Suppression of Human Platelet Activation via Integrin αIIbβ3 Outside-In Independent Signal and Reduction of the Mortality in Pulmonary Thrombosis by Auraptene

Auraptene is the most abundant coumarin derivative from plants. The pharmacological value of this compound has been well demonstrated, especially in the prevention of cancer and neurodegenerative diseases. Platelet activation is a major factor contributing to arterial thrombosis. Thus, this study evaluated the influence of auraptene in platelet aggregation and thrombotic formation. Auraptene inhibited platelet aggregation in human platelets stimulated with collagen only. However, auraptene was not effective in inhibiting platelet aggregation stimulated with thrombin, arachidonic acid, and U46619. Auraptene also repressed ATP release, [Ca2+]i mobilization, and P-selectin expression. Moreover, it markedly blocked PAC-1 binding to integrin αIIbβ3. However, it had no influence on properties related to integrin αIIbβ3-mediated outside-in signaling, such as the adhesion number, spreading area of platelets, and fibrin clot retraction. Auraptene inhibited the phosphorylation of Lyn-Fyn-Syk, phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), Akt, and mitogen-activated protein kinases (MAPKs; extracellular-signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK1/2), but not p38 MAPK). Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the auraptene-mediated inhibition of platelet aggregation. Auraptene reduced mortality caused by adenosine diphosphate (ADP)-induced pulmonary thromboembolism. In conclusion, this study provides definite evidence that auraptene signifies a potential therapeutic agent for preventing thromboembolic disorders.


Inhibitory Profiles of Auraptene in Agonist-Stimulated Washed Human Platelets
Auraptene is a coumarin-derived compound from citrus plants, and it possesses a geranyloxyl moiety at the C-7 position ( Figure 1A). Teng et al. [9] reported that auraptene (100-200 µM) concentration dependently suppressed collagen, thrombin, ADP, AA, U46619 (a thromboxane A 2 receptor agonist), and platelet-activating factor-stimulated rabbit platelet aggregation. No further evidence has been provided after that study. In this study, auraptene markedly inhibited collagen (1 µg/mL)-stimulated human platelet aggregation at 10 to 50 µM concentrations. These concentrations are lower than those employed for rabbit platelets in a previous study [9]. However, auraptene slightly inhibited platelet aggregation, and the inhibition was not significant in platelets stimulated with either AA, thrombin, or U46619, even with concentrations up to 100 µM ( Figure 1B,C). These results indicate that auraptene exhibited differences on its potency and mechanisms between the human and rabbit platelets. The IC 50 of auraptene in collagen-induced platelet aggregation was approximated at 35 µM ( Figure 1C). The solvent control (0.1% DMSO) did not exhibit any significant effects on platelet aggregation ( Figure 1B) In addition, auraptene (50 µM) inhibited ADP (20 µM)-induced platelet aggregation by approximately about 20% in platelet-rich plasma (data not shown). Furthermore, the lactate dehydrogenase (LDH) assay revealed that auraptene (35, 50, and 100 µM) pretreatment for 20 min did not alter LDH release and did not cause any observable cytotoxic effects in platelets ( Figure 1D). This result demonstrates that auraptene neither affects platelet permeability nor induces platelet cytolysis.

