Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP)
Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP)
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DOI：
10.1042/bj20041021
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发表时间：
2005-02-01
影响因子：
4.1
通讯作者：
Preissner, KT
中科院分区：
文献类型：
--
作者：
Nakazawa, F;Kannemeier, C;Preissner, KT
凝血因子VII激活蛋白水解酶(FSAP)是一种新型的血浆来源丝氨酸蛋白酶，具有激活凝血因子VII和产生尿型纤溶酶原激活物(尿激酶)的双重止血功能。FSAP的(自动)激活是由多阴离子糖胺多聚糖促进的，例如在肝素或葡聚糖硫酸盐中，而钙离子稳定FSAP的活性形式。在本研究中，细胞外RNA被鉴定为一种新的FSAP辅助因子。来自各种细胞类型的条件培养液，如平滑肌细胞、内皮细胞、骨肉瘤细胞或CHO(中国仓鼠卵巢)细胞，都含有启动(自身)激活FSAP的酸性因子。RNase A，而不是其他水解酶(蛋白水解酶、聚糖酶和DNase)，取消了FSAP辅因子的活性，该辅因子随后被阴离子交换层析分离，并被明确地鉴定为RNA。在纯化体系中，以及在血浆中，不同形式的天然RNA(Rena、tRNA、病毒RNA和人工RNA)能够(自动)将FSAP激活为双链酶形式。通过迁移率改变实验和紫外光交联实验，证明FSAP是一种新的胞外RNA结合蛋白，其Kn约为170-350 nM。FSAP的激活通过RNA依赖的模板机制发生，涉及至少100个核苷酸大小的核酸。在一个纯化的系统中，天然RNA将FSAP依赖的因子VII的激活增加了几倍(如随后的因子Xa的产生所示)，以及FSAP介导的尿激酶的产生。我们的结果首次证明，存在于细胞损伤或血管损伤部位的细胞外RNA可以通过一种新的表面依赖机制诱导(自动)激活FSAP，从而在止血中发挥重要的辅助因子功能。
FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as In heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracelfular RNA was identified and characteized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscular cells, endothelial cells, osteosarcoma cells or CHO (Chinese -hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anionexchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rENA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the Kn estimated to be 170-350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation severalfold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.