Inhibition of platelet aggregation and thrombosis by indole alkaloids isolated from the edible insect Protaetia brevitarsis seulensis (Kolbe)

Abstract Protaetia brevitarsis seulensis (Kolbe) has been temporarily registered as a food material by the Ministry of Food and Drug Safety of Korea (MFDS). The current study aimed to discover small antithrombotic molecules from this edible insect. Five indole alkaloids, 5‐hydroxyindolin‐2‐one (1), (1R,3S)‐1‐methyl‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid (2), (1S,3S)‐1‐methyl‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid (3), (3S)‐1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid (4) and L‐tryptophan (5), were isolated from the insect. Among them, compounds 1 and 2 prolonged aPTT and PT and impaired thrombin and FXa generation on HUVEC surface. Moreover, these compounds inhibited platelet aggregation. Antithrombotic effects of compounds 1 and 2 were further confirmed in pre‐clinical models of pulmonary embolism and arterial thrombosis. Collectively, these results demonstrated that compounds 1 and 2 could be effective antithrombotic agents and serve as new scaffolds for the development of antithrombotic drug.


Introduction
The leading causes of death worldwide are diseases that involve the heart and blood vessels and, consequently, thrombosis [1]. Most thromboembolic processes require anticoagulant therapy. This explains the current effort to develop specific and potent anticoagulant and antithrombotic agents [1]. Thrombus formation due to an abnormal coagulation process is often observed in arteries or veins and may result in reduced blood flow or ischaemia [1]. Platelet activation in atherosclerotic arteries is central to the development of arterial thrombosis; therefore, a precise control of platelet function is imperative in preventing thrombotic events [2]. Because of the central role of platelets in cardiovascular thrombosis, current and investigational antiplatelet therapies target key pathways of platelet activation [3]. These targets include platelet surface receptors (e.g. P2Y purinoceptor 12 (P2Y12), integrin aIIbb3, protease activated receptor (PAR)-1, glycoprotein 1b, P-selectin and the thromboxane prostanoid receptor), signalling molecules (e.g. cyclooxygenase 1 (COX1)) and endothelial products (nitric oxide) [3]. Therefore, antiplatelet therapy is a well-established part of the treatment of cardiovascular disease including the COX 1 inhibitor aspirin, the P2Y12 antagonist (clopidogrel, ticagrelor or prasugrel) and integrin aIIbb3 antagonists [3]. However, limitations of current therapies include weak inhibition of platelet function (by aspirin), blockade of only one pathway of ADPmediated signalling (by clopidogrel), slow onset of action (of clopidogrel), interpatient response variability with poor inhibition of platelet response in some patients (to clopidogrel), the inability to transform the success of intravenous integrin aIIbb3 antagonist therapy into oral therapy and the inability to completely separate a reduction in thrombotic events from an increase in bleeding events [3][4][5][6]. The insufficient antithrombus and antiplatelet effect of the present armamentarium might explain the vascular relapses. Most thromboembolic processes require anticoagulant therapy. This explains the current effort to develop specific and potent anticoagulant and antithrombotic agents. Research on novel bioactive compounds and drugs with different mechanisms of action, increased efficacy, safety and pharmacokinetics, and low toxicity is highly needed [1].
Insects have been considered as potential food and drug resources. In a recent study, we revealed the anticoagulant activity of small-molecule alkaloids from Scolopendra subspinipes mutilans, lactams which have been applied for cardiovascular disorders [7]. Bioactive compounds including new norepinephrine derivatives, sesquiterpenoids and lactams were also discovered from the edible insect Aspongopus chinensis Dallas and were effective against pain, dyspepsia and kidney diseases [8]. The venom derived from samsum ant (Pachycondyla sennaarensis) was known to have antitumour and anti-inflammatory effects, and could improve the immune system [9]. In Korea, mealworms, the larvae of Tenebrio molitor that are used to treat liver disease and dementia have been approved as a food material by the MFDS [10]. The larvae of Protaetia brevitarsis seulensis (Kolbe) have also been temporarily approved by the MFDS as a food material since September 2014 [11]. P. brevitarsis seulensis, belonging to the Cetoniidae family, is a white-spotted flower chafer that is widely distributed in Korea, China, Japan, Taiwan and Europe [12]. The insect has been used as a functional food and traditional medicine to treat breast cancer, inflammatory disease, hepatic cancer, liver cirrhosis and hepatitis [11]; particularly, peptides from its larvae have been shown to possess antibacterial activity [13]. A composition analysis of the larvae revealed the presence of fatty acids, volatile constituents and nutrient substances [14]. Its fatty acids have been shown to induce apoptosis mediated by caspase-3 activation in tumour cells [15]. Up to date, there are limited studies characterizing the small-molecule metabolites of P. brevitarsis seulensis. In our preliminary study, the whole body extract of the larvae extended blood coagulation time with no toxicity in human umbilical vein endothelial cells (HUVECs). This study thus aimed to discover the antithrombotic compounds in the insect, which resulted in the isolation of five indole alkaloids. The indole alkaloids 1-5 were tested for their antithrombotic activity by monitoring the prothrombin time (PT), activated partial thromboplastin time (aPTT) and platelet aggregation. Furthermore, we examined the antithrombotic effect using the ferric chloride (FeCl 3 )-induced thrombosis animal models.