Role of Auraptene in Integrin α IIb β 3 Activation
Platelet aggregation is dependent on fibrinogen-integrin α IIb β 3 interaction. Integrin α IIb β 3 inhibition results in the disaggregation of accumulated platelets [12]. To know whether auraptene disturbs integrin α IIb β 3 activation, the binding of the fluorescein isothiocyanate (FITC)-conjugated PAC-1 mAb that responds to the stimulation-induced conformational epitope of integrin α IIb β 3 was analyzed by flow cytometry ( Figure 3A). Auraptene (35 and 50 µM) considerably inhibited integrin α IIb β 3 activation stimulated by collagen. This finding indicates that auraptene may influence the binding of PAC-1 to the activated integrin α IIb β 3 . Furthermore, the quantity of immobilized fibrinogen to which platelets adhered was significantly greater than that of immobilized bovine serum albumin (BSA) (Figure 3Ba,b), as indicated by the presence of platelets stained with FITC-conjugated phalloidin. No significant differences were found in platelet adhesion and spreading on immobilized fibrinogen between 0.1% DMSO and auraptene (35 and 50 µM)-treated platelets (Figure 3Bc,d). Figure 3C shows resting platelets were fixed to immobilized fibrinogen (106.0 ± 9.3 platelets/0.01 mm 2 ; n = 4) compared to the immobilized BSA (BSA, 44.8 ± 10.6 platelets/0.01 mm 2 ; n = 4). However, platelets that had been treated with auraptene showed similar binding effects to the fibrinogen-coated surface (35 µM, 105.3 ± 14.3 platelets/0.01 mm 2 ; 50 µM, 94.8 ± 13.1 platelets/0.01 mm 2 , n = 4). Moreover, no significant difference was found in the surface coverage of a single platelet between 0.1% DMSO and auraptene-treated platelets (0.1% DMSO, 26.1 ± 1.3 µm 2 ; auraptene 35 µM, 23.6 ± 2.0 µm 2 and 50 µM, 22.4 ± 1.7 µm 2 ; n = 4). Furthermore, clot retraction of fibrin polymers, the end process in thrombus formation, is necessary in aggregate stabilization [3], and it is paradigmatic of integrin α IIb β 3 outside-in signaling. A clot retraction study was performed by adding thrombin to a solution enclosing fibrinogen in the presence of either auraptene-or 0.1% DMSO-treated human platelets. The results show that clot retraction was more apparent after the 30-min incubation of 0.1% DMSO than that of the 15-min incubation in platelets ( Figure 3E). However, clot retraction was not significantly abridged in platelets treated in 35 and 50 µM auraptene. This finding indicates that auraptene has no significant ability to reduce fibrin clot retraction. Taken together, the results indicate that auraptene does not restrict clot retraction as well as the integrin α IIb β 3 -mediated outside-in signaling of cell adhesion and spreading.

Effects of Auraptene on Fyn, Lyn, Syk, and PLCγ2/PKC Signaling
The binding of collagen with glycoprotein (GP) VI is reported to be mediated by the Src-family kinases (SFKs) Fyn and Lyn [13], resulting in the activation of the cytosolic tyrosine kinase Syk. As shown in Figure 4A-C, the collagen-induced phosphorylation of Fyn, Lyn, and Syk was significantly inhibited by auraptene. Moreover, PLC comprises a family of kinases that hydrolyze phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to produce two second messengers, diacylglycerol (DAG) and inositol trisphosphate (IP 3 ). DAG activates PKC-stimulating protein phosphorylation (p47 protein; pleckstrin) and ATP release in activated platelets; IP 3 elevates calcium influx [14]. In the present study, the effect of auraptene on the phosphorylation of the PLCγ2-PKC signaling cascade was examined. Auraptene treatment (35 and 50 µM) concentration dependently diminished the phosphorylation of PLCγ2 in collagen-activated platelets ( Figure 4D). As illustrated in Figure 4E, a similar molecular weight protein p47 (47 kDa) was apparently phosphorylated in collagen-activated platelets, which was concentration-dependently reduced by auraptene. In addition, neither 35 nor 50 µM of auraptene reduced PDBu (PKC activator)-induced platelet aggregation ( Figure 4F). This result indicates that auraptene inhibits both SFKs and PLCγ2/PKC activation.
Moreover, the antithrombotic effects of auraptene on acute pulmonary embolism-induced mortality in mice are summarized in Table 1. The results revealed that auraptene at 7.5 and 15 mg/kg significantly reduced ADP-induced mortality and significantly reversed platelet numbers in mice. Specifically, the reduction rate was from 87.5% (7 dead, n = 8 in total) in 0.1% DMSO-treated mice to 62.5% (5 dead, n = 8 in total) and 25% (2 dead, n = 8 in total) in auraptene-treated mice, respectively. Furthermore, aspirin (20 mg/kg), an effective antiplatelet drug prescribed for preventing or treating cardiovascular diseases, also reduced mortality and reversed platelet numbers in this study (Table  1).  Moreover, the antithrombotic effects of auraptene on acute pulmonary embolism-induced mortality in mice are summarized in Table 1. The results revealed that auraptene at 7.5 and 15 mg/kg significantly reduced ADP-induced mortality and significantly reversed platelet numbers in mice. Specifically, the reduction rate was from 87.5% (7 dead, n = 8 in total) in 0.1% DMSO-treated mice to 62.5% (5 dead, n = 8 in total) and 25% (2 dead, n = 8 in total) in auraptene-treated mice, respectively. Furthermore, aspirin (20 mg/kg), an effective antiplatelet drug prescribed for preventing or treating cardiovascular diseases, also reduced mortality and reversed platelet numbers in this study (Table 1).

