancies may explain why MK571 does not affect thrombin- and U46619-induced platelet aggregation. Moreover, these observations may indicate that MRP4 does not participate in GPCR signaling pathway.

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EJP-38619-R3 The MRP4 inhibition by MK571 has been reported to enhance the inhibitory effects of cAMP and cGMP on platelet activation (Borgognone and Pulcinelli, 2012). In the present study, our results revealed that cyclic nucleotide production are not involved in the MK571-mediated inhibition of platelet activation (Fig. 2A, B). However, H89 (a PKA inhibitor) could partially reverse the MK571-mediated inhibition of platelet aggregation (Fig. 2C), indirectly suggesting that the levels of cAMP in platelets may be maintained by the inhibition of MRP4, thereby continuously activating PKA and partially blocking platelet activation. These findings are consistent with previous studies, which reported that the inhibition of MRP4 blocks the transport of cAMP (Copsel et al., 2011; Borgognone and Pulcinelli, 2012). These observations also indicate that MRP4 may be involved in the regulation of platelet activation by other mechanisms. However, like MRP4, MRP1 is also inhibited by the leukotriene D4 (LTD4) receptor antagonist MK571 (Reid et al., 2003). Moreover, Mehta et al. (1986) reported that LTs (LTC4, LTD4, and LTE4) potentiated epinephrine-induced platelet aggregation by modulating TxA2 synthetase activity; on the other hand, many ABC transporters (MRP) are also identified in platelets, including MRP1, MRP3, and MRP4. It was suggested that proposed substrates of MRP1 are amphiphilic anions and LTC4 (Jedlitschky et al., 2012). Thus, the possibility that the block of MRP1 or LTD4 receptor by MK571 may play a role in regulating platelet activation must be considered. The binding fibrinogen to activated DIIbE3 integrin is the final common pathway in platelet aggregation. Triflavin contains the platelet binding motif, Arg-Gly-Asp. It displays a higher binding affinity (Kd, 7 × 10-8 M) and has a lower molecular weight (7.6 kDa) (Sheu et al., 1999). The triflavin-binding site is specifically located in the E3 subunit, which is an important binding domain for adhesion proteins on platelets

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EJP-38619-R3 (Sheu et al., 1992). Therefore, we used FITC-triflavin to determine whether the inhibition of MRP4 by MK571 interfered with the conformation of the DIIbE3 integrin. We found that MRP4 inhibition did not influence the conformation of the integrin DIIbE3. The activation of platelets by agonists, such as collagen, significantly alters phospholipase activation. The activation of PLC results in the production of IP3 and DAG, which induces calcium mobilization and PKC activation, respectively. The activated PKC subsequently induces the phosphorylation of the p47 protein (Mangin et al., 2003). The activation of PKC allows select responses to specific activating signals in distinct cellular compartments (Pascale et al., 2007). In this study, MK571 inhibited both calcium mobilization and p47 phosphorylation in collagen-stimulated human platelets. We suggest that MK571 exert an indirect effect on PKC activation because MK571 does not inhibit PDBu-induced platelet aggregation (Fig. 3C). These findings indicate that MK571 inhibits collagen-induced platelet activation, in part, through the inhibition of calcium mobilization and PKC activation. The MAPKs consist of the ERK, p38, and JNK, which are involved in cell proliferation, migration, differentiation, and apoptosis. Although much is known about MAPKs in nucleated cells, their functions in platelets remain unclear. However, it is a well-established fact that ERK, p38 and JNK are present in platelets, activated by various agonists, and involved in thrombosis (Adam et al., 2008). The ERK and p38 play an important role in stimulating secretion of granules and in facilitating clot retraction (Flevaris et al., 2009). JNK1 reportedly is involved in collagen-induced platelet aggregation and thrombus formation (Kauskot et al., 2007). We found that MK571 attenuated the collagen-induced phosphorylation of JNK, but not that of ERK and p38. These observations reveal that JNK may be involved in MK571-mediated

