A naphthalimide derivative exerts potent antiplatelet and antithrombotic activities without a bleeding tendency

Abstract

Background: Bleeding is the inherent adverse effect of antiplatelet drugs that has limited their use in the prevention of secondary heart attack and stroke. Thus, finding a novel antiplatelet drug with antithrombotic activities while preserving hemostatic function remains a crucial issue. Here, we screened naphthalimide derivatives that we previously synthesized and identified a novel derivative compound 6, which has a more potent antithrombotic effect and has no effect on bleeding cessation. This study is aimed to determine the antiplatelet mechanism of compound 6 and further test whether compound 6 is a safer and more potent antithrombotic agent.

Methods: Platelet aggregation, flow cytometry and immunoblotting were used to determine the in vitro antiplatelet effect of compound 6. The study of thrombus formation of mesenteric venules in mice was used to evaluate the antithrombotic effect of compound 6.

Results: Compound 6 selectively inhibited collagen-mediated platelet aggregation and markedly prevented thrombus formation without bleeding tendency. Compound 6 also inhibited glycoprotein VI (GPVI) downstream signaling, such as Fyn and Lyn, phospholipase C gamma 2, protein kinase C. Moreover, a surface plasmon resonance assay indicated that compound 6 may directly bind to GPVI, thereby interrupting the interaction of collagen and GPVI. Compound 6 also effectively attenuates collagen-induced granule release, calcium mobilization, and GPIIbIIIa activation.

Conclusion: These findings indicate that compound 6 can selectively inhibit GPVI, eventually suppressing platelet activation and thrombus formation while preserving hemostasis. Compound 6 is a GPVI antagonist and safe antiplatelet agent. Compound 6 also has therapeutic potential for treating cardiovascular diseases.

Keywords: GPVI; antiplatelet agent; naphthalimide derivative; platelet activation; thrombus formation.

FIGURE 1

Structure of compounds 5 and 6.

FIGURE 2

Effects of compound 6 on human platelet aggregation triggered by various agonists. (A–C) Washed platelets (3.6 × 10⁸ cells/mL) were treated with compound 6 (1–50 μM) or dimethyl sulfoxide (DMSO; solvent control) before adding 1 μg/mL collagen (A), 0.02 U/mL thrombin (B), and 1 μM U46619 (C). Panels A–C present the curve of platelet aggregation, and panel D presents the statistical analysis of panels A–C. Data (D) are presented as means ± SEM (n = 4). ###p < 0.001 compared with the DMSO group.

FIGURE 3

Effects of compound 6 on thrombosis and hemostasis in mice. Mice were treated with compound 6 (0.3, 0.6, or 1.2 mg/kg), DMSO (solvent control), or aspirin (20 mg/kg; positive control) via intravenous route for 10 min. (A) The microthrombus formation (arrows) in mesenteric venules. Scale bar = 30 μm. (B) The bleeding time was recorded after cutting the tail until no bleeding sign was observed for at least 10 s. Each point on the plot indicates a mouse (n = 6). Data (A,B) are presented as means ± SEM (n = 6). *p < 0.05 and ***p < 0.001 compared with the DMSO group.

FIGURE 4

Effects of compound 6 on collagen/epinephrine-induced pulmonary thrombosis and FeCl₃-induced thrombosis in mesenteric artery. Mice were intravenously administered with DMSO (solvent control), compound 6 (1.2 mg/kg), or aspirin (20 mg/kg, positive control) for 10 min (A) Mice were injected with collagen/epinephrine to induce pulmonary embolism, which was detected through staining with Evans blue (left panel). The survival rate was recorded for 24 h (right panel). Data are presented as means ± SEM (n = 6). **p < 0.01 compared with the sham group. #p < 0.05 compared with the DMSO (solvent control) group. (B) FeCl₃-induced thrombosis in mesenteric artery as observed under real-time fluorescent microscopy. Thrombus was observed for the indicated time (0, 10, and 20 min). Scale bar = 100 µm.

FIGURE 5

Effects of compound 6 on the phosphorylation of Fyn, Lyn, PLCγ2, and PKC substrates (p47) induced by collagen in human platelets. (A–D) Washed platelets (3.6 × 10⁸ cells/mL) were treated with compound 6 (2.5 and 5 μM) or DMSO before adding collagen (1 μg/mL). Proteins of these samples were separated by Western blotting. These targets (Fyn, Lyn, PLCγ2, and p47) were detected using specific antibodies. Data (A–D) are presented as means ± SEM (n = 5). **p < 0.01 and ***p < 0.001 compared with the resting group. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the DMSO (solvent control) group.

FIGURE 6

Effects of compound 6 on the phosphorylation of Akt, and MAPKs induced by collagen in human platelets. Washed platelets (3.6 × 10⁸ cells/mL) were treated with compound 6 (2.5 and 5 μM) or DMSO before adding collagen (1 μg/mL). Proteins of these samples were separated by Western blotting. Akt (A), p38 MAPK (B), ERK (C), and JNK (D) were detected using specific antibodies. Data (A–D) are presented as means ± SEM (n = 4). **p < 0.01 and ***p < 0.001 compared with the resting group. #p < 0.05 and ##p < 0.01 compared with the DMSO (solvent control) group.

FIGURE 7

Direct binding of compound 6 on GPVI. The recombinant GPVI proteins were coated on the nitrilotriacetic acid sensor chip, followed by perfusing the solutions of (A) compound 5 (C5, 0.1 and 1 μM) or (B) compound 6 (C6, 0.1 and 1 μM) on the chip. Equilibrium dissociation constant (KD) were determined using the OpenSPR system. (C) The prediction of binding site of ligand and receptor. 3D (A,B) and 2D (C) representations of the binding interaction between compound 6 and GPVI using ChimiraX, Autodock 4, and Discovery Studio Visualizer, respectively.

FIGURE 8

Effects of compound 6 on ATP release, calcium mobilization, and GPIIb–IIIa activation triggered by collagen in human platelets. Washed platelets (3.6 × 10⁸ cells/mL) were treated with compound 6 (C6, 2.5 and 5 μM) or DMSO and then stimulated with collagen (1 μg/mL) to trigger ATP release (A), calcium mobilization (B), and GPIIb–IIIa activation (C) that were detected using luciferase/luciferin, Fura-2 AM, and APC–PAC1 antibodies, respectively. Data (A–C) are presented as means ± SEM (n = 4). **p < 0.01 and ***p < 0.001 compared with the resting group. #p < 0.05, ##p < 0.01, and ###p < 0.001 compared with the DMSO (solvent control) group.