A novel naphthalimide derivative reduces platelet activation and thrombus formation via suppressing GPVI

Abstract Naphthalimide derivatives have multiple biological activities, including antitumour and anti‐inflammatory activities. We previously synthesized several naphthalimide derivatives; of them, compound 5 was found to exert the strongest inhibitory effect on human DNA topoisomerase II activity. However, the effects of naphthalimide derivatives on platelet activation have not yet been investigated. Therefore, the mechanism underlying the antiplatelet activity of compound 5 was determined in this study. The data revealed that compound 5 (5–10 μM) inhibited collagen‐ and convulxin‐ but not thrombin‐ or U46619‐mediated platelet aggregation, suggesting that compound 5 is more sensitive to the inhibition of glycoprotein VI (GPVI) signalling. Indeed, compound 5 could inhibit the phosphorylation of signalling molecules downstream of GPVI, followed by the inhibition of calcium mobilization, granule release and GPIIb/IIIa activation. Moreover, compound 5 prevented pulmonary embolism and prolonged the occlusion time, but tended to prolong the bleeding time, indicating that it can prevent thrombus formation but may increase bleeding risk. This study is the first to demonstrate that the naphthalimide derivative compound 5 exerts antiplatelet and antithrombotic effects. Future studies should modify compound 5 to synthesize more potent and efficient antiplatelet agents while minimizing bleeding risk, which may offer a therapeutic potential for cardiovascular diseases.


| INTRODUC TI ON
Normal haemostasis is a complicated process involving platelets and the coagulation cascade. 1 When blood vessels are injured, circulating platelets adhere to exposed extracellular matrix proteins, such as von Willebrand factor and collagen. The exposed collagen can activate platelets through glycoprotein VI (GPVI). GPVI, a collagen receptor present on platelets, is associated with an immunorecep- downstream of GPVI, which regulates platelet secretion and calcium mobilization. 5,6 These activation cascades finally result in the formation of a firm platelet plug at the injury site to stop blood loss. Inappropriate regulation of the haemostatic process may lead to pathological consequences such as bleeding and thrombosis.
Antiplatelet agents are used to prevent or treat secondary ischaemic stroke and myocardial infarction. However, the clinically significant bleeding has limited their use, 7,8 necessitating the development of new antiplatelet agents with minimal bleeding risk.
Most compounds containing the naphthalimide moiety are fluorescent and have multiple biological activities including antitumuor, anti-inflammatory and antiviral avtivities. 9 Naphthalimide derivatives can easily intercalate into DNA and block cell division because of their flat structures. 9,10 Moreover, these derivatives can intercalate into topoisomerase II (topo II), an enzyme with DNA breakage-reunion activity. 10 Because of the aforementioned properties, naphthalimide derivatives can be potential antitumour agents. Moreover, many researchers have synthesized novel naphthalimide derivatives to develop more potent and efficient antitumour drugs with low toxicity. [11][12][13][14] Previously, we synthesized several naphthalimide derivatives 15,16 and evaluated their anticancer effect. Among these derivatives, compound 5 ( Figure 1) exhibited the highest potency in inhibiting human DNA topo II activity (IC 50 = 2.6 ± 0.1 μM) in murine B16F10 melanoma cells. 16 In addition, 7b, a naphthalimide derivative, could inhibit lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation in RAW264.7 macrophages. 17 Moreover, naphthalimides antagonize NS1 and influenza virus and thus inhibit viral replication; this process might be mediated by REDD1. 18 Although these naphthalimide derivatives have been reported to be involved in several pathophysiological processes, their effects on platelet activation have not been investigated. Thus, in the present study, we examined the antiplatelet mechanism of compound 5. We expect that compound 5 be a lead compound for the development of novel antiplatelet agents for treating cardiovascular diseases.

