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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 组织因子途径抑制物(TFPI)可阻断组织因子VIIa(Tf-FVIIa)复合体对因子X(FX)和因子IX的激活。观察到TFPI基因缺失导致胚胎死亡，部分原因是凝血功能障碍，这表明了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因子Xa与TFPI结合，将TFPI集中在带负电荷的膜表面，从而促进对Tf-FVIIa的抑制。在体内，大多数TFPI与内皮细胞(EC)相关，在那里它处于调节TF-FVIIa活性的理想位置，可能绕过了FXA浓缩效应的需要。细胞TFPI至少存在于两个池中，一个可逆地与未知的“受体”结合，另一个可能由糖基磷脂酰肌醇(GPI)锚点(即对磷脂酶C敏感)介导。这个应用程序的目标是确定 TFPI通过其与膜表面结合。虽然TFPI是由磷脂酶C从EC中释放出来的，但TFPI缺乏其他GPI锚定蛋白中经典的跨膜序列，这表明要么是一种新的GPI结合机制，要么是通过GPI锚定的TFPI受体结合。致信地址 用免疫亲和层析法从人胎盘或培养的EC中分离出TFPI。指示GPI锚定的疏水形式将通过反相高效液相色谱分离、酶消化和质谱分析来鉴定GPI连接的多肽。我们还将通过固定化TFPI亲和层析来分离候选TFPI受体。从胎盘或培养的EC中提取的洗涤剂将被用作推测受体的来源。结合的蛋白质将被分离，测试TFPI结合活性，进行显微测序，并通过搜索数据库进行鉴定。潜在的GPI锚地将进行如上所述的测试。最后，将GPI结合或跨膜区的经典序列融合到TFPI的C-末端区域后，将研究这种修饰对TF-FVIIa抑制的影响和突变体的细胞分布。如果成功，这些研究将为细胞结合TFPI在不同病理条件下控制TF活性的作用和机制提供新的数据。

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.