Materials and methods
General experimental procedures IR data were recorded on a Thermo Electron US/Nicolet 380 (Madison, WI, USA). Nuclear Magnetic Resonance (NMR) experiments were carried out using a Bruker Avance III (600 MHz) spectrometer. HRESIMS data were obtained on a JMS 700 high-resolution mass spectrometer (JEOL, Tokyo, Japan). Vacuum liquid chromatography (VLC) was conducted on Merck silica gel (70-230 mesh), and medium-pressure liquid chromatography (MPLC) was carried out utilizing a Biotage Isolera apparatus equipped with a reversed-phase C18 SNAP Cartridge KPC18-HS (340 g; Biotage AB, Uppsala, Sweden). Preparative reversed-phase HPLC (prep HPLC) separation was carried out on an YMC C 18 column (250 9 20.0 mm, 5 lm) or on a Kinetex Biphenyl column (250 9 21.5 mm, 5 lm) at 5 ml/min. flow rate.

Cell culture
Primary HUVECs were obtained from Cambrex Bio Science (Charles City, IA, USA) and were maintained as described previously [16]. Briefly, cells were cultured in EBM-2 basal media supplemented with growth supplements (Cambrex Bio Science, Charles City, IA, USA) at 37°C under 5% CO 2 atmosphere until confluent. All experiments were performed with HUVECs at passage 3-5.

In vitro and ex vivo platelet aggregation assay
The in vitro platelet aggregation study was performed according to a previously reported method [17,18]  1, 3, 5 or 10 min. They were subsequently stimulated by U46619 (2 lM) in 0.9% saline solution or collagen (1 lg/ml) at 37°C for 5 min. Platelet aggregation was recorded using an aggregometer (Chronolog, Havertown, PA, USA). For the ex vivo aggregation assay, male mice were fasted overnight and the indicated concentration of each compound in DMSO was administered by intravenous (i.v.) injection. After 24 hrs, PRP (10 9 platelets/ml) in a volume of 240 ll was incubated at 37°C for 1.5 min. in the aggregometer under continuous stirring at 1000 r.p.m. and subsequently stimulated with U46619 (2 lM). Platelet aggregation was recorded as described above.

Coagulation assay
The aPTT and PT were determined using a Thrombotimer (Behnk Elektronik, Norderstedt, Germany) as per the manufacturer's instructions and as described previously [19]. Briefly, citrated normal human plasma (90 ll) was mixed with 10 ll of heparin or of each compound and was incubated for 1 min. at 37°C. Subsequently, the aPTT assay reagent (100 ll) was added and the plasma sample was incubated for an additional 1 min. at 37°C, followed by the addition of 20 mM CaCl 2 (100 ll). The clotting times were recorded. For the PT assays, citrated normal human plasma (90 ll) was mixed with 10 ll of each compound's stock solution and was incubated for 1 min. at 37°C. The PT assay reagent (200 ll), which had been pre-incubated for 10 min. at 37°C, was subsequently added and the clotting time was recorded. The

In vivo bleeding time
Tail bleeding times were measured using the method described by Dejana et al. [19]. Briefly, C57BL/6 mice were fasted overnight prior to the experiments. One hour after the i.v. administration of each compound, the tails of the mice were transected at 2 mm from their tips. The bleeding time was defined as the time elapsed until the bleeding stopped. Bleeding times exceeding 15 min. were recorded as lasting for 15 min.

