丹酚酸A通过抑制3羧基磷脂酰肌醇激酶（PI3K）途径降低血小板激活和动脉血栓形成
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摘要
孝景和目的:动脉粥样硬化疾病是西方发达国家发病率和死亡率最高的疾病,而且也是我国主要的死亡原因。血小板在动脉粥样硬化发生发展过程中起着非常重要的作用。丹参是亚洲广泛用于治疗动脉粥样硬化血栓性疾病的一种中药,而丹酚酸A是其中的一种主要水溶性成分。本研究探索了丹酚酸A对血小板活性的影响及其可能的作用机制。方法和结果:重要的血小板激活刺激剂ADP,凝血酶,胶原和血栓烷A2类似物U46619所引起血小板聚集能被丹酚酸A显著抑制,而且丹酚酸A的抑制作用呈浓度依赖性。另外应用流式细胞术检测单个血小板激活时,发现丹酚酸A对ADP和凝血酶刺激引起的P选择素表达和纤维蛋白原结合有显著的抑制作用。流式细胞术的检测同时也证明丹酚酸A可以抑制血小板-白细胞聚集体的形成。丹酚酸A还可以抑制血小板在固定的纤维蛋白原上的铺展,这说明丹酚酸A可以抑制血小板从外到内的信号通路(Outside-in signaling)。在相当于体内动脉流速的剪切力(1000s-1)下,丹酚酸A可以抑制血小板在固定胶原上的粘附,抑制率大概为40%。免疫印迹分析显示,丹酚酸A抑制了血小板Akt的磷酸化,因此它的作用靶点可能是3羧基磷脂酰肌醇激酶(PI3K)。通过应用PI3K的抑制剂LY294002和TGX-221进行对比和叠加实验,证实了丹酚酸A的作用靶点是PI3K的p亚型。最后体外实验的作用在我们体内实验得到了验证。在体内动脉血栓模型中,丹酚酸A可以显著延长野生型小鼠肠系膜动脉血管堵塞的时间(对照组：35±2 min,丹酚酸A处理组：56±4 min；P<0.01)。而且,丹酚酸A纠正了因为低密度脂蛋白受体敲除导致的高血脂所引起的促血栓表型,在低密度脂蛋白受体敲除的小鼠也显著地延长了肠系膜动脉血管堵塞时间(对照组：21±2 min,丹酚酸A处理组45±4 min with SAA；P<0.01).结论:丹酚酸A通过抑制PI3K途径降低血小板激活和抑制体内动脉血栓形成。我们的数据提示了丹酚酸A可能发展成为一种新的药物用来阻止血栓的形成。
Summary. Background and objective:Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms. Methods and Results:SAA dose-dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP-enhanced platelet P-selectin expression and fibrinogen binding, which consequently hampered ADP-induced platelet-leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside-in signaling. Under an arterial shear rate of 1000 s-1, SAA decreased platelet adhesion on collagen surfaces by 40%. Western blot analysis showed that SAA, like the phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and TGX-221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild-type mice (35±2 min without SAA and 56±4 min with SAA; P <0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in LdlrtmlHer mutant mice (21±2 min without SAA and 45±4 min with SAA; P< 0.01). Conclusions:SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.
Summary. Background and objective:Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia miltiorrhiza Bunge, a herb that is widely used for atherothrombotic disease treatment in Asian medicine. As platelets play pivotal roles in atherothrombogenesis, we studied the effect of SAA on platelet activation and its underlying mechanisms. Methods and Results:SAA dose-dependently inhibited platelet aggregation induced by ADP, thrombin, collagen and U46619. It reduced ADP-enhanced platelet P-selectin expression and fibrinogen binding, which consequently hampered ADP-induced platelet-leukocyte aggregation. SAA also inhibited platelet spreading on fibrinogen, a process mediated by outside-in signaling. Under an arterial shear rate of 1000 s-1, SAA decreased platelet adhesion on collagen surfaces by 40%. Western blot analysis showed that SAA, like the phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and TGX-221, potently inhibited PI3K, as shown by reduced Akt phosphorylation. The in vitro findings were further evaluated in the mouse model of arterial thrombosis, in which SAA prolonged the mesenteric arterial occlusion time in wild-type mice (35±2 min without SAA and 56±4 min with SAA; P <0.01). Interestingly, SAA could even counteract the shortened arterial occlusion time in LdlrtmlHer mutant mice (21±2 min without SAA and 45±4 min with SAA; P< 0.01). Conclusions:SAA inhibits platelet activation via the inhibition of PI3K, and attenuates arterial thrombus formation in vivo. Our data suggest that SAA may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.
引文
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2. Tunstall-Pedoe H, Kuulasmaa K, Mahonen M, et al. Contribution of trends in survival and coronary-event rates to changes in coronary heart disease mortality: 10-year results from 37 WHO MONICA project populations. Monitoring trends and determinants in cardiovascular disease. Lancet.1999;353(9164):1547-57.
3. Cheng TO. Cardiovascular effects of Danshen. International journal of cardiology. 2007;121(1):9-22.
4. Han JY, Fan JY, Horie Y, et al. Ameliorating effects of compounds derived from Salvia miltiorrhiza root extract on microcirculatory disturbance and target organ injury by ischemia and reperfusion. Pharmacology & therapeutics. 2008;117(2):280-95.
5. Zhou L, Zuo Z, Chow MS. Danshen:an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. Journal of clinical pharmacology. 2005;45(12):1345-59.
6. Yao Y, Wu WY, Liu AH, et al. Interaction of salvianolic acids and notoginsengnosides in inhibition of ADP-induced platelet aggregation. Am J Chin Med.2008;36(2):313-28.
7. Wu YP, Zhao XM, Pan SD, et al. Salvianolic acid B inhibits platelet adhesion under conditions of flow by a mechanism involving the collagen receptor alpha2betal. Thromb Res.2008;123(2):298-305.
8. Liu P, Hu Y, Liu C, et al. Effects of salviainolic acid A (SA-A) on liver injury: SA-A action on hepatic peroxidation. Liver.2001;21(6):384-90.
9. Xing JJ, Chen X, Tu PF, et al. Effects of salvianolic acids on erythrocyte deformability in oleic acid induced acute lung injury in rabbits. Clinical hemorheology and microcirculation.2006;34(4):507-17.
10. Lin TJ, Zhang KJ, Liu GT. Effects of salvianolic acid A on oxygen radicals released by rat neutrophils and on neutrophil function. Biochemical pharmacology. 1996;51(9):1237-41.
11. Hirsch E, Bosco O, Tropel P, et al. Resistance to thromboembolism in PI3Kgamma-deficient mice. FASEB J.2001;15(11):2019-21.
12. Li Z, Zhang G, Le Breton GC, et al. Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases. The Journal of biological chemistry.2003;278(33):30725-31.
13. Watanabe N, Nakajima H, Suzuki H, et al. Functional phenotype of phosphoinositide 3-kinase p85alpha-null platelets characterized by an impaired response to GP VI stimulation. Blood.2003;102(2):541-8.
14. Cosemans JM, Munnix IC, Wetzker R, et al. Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization. Blood.2006;108(9):3045-52.
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16. Kennedy SG, Wagner AJ, Conzen SD, et al. The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes & development. 1997;11(6):701-13.
17. Chen J, De S, Damron DS, et al. Impaired platelet responses to thrombin and collagen in AKT-1-deficient mice. Blood.2004; 104(6):1703-10.
18. Woulfe D, Jiang H, Morgans A, et al. Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2. The Journal of clinical investigation.2004;113(3):441-50.
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