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EJP-38619-R3 inhibition of collagen-induced platelet activation. Several studies showed that PI3K/Akt plays an important role in regulating platelet aggregation and thrombus formation (Cosemans et al., 2006; O'Brien et al., 2011). Our data revealed that MK571 also inhibits Akt phosphorylation. Previous studies reported that Akt activation by convulxin, a selective agonist of glycoprotein VI, is partially dependent on ADP release (Kim et al., 2009). Moreover, MRP4 has been reported to remove ADP from platelets (Jedlitschky et al., 2004). Our results also showed that MK571 attenuates collagen-induced ATP release (Fig. 1A), indicating that the release of ADP/ATP from the dense granules in platelets is impaired by the inhibition of MRP4. Thus, the inhibition of MRP4 may prevent the phosphorylation of Akt and JNK, at least in part, through the inhibition of ADP secretion. The collagen-induced reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals, in platelets have been shown to act as second messengers that stimulate the AA metabolism and PLC pathway and enhance ADP release, resulting in increased platelet recruitment (Pignatelli et al., 1998; Krotz et al., 2002). Our previous study also showed that the collagen-induced hydroxyl radicals were diminished by hydroxyl radical scavengers (Lu et al., 2011). Recently, NADPH oxidases (NOXs) were reported to support the collagen-dependent thrombus formation (Vara et al., 2013). Although the mechanism of collagen-induced ROS production is not complete clear, these evidences indicate that ROS play critical roles in collagen-induced platelet activation. In the present study, the results of our ESR analysis provide direct evidence that the inhibition of MRP4 reduces the formation of hydroxyl radicals. Thus, MK571 inhibit collagen-induced platelet activation, in part, through the reduction of free radical formation. However, how MRP4 involve the

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EJP-38619-R3 signal transduction of collagen-induced ROS production needs to be further clarified. In addition, we assessed platelet function under conditions that mimic blood vessel injury in vivo, and found that 50 μM MK571 prolonged the time required to form a thrombus in the presence of collagen and ADP. Moreover, we found that treatment with 5 mg/kg MK571 significantly delayed thrombus formation in the irradiated blood vessel of fluorescein sodium-pretreated mice. Therefore, MK571 may prevent thrombus formation in vitro and in vivo. In conclusion, our results collectively indicate for the first time the possible involvement of MRP4 in regulating thrombus formation in vitro and in vivo. The possible mechanism is that MK571 may inhibit PLCJ2 and subsequent PKC and calcium mobilization. In addition, MK571 also inhibits Akt and JNK phosphorylation, and ROS formation, finally preventing platelet activation and thrombus formation. Our findings also support the idea that MRP4 may represent a potential target for the development of novel therapeutic interventions for the treatment of thromboembolic disorders.

Acknowledgements This work was supported by grants from the Shin Kong Wu Ho-Su Memorial Hospital (SKH-8302-101-DR-12, SKH-8302-102-NDR-04, and SKH-8302-103-NDR-05); National Science Council, Taiwan (NSC102-2320-B-341-001-MY3, NSC102-2811-B-038-026, NSC101-2811-B-038-002, and NSC101-2811-B-038-006); and Chi-Mei Medical Center-Taipei Medical University (101CM-TMU-11).

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EJP-38619-R3

Conflict of interest The authors declare that they have no conflicts of interest. References Adam, F., Kauskot, A., Rosa, J.P., Bryckaert, M., 2008. Mitogen-activated protein kinases in hemostasis and thrombosis. J. Thromb. Haemost. 6, 2007-2016. Borgognone, A., Pulcinelli, F.M., 2012. Reduction of cAMP and cGMP inhibitory effects in human platelets by MRP4-mediated transport. Thromb. Haemost. 108, 955-962. Chen, Z.S., Lee, K., Kruh, G.D., 2001. Transport of cyclic nucleotides and estradiol 17-beta-D-glucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine. J. Biol. Chem. 276, 33747-33754. Chou, D.S., Hsiao, G., Shen, M.Y., Tsai, Y.J., Chen, T.F., Sheu, J.R., 2005. ESR spin trapping of a carbon-centered free radical from agonist-stimulated human platelets. Free Radic. Biol. Med. 39, 237-248. Copsel, S., Garcia, C., Diez, F., Vermeulem, M., Baldi, A., Bianciotti, L.G., Russel, F.G., Shayo, C., Davio, C., 2011. Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation. J. Biol. Chem. 286, 6979-6988. Cosemans, J.M., Munnix, I.C., Wetzker, R., Heller, R., Jackson, S.P., Heemskerk, J.W., 2006. Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization. Blood 108, 3045-3052. Flevaris, P., Li, Z., Zhang, G., Zheng, Y., Liu, J., Du, X., 2009. Two distinct roles of mitogen-activated protein kinases in platelets and a novel