Ex vivo clotting time
Male C57BL/6 mice were fasted overnight and each compound in 0.2% dimethyl sulfoxide (DMSO) was administered by i.v. injection. One hour after the administration, arterial blood samples (0.1 ml) were collected in 3.8% sodium citrate (1/10, v/v) for the ex vivo aPTT and PT determination. The clotting times were determined as described above.

Thrombin activity assay
Each compound in 50 mM Tris-HCl buffer (pH 7.4) containing 7.5 mM EDTA and 150 mM NaCl was mixed. Following a 2-min incubation at 37°C, thrombin solution (150 ll, 10 U/ml) was added, followed by incubation at 37°C for 1 min. S-2238 (a thrombin substrate. 150 ll, 1.5 mM) solution was subsequently added and the absorbance at 405 nm was monitored for 120 sec. using a spectrophotometer (TECAN, M € annedorf, Switzerland).

Factor Xa activity assay
The FXa assay was performed using the same method as described for the thrombin activity assay, except for the use of FXa (1 U/ml) and S-2222 as substrates instead. *Please see Data S1 for more methods.

Results
Isolation and structural determination of smallmolecule alkaloids from P. brevitarsis seulensis Chemical investigation of the EtOH extract of P. brevitarsis seulensis larvae resulted in the isolation of a series of indole alkaloids 1-5 (Fig. S1). The structures of the isolated compounds were determined by MS, 1D and 2D NMR analysis.

Effect of the isolated compounds on platelet aggregation
The effect of each compound on a U46619 (a stable thromboxane A2 analogue/aggregation agonist) or collagen-induced platelet aggregation was performed as described in the 'Materials and methods' section. As shown in Figure 1A and B, treatment with compounds 1 and 2 significantly inhibited human platelet aggregation induced by U46619 (final concentration: 2 lM) or collagen (final concentration: 1 lg/ml) in a concentration-dependent manner. These in vitro results were confirmed in an ex vivo platelet aggregation assay (i.v. injection, Fig. 1C). The average circulating blood volume for mice is 72 ml/kg [21]. Because the average weight of the mouse used in this study was 27 g and the average blood volume is 2 ml, the amount of compound 1 (2.9, 7.5, or 14.9 lg per mouse) and 2 (4.6, 11.5, or 23.0 lg per mouse) equalled a peripheral blood concentration of approximately 10, 25 or 50 lM, respectively.

Effects of the isolated compounds on protein kinase C activation and intracellular Ca 2+ mobilization
We next investigated the selectivity of each compound for the signalling pathways that regulate platelet aggregation. Upon platelet stimulation by agonists, phospholipase C (PLC) hydrolyses phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol-1,4,5triphosphate, which promote the activation of protein kinase C (PKC) and increase cytosolic Ca 2+ , respectively [22]. PKC and Ca 2+ act synergistically to induce the granular secretion and activation of glycoprotein (GP) IIb/IIIa, the receptor responsible for the final step of platelet aggregation [22]. In this study, the effect of each compound on PKC activation was determined by measuring the phosphorylation of MARCKS, which is a major substrate of PKC in human platelets [23]. At the concentrations required to prevent platelet aggregation, compounds 1 and 2 inhibited U46619 (left)-and thrombin-induced (right) MARCKS phosphorylation ( Fig. 2A). The changes in intracellular [Ca 2+ ] i were measured in U46619-and thrombin-stimulated, fura-2-loaded platelets by monitoring the fluorescence of fura-2. Compounds 1 and 2 inhibited the U46619 (Fig. 2B)-and thrombininduced increases (Fig. 2C)

Effect of the isolated compounds on the clotting time
Incubation of human plasma with compounds 1 and 2 affected coagulation. The anticoagulant activity of each compound was evaluated using aPTT and PT assays and human plasma ( Table 1). As shown in Table 1, the aPTT and PT were significantly prolonged by each compound. Furthermore, the anticoagulant activity of compound 1 was higher than that of heparin or low molecular weight heparin (LMWH). However, (1S,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid (3), (3S)-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid (4) and L-tryptophan (5) did not alter the in vitro coagulation time (Table S3). Compounds 1 and 2 at 24.61 and 35.86 lM, respectively, doubled the clotting time in the aPTT assay and at concentrations of 17.06 and 46.46 lM, respectively, doubled the clotting time in the PT assay. Therefore, our results indicate that compounds 1 and 2 can inhibit the blood coagulation pathway.

