COBRE: OK MED RES FOUND: P3: GPI ANCHORING & TISSUE FACTOR PATHWAY INHIBITOR

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 组织因子途径抑制剂（TFPI）通过组织因子-因子VIIa（TF-FVIIa）复合物阻断因子X（FX）和IX的活化。TFPI基因缺失导致胚胎死亡的观察结果部分由凝血功能障碍引起，表明TFPI的生理重要性。在体外，可溶性TFPI的有效TFVIIa抑制需要产物Xa因子与TFPI结合，将TFPI浓缩在带负电荷的膜表面上并促进TF-FVIIa的抑制。在体内，大多数TFPI与内皮细胞（EC）相关，在那里它理想地位于调节TF-FVIIa活性的位置，可能避免了对FXa浓缩作用的需要。细胞TFPI存在于至少两个库中，一个库可逆地结合至未鉴定的“受体”，另一个库可能由糖基磷脂酰肌醇（GPI）锚介导（即对磷脂酶C敏感）。本应用程序的目标是确定 TFPI通过其与膜表面结合。尽管TFPI通过磷脂酶C从EC释放，但TFPI缺乏在其他GPI锚定蛋白中发现的经典跨膜序列，这表明了新的GPI附着机制或通过GPI锚定的TFPI受体结合。解决 因此，TFPI将通过免疫亲和层析从人胎盘或培养的EC中分离。 将通过反相HPLC分离指示GPI锚定的疏水形式，酶促消化并通过质谱法分析以鉴定GPI连接肽。我们还将通过亲和色谱法在固定的TFPI上分离候选TFPI受体。来自胎盘或培养EC的洗涤剂提取物将用作推定受体的来源。将分离结合蛋白，检测TFPI结合活性，进行微量测序，并通过检索数据库进行鉴定。 将按照上述方法对潜在的GPI锚固进行测试。最后，将GPI连接或跨膜结构域的经典序列融合至TFPI的C末端区域后，将研究该修饰对TF-FVIIa抑制和突变体的细胞分布的影响。如果成功，这些研究将提供新的数据的作用和机制的功能，细胞结合的TFPI在控制TF在各种病理条件下的活动。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tissue factor pathway inhibitor (TFPI) blocks activation of factors X (FX) and IX by the tissue factorfactor VIIa (TF-FVIIa) complex. The observation that TFPI gene deletion results in embryonic lethality caused in part by a coagulopathy demonstrates the physiological importance of TFPI. In vitro, effective TFVIIa inhibition by soluble TFPI requires the product, factor Xa, to bind to TFPI, concentrating TFPI on negatively charged membrane surfaces and facilitating inhibition of TF-FVIIa. In vivo, most of the TFPI is associated with endothelial cells (EC) where it is ideally situated to regulate TF-FVIIa activity, perhaps circumventing the need for FXa concentrating effect. Cellular TFPI exists in at least two pools, one bound reversibly to unidentified "receptors", and another probably mediated by a glycosyl phosphatidylinositol (GPI) anchor (i.e. sensitive to phospholipase C). The goal of this application is to determine the mechanisms by which TFPI associates with membrane surfaces. Although TFPI is released from EC by phospholipase C, TFPI lacks the classical membrane spanning sequence found in other GPI anchored proteins, suggesting either a novel GPI attachment mechanism or binding through a GPI anchored TFPI receptor. To address this, TFPI will be isolated from human placenta or cultured EC by immunoaffinity chromatography. Hydrophobic forms indicative of GPI anchorage will be separated by reversed phase HPLC, enzymatically digested and analyzed by mass spectrometry to identify the GPI-linked peptide. We will also isolate candidate TFPI receptors by affinity chromatography on immobilized TFPI. Detergent extracts from placenta or cultured EC will be used as a source for putative receptors. Bound proteins will be separated, tested for TFPI binding activity, subjected to micro sequencing and identified by searching databases. Potential GPI anchorage will be tested as described above. Finally, after classical sequences for GPI attachment or transmembrane domains are fused to the C-terminal region of TFPI, the impact of this modification on TF-FVIIa inhibition and the cellular distribution of the mutants will be studied. If successful, these studies will provide novel data about the role and mechanism of function of cell-bound TFPI in controlling the activity of TF in various pathological conditions.