K E Y W O R D S
antiplatelet activity, bleeding risk, collagen, naphthalimide derivatives, platelet activation, thrombus formation F I G U R E 1 Structure of compound 5. Compound 5 consists of a naphthalimide framework (green box) in which the C4 position is linked with an ethylenediamine group (blue box) where the amino group couples with a 2-piconic acid moiety (red box) Anti-phospho-(Ser) PKC substrate, anti-phospho-JNK (Thr 183 /Tyr 185 ), anti-phospho-p38 MAPK (Thr 180 /Tyr 182 ) and anti-JNK pAbs; and anti-Akt, anti-p38 MAPK and anti-ERK mAbs were purchased from Cell Signaling. Horseradish peroxidase (HRP)-conjugated AffiniPure goat antirabbit, AffiniPure goat antimouse and AffiniPure donkey antigoat immunoglobulin G (IgG) were purchased from Jackson ImmunoResearch. Allophycocyanin (APC)-conjugated PAC-1 antibodies and anti-P-selectin were purchased from Biolegend. Hybond-P polyvinylidene difluoride (PVDF) membrane was purchased from GE Healthcare Life Sciences. A SuperLight Chemiluminescent HRP kit was purchased from Bionovas.

| Synthesis of compound 5
Compound 5 was synthesized as described previously. 16 Compound 5 consists of a naphthalimide framework in which the C4 position is linked with an ethylenediamine group where the amino group couples with a 2-piconic acid moiety ( Figure 1). Compound 5 was dissolved in DMSO and stored at 4°C until use.

| Preparation of platelet suspensions
This study was approved by the Taipei Medical University-Joint Institutional Review Board (TMU-JIRB-No. N202003148) and conformed to principles outlined in the Declaration of Helsinki.
All volunteers provided informed consent prior to participation in this study. Washed human platelets were prepared as described previously. 19 In brief, whole blood was obtained from healthy participants who had not received any medicines such as nonsteroidal anti-inflammatory drugs and aspirin during the preceding 2 weeks.
Whole blood was drawn into polypropylene plastic tubes filled with an acid citrate/dextrose solution (A.C.D; 9:1, v/v). After mixing the blood samples at 120g for 10 min, the platelet-rich plasma (PRP; upper layer) was collected and supplemented with prostaglandin E 1 and heparin. After further centrifugation at 500 g for 10 min, platelet pellets were washed twice. The washed platelets were resuspended in Tyrode's solution supplemented with 3.5 mg/ml bovine serum albumin (BSA) to obtain platelet suspensions. The final Ca 2+ concentration in platelet suspensions (3.6 × 10 8 cells/ml) was 1 mM.

| Western blotting
Western blotting was performed as described previously. 20 In brief, platelet suspensions (3.6 × 10 8 cells/ml) were pretreated with compound 5 (5 and 10 μM) or 0.1% DMSO for 3 min and then treated again with collagen for 6 min. After centrifugation, platelet pellets were immediately resuspended in lysis buffer (200 μl) for 1 h. The supernatants were collected after centrifugation at 5000 g for 5 min.

| ATP release and calcium mobilization
This method was performed as described previously. 21 In brief, luciferase/luciferin and Fura 2-AM were used to detect ATP release and calcium mobilization respectively. The intensity of luminescence (ATP release) and the ratio (wavelength = 340/380 nm) of fluorescence (calcium mobilization) were measured using a Hitachi Spectrometer F-7000 in accordance with the manufacturer's instructions.

| Flow cytometry
Flow cytometry was performed as described previously. 20 In brief, 20 min after collagen stimulation, platelets were fixed and labelled with P-selectin or PAC-1 antibodies conjugated with APC for 30 min to detect the surface expression of P-selectin and the level of GPIIb/IIIa activation respectively. After centrifugation and washing, platelets were suspended in 1 ml of phosphate-buffered saline and measured using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences). In the flow cytometry setting, platelets were gated by a forward scatter and a side scatter, and the number of events at 10,000 counts was stopped. All experiments were performed at least three times to ensure reliability.

