Cystine 186-cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption
Cystine 186-cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption
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DOI：
10.1182/blood-2009-09-241356
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发表时间：
2010-05-27
期刊：
影响因子：
20.3
通讯作者：
Pendurthi, Usha R.
中科院分区：
文献类型：
--
作者：
Kothari, Hema;Nayak, Ramesh C.;Pendurthi, Usha R.
细胞表面的组织因子(Tf)大多以隐蔽状态存在。目前还不完全清楚隐秘的TF与促凝血的活性的TF有何不同，以及解密是如何发生的。在这里，我们严格评估了半胱氨酸186-半胱氨酸209(Cys186-Cys209)键的形成对TF促凝活性及其解密性的重要性。转TFC186S、TFC209S或TFC186S/C209S的中国仓鼠卵巢细胞在细胞表面几乎没有凝血活性。Tf单抗和激活因子VII(FVIIa)的结合研究表明，在表达突变型Tf的细胞中，细胞表面几乎不存在Tf蛋白。在转导TFC186S、TFC209S或TFC186S/C209S的人脐静脉内皮细胞(HUVECs)中也获得了类似的数据。对表达相似水平的野生型Tf和TFC186S/C209S的HUVECs的Tf活性分析表明，在FVIIa饱和浓度存在下，Tf突变体具有与野生型Tf相似的凝血活性。更重要的是，用HgCl2或离子霉素处理表达TFC186S/C209S的人脐静脉内皮细胞，其细胞表面转铁蛋白的活性与野生型转铁蛋白的水平相同。我们的数据提供了明确的证据，缺乏Cys186-Cys209键的Tf一旦与FVIIa络合就具有凝血活性，并且Tf解密不需要形成Cys186-Cys209二硫键。(血。2010；115(21)：4273-4283)
Tissue factor (TF) on cell surfaces resides mostly in a cryptic state. It is not entirely clear how cryptic TF differs from procoagulantly active TF and how deencryption occurs. Here, we critically evaluated the importance of cystine 186-cystine 209 (Cys186-Cys209) bond formation for TF procoagulant activity and its de-encryption. Chinese hamster ovary cells transfected with TFC186S, TFC209S, or TFC186S/C209S expressed little procoagulant activity at the cell surface. TF monoclonal antibody and activated factor VII (FVIIa) binding studies showed that little TF protein was present at the cell surface in cells expressing mutant TF. Similar data were obtained in human umbilical vein endothelial cells (HUVECs) transduced to express TFC186S, TFC209S, or TFC186S/C209S. Analysis of TF activity in HUVECs expressing similar levels of wildtype TF and TFC186S/C209S showed that TF mutant in the presence of saturating concentrations of FVIIa exhibited similar coagulant activity as that of wild-type TF. More importantly, treatment of HUVECs expressing TFC186S/C209S with HgCl2 or ionomycin increased the cell-surface TF activity to the same extent as that of the wild-type TF. Our data provide clear evidence that TF lacking the Cys186-Cys209 bond is coagulantly active once it is complexed with FVIIa, and TF deencryption does not require Cys186-Cys209 disulfide bond formation. (Blood. 2010; 115(21): 4273-4283)