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EJP-38619-R3 Rac1-MAPK-dependent integrin outside-in retractile signaling pathway. Blood 113, 893-901. Hara, Y., Sassi, Y., Guibert, C., Gambaryan, N., Dorfmuller, P., Eddahibi, S., Lompre, A.M., Humbert, M., Hulot, J.S., 2011. Inhibition of MRP4 prevents and reverses pulmonary hypertension in mice. J. Clin. Invest. 121, 2888-2897. Hsiao, G., Lin, K.H., Chang, Y., Chen, T.L., Tzu, N.H., Chou, D.S., Sheu, J.R., 2005. Protective mechanisms of inosine in platelet activation and cerebral ischemic damage. Arterioscler. Thromb. Vasc. Biol. 25, 1998-2004. Jedlitschky, G., Tirschmann, K., Lubenow, L.E., Nieuwenhuis, H.K., Akkerman, J.W., Greinacher, A., Kroemer, H.K., 2004. The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage. Blood 104, 3603-3610. Jedlitschky, G., Greinacher, A., Kroemer, H.K., 2012. Transporters in human platelets: physiologic function and impact for pharmacotherapy. Blood 119, 3394-3402. Jilma, B., 2001. Platelet function analyzer (PFA-100): a tool to quantify congenital or acquired platelet dysfunction. J. Lab. Clin. Med. 138, 152-163. Kauskot, A., Adam, F., Mazharian, A., Ajzenberg, N., Berrou, E., Bonnefoy, A., Rosa, J.P., Hoylaerts, M.F., Bryckaert, M., 2007. Involvement of the mitogen-activated protein kinase c-Jun NH2-terminal kinase 1 in thrombus formation. J. Biol. Chem. 282, 31990-31999. Kim, S., Mangin, P., Dangelmaier, C., Lillian, R., Jackson, S.P., Daniel, J.L., Kunapuli, S.P., 2009. Role of phosphoinositide 3-kinase beta in glycoprotein VI-mediated Akt activation in platelets. J. Biol. Chem. 284, 33763-33772. Krotz, F., Sohn, H.Y., Gloe, T., Zahler, S., Riexinger, T., Schiele, T.M., Becker, B.F., Theisen, K., Klauss, V., Pohl, U., 2002. NAD(P)H oxidase-dependent platelet