Effects of the isolated compounds on thrombin and FXa activity
To elucidate the mechanism responsible for the inhibition of coagulation by compounds 1 and 2, the inhibition of thrombin and FXa activity was determined using chromogenic substrates. Treatment with compounds 1 and 2 resulted in a dose-dependent inhibition of the amidolytic activity of thrombin, indicating a direct inhibition of thrombin activity by compounds 1 and 2 (Fig. 3A). The direct thrombin inhibitor, argatroban, was used as a positive control. In addition, compounds 1 and 2 inhibited the activity of FXa (Fig. 3B). The direct FXa inhibitor, rivaroxaban, was used as a positive control. These results are consistent with those of our antithrombin assay and therefore suggest that the antithrombotic mechanism underlying the compound 1 and 2's actions involves the inhibition of the blood coagulation pathway.

Effects of the isolated compounds on thrombin and FXa production on HUVECs
In a previous study, Sugo et al. reported that endothelial cells support prothrombin activation by FXa [24]. In the current study, pre-incubation of HUVECs with FVa and FXa in the presence of CaCl 2 before the addition of prothrombin resulted in thrombin production (Fig. 3C). In addition, treatment with compounds 1 and 2 caused a dose-dependent inhibition of prothrombin-produced thrombin (Fig. 3C). According to findings reported by Rao et al., the endothelium provides the functional equivalent of pro-coagulant phospholipids and supports the activation of FX [25]. Furthermore, in TNF-a-stimulated HUVECs, the activation of FX by FVIIa was dependent on TF expression [26]. Thus, we investigated the effects of compounds 1 and 2 on the activation of FX by FVIIa. HUVECs were stimulated with TNF-a to induce TF expression, causing a 13-fold increase in the rate of FX activation by FVIIa in the stimulated HUVECs (101.5 AE 7.5 nM) compared with that in the non-stimulated HUVECs (7.8 AE 0.8 nM); these effects were abrogated by anti-TF IgG (15.9 AE 1.5 nM; Fig. 3D). In addition, pre-incubation with compounds 1 and 2 resulted in a dose-dependent inhibition of FX activation by FVIIa (Fig. 3D). The results of a chromogenic substrate assay demonstrate that compound 1 is a potent inhibitor of human thrombin and FXa with a Ki∇″>À+ lM and ″>À°l M, respectively (Table 2). And, compound 2 is also a potent inhibitor of human thrombin and FXa with a Ki∇″>9 0 lM and ″>Ä9 lM, respectively (Table 2). Therefore, these results suggest that compounds 1 and 2 inhibit the production of thrombin and FXa.

