ANTIADHESIVE AND ANTICOAGULANT ACTIVITY OF KININOGENS

激肽原的抗粘连和抗凝血活性

• 批准号: 6485294
• 负责人: Robert W Colman
• 金额: $ 29万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6485294
• 关键词: allosteric site; binding sites; bradykinin; chemical binding; fibrinolysis; gene deletion mutation; human subject; kallikreins; kininogens; laboratory rat; neuropeptide receptor; neutrophil; phlebotomy; platelet aggregation; platelets; protein structure function; receptor binding; thrombin; urokinase; vascular endothelium

The biochemistry, cell and molecular biology of high (HK) and low (LK) molecular weight kininogens will be studied to learn about structure-function correlates as they relate to binding to and effects on blood and vascular cells. D5 has been identified as the region that interacts with anionic surfaces and as a binding site for HKA to neutrophils. Deletion mutants as well as synthesized peptides should allow mapping of the sequence(s) required for binding to neutrophils and endothelial cells. Physical studies of D5 with and without Zn plus plus will be performed. A 31 amino acid sequence in D6 contains sufficient information to bind to two noncontinuous sites on prekallikrein. Using peptides and deletion mutagenesis of D6, the minimal sequences for binding and the topology of HK binding to prekallikrein will be determined. We use these peptides and cognate peptides of prekallikrein to down-regulate fibrinolysis on endothelial cells. D3 has previously been identified as one cell binding region for platelets, endothelial cells and neutrophils. Exons 7, 8, and 9 coding for D3 will be expressed to test the hypothesis that one of the exon products contains all of the information for neutrophil binding on the heavy chain, while another is responsible for inhibiting the binding of thrombin to platelets and endothelial cells. Synthesized, conformationally restrained peptides will be used for fine mapping based on surface-accessible regions of a molecular model of D3 of HK. HK binds to neutrophils on Mac-1, and we will further map the ligand and receptor to better define the interaction. We have recently shown that fibrinogen does not compete with HK binding to endothelial cells, and an antibody to alpha nu beta 3 does not inhibit HK binding, ruling out alpha nu beta 3 as the receptor for HK. However, we found that vitronectin and soluble urokinase receptor inhibit HK binding, suggesting that the UK receptor is the binding site for HK. We will further test this hypothesis by mapping the HK binding site on the UK receptor and map the binding site on HK to the receptor. The mechanism by which HK blocks thrombin binding to platelets by interaction with the thrombin receptor or GPIb will be explored. Kininogen-deficient rats will be used to verify whether HK or LK is an important anti-thrombotic protein. Peptides from kininogen domains will be tested in rats for their antiadhesive and antiplatelet potential. Such polypeptides could serve as templates for peptidomimetic compounds, which should be therapeutic in sepsis, arthritis, hereditary angioedema, and gingival disease in addition to reocclusion after thrombolytic therapy.

高（HK）和低的生物化学、细胞和分子生物学 (LK)分子量激肽原将被研究以了解 结构-功能相关，因为它们与结合和作用有关 血液和血管细胞。 D5 已被确定为区域 与阴离子表面相互作用并作为 HKA 的结合位点 中性粒细胞。 缺失突变体以及合成肽应该 允许绘制与中性粒细胞结合所需的序列 和内皮细胞。 添加和不添加 Zn 的 D5 的物理研究 plus 将被执行。 D6 中的 31 个氨基酸序列包含 足够的信息绑定到两个不连续的站点 前激肽释放酶。 利用 D6 的肽和缺失诱变， 最小结合序列和 HK 结合的拓扑结构 将测定前激肽释放酶。 我们使用这些肽和同源 前激肽释放酶肽下调内皮细胞纤维蛋白溶解 细胞。 D3 先前已被确定为一个细胞结合区域 血小板、内皮细胞和中性粒细胞。 外显子 7、8 和 9 编码 对于 D3 将被表达以检验外显子之一的假设 产品包含中性粒细胞结合的所有信息 重链，而另一个负责抑制结合 凝血酶作用于血小板和内皮细胞。 合成的， 构象限制肽将用于精细作图 基于 D3 分子模型的表面可及区域 香港。 HK 与 Mac-1 上的中性粒细胞结合，我们将进一步绘制 配体和受体以更好地定义相互作用。 我们最近有 表明纤维蛋白原不与 HK 竞争结合 内皮细胞，α nu beta 3 抗体不会抑制 HK 结合，排除 alpha nu beta 3 作为 HK 受体。 然而，我们发现玻连蛋白和可溶性尿激酶受体 抑制 HK 结合，表明 UK 受体是结合位点 对于香港。 我们将通过绘制香港的地图来进一步检验这一假设 UK 受体上的结合位点并将 HK 上的结合位点映射到 受体。 HK 阻断凝血酶结合的机制 血小板通过与凝血酶受体或 GPIb 相互作用 探索过。 将使用缺乏激肽原的大鼠来验证HK是否 或LK是一种重要的抗血栓蛋白。 肽来自 激肽原结构域将在大鼠中测试其抗粘附和 抗血小板潜力。 此类多肽可以作为模板 拟肽化合物，应该可以治疗脓毒症， 关节炎、遗传性血管性水肿和牙龈疾病 溶栓治疗后再闭塞。