DataSheet2_Matrine Impairs Platelet Function and Thrombosis and Inhibits ROS Production.docx

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.

苦参碱（Matrine）是一种天然存在的生物碱，具有广泛的药理学活性，涵盖抗肿瘤、抗氧化、抗炎等多种功效。然而其对血小板功能与血栓形成的影响尚未明确。本研究旨在评估苦参碱对血小板功能及血栓形成的作用。研究人员将人血小板以0~1mg/ml浓度的苦参碱在37℃下孵育1小时，随后通过流式细胞术（flow cytometry）检测血小板聚集、颗粒分泌、受体表达水平，并观察血小板铺展与血块回缩情况。此外，向小鼠腹腔注射10mg/kg的苦参碱，检测其尾部出血时间、动脉及静脉血栓形成能力。实验结果表明，苦参碱可呈剂量依赖性地抑制胶原相关肽（Collagen-related peptide, CRP，0.1μg/ml）或凝血酶（0.04U/mL）刺激诱导的血小板聚集与ATP释放，且不会改变P选择素（P-selectin）、糖蛋白Ibα（glycoprotein Ibα）、GPVI以及αIIbβ3的表达水平。经苦参碱处理的血小板在纤维蛋白原或胶原表面的铺展能力显著降低，血块回缩现象减弱，同时c-Src的磷酸化水平下降。此外，苦参碱给药可显著损害小鼠体内血小板的止血功能，抑制动脉及静脉血栓形成。在经CRP或凝血酶刺激的血小板中，苦参碱可显著减少活性氧（Reactive oxygen species, ROS）的生成，抑制ERK1/2（Thr202/Tyr204）、p38（Thr180/Tyr182）及AKT（Thr308/Ser473）的磷酸化水平，同时升高血管扩张刺激磷蛋白（VASP，Ser239）的磷酸化程度与细胞内环磷酸鸟苷（cGMP）含量。综上，苦参碱可抑制血小板功能及动、静脉血栓形成，其潜在机制可能与抑制ROS生成有关，提示苦参碱有望作为抗血小板药物应用于血栓性疾病或心血管疾病的治疗。