Effects of the isolated compounds on the secretion of PAI-1 and t-PA
TNF-a is known to inhibit fibrinolysis in HUVECs by inducing the production of PAI-I. The alteration of the balance between t-PA and PAI-1 is known to modulate coagulation and fibrinolysis [27]. To determine the direct effects of each compound on TNF-a-stimulated secretion of PAI-1, HUVECs were cultured in media with or without each compound and in the absence or presence of TNF-a for 18 hrs. As shown in Figure 4A, treatment with compounds 1 and 2 resulted in a dosedependent inhibition of TNF-a-induced secretion of PAI-1 from HUVECs, which was significant at 10-50 lM.
TNF-a does not have a significant effect on t-PA production [28] and the balance between plasminogen activators and their inhibitors   reflects the net plasminogen-activating capacity [29]; therefore, we investigated the effect of the combination of TNF-a and compounds 1 and 2 on the secretion of t-PA by HUVECs. Our results were consistent with those of a previous study reporting a modest decrease in the production of t-PA by TNF-a in HUVECs [30]. This decrease was not significantly altered by treatment with compounds 1 and 2 (Fig. 4B). Collectively, these results indicate that TNF-a increased the PAI-1/t-PA ratio, which was inhibited by compounds 1 and 2 (Fig. 4C).
In vivo effects of the isolated compounds in an arterial thrombosis, a pulmonary thrombosis model and bleeding time The mouse model of ferric chloride (FeCl 3 )-induced carotid artery thrombosis [31] has been commonly used to assess antiplatelet effects. The time to thrombus formation and the size of the resulting thrombi are summarized in Figure 5. Data showed that endothelial injury after FeCl 3 treatment in control mice led to the growth of large thrombi at 8.2 AE 0.8 min. and the antiplatelet GP IIb/IIIa inhibitor, tirofiban, significantly slowed the growth of large thrombi at 54.1 AE 5.7 min. Compounds 1 and 2 significantly slowed the growth of thrombi (Fig. 5A). We also examined the effect of each compound on thrombus size at 60 min. after FeCl 3 -induced endothelial injury (Fig. 5B). Results showed that compounds 1 and 2 reduced FeCl 3induced thrombus formation. In addition, the results of the in vivo pulmonary thrombosis model are shown in Figure 5C. An intravenous injection of a mixture of collagen and epinephrine into mice induced massive pulmonary thrombosis, causing acute paralysis and sudden deaths (90-95% mortality). The mortalities in compound 1-and compound 2-treated groups decreased significantly compared to that in collagen-and epinephrine-treated group (Fig. 5C). To confirm above-mentioned antithrombotic and antiplatelet functions, the in vivo tail bleeding times were determined. As shown in Table 1, the tail bleeding times were significantly prolonged by compounds 1 and 2 (i.v. injection) in comparison with those of the controls. The anticoagulation effect of each compound was observed ex vivo in mice as demonstrated by the dose-dependent prolongation of the aPTT and PT (Table S4). However, the ex vivo coagulation time was not altered by other compounds 3, 4 and 5 (Table S4).

