Li Min, Wu Wei-kang, Liu Liang, Liao Fu-long, Shinohara Yukito, Handa Shunnosuke, Goto Shinya
School of Chinese Medicine, Hong Kong Baptist University Kowloon Tong, Hong Kong, China.
Chin Med J (Engl). 2004 Feb;117(2):241-6.
Ilexonin A (IA), purified from the Chinese herbal medicine Maodongqing (Ilex pubescens Hook, et Arn) has been commonly used in south China to treat thrombotic disorders. In this study, we aimed to study the inhibiting effects and mechanism of IA on von Willebrand factor (vWF)-dependent high shear-induced platelet aggregation.
vWF-dependent high shear (10,800 s(-1)) induced aggregation of platelets obtained from normal donors in the presence or absence of IA was measured by a modified cone-plate viscometer and shear-induced vWF binding was measured by quantitative flow cytometry with monoclonal antibody known to bind exclusively to the C-terminal domain of vWF (LJ-C3) directly labeled with fluorescein isothiocyanate (FITC). P-selectin surface expression was also measured by a similar method with FITC conjugated anti-P-selectin monoclonal antibody (WGA1).
Shear-induced platelet aggregation was inhibited by IA in a dose-dependent manner. The extent of aggregation decreased from (78.6 +/- 4.6)% in the absence of IA to (36.5 +/- 2.1)% in the presence of IA (3.3 mmol/L) (P < 0.0001, n = 9) with a high shear rate of 10800 s(-1). vWF binding and P-selectin expression were also inhibited by IA in a dose dependent manner. The number of binding FITC-LJ-C3 molecules increased after exposure of platelet-rich plasma to a high shear rate of 10800 s(-1) for 6 minutes, but this shear-induced increased binding platelet surface vWF molecules and P-selectin expression can be decreased in the presence of IA.
vWF binding and vWF mediated platelet activation, aggregation occurring under high shear rate were inhibited by IA. IA may be a unique antithrombotic drug inhibiting the vWF-GP Ibalpha interaction, and may thus facilitate drug design targeting arterial thrombosis.
从中国草药毛冬青（Ilex pubescens Hook, et Arn）中提纯的冬青素A（IA）在中国南方常用于治疗血栓性疾病。在本研究中，我们旨在研究IA对血管性血友病因子（vWF）依赖的高剪切力诱导的血小板聚集的抑制作用及其机制。
使用改良的锥板式粘度计测量在有或无IA存在的情况下，vWF依赖的高剪切力（10,800 s(-1)）诱导的正常供体血小板聚集，并使用直接用异硫氰酸荧光素（FITC）标记的、已知仅与vWF C末端结构域结合的单克隆抗体（LJ-C3）通过定量流式细胞术测量剪切诱导的vWF结合。还使用与FITC偶联的抗P-选择素单克隆抗体（WGA1）通过类似方法测量P-选择素表面表达。
IA以剂量依赖性方式抑制剪切诱导的血小板聚集。在高剪切速率10800 s(-1)下，聚集程度从无IA时的（78.6±4.6）%降至有IA（3.3 mmol/L）时的（36.5±2.1）%（P < 0.0001，n = 9）。IA还以剂量依赖性方式抑制vWF结合和P-选择素表达。富含血小板血浆暴露于10800 s(-1)的高剪切速率6分钟后，结合FITC-LJ-C3分子的数量增加，但在有IA存在的情况下，这种剪切诱导的血小板表面vWF分子结合增加和P-选择素表达可降低。
IA抑制vWF结合以及vWF介导的血小板活化、在高剪切速率下发生的聚集。IA可能是一种独特的抗血栓药物，可抑制vWF-GP Ibalpha相互作用，因此可能有助于针对动脉血栓形成的药物设计。