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EJP-38619-R3 superoxide anion release increases platelet recruitment. Blood 100, 917-924. Li, Z., Delaney, M.K., O'Brien, K.A., Du, X., 2010. Signaling during platelet adhesion and activation. Arterioscler. Thromb. Vasc. Biol. 30, 2341-2349. Lu, W.J., Lee, J.J., Chou, D.S., Jayakumar, T., Fong, T.H., Hsiao, G., Sheu, J.R., 2011. A novel role of andrographolide, an NF-kappa B inhibitor, on inhibition of platelet activation: the pivotal mechanisms of endothelial nitric oxide synthase/cyclic GMP. J. Mol. Med. 89, 1261-1273. Mangin, P., Yuan, Y., Goncalves, I., Eckly, A., Freund, M., Cazenave, J.P., Gachet, C., Jackson, S.P., Lanza, F., 2003. Signaling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganization. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA. J. Biol. Chem. 278, 32880-32891. Mattiello, T., Guerriero, R., Lotti, L.V., Trifiro, E., Felli, M.P., Barbarulo, A., Pucci, B., Gazzaniga, P., Gaudio, C., Frati, L., Pulcinelli, F.M., 2011. Aspirin extrusion from human platelets through multidrug resistance protein-4-mediated transport: evidence of a reduced drug action in patients after coronary artery bypass grafting. J. Am. Coll. Cardiol. 58, 752-761. Mehta, P., Mehta, J., Lawson, D., Krop, I., Letts, L.G., 1986. Leukotrienes potentiate the effects of epinephrine and thrombin on human platelet aggregation. Thromb. Res. 41, 731-738. Niessen, J., Jedlitschky, G., Grube, M., Kawakami, H., Kamiie, J., Ohtsuki, S., Schwertz, H., Bien, S., Starke, K., Ritter, C., Strobel, U., Greinacher, A., Terasaki, T., Kroemer, H.K., 2010. Expression of ABC-type transport proteins in human platelets. Pharmacogenet Genomics 20, 396-400. Norris, M.D., Smith, J., Tanabe, K., Tobin, P., Flemming, C., Scheffer, G.L., Wielinga,

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EJP-38619-R3 P., Cohn, S.L., London, W.B., Marshall, G.M., Allen, J.D., Haber, M., 2005. Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro. Mol. Cancer Ther. 4, 547-553. O'Brien, K.A., Stojanovic-Terpo, A., Hay, N., Du, X., 2011. An important role for Akt3 in platelet activation and thrombosis. Blood 118, 4215-4223. Pascale, A., Amadio, M., Govoni, S., Battaini, F., 2007. The aging brain, a key target for the future: the protein kinase C involvement. Pharmacol. Res. 55, 560-569. Pignatelli, P., Pulcinelli, F.M., Lenti, L., Gazzaniga, P.P., Violi, F., 1998. Hydrogen peroxide is involved in collagen-induced platelet activation. Blood 91, 484-490. Reid, G., Wielinga, P., Zelcer, N., van der Heijden, I., Kuil, A., de Haas, M., Wijnholds, J., Borst, P., 2003. The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc. Natl. Acad. Sci. U. S. A. 100, 9244-9249. Russel, F.G., Koenderink, J.B., Masereeuw, R., 2008. Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules. Trends Pharmacol. Sci. 29, 200-207. Sheu, J.R., Teng, C.M., Huang, T.F., 1992. Triflavin, an RGD-containing antiplatelet peptide, binds to GpIIIa of ADP-stimulated platelets. Biochem. Biophys. Res. Commun. 189, 1236-1242. Sheu, J.R., Hung, W.C., Wu, C.H., Ma, M.C., Kan, Y.C., Lin, C.H., Lin, M.S., Luk, H.N., Yen, M.H., 1999. Reduction in lipopolysaccharide-induced thrombocytopenia by triflavin in a rat model of septicemia. Circulation 99, 3056-3062.

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EJP-38619-R3 Sheu, J.R., Lee, C.R., Lin, C.H., Hsiao, G., Ko, W.C., Chen, Y.C., Yen, M.H., 2000. Mechanisms involved in the antiplatelet activity of Staphylococcus aureus lipoteichoic acid in human platelets. Thromb. Haemost. 83, 777-784. Singer, W.D., Brown, H.A., Sternweis, P.C., 1997. Regulation of eukaryotic phosphatidylinositol-specific phospholipase C and phospholipase D. Annu. Rev. Biochem. 66, 475-509. Sinha, C., Ren, A., Arora, K., Moon, C.S., Yarlagadda, S., Zhang, W., Cheepala, S.B., Schuetz, J.D., Naren, A.P., 2013. Multi-drug resistance protein 4 (MRP4)-mediated regulation of fibroblast cell migration reflects a dichotomous role of intracellular cyclic nucleotides. J. Biol. Chem. 288, 3786-3794. Vara, D., Campanella, M., Pula, G., 2013. The novel NOX inhibitor 2-acetylphenothiazine impairs collagen-dependent thrombus formation in a GPVI-dependent manner. Br. J. Pharmacol. 168, 212-224.

