Data are presented as means SD. [4,5], anticancer [6,7,8], and antithrombotic [9]. Antithrombotic effects of curcumin are mediated by complex reactions with endothelial cells, with the blood coagulation system, and by inhibition of platelet aggregation. Inhibitory effects EBI-1051 of curcumin on platelet aggregation induced by several agonists were characterized in several reports [9,10,11]. Prevention of platelet activation and aggregation by curcumin [10,12,13] includes inhibition of cyclooxygenase and lipoxygenase activity, and consequently thromboxane B2 and 12-HETE generation [14,15]. However, prevention of collagen-induced platelet activation and aggregation was independent IL2R of cyclooxygenase activity and associated with inhibition of Syk kinase and of the subsequent activation of PLC2. Curcumin alone or in combination with anticancer drugs is often used for the treatment of different types of cancer [16,17]. In cellular models [18,19,20] and in vivo studies [21,22], including several clinical trials [23,24], the beneficial effects of curcumin on cancer development were described. Clinical applications of curcumin are restricted because of low bioavailability, poor solubility, low intestinal absorption, and rapid metabolism [25]. Therefore, EBI-1051 curcumin is currently used as an adjuvant to anticancer compounds formulated in nanoparticles [25,26,27]. Curcumin induces apoptosis through different pathways, including activation of caspase 3 in several cancer cell lines [28,29,30]. Inhibition of platelet activation by curcumin is well documented [9,31]. However, it is still unknown whether curcumin induces apoptosis or autophagy, the formation of procoagulant platelets, or whether it influences platelet apoptosis induced by the precursor of the anticancer drug ABT-737. In this study, we showed that curcumin induces procoagulant platelet formation that results in strong surface exposure of anionic phospholipids such as phosphatidylserine (PS), loss of mitochondrial membrane potential, and microparticle formation. Curcumin inhibited P-gp, strongly activated AMP kinase (AMPK), inhibited even basal protein kinase B (PKB) activity, and induced autophagy expressed by conversion of LC3I to LC3II. Curcumin itself did not activate caspase 3-dependent apoptosis; however, curcumin at low doses potentiated, and at high doses inhibited ABT-737-induced platelet apoptosis. 2. Results 2.1. Curcumin Inhibits Thrombin-Induced Platelet Activation but Does Not Stimulate Caspase 3-Dependent Apoptosis Curcumin, by activation of several apoptotic pathways, can induce apoptosis in cancer cells [19,28,32]. Apoptosis significantly prevents platelet activation [33,34]. Therefore, we tested whether curcumin-mediated platelet inhibition results in activation of apoptotic pathways in platelets. Curcumin itself, even at a high concentration (50 M), had no effect on platelet activation after 10 and 60 min of incubation (Figure S1). In contrast, 50 M of curcumin significantly inhibited thrombin-induced integrin IIb3 activation (Figure 1A) and thrombin-induced intracellular Ca2+-mobilization (Figure S2). Platelets incubated with 50 M curcumin showed substantial autofluorescence in the flow cytometric FL1 channel (data not shown). To quantify the specific Fluo-3 signal, representing intracellular Ca2+-mobilization, the autofluorescent signal of 50 M curcumin samples was subtracted from Fluo-3 signals prior to and after the addition of thrombin, respectively (Figure S2B,C). Platelet inhibition was strongly associated with the increase of annexin-V-binding (Figure 1B), microparticle formation (Figure 1C,D), and decrease of mitochondrial membrane potential (Figure 1E,F). Open in a separate window Figure 1 Curcumin inhibits thrombin-induced platelet IIb3 integrin activation and does not stimulate caspase 3-dependent apoptosis. (A) Flow cytometric analysis of IIb3 integrin activation (PAC-1-FITC binding), (B) PS surface exposure (annexin-V-PE binding), (C,D) microparticle formation, (E,F) mitochondrial membrane potential changes (TMRE fluorescence), and (G) Western blot of caspase 3 activation. Washed platelets (WP 1 108 /mL in A-F and 3 108/mL in G) were incubated with the indicated concentrations of curcumin for 10 and 60 min. (A) Thrombin (0.01 U/mL) was added for 2 min, followed by PAC-1-FITC antibody (1:10 dilution) for 10 min, and the reaction was stopped by dilution (10 volumes) with PBS. (B) WP were incubated with the indicated concentrations/time of curcumin, then annexin-V-PE (dilution 1:10) was added for an additional 10 min, and the reaction was stopped by dilution (10 volumes) with the annexin-V-binding solution. (C) Representative (from four independent experiments) dot plot of microparticle formation (upper panel), and annexin-V-PE positive platelets and microparticles (lower panel). Annexin-V-PE was analyzed as shown in B. (D) Quantification of platelet microparticle formation. Microparticles were quantified as CD42a EBI-1051 positive events in the gate B. (E,F) WP were incubated with curcumin (50 M, 10 and 60 min), TMRE dye (dilution 1:10) was added for an additional 10 min, and samples were diluted (10 volumes) with PBS. (G) WP were incubated with the indicated concentrations/time of curcumin and processed for Western blotting with caspase 3 antibody (1:1000). ABT-737 was used as positive control and actin blot served as a loading control. All data are presented as means SD. Data in A are presented as % of MFI (thrombin sample represents 100%, one-way.