| Washed mouse platelet preparation and aggregation study
Mouse blood was collected through cardiac puncture into a tube containing 100 µl of sodium citrate followed by gentle mixing. After centrifugation at 180 g for 5 min, PRP was obtained and mixed with A.C.D. (9:1, v:v). The platelet pellet was obtained after centrifugation at 1300 g for 15 min and then resuspended in Tyrode's solution. A lumi-aggregometer (Payton Associates) was used to measure platelet aggregation as described previously. 19 Platelet suspensions (1.5 × 10 8 /ml) were stimulated using collagen (1 µg/ml) for 10 min; the extent of aggregation is expressed in light transmission units.

| Pulmonary embolism in mice induced by collagen/epinephrine
Pulmonary thromboembolism was induced using collagen and epinephrine in male ICR mice according to the method described by (wavelength <520 nm) to induce endothelial damage-causing thrombus formation and subsequent vessel occlusion. 24 The time required to occlude a microvessel was recorded. A formula for dose translation based on the body surface area was used to calculate the dose for use in the mice. 23

| Tail-bleeding assay
Mice were anaesthetized using 3% isoflurane with oxygen-air mixture at a gas flow rate of 1.5-2 L/min. A bolus dose of compound 5 (1.2, 2.3 and 4.5 mg/kg), DMSO (solvent control) or aspirin (20 mg/ kg, positive control) was intravenously administered for 30 min.
Next, a 3-mm incision was made from the tail tip to induce tail bleeding. The bleeding tail stump was immediately immersed in saline, and the bleeding time, which was defined as the time until no sign of bleeding was observed for at least 10 s, was recorded. 20 A formula for dose translation based on the body surface area was used to calculate the dose for use in the mice. 23

| Statistical analysis
Data were analysed using analysis of variance with a post hoc analysis performed using the Newman-Keuls test. For survival analysis, survival curves were plotted using the Kaplan-Meier curves and analysed using the log-rank test, and all pair-wise multiple comparison procedures were performed by the Holm-Sidak method. Results are expressed as the mean ± standard error of the mean (SEM). p < 0.05 was considered statistically significant.

| Compound 5 blocks collagen-induced platelet aggregation
Various platelet agonists were used to investigate the effect of com-  Figure 2G (washed platelets) and 2H (PRP). Compound 5 alone did not cause platelet activation as evidenced by observing shape change ( Figure S1) and did not cause platelet cytotoxicity as detected by LDH cytotoxicity assay ( Figure S2). On the basis of these results, we investigated the effect of compound 5 on collagen receptor downstream signalling in subsequent experiments.

| Compound 5 attenuates platelet activation by suppressing GPVI signalling
The binding of collagen to GPVI can activate SFKs Fyn and Lyn and subsequently activate PLCγ2-PKC and MAPKs. 2-4 Thus, we examined the effect of compound 5 on GPVI downstream signalling. As illustrated in Figure 3A and Figure  and not directly inhibiting PKC ( Figure 3E). Collectively, these findings indicated that compound 5 can inhibit GPVI signalling.
Other signalling pathways responsible for collagen-induced platelet activation were examined. As shown in Figure 4, compound 5 did not affect collagen-induced Akt or p38 activation but significantly inhibited ERK and JNK activation, suggesting that compound 5 reduced collagen-induced platelet activation partly through inhibiting the activation of ERK and JNK.

| Compound 5 blocked granule release, calcium mobilization and GPIIb/IIIa activation
Platelet activation signalling can result in substance release from alpha and dense granules, such as ADP and fibrinogen that regulate  25 Moreover, calcium release can promote granule release. Eventually, the inside-out signalling can activate GPIIb/IIIa. 3 Thus, these activation events including granule release, calcium mobilization and GPIIb/IIIa are considered to be indicators of platelet activation. We investigated the effect of compound 5 on these activation events. Granule release was detected by measuring the release of ATP and the surface expression of P-selectin, which represent the release of dense and alpha granules respectively. As illustrated in Figure 5A,B, collagen markedly induced the release of ATP and the expression of P-selectin; this release was reversed by  Figure S4). These findings indicate that compound 5 (5 and 10 μM) could attenuate collagen-induced platelet activation.