Discussion
This study is the first to demonstrate that in addition to its well-known properties, auraptene possesses marked antiplatelet activity against human platelets. Because it is a hydrophobic molecule, auraptene is sparingly soluble in aqueous buffers. Thus, when administered orally, it undergoes rapid absorption and is rapidly distributed to all tissues; it is especially able to penetrate the blood-brain barrier [7]. Furthermore, auraptene is widely and easily available in large amounts through chemical synthesis. Therefore, auraptene is a potentially new therapeutic agent for the treatment of thrombotic diseases in humans.
In this study, auraptene potently inhibited collagen-induced platelet aggregation. However, inhibition was slight and not statistically significant for platelets stimulated with other agonists. This finding implies that auraptene-mediated inhibition of platelet aggregation occurs through a distinct PLC-dependent mechanism. The collagen-induced platelet activation noticeably modifies phospholipase activation. The triggering of PLC causes IP 3 and DAG formation, which in turn activates PKC and subsequently induces the phosphorylation of the p47 protein [17]. The IP 3 -triggering calcium release from the intracellular calcium store is only a minor part; however, the predominant part is regulated by the calcium channels on the plasma membrane of platelets [18]. Therefore, auraptene seems not only to affect IP 3 -mediated calcium exflux but also affects calcium influx from calcium channels. The PLC enzyme comprises six families: PLCβ, PLCγ, PLCδ, PLCε, PLCζ, and PLCη [19]. The PLCγ family comprises isozymes 1 and 2. PLCγ2 participates in collagen-dependent signaling in platelets [19]. In our current study, auraptene weakened collagen-activated PLCγ2. However, auraptene exerted no direct effects on PKC activation because it did not reduce PDBu-induced platelet aggregation. This finding suggests that PLCγ2 downstream signaling involves auraptene-mediated inhibition of platelet activation.
GP VI belongs to a membrane of the immunoglobulin superfamily, which forms a complex with the Fc receptor γ-chain (FcRγ) containing immunoreceptor tyrosine-based activation motifs (ITAM) and is phosphorylated by SFKs, such as Fyn and Lyn [2]. In platelets, SFKs, particularly Lyn and Fyn, play a crucial role downstream of collagen receptors [13]. Studies have also proven that SFKs play a role in thromboxane generation, shape change, as well as the regulation of phosphorylation of Akt [20] and ERK [21]. In addition, MAPKs constitute a family of serine-threonine protein kinases that convert extracellular stimuli into a wide range of cellular responses. Previous studies using specific inhibitors or knockout mice have identified ERK1/2, JNK1/2, and p38 MAPK in platelets and showed their participation in platelet activation [22]. All these MAPKs were triggered by specific MAPK kinases (MEKs). The physiopathological aspects of JNK1/2 and ERK1/2 in platelets remain largely unknown. However, proof advocates that the destruction of α IIb β 3 integrin activation may be intricate in their roles [23]. Moreover, ERK1/2 activation is essential for collagen-induced platelet aggregation [24]. Cytosolic phospholipase A 2 (cPLA 2 ), which catalyzes AA release to produce thromboxane A 2 , is an important substrate of p38 MAPK activation and is tempted by different stimulators, including thrombin [25]. The present results indicate that auraptene-promoted inhibition of collagen-stimulated platelet activation involves ERK1/2 and JNK1/2 but not p38 MAPK. This might explain why auraptene displays potency in inhibiting platelets stimulated with collagen compared to AA, U46619, and thrombin. Moreover, Fan et al. [22] recently reported that ERK1/2 and JNK1/2, but not p38 MAPK, are the major MEK kinase (MEKK)3 downstream signaling molecules in platelet activation. Thus, we speculate that auraptene may affect MEEK3, resulting in the inhibition of ERK1/2 and JNK1/2 phosphorylation. However, this needs to be proven in a future study.
