Deguchi Hiroshi, Yegneswaran Subramanian, Griffin John H
Department of Molecular and Experimental Medicine, The Scripps Research Institute, MEM 180, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.
J Biol Chem. 2004 Mar 26;279(13):12036-42. doi: 10.1074/jbc.M302531200. Epub 2004 Jan 13.
Sphingolipids contribute to modulation of two opposing cell processes, cell growth and apoptotic cell death; ceramide and sphingosine promote the latter and sphingosine-1-phosphate triggers the former. Thrombin, a pro-inflammatory protease that is regulated by the blood coagulation cascade, exerts similar effects depending on cell type. Here we report a new mechanism for cross-talk between sphingolipid metabolism and thrombin generation. Sphingosine and sphinganine, but not ceramide or sphingosine-1-phosphate, down-regulated thrombin generation on platelet surfaces (IC(50) = 2.4 and 1.4 microm for sphingosine and sphinganine, respectively) as well as in whole plasma clotting assays. Thrombin generation was also inhibited by glucosylsphingosine, lysosphingomyelin, phytosphingosine, and primary alkylamines with >10 carbons. Acylation of the amino group ablated anticoagulant activities. Factor Va was required for the anticoagulant property of sphingosine because prothrombin activation was inhibited by sphingosine, sphinganine, and stearylamine in the presence but not in the absence of factor Va. Sphingosine did not inhibit thrombin generation when Gla-domainless factor Xa was used in prothrombinase assays, whereas sphingosine inhibited activation of Gla-domainless prothrombin by factor Xa/factor Va in the absence of phospholipids (IC(50) = 0.49 microm). Fluorescence spectroscopy studies showed that sphingosine binds to fluorescein-labeled factor Xa and that this interaction required the Gla domain. These results imply that sphingosine disrupts interactions between factor Va and the Gla domain of factor Xa in the prothrombinase complex. Thus, certain sphingolipids may be bioactive lipid mediators of thrombin generation such that certain sphingolipid metabolites may modulate proteases that affect cell growth and death, blood coagulation, and inflammation.
鞘脂有助于调节两种相反的细胞过程，即细胞生长和凋亡性细胞死亡；神经酰胺和鞘氨醇促进后者，而1-磷酸鞘氨醇触发前者。凝血酶是一种受血液凝固级联反应调节的促炎蛋白酶，根据细胞类型发挥类似的作用。在这里，我们报告了一种鞘脂代谢与凝血酶生成之间相互作用的新机制。鞘氨醇和二氢鞘氨醇，但不是神经酰胺或1-磷酸鞘氨醇，下调血小板表面的凝血酶生成（鞘氨醇和二氢鞘氨醇的IC50分别为2.4和1.4微摩尔）以及全血浆凝血试验中的凝血酶生成。凝血酶生成也受到葡萄糖鞘氨醇、溶血磷脂酰胆碱、植物鞘氨醇和碳数大于10的伯烷基胺的抑制。氨基的酰化消除了抗凝活性。鞘氨醇的抗凝特性需要因子Va，因为在有因子Va存在但无因子Va时，凝血酶原激活受到鞘氨醇、二氢鞘氨醇和硬脂胺的抑制。当在凝血酶原酶试验中使用无Gla结构域的因子Xa时，鞘氨醇不抑制凝血酶生成，而在没有磷脂的情况下，鞘氨醇抑制无Gla结构域的凝血酶原被因子Xa/因子Va激活（IC50 = 0.49微摩尔）。荧光光谱研究表明，鞘氨醇与荧光素标记的因子Xa结合，这种相互作用需要Gla结构域。这些结果表明，鞘氨醇破坏了凝血酶原酶复合物中因子Va与因子Xa的Gla结构域之间的相互作用。因此，某些鞘脂可能是凝血酶生成的生物活性脂质介质，使得某些鞘脂代谢物可能调节影响细胞生长和死亡、血液凝固及炎症的蛋白酶。
