Shimada K, Ozawa T
Thromb Res. 1985 Aug 15;39(4):387-97. doi: 10.1016/0049-3848(85)90162-8.
Anticoagulant heparan sulfate proteoglycans have been shown to be released from cultured endothelial cells. The effect of thrombin on their release was investigated. Thrombin at more than one unit/ml accelerated the release of [35S] sulfate-labeled glycosaminoglycans from cultured porcine aortic endothelial cells. The effect of thrombin reached a maximum after one hour of incubation, and was dependent on the enzyme concentration. 10 unit/ml of thrombin released approximately twice as much amount of 35S-glycosaminoglycans as did Hanks' balanced salt solution alone. When the active site of thrombin was blocked by either diisopropylfluorophosphate or hirudin, the enzyme effect was completely abolished. Released glycosaminoglycans were resistant to chondroitin ABC lyase digestions, but degraded by either heparitinase or nitrous acid treatments. Released 35S-materials were precipitated with trichloroacetic acid and shown to be degraded into smaller molecules after alkali treatment on Sepharose CL-6B gel filtration chromatography. On the other hand, thrombin treatment of 51Cr-labeled cells did not cause the release of radioactivity. These results indicate that thrombin potentiates the release of heparan sulfate proteoglycans from cultured aortic endothelial cells without causing an appreciable damage to the cells. The effect of thrombin is active site-dependent and requires a relatively high enzyme concentration.
抗凝硫酸乙酰肝素蛋白聚糖已被证明可从培养的内皮细胞中释放出来。研究了凝血酶对其释放的影响。每毫升超过一个单位的凝血酶加速了[35S]硫酸盐标记的糖胺聚糖从培养的猪主动脉内皮细胞中的释放。凝血酶的作用在孵育一小时后达到最大值，并且依赖于酶浓度。每毫升10单位的凝血酶释放的35S-糖胺聚糖量大约是单独的汉克斯平衡盐溶液的两倍。当凝血酶的活性位点被二异丙基氟磷酸或水蛭素阻断时，酶的作用完全消失。释放的糖胺聚糖对软骨素ABC裂解酶消化有抗性，但可被乙酰肝素酶或亚硝酸处理降解。释放的35S物质用三氯乙酸沉淀，并在Sepharose CL-6B凝胶过滤色谱上经碱处理后显示降解为较小的分子。另一方面，用凝血酶处理51Cr标记的细胞不会导致放射性物质的释放。这些结果表明，凝血酶增强了培养的主动脉内皮细胞中硫酸乙酰肝素蛋白聚糖的释放，而不会对细胞造成明显损伤。凝血酶的作用依赖于活性位点，并且需要相对较高的酶浓度。