Discussion
As a new biological resource in the development of the food and the pharmaceutical industries, the value of insects has been highly increased. Particularly, edible insects have attracted attention in the development of functional foods. The current study focused on the isolation of anticoagulant secondary metabolites from the larvae of P. brevitarsis seulensis and on the evaluation of isolated compounds by monitoring the clotting and bleeding time, platelet aggregation, and production of thrombin and FXa. Five indole alkaloids, 5-hydroxyindolin-2-one (1), (1R,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid (2), (1S,3S)-1-methyl-1,2,3,4-tetrahydro-bcarboline-3-carboxylic acid (3), (3S)-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid (4) and L-tryptophan (5) were isolated from the larvae. Compound 1 was synthetically reported [32], and compounds 2 and 3 were discovered from plants [20]. However, this is the first report to show that they are chemical constituents of insects and that  they exhibit antiplatelet activity. The indole alkaloid 1 was demonstrated to have a more potent antithrombotic activity than heparin. Tetrahydro-b-carbolines 2-4 could be produced through a Pictet-Spengler condensation of tryptophan with aldehydes or a-oxo acids [33]. Although the antiplatelet activity of one of the tetrahydro-b-carbolines has been reported [34], intravital microscopic technique to study thrombus formation in real time has enabled us to establish a role for this molecule in the regulation of thrombus formation in vivo. One of the most widely used procedures employs topical application of ferric chloride (FeCl 3 ) to an artery [35]. The mouse model of FeCl 3 -induced carotid artery thrombosis is an arterial thrombosis and a pulmonary thrombosis model and one of the most established and commonly used preparations to determine the efficacy of novel antithrombotic drugs in vivo [31]. This is a simple and well-established model known to be sensitive to both anticoagulant and antiplatelet drugs [35]. In our experiment, a mouse model of acute arterial thrombosis was developed by applying FeCl 3 to the outer tissues of arteries that had lost endothelial cell protection due to circulating platelets and components of the coagulation cascade, which was used to find both anticoagulant and antiplatelets drugs [36]. A hallmark of the FeCl 3 injury model is its sensitivity to thrombin inhibitors [37]. In this study, the FeCl 3 injury model produced an occlusive thrombosis quickly and stably (Fig. 5), which was suppressed by administering compounds 1 and 2.
The PT, aPTT and platelet aggregation are the most established and commonly used methods to determine the efficacy of novel antithrombotic drugs [38]. In our experiments, the PT and aPTT assays were performed using human plasma to evaluate the antithrombotic effects of compounds 1 and 2, while platelet aggregation was evaluated to determine the antiplatelet activity of compounds 1 and 2. LMWH and heparin could effectively inhibit aPPT and aPTT, and PT, respectively, and compounds 1 and 2 prolonged the PT and aPTT (Table 1). Furthermore, the antithrombotic effect of compound 1 was better than that of LMWH and heparin, indicating that compound 1 might be used as a novel strong antithrombotic drug. In addition, compounds 1 and 2 caused a significant inhibited platelet aggregation (Fig. 1).
Mode of action of compounds 1 and 2 for antithrombotic and antiplatelet effects was inhibiting (i) platelet aggregation, (ii) phosphorylation of MARCKS by PKC pathway, (iii) cytosolic Ca 2+ mobilization, (iv) the activation and production of thrombin and FXa, (v) intrinsic and extrinsic coagulation times, and (vi) reducing PAI-1/t-PA ratio. The mechanism of coagulation involves activation, adhesion and aggregation of platelets [39][40][41]. Coagulation begins almost instantly after an injury to the blood vessel has damaged the endothelium lining the vessel. Exposure of blood to the space under the endothelium initiates two processes: changes in platelets and the exposure of subendothelial tissue factor (TF) to factor VII (FVII), which ultimately leads to fibrin formation [39][40][41]. Disruption of the endothelium exposes platelets to collagen in the vessel wall and FVIIa to TF. Subsequently, propagation of the thrombus involves recruitment of additional platelets and amplification of the coagulation cascade by the intrinsic pathway of blood coagulation, which includes the haemophilia factors FVIII and FIX [41]. Importantly, platelets and endothelial cells play a critical role in the amplification of the coagulation cascade by providing a thrombogenic surface. Therefore, the activation and behaviours of platelets are the mechanistic targets of both pathways of coagulation and aggregation, suggesting that the inhibitory effects of compounds 1 and 2 on the activation and behaviours of platelets could be the most important functions of antithrombotic and antiplatelet effects of compounds 1 and 2.
Platelets are activated by several agonists that bind to specific platelet membrane receptors [42]. Serotonin (5-hydroxytryptamine, 5-HT) facilitates the development of platelets with increased pro-coagulant activity and potentiates platelet activation, resulting in converging signal transduction pathways that would be responsible for blood coagulation [43,44]. This implies that modulation of serotoninmediated responses may offer a therapeutic target for the development of anticoagulant agent. Among the known 5-HT receptors, 5-HT 2A (formerly termed 5-HT 2 ) receptors on vascular smooth muscle and platelets play important roles in the cardiovascular system [45][46][47]. Several studies suggest a connection between serotonergic mechanisms and cardiovascular events. Nishihira et al., reported that intravenous injection of sarpogrelate, an inhibitor of the 5-HT 2A receptor (serotonin receptor), in a rabbit model significantly reduced the ex vivo platelet aggregation induced by ADP, thrombin and collagen alone as well as with 5-HT and significantly prevented occlusive thrombus formation in vivo [48]. Another study also reported that increased platelet 5-HT could be effective in the control of bleeding in idiopathic thrombocytopenic purpura [49]. Considering the structure of compounds 1 and 2 possessing indole core structure of serotonin, they may act as serotonin antagonists in platelet aggregation and thrombus formation.
As a commercial anticoagulant, heparin has been used for the prevention of venous thromboembolic diseases for more than 60 years [50]. However, heparin has side effects such as the inability to inhibit fibrin-bound thrombin activity, ineffectiveness in congenital or acquired antithrombin deficiencies, the development of thrombocytopenia, an increased risk of thromboembolic disease if the therapeutic response is not achieved and an increased risk of bleeding if the therapeutic range is exceeded [51]. Furthermore, the amounts of available heparin are low in bovine lungs or pig intestines, from which heparin is primarily extracted [51]. Therefore, the need for discovering alternative sources of anticoagulants has increased owing to the demand for a safer anticoagulant therapy. Based on the current findings, compounds 1 and 2 may provide new chemotypes for the development of anticoagulants provided their therapeutic effects are established.
In conclusions, this study demonstrated that compounds 1 and 2 inhibited the blood coagulation pathways by inhibiting FXa and thrombin production in HUVECs. They also inhibited the TNF-ainduced secretion of PAI-1 and platelet aggregation in vitro and ex vivo. These results add to those from previous works on this topic and may be of interest to those designing pharmacological strategies for the treatment or prevention of coagulation-related vascular diseases.

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Data S1 Supplementary Materials and Methods.