| Compound 5 inhibits ex vivo platelet aggregation and pulmonary embolism in mice
Platelets are involved in thrombosis, and antiplatelet agents are commonly used in clinical practice to prevent secondary thromboembolic events. 7,8 The findings of this study demonstrated that compound 5 exhibited antiplatelet activity in vitro. Thus, we investigated whether compound 5 exerts antithrombotic effects in vivo. To examine platelet aggregation ex vivo, whole blood was obtained from mice after they were injected with compound 5 or aspirin for 10 min. Subsequently, washed mouse platelets were prepared. Platelet aggregation was recorded for 10 min upon collagen treatment. As shown in Figure  To examine pulmonary embolism in vivo, after mice were injected with compound 5 or aspirin for 10 min, collagen/epinephrine was injected to induce pulmonary embolism, which was observed through staining with Evans blue. The survival rate of mice was evaluated for 24 h. As shown in Figure 6B

| Compound 5 delayed thrombus formation in mesenteric microvessels in mice
We employed another mouse thrombosis model to confirm the antithrombotic effect of compound 5. In this model, the endothelium was damaged through UV irradiation, resulting in vessel occlusion, which was observed and recorded using a real-time monitor. As illustrated in Figure 7A

| DISCUSS ION
This is the first study to demonstrate that compound 5 is more sensitive to the inhibition of collagen-mediated platelet activation, partly through suppressing GPVI signalling, followed by the inhibition of granule release, calcium mobilization and GPIIb/IIIa activation, eventually blocking platelet activation and thrombus formation (Figure 8).
These findings indicate that naphthalimide-based compounds have antiplatelet and antithrombotic activities. Therefore, compound 5 may serve as a lead compound that can be further modified to synthesize more potent and efficient antiplatelet agents with minimal bleeding risk.
Naphthalimides have been used as a core scaffold for the development of antitumour and anti-inflammatory agents. 9,10 They can intercalate with DNA and inhibit topo II due to their planar and heteroaromatic structure. Thus, many new naphthalimide derivatives have been developed as anticancer agents. 9, 10 We previously synthesized several naphthalimide derivatives and found them to exhibit cytotoxic effects on B16F10 melanoma cells and reduce lung metastasis at least partly through the inhibition of topo II activity. 15,16 Among the naphthalimide derivatives we synthesized, compound 5 was found to exert the strongest inhibitory effect on topo II activity. 16 At a concentration of 10 μM, compound 5 almost completely inhibited topo II activity. In the present study, we found that compound 5 (10 μM) blocked platelet aggregation and platelet activation events including granule release, calcium mobilization and GPIIb/IIIa activation. This finding implies that naphthalimides can serve as a core scaffold for antiplatelet agents. In addition, com- However, whether naphthalimide-based compound 5 exerts antiinflammatory effect remains to be determined.
Akt is essential for collagen-induced platelet aggregation, 32 and an SFK inhibitor was reported to completely inhibit GPVI- Therefore, although compound 5 may act as a lead compound for the design of new naphthalimide-based antiplatelet agents, the bleeding side effect must be eliminated or minimized during future drug development.
Glycoprotein VI was recently reported to promote metastasis by interacting with cancer cell-derived galectin-3. 41 Platelet activation causing the formation of aggregates on the surface of circulating tumour cells may protect against immune cell attack. 42 Previously, we demonstrated that naphthalimide derivatives could prevent lung metastasis of melanoma cells through the inhibition of topo II. 15,16 Our present data revealed that a naphthalimide derivative (compound 5) could prevent platelet activation, likely through the inhibition of GPVI signalling. In addition to the inhibition of topo II activity, whether the antimetastatic effect of naphthalimide derivatives can be attributed to their ability to inhibit platelet activation should be examined in future studies.
In conclusion, our findings indicated that the naphthalimide derivative compound 5 could exert antiplatelet and antithrombotic effects, at least in part, through the suppression of GPVI signalling.
This naphthalimide derivative can serve as a core scaffold for developing novel antiplatelet agents to treat patients with cardiovascular diseases if the potential adverse effect of bleeding is eliminated or minimized.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest. Writing-original draft (lead); Writing-review & editing (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.