Nadir Yona, Brenner Benjamin, Gingis-Velitski Sveta, Levy-Adam Flonia, Ilan Neta, Zcharia Eyal, Nadir Erez, Vlodavsky Israel
Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel.
Thromb Haemost. 2008 Jan;99(1):133-41.
Heparanase activity is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we have reported that heparanase stimulates tissue factor (TF) expression in endothelial and cancer cells, resulting in elevation of coagulation activity. We hypothesized that heparanase regulates other coagulation modulators, and examined the expression and localization of tissue factor pathway inhibitor (TFPI) following heparanase over-expression or exogenous addition. Primary human umbilical vein endothelial cells (HUVEC) and human tumor-derived cell lines were incubated with heparanase, or were stably transfected with heparanase gene-constructs, and TFPI expression and secretion were examined. Heparanase over-expression or exogenous addition stimulated TFPI expression by 2-3 folds. TFPI accumulation in the cell culture medium exceeded in magnitude the observed induction of TFPI gene transcription reaching 5- to 6-fold increase. Extracellular accumulation of TFPI was evident already 60 min following heparanase addition, prior to TFPI protein induction, and correlated with increased coagulation activity. This effect was found to be independent of heparanase enzymatic activity and interaction with heparan-sulfate, and correlated with reduced TFPI levels on the cell surface. Data were verified in heparanase transgenic mice tissues and plasma. Interaction between heparanase and TFPI was evident by co-immunoprecipitation. Interaction of heparanase with TFPI resulted in its displacement from the surface of the vascular endothelium and in increased pro-coagulant activity. Thus, heparanase facilitates blood coagulation on the cell surface by two independent mechanisms: dissociation of TFPI from the vascular surface shortly after local elevation of heparanase levels, and subsequent induction of TF expression.
乙酰肝素酶活性与细胞侵袭、肿瘤转移和血管生成有关。最近，我们报道了乙酰肝素酶刺激内皮细胞和癌细胞中组织因子（TF）的表达，导致凝血活性升高。我们推测乙酰肝素酶调节其他凝血调节因子，并在乙酰肝素酶过表达或外源添加后检测了组织因子途径抑制物（TFPI）的表达和定位。将原代人脐静脉内皮细胞（HUVEC）和人肿瘤来源的细胞系与乙酰肝素酶一起孵育，或用乙酰肝素酶基因构建体进行稳定转染，然后检测TFPI的表达和分泌。乙酰肝素酶过表达或外源添加可使TFPI表达增加2至3倍。细胞培养基中TFPI的积累量在幅度上超过了观察到的TFPI基因转录诱导量，达到了5至6倍的增加。在添加乙酰肝素酶后60分钟，在TFPI蛋白诱导之前，TFPI的细胞外积累就很明显，并且与凝血活性增加相关。发现这种作用独立于乙酰肝素酶的酶活性以及与硫酸乙酰肝素的相互作用，并且与细胞表面TFPI水平降低相关。在乙酰肝素酶转基因小鼠的组织和血浆中验证了这些数据。通过共免疫沉淀可明显看出乙酰肝素酶与TFPI之间的相互作用。乙酰肝素酶与TFPI的相互作用导致其从血管内皮表面移位，并增加促凝血活性。因此，乙酰肝素酶通过两种独立机制促进细胞表面的血液凝固：在乙酰肝素酶水平局部升高后不久，TFPI从血管表面解离，以及随后诱导TF表达。
