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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。组织因子途径抑制剂（TFPI）阻断组织因子VIIa （TF-FVIIa）复合物对因子X （FX）和因子IX的激活。观察到TFPI基因缺失导致部分由凝血功能障碍引起的胚胎死亡，证明了TFPI的生理重要性。在体外，可溶性TFPI对TFVIIa的有效抑制需要产物Xa因子与TFPI结合，将TFPI集中在带负电荷的膜表面，促进对TF-FVIIa的抑制。在体内，大多数TFPI与内皮细胞（EC）有关，其理想位置是调节TF-FVIIa活性，可能绕过了对FXa浓缩作用的需要。细胞内的TFPI至少存在于两个库中，一个库可逆地与未知的“受体”结合，另一个库可能由糖基磷脂酰肌醇（GPI）锚定介导（即对磷脂酶C敏感）。本应用程序的目的是确定TFPI与膜表面相关的机制。虽然TFPI通过磷脂酶C从EC中释放，但TFPI缺乏其他GPI锚定蛋白中发现的经典膜跨越序列，这表明一种新的GPI附着机制或通过GPI锚定的TFPI受体结合。为了解决这个问题，TFPI将通过免疫亲和层析从人胎盘或培养EC中分离出来。指示GPI锚定的疏水形式将通过反相高效液相色谱分离，酶解并通过质谱分析以鉴定GPI连接肽。我们还将在固定化TFPI上通过亲和层析分离候选TFPI受体。从胎盘或培养EC中提取的洗涤剂将被用作假定受体的来源。结合蛋白将被分离，测试TFPI结合活性，进行微测序，并通过检索数据库进行鉴定。潜在的GPI锚定将按照上述方法进行测试。最后，将GPI附着或跨膜结构域的经典序列融合到TFPI的c端区域后，将研究这种修饰对TF-FVIIa抑制和突变体细胞分布的影响。如果成功，这些研究将提供关于细胞结合的TFPI在各种病理条件下控制TF活性的作用和功能机制的新数据。

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.