The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbβ3-Mediated Inside-Out and Outside-In Signals in Human Platelets

Abstract

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2-8 μM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin α_IIbβ₃ activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin α_IIbβ₃-mediated outside-in signaling, such as integrin β₃, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin α_IIbβ₃-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

Keywords: arterial thrombosis; hydroxyl radicals; integrin αIIbβ3; platelet aggregation; pterostilbene; resveratrol derivative.

Figure 1

Inhibitory profiles of the effects of pterostilbene (PTE) on platelet aggregation stimulation by collagen and thrombin. (A) Chemical structures of PTE (C₁₆H₁₆O₃) and resveratrol (C₁₄H₁₂O₃). (B) Washed human platelets (3.6 × 10⁸ cells/mL) were preincubated with a solvent control (0.1% DMSO) or PTE (2–20 μM) and subsequently treated with either collagen (1 μg/mL) or thrombin (0.01 U/mL) to stimulate platelet aggregation. (C,D) Concentration–response histograms of PTE demonstrating its inhibitory activity for platelet aggregation stimulated by collagen (%). Data are presented as the mean ± standard deviation (n = 4).

Figure 2

Effectiveness of pterostilbene (PTE) in Lyn, Fyn, and Syk activation in human platelets. Washed platelets (1.2 × 10⁹ cells/mL) were preincubated with 0.1% DMSO or PTE (3.5 and 6 µM), followed by the addition of collagen (1 μg/mL) to trigger (A) Lyn, (B) Fyn, and (C) SyK phosphorylation. Platelets were collected, and subcellular extracts were examined to determine the levels of protein phosphorylation. Data are presented as the mean ± standard deviation (n = 4). ** p < 0.01 and *** p < 0.001, compared with the resting control; ^## p < 0.01 and ^### p < 0.001, compared with the 0.1% DMSO-treated group.

Figure 3

Inhibitory property of pterostilbene (PTE) in integrin α_IIbβ₃ activation stimulated by either collagen or thrombin. For the flow cytometry analysis, resting platelets (a) or platelets were preincubated with the solvent control (b, 0.1% DMSO) or PTE (c, 3.5 µM; d, 6 µM), and fluorescein isothiocyanate-conjugated anti-PAC-1 mAb (2 µg/mL) was added before the addition of (A) collagen (1 µg/mL) or (B) thrombin (0.01 U/mL). Data are presented as the mean ± standard deviation (n = 4). *** p < 0.001, compared with the resting control; ^### p < 0.001, compared with the 0.1% DMSO-treated group.

Figure 4

Effects of pterostilbene (PTE) on integrin β₃, Src, and FAK phosphorylation in platelets exposed to immobilized fibrinogen. Washed platelets were preincubated with the solvent control (0.1% DMSO) or PTE (3.5 and 6 µM) and subsequently activated with immobilized fibrinogen (100 μg/mL) for determining the levels of (A) integrin β₃, (B) Src, and (C) FAK phosphorylation, as described in the Materials and Methods section. Data are presented as the mean ± standard deviation (n = 4). ** p < 0.01 and *** p < 0.001, compared with the bovine serum albumin (BSA; control); ^## p < 0.01 and ^### p < 0.001, compared with the 0.1% DMSO-treated group.

Figure 5

Inhibitory activity of pterostilbene (PTE) on platelet adhesion and spreading on immobilized fibrinogen as well as fibrin clot retraction. (A) Washed platelets were allowed to spread on the (a) BSA- or (b–d) fibrinogen-coated surfaces in the presence of the (b) solvent control (0.1% DMSO) or PTE (c, 3.5 μM; d, 6 μM) and subsequently labeled with fluorescein isothiocyanate–phalloidin, as described in the Materials and Methods section. Scale bar, 10 µm. Plot of (B) the number of adherent platelets per 0.01 mm² (a), and the average spreading surface area of individual platelets in six sight views (b). (C) Washed platelets (3.6 × 10⁸ cells/mL) were suspended in Tyrode’s solution containing 2 mg/mL fibrinogen and 1 mM CaCl₂ with the solvent control (0.1% DMSO) or PTE (3.5 and 6 μM). Clot retraction was initiated with thrombin (0.01 U/mL) at 37 °C. Images were photographed at 15 and 30 min intervals by using a digital camera. Profiles in (C) are representative of four similar experiments. Data are presented as the mean ± standard deviation (n = 4). *** p < 0.001, compared with the BSA (control); ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001, compared with the 0.1% DMSO-treated group.

Figure 6

Activity of pterostilbene (PTE) on hydroxyl radical formation in human platelets. Washed platelets were incubated with (a) Tyrode’s solution only (resting group) or preincubated with (b) 0.1% DMSO, PTE (c, 3.5 μM; d, 6 μM), (e) aspirin (100 μM) followed by the addition of collagen (1 μg/mL) to trigger hydroxyl radical (*) formation. Data are presented as the mean ± standard deviation (n = 4). *** p < 0.001, compared with the Tyrode’s solution only (resting group); ^### p < 0.001, compared with the 0.1% DMSO-treated group.

Figure 7

Effect of pterostilbene (PTE) on vascular thrombosis in the mesenteric venules of mice. Mice were administered an intravenous bolus of the solvent control (0.1% DMSO) or PTE (1 and 2 mg/kg), and the mesenteric venules were irradiated to induce microthrombus formation (occlusion time). Microscopic images (400× magnification) of 0.1% DMSO-treated controls and 1 and 2 mg/kg PTE-treated groups were recorded at 5 and 200 s after irradiation, respectively. The photographs shown are representative of eight similar experiments, and white arrows indicate platelet plug formation. Data are presented as the mean ± standard deviation (n = 8). *** p < 0.001, compared with the 0.1% DMSO-treated group.