Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, the Netherlands.
Haematologica. 2013 Jan;98(1):153-8. doi: 10.3324/haematol.2012.069997. Epub 2012 Jul 16.
Tissue factor activation (decryption) has been proposed to be dependent on the cysteine 186-cysteine 209 allosteric disulfide in the tissue factor extracellular domain. Tissue factor procoagulant activity is under the control of protein disulfide isomerase-dependent modulation and nitrosylation of this disulfide. Human tissue factor disulfide mutants have been proposed as a model for encrypted tissue factor, but poor expression of these mutants hampers research into tissue factor decryption. We, therefore, investigated whether mouse tissue factor cysteine 186-cysteine 209 disulfide bond mutants form a better suited model for tissue factor decryption. Stable mouse wild-type tissue factor, tissue factor(C190A), tissue factor(C213A) and tissue factor(C190/213A) disulfide mutant-expressing baby hamster kidney cells with equal levels of surface tissue factor were established. Tissue factor coagulant activity on these cells was determined using an active factor Xa-dependent chromogenic assay. The effect of nitrosylation on tissue factor function was also assessed. A tissue factor(C190/213A) mutant exerted marginal procoagulant activity, also after addition of supraphysiological concentration of factor VIIa. Tissue factor(C190A) and tissue factor(C213A) mutants showed reduced activity and the presence of tissue factor dimers. Nitrosylation of wild-type tissue factor cells decreased procoagulant function, an effect which was reversed by incubation with bacitracin, an inhibitor of protein disulfide isomerase, suggesting that this isomerase promotes de-nitrosylation of tissue factor. Mouse tissue factor procoagulant function is dependent on the Cys190-Cys213 disulfide bond and is modulated by nitrosylation. The murine model of disulfide-mutated tissue factor is more suitable for studying tissue factor decryption than are human tissue factor mutants.
组织因子激活（解密）被认为依赖于组织因子细胞外结构域中半胱氨酸 186-半胱氨酸 209 之间的变构二硫键。组织因子促凝活性受到蛋白二硫键异构酶依赖性调节和该二硫键的硝化作用的控制。已经提出了人组织因子二硫键突变体作为加密组织因子的模型，但这些突变体的表达不良阻碍了对组织因子解密的研究。因此，我们研究了小鼠组织因子半胱氨酸 186-半胱氨酸 209 二硫键突变体是否形成了更适合组织因子解密的模型。用等量表面组织因子建立了稳定表达野生型组织因子、组织因子（C190A）、组织因子（C213A）和组织因子（C190/213A）二硫键突变体的仓鼠肾细胞系。使用依赖活性因子 Xa 的显色测定法测定这些细胞上的组织因子凝血活性。还评估了硝化作用对组织因子功能的影响。组织因子（C190/213A）突变体仅表现出轻微的促凝活性，即使加入超生理浓度的因子 VIIa 也是如此。组织因子（C190A）和组织因子（C213A）突变体显示出活性降低和组织因子二聚体的存在。野生型组织因子细胞的硝化作用降低了促凝功能，而用 bacitracin（一种蛋白二硫键异构酶抑制剂）孵育可逆转该作用，这表明该异构酶促进了组织因子的去硝化作用。小鼠组织因子的促凝功能依赖于 Cys190-Cys213 二硫键，并受到硝化作用的调节。与人类组织因子突变体相比，二硫键突变体的鼠模型更适合研究组织因子解密。