Akt is a downstream regulator of PI3K, and Akt-deleted mice showed weaknesses in platelet activation [26]. Hence, Akt activation through protein kinases, particularly PI3K, may be attractive targets for the development of antithrombotic therapeutics. Although the effectors through which Akt contributes to platelet activation are not definitively known, several candidates, including glycogen synthase kinase 3β, phosphodiesterase 3A, and integrin β 3 , have been identified [26]. In addition, both PI3K/Akt and MAPKs are mutually activated in platelets, and PKC's role as an upstream regulator of them has also been reported [27].
Cyclic AMP and cyclic GMP elevation in platelets activates their respective protein kinase A and protein kinase G. This in turn regulates platelet activation by phosphorylating intracellular protein substrates, such as VASP. Such protein substrates are involved in the inhibition of agonist-induced platelet aggregation and the adhesion of platelets to the vascular wall [28]. Increased levels of cyclic nucleotides hinder several platelet responses and diminish the levels of [Ca 2+ ]i through Ca 2+ uptake into the dense tubular system. This inhibits the stimulation of PLC/PKC signaling. Thus, increased cyclic AMP/GMP mutually work in the inhibition of platelet triggering. In this study, both SQ22536 and ODQ did not significantly reverse the auraptene-mediated reduction of platelet aggregation. Auraptene also had no effect on VASP phosphorylation. Therefore, the intracellular cyclic nucleotides-VASP pathway is not involved for the noted antiplatelet effects of auraptene.
The binding of fibrinogen to integrin α IIb β 3 is a major component of the platelet aggregation response. Integrin α IIb β 3 undergoes conformational changes upon activation. It generates specific ligand-binding sites for fibrinogen, von Willebrand factor, and fibronectin [3]. Platelet adheres to immobilized fibrinogen and mediates clot retraction, which are involved in integrin α IIb β 3 outside-in signaling and cytoskeleton reorganization [3]. Here, auraptene reduced the binding of PAC-1 to activated integrin α IIb β 3 . However, it had no effects on clot retraction and abolishing platelet adhesion and spreading. This indicates that auraptene influences integrin α IIb β 3 in inside-out, but not outside-in, signaling. Animal models of vascular thrombosis are necessary to understand the effectiveness of test compounds to prevent or treat these diseases. An ideal mouse model should be technically simple, fast to establish, and easily reproducible. An intravenous injection of platelet agonist ADP results in the formation of acute occlusive thrombi; the mortality rate of ADP-treated mice is believed to result in the formation of thrombi in lungs [29]. In this study, the mortality rate of auraptene-treated animals was markedly lower than that of solvent-treated animals. This indicates that auraptene is a natural compound that potentially treats thromboembolic disorders.
In conclusion, auraptene shows a unique property of inhibiting human platelet activation. Thus, it has potential therapeutic applications. Specifically, auraptene significantly inhibits platelet triggering by delaying the SFKs and PLCγ2-PKC cascade. This can lead to diminished activation of Akt and ERK1/2/JNK1/2. This inhibits the release of substances, such as P-selectin, ATP, and [Ca 2+ ]i, which in turn influences integrin α IIb β 3 inside-out signaling and eventually inhibits platelet aggregation. The results of this study provide new insights into the role of auraptene in human platelet activation.

