Matrine Impairs Platelet Function and Thrombosis and Inhibits ROS Production

Abstract

Matrine is a naturally occurring alkaloid and possesses a wide range of pharmacological properties, such as anti-cancer, anti-oxidant, anti-inflammatory effects. However, whether it affects platelet function and thrombosis remains unclear. This study aims to evaluate the effect of matrine on platelet function and thrombus formation. Human platelets were treated with matrine (0-1 mg/ml) for 1 h at 37°C followed by measuring platelet aggregation, granule secretion, receptor expression by flow cytometry, spreading and clot retraction. In addition, matrine (10 mg/kg) was injected intraperitoneally into mice to measure tail bleeding time, arterial and venous thrombus formation. Matrine dose-dependently inhibited platelet aggregation and ATP release in response to either collagen-related peptide (Collagen-related peptide, 0.1 μg/ml) or thrombin (0.04 U/mL) stimulation without altering the expression of P-selectin, glycoprotein Ibα, GPVI, or αIIbβ3. In addition, matrine-treated platelets presented significantly decreased spreading on fibrinogen or collagen and clot retraction along with reduced phosphorylation of c-Src. Moreover, matrine administration significantly impaired the in vivo hemostatic function of platelets, arterial and venous thrombus formation. Furthermore, in platelets stimulated with CRP or thrombin, matrine significantly reduced Reactive oxygen species generation, inhibited the phosphorylation level of ERK1/2 (Thr202/Tyr204), p38 (Thr180/Tyr182) and AKT (Thr308/Ser473) as well as increased VASP phosphorylation (Ser239) and intracellular cGMP level. In conclusion, matrine inhibits platelet function, arterial and venous thrombosis, possibly involving inhibition of ROS generation, suggesting that matrine might be used as an antiplatelet agent for treating thrombotic or cardiovascular diseases.

Keywords: ROS; hemostasis; matrine; platelet; thrombosis.

FIGURE 1

Platelet aggregation and granules release. Washed human platelets were treated with different doses of matrine (0, 0.25, 0.5, and 1 mg/ml) at 37°C for 1 h to measure platelet aggregation induced by CRP (0.1 μg/ml) (A) or thrombin (0.04 U/ml) (B) in a Lumi-Aggregometer. At the same time, ATP release was monitored simultaneously using luciferin/luciferase reagent and presented as a relative to 0 mg/ml matrine which was defined as 100%. After matrine treatment, platelet P-selectin expression (α-granule) (C) and integrin αIIbβ3 activation (PAC-1 antibody binding) (D) was measured after collagen or thrombin stimulation using PE-conjugated anti-P-selectin antibody or FITC-conjugated PAC-1 antibody by flow cytometry. Data were presented as mean ± SE (n = 3–5) and analyzed by one-way ANOVA. Compared to 0, ^* p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Surface expression level of platelet membrane receptors. After matrine treatment, the expression of platelet receptors α_IIbβ₃ (A), GPIbα (B) and GPVI (C) was detected by flow cytometry. Data were presented as mean ± SE (n = 6–7) and analyzed by one-way ANOVA.

FIGURE 3

Platelet spreading and clot retraction. After matrine treatment, platelets were placed on fibrinogen or collagen coated glass coverslips and allowed to spread at 37°C for 90 min followed by staining with Alexa Fluor-546-labelled phalloidin (A) (mean ± SE, n = 3) or measurement of c-Src phosphorylation by western blot (mean ± SD, n = 3) (B). Clot retraction was initiated in matrine-treated platelets in the presence of 2 mM Ca²⁺ and 0.5 mg/ml fibrinogen after addition of thrombin (1 U/ml). Images were captured every 30 min (mean ± SD, n = 3) (C). Under clot retraction condition, the phosphorylation level of c-Src was measured by western blot and represented as a ratio relative to the total level (mean ± SD, n = 3) (D). BSA: bovine serum albumin. Compared with 0, ^* p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

Effect of matrine on hemostasis and arterial thrombosis in mice. Mice received intraperitoneal injection of matrine (10 mg/kg) followed by analysis of platelet count (A), tail bleeding time (B), and arterial thrombus formation induced by FeCl₃ which was monitored by a fluorescence microscopy (Olympus BX53) (mean, n = 6) (C). Human blood labelled with mepacrine (100 μM) was perfused through Bioflux plates in a microfluidic whole-blood perfusion assay followed by monitoring the thrombus formation under a fluorescence microscopy. The platelet-covered area was quantified using Bioflux software (Fluxion) (D). Scale bar = 100 μm. Data were presented as mean ± SE (n = 6–9). **p < 0.01 and ****p < 0.0001. Compared with 0, ***p < 0.001.

FIGURE 5

Deep vein thrombus formation and coagulation analysis. After intraperitoneal injection of matrine (10 mg/kg) or vehicle, mice underwent ligation of inferior vena cava (IVC) to induce venous thrombus formation. After 24 h, the IVC samples were isolated to measure thrombus weight (A) and length (B). Meanwhile, peripheral blood was obtained from matrine or vehicle treated mice to detect the coagulation factor FVIII (C), FIX (D), activated partial thromboplastin time (APTT) (E) and prothrombin time (F). Data were presented as mean ± SE (n = 4–6). **p < 0.01; ***p < 0.001.

FIGURE 6

ROS generation, phosphorylation of ERK1/2, p38, AKT and VASP and cGMP level. After matrine treatment, platelet ROS generation was measured using H2DCF-DA by flow cytometry (A). Matrine-treated platelets were stimulated with 5 μg/ml CRP or 1 U/ml thrombin for 5 min to measure the phosphorylation level of ERK1/2, p38, AKT (B and C) or VASP (D) by western blot (mean ± SD, n = 3), cGMP level by ELISA (mean ± SE, n = 3) (E). Compared with 0, ^* p < 0.05; **p < 0.01; ***p < 0.001.