Ünlü B, Bogdanov V Y, Versteeg H H
Einthoven Laboratory for Experimental Vascular Medicine, Department of Internal Medicine, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.
Division of Hematology/Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, United States.
Thromb Res. 2017 Aug;156:1-7. doi: 10.1016/j.thromres.2017.05.028. Epub 2017 May 25.
Full length Tissue factor (flTF) is a key player in hemostasis and also likely contributes to venous thromboembolism (VTE), the third most common cardiovascular disease. flTF and its minimally coagulant isoform, alternatively spliced TF (asTF), have been detected in thrombi, suggesting participation of both isoforms in thrombogenesis, but data on participation of asTF in hemostasis is lacking. Therefore, we assessed the role of asTF in flTF cofactor activity modulation, using a co-expression system.
To investigate the interplay between flTF and asTF in hemostasis on endothelial cell surface.
Immortalized endothelial (ECRF) cells were adenovirally transduced to express asTF and flTF, after which flTF cofactor activity was measured on cells and microvesicles (MVs). To study co-localization of flTF/asTF proteins, confocal microscopy was performed. Finally, intracellular distribution of flTF was studied in the presence or absence of heightened asTF levels.
Levels of flTF antigen and cofactor activity were not affected by asTF co-expression. asTF and flTF were found to localize in distinct subcellular compartments. Only upon heightened overexpression of asTF, lower flTF protein levels and cofactor activity were observed. Heightened asTF levels also induced a shift of flTF from non-raft to lipid raft plasma membrane fractions, and triggered the expression of ER stress marker BiP. Proteasome inhibition resulted in increased asTF - but not flTF - protein expression.
At moderate levels, asTF appears to have negligible impact on flTF cofactor activity on endothelial cells and MVs; however, at supra-physiological levels, asTF is able to reduce the levels of flTF protein and cofactor activity.
全长组织因子（flTF）是止血过程中的关键因子，也可能与静脉血栓栓塞症（VTE）有关，VTE是第三大常见心血管疾病。在血栓中已检测到flTF及其最小凝血同工型，可变剪接组织因子（asTF），这表明两种同工型均参与血栓形成，但缺乏关于asTF参与止血的数据。因此，我们使用共表达系统评估了asTF在flTF辅因子活性调节中的作用。
研究flTF和asTF在内皮细胞表面止血过程中的相互作用。
将永生化内皮（ECRF）细胞用腺病毒转导以表达asTF和flTF，然后在细胞和微泡（MVs）上测量flTF辅因子活性。为了研究flTF / asTF蛋白的共定位，进行了共聚焦显微镜检查。最后，在asTF水平升高或不升高的情况下研究flTF的细胞内分布。
flTF抗原水平和辅因子活性不受asTF共表达的影响。发现asTF和flTF定位于不同的亚细胞区室。仅在asTF过度表达增强时，才观察到较低的flTF蛋白水平和辅因子活性。asTF水平升高还导致flTF从非筏状向脂筏质膜部分转移，并触发内质网应激标志物BiP的表达。蛋白酶体抑制导致asTF蛋白表达增加，但flTF蛋白表达未增加。
在中等水平时，asTF似乎对内皮细胞和MVs上的flTF辅因子活性影响可忽略不计；然而，在超生理水平时，asTF能够降低flTF蛋白水平和辅因子活性。