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EJP-38619-R3

Figure legends Fig. 1. Effects of MK571 in the inhibition of platelet aggregation, ATP release, and calcium mobilization in washed human platelets. (A) Washed platelets (3.6 × 108 cells/ml) were pre-incubated with MK571 (10~50 M), followed by treatment with 1 g/ml collagen to stimulate platelet aggregation and the ATP-release reaction. The delta T means the change of transmission. (B and C) Washed platelets (3.6 × 108 cells/ml) were pre-incubated with MK571 (10~100 M), followed by treatment with 60 M arachidonic acid (AA), 0.05 IU/ml thrombin, or 1 M U46619 to stimulate platelet aggregation. The delta T means the change of transmission. (D) Washed platelets were pre-incubated with MK571 (20 or 50 M), followed by treatment with 1 g/ml collagen to induce the cytoplasmic influx of calcium from intracellular stores. The profiles are representative of 4 independent experiments.

Fig. 2. Effects of MK571 on platelet aggregation independent of cyclic nucleotide production in washed human platelets. Washed platelets (3.6 × 108 cells/ml) were pre-incubated with (A) PGE1 (0.1 nM) or NTG (10 PM), and (B) MK571 (50 M), followed by treatment with 1 g/ml collagen to induce platelet aggregation in the absence or presence of the AC inhibitor SQ22536 (100 PM) or the GC inhibitor ODQ (10 PM). (C) Washed platelets (3.6 × 108 cells/ml) were pre-incubated with PGE1 (0.1 nM) or MK571 (50 M), followed by treatment with 1 g/ml collagen to induce platelet aggregation in the absence or presence of the PKA inhibitor H89 (5 PM). The delta T means the change of transmission. The profiles are representative of 4 independent experiments.

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EJP-38619-R3 Fig. 3. The influence of MK571 on FITC-triflavin binding to the IIb3 integrin and PKC activation in activated platelets. (A) A flow cytometry analysis was used to assess the binding of MK571 to the IIb3 integrin. Platelet suspensions (1 × 106 cells/ml) were pre-incubated with 5 mM EDTA, 20 M MK571, or 50 M MK571 for 3 min, followed by the addition of 2 g/ml FITC-triflavin. The solid line represents the fluorescence profiles of FITC-triflavin (2 g/ml) in the absence of MK571 as a positive control; the dashed line represents the fluorescence profiles of FITC-triflavin in the presence of 5 mM EDTA as a negative control; the dotted line represents the fluorescence profiles of FITC-triflavin in the presence of 20 M or 50 M MK571. (B) Washed platelets were pre-incubated with MK571 (10~50 M), followed by treatment with 1 g/ml collagen to induce platelet activation. Platelets were collected, and subcellular extracts were analyzed for p47 phosphorylation. (C) Washed platelets were pre-incubated with MK571 (50 and 100 M), followed by treatment with 150 nM PDBu to induce platelet aggregation. The profiles (A and C) are representative of 4 independent experiments. The data (B) are presented as the mean ± S.E.M. (n = 3). **

P < 0.01, compared with the control (resting) platelets; #P < 0.05, compared with

the positive control (collagen treatment only)

Fig. 4. Effects of MK571 on Akt, JNK, ERK, and p38 phosphorylation in collagen-activated platelets. Washed platelets (1.2 × 109 cells/ml) were pre-incubated with MK571 (10~50 M), followed by treatment with 1 g/ml collagen to induce platelet activation. Platelets were collected, and subcellular extracts were analyzed for (A) Akt, (B) JNK, (C) ERK, and (D) p38 phosphorylation. The data are presented as the mean ± S.E.M. (n = 3). **P < 0.01 and ***P < 0.001, compared with the control (resting) platelets; #P < 0.05 and ##P < 0.01, compared with the positive control

