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

COBRE：确定医学研究结果：P3：GPI 锚定

• 批准号: 7382046
• 负责人: Cristina Lupu
• 金额: $ 28.19万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7382046
• 关键词: COBRE; OK; MED; RES; FOUND

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。组织因子途径抑制剂 (TFPI) 可阻断组织因子 VIIa (TF-FVIIa) 复合物对 X 因子 (FX) 和 IX 因子的激活。 TFPI 基因缺失导致部分由凝血病引起的胚胎死亡的观察结果证明了 TFPI 的生理重要性。在体外，可溶性 TFPI 有效抑制 TFVIIa 需要产物 Xa 因子与 TFPI 结合，将 TFPI 集中在带负电荷的膜表面并促进 TF-FVIIa 的抑制。在体内，大部分 TFPI 与内皮细胞 (EC) 相关，它非常适合调节 TF-FVIIa 活性，或许可以绕过 FXa 浓缩作用的需要。细胞 TFPI 至少存在于两个池中，一个可逆地与未识别的“受体”结合，另一个可能由糖基磷脂酰肌醇 (GPI) 锚介导（即对磷脂酶 C 敏感）。本应用的目标是确定 TFPI 与膜表面结合的机制。尽管 TFPI 是通过磷脂酶 C 从 EC 释放的，但 TFPI 缺乏其他 GPI 锚定蛋白中发现的经典跨膜序列，这表明存在一种新的 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，将通过免疫亲和层析从人胎盘或培养的 EC 中分离 TFPI。表明 GPI 锚定的疏水形式将通过反相 HPLC 分离、酶消化并通过质谱分析来鉴定 GPI 连接的肽。我们还将通过固定化 TFPI 上的亲和层析分离候选 TFPI 受体。来自胎盘或培养的 EC 的洗涤剂提取物将用作推定受体的来源。结合的蛋白质将被分离、测试 TFPI 结合活性、进行微测序并通过搜索数据库进行鉴定。潜在的 GPI 锚定将按上述方法进行测试。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，这些研究将提供有关细胞结合 TFPI 在控制各种病理条件下 TF 活性方面​​的作用和功能机制的新数据。