Platelet Preparation, Aggregation, and ATP Release
This study conformed to the directives of the Helsinki Declaration and was approved by the Institutional Review Board of Taipei Medical University (TMU-JIRB-N201812024,09 January 2019). An informed consent form was provided to all human blood donors involved this study. Washed human platelets were prepared as described previously [30]. Either auraptene (10-100 µM) or solvent control (0.1% DMSO) was incubated with platelets for 3 min before stimulation. ATP release was assayed using a Hitachi Spectrometer F-7000 (Tokyo, Japan) based on the manufacturer's procedure.

Intracellular [Ca 2+ ] Mobilization Using Fura 2-AM Fluorescence
To measure the level of [Ca 2+ ]i, citrated whole blood was centrifuged and the supernatant was incubated with 5 µM Fura 2-AM. The Fura 2-AM fluorescence was measured using the Hitachi Spectrometer F-7000. The intracellular calcium ([Ca 2+ ]i) level was calculated at the excitation wavelengths of 340 and 380 nm and the emission wavelength of 500 nm [31].

Lactate Dehydrogenase Assay
The cytotoxic effect was examined by determining the level of lactate dehydrogenase (LDH). Washed platelets (3.6 × 10 8 cells/mL) were preincubated with either auraptene (35, 50, and 100 µM) or 0.1% DMSO for 20 min at 37 • C. An aliquot of the supernatant (10 µL) was deposited on a Fuji Dri-Chem slide LDH-PIII (Tokyo, Japan) and read by a spectrophotometer (UV-160; Shimazu, Japan). The maximal level of LDH was observed in triton-treated platelets.

Platelet Adhesion and Spreading Analysis on Immobilized Fibrinogen
Analysis of platelet spreading on immobilized fibrinogen was performed by using confocal microscopy as described previously [32]. Platelets were stained with FITC-labeled phalloidin and imaged using a Leica TCS SP5 microscope equipped with a 100×, 1.40 NA oil immersion objective (Leica, Wetzlar, Germany). Platelet adhesion and the platelet spreading were analyzed using NIH ImageJ software (NIH, Bethesda, MD, USA; http://rsbweb.nih.gov/ij/).

Platelet-Mediated Clot Retraction
Washed platelets were remixed in Tyrode's solution that contained 2 mg/mL fibrinogen and 1 mM CaCl 2 in tubes designed for aggregation [33]. The platelet suspensions were preincubated with auraptene (35 and 50 µM) or 0.1% DMSO for 3 min prior to thrombin (0.01 U/mL)-induced clot retraction without stirring. The reaction was developed at 37 • C in an aggregometer tube and photographed at 15 and 30 min, respectively.

Immunoblotting
Washed platelets (1.2 × 10 9 cells/mL) were pretreated with either auraptene (35 and 50 µM) or 0.1% DMSO, and collagen was subsequently added to trigger platelet activation. The platelet suspensions were lysed and separated through 12% SDS-PAGE. Several proteins were detected using specific primary antibodies. The respective semi quantitative results were obtained by quantifying the optical density of protein bands on a video densitometer and through Bio-profil Biolight software, Version V2000.01 (Vilber Lourmat, Marne-la-Vallée, France).

Acute Pulmonary Thromboembolism Stimulated by ADP in Mice
Acute pulmonary thromboembolism was induced in mice following a previous method [34]. Mice were intraperitoneally administered different doses of auraptene (7.5 and 15 mg/kg) and aspirin (20 mg/kg) or 0.1% DMSO (50 µL for all). After 5 min of auraptene treatment, a 0.7 mg/g dose of ADP was injected via the tail vein. Blood (0.5 mL) was collected by cardiac puncture and transferred into BD microtainer ® tubes with K 2 EDTA for the determination of platelet counts using an automatic cell counter (Procyte Dx; IDEXX Laboratories Inc., Westbrook, ME, USA). The rate of mortality was determined in each group.

Statistical Analysis
The data are presented as mean ± SEM, and convoyed by the number of interpretations (n). Specifically, n represents the number of investigates, and each investigation was accompanied using different blood donors. The unpaired Student's t-test and analysis of variance (ANOVA) were used to determine the significance of differences among the groups. Groups with significant differences in analysis were compared using the Student-Newman-Keuls method. p < 0.05 indicated statistical significance.