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EJP-38619-R3 (collagen treatment only). Fig. 5. Effects of MK571 on the collagen-induced hydroxyl radical (HO•) formation, and thrombus formation in vitro and in mice. (A) For the electron spin resonance (ESR) analysis, washed platelets (3.6 × 108 cells/ml) were incubated with (a) Tyrode’s solution only (resting group); or the platelets were pre-incubated with (b) Tyrode’s solution, (c) 20 M MK571, or (d) 50 M MK571, followed by treatment with 1 g/ml collagen to induce hydroxyl radical formation in platelets. (B) Whole blood samples, pretreated with saline (control) or MK571 (20 or 50 M), were applied to a collagen-ADP (CADP) or a collagen-epinephrine (CEPI) cartridge in a PFA-100 device, and the closure time (platelet plug formation) was recorded. (C) Mice were administered saline (control; ctl) or MK571 (2.5 or 5 mg/kg), and selected mesenteric venules were irradiated to induce microthrombus formation. The profiles (A) are representative of 4 independent experiments, and an asterisk (*) indicates the formation of hydroxyl radicals. The data (B and C) in the bar graphs are presented as the mean ± S.E.M. of the in-vitro closure time (n = 4) or the in-vivo occlusion time (n = 5) is seconds. **P < 0.01 compared with the relevant control group.

Fig. 6. Schematic illustration of the MK571-mediated inhibition of platelet activation. In addition to blocking the transport of cyclic nucleotides and ADP, the inhibition of MRP4 by MK571 prevents the collagen-induced the phosphorylation of PKC, Akt, and JNK, and the formation of hydroxyl radicals, followed by the suppression of intracellular calcium mobilization. Finally, MRP4 inhibition by MK571 suppresses platelet activation and subsequent thrombus formation.

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Fig. 1-R1



MK571 10

MK571 20

MK571 50

T(10%)

ATP release

A

min

collagen

B MK571 10

MK571 20

MK571 50

MK571 100

AA

C MK571 100

T(10%)

MK571 100

min

U46619

thrombin

Fluorescence intensity

D

MK571 20

collagen

50

Fig. 1-R1

Fig. 2-R1



PGE1

SQ22536 PGE1

NTG

ODQ NTG

T(10%)

A

min

collagen

MK571

SQ22536 MK571

ODQ MK571

T(10%)

B

min

collagen

PGE1

H89 PGE1

MK571

H89 MK571

T(10%)

C

min

collagen

collagen

Fig. 2-R1

Fig. 3

A

Triflavin

EDTA + Triflavin

MK571 (20 PM) + Triflavin

MK571 (50 PM) + Triflavin

B p-p47 p47 3.0

p47 phosphorylation (folds/basal)

** 2.5

#

2.0

#

1.5 1.0 0.5 0.0

DMSO

10

20 MK571 collagen

50

MK571 50

MK571 100

ΔT(10%)

C

min

PDBu

Fig. 3

Fig. 4



A

B p-Akt

p-JNK

Akt

JNK 3.0

***

4

# #

3

##

2 1 0

DMSO

10

20 MK571 collagen

JNK phosphorylation (folds/basal)

Akt phosphorylation (folds/basal)

5

2.5

# ##

2.0 1.5 1.0 0.5 0.0

50

***

DMSO

10

20 MK571 collagen

50

10

50

D

C p-ERK

p-p38

ERK

p38 2.5

**

3.0

p38 phosphorylation (folds/basal)

ERK phosphorylation (folds/basal)

3.5

2.5 2.0 1.5 1.0 0.5 0.0

DMSO

10

20 MK571 collagen

50

**

2.0 1.5 1.0 0.5 0.0

DMSO

20 MK571 collagen

Fig. 4

Fig. 5

A

*

a

15 G

*

*

* *

*

*

*

b

*

*

c

*

* *

*

d

*

*

B 120

**

Closure time (s)

100 80 60 40 20 0

ctl

20

50

ctl

CADP

20

50

CEPI

C Occlusion time (s)

120

**

100 80 60 40 20 0

ctl 2.5

ctl

5

Fig. 5

Fig. 6-R3



Fig. 6-R3

Graphical Abstract-R3



Graphical abstract-R3