Structure and Function of Heparin Cofactor II

肝素辅因子 II 的结构和功能

• 批准号: 7143559
• 负责人: DOUGLAS M TOLLEFSEN
• 金额: $ 53.55万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7143559
• 关键词: animal infant mortality; antithrombins; atherosclerosis; blood coagulation disorders; blood vessels; carbohydrate structure; coagulation factor V; congenital blood disorder; disease /disorder etiology; enzyme activity; fibrin; gene mutation; gene targeting; genetically modified animals; immunocytochemistry; laboratory mouse; macrophage; mucopolysaccharides; nutrition related tag; pathologic process; platelets; protein binding; protein deficiency; protein structure function; recombinant proteins; thrombin; thrombosis; vascular smooth muscle

DESCRIPTION (provided by applicant): Heparin cofactor II (HCII) is an inhibitor of thrombin in plasma that can be activated by dermatan sulfate (DS) proteoglycans on vascular smooth muscle cells and fibroblasts. In a murine model of HCII deficiency, we showed that HCII modulates the response to arterial injury. In comparison with wild-type mice, HCHII-/- mice require less time to form a thrombus in the carotid artery following endothelial injury and have a greater degree of neointimal smooth muscle cell proliferation. HC-/- mice also develop more atherosclerotic lesions in the aorta when hypercholesterolemic. To build upon these observations, we propose the following specific aims: (1) Investigate the cause of neonatal mortality in HCII-deficient 129/SvJ mice and the phenotype of mice with combinations of HCII deficiency and other thrombophilic mutations. Although HCII-/- mice in the C57BL/6 background produce litters of normal size and viability, HCII-/- 129/SvJ mice do not produce viable offspring. Breeding studies will be done to determine whether neonatal mortality is related to maternal, paternal, or fetal HCII deficiency, and pathologic abnormalities will be identified. C57BL/6 mice with HCII deficiency in combination with factor V Leiden or protein Z deficiency will be observed for evidence of spontaneous thrombosis or other abnormalities. (2) Obtain evidence for activation of HCII upon binding to DS in the vessel wall, and identify HCII-binding sites in other tissues. HCII-/- mice will be reconstituted with recombinant HCII variants having specific defects in binding to heparin or DS to determine the importance of HCII-glycosaminoglycan interactions in models of arterial and venous thrombosis. Immunohistochemical methods will be used to localize HCII and glycosaminoglycans in the vessel wall after injury, and HCII-binding sites in other tissues will be identified. (3) Determine the location and amount of thrombin activity and the time course of smooth muscle cell and macrophage accumulation in neointimal and atherosclerotic lesions of HCII+/+ and HCII-/- mice. Hirudin-binding and chromogenic substrate assays will be used to examine arteries of HCII-/- mice for evidence of increased thrombin activity during neointima formation or development of atherosclerotic lesions. Rates of accumulation of fibrin(ogen), platelets, smooth muscle cells, and macrophages in these lesions will be determined in HCII-/- and wild-type mice. (4) Compare the structures of HCII-binding DS oligosaccharides derived from porcine skin and mucosa with those of vascular smooth muscle cells and fibroblasts. Size-fractionated DS oligosaccharides will be subjected to HCII affinity chromatography, and the structures of selected high- and low-affinity oligosaccharides will be determined. Metabolically-labeled vascular smooth muscle cells and fibroblasts will be assayed for the presence of HCII-binding oligosaccharides. This work will provide insight into the mechanism of activation of HCII by vascular glycosaminoglycans and the pathogenesis of human diseases such as thrombosis and atherosclerosis.

描述（由申请方提供）：肝素辅因子II（HCII）是血浆中凝血酶的抑制剂，可被血管平滑肌细胞和成纤维细胞上的硫酸皮肤素（DS）蛋白聚糖激活。在HCII缺乏的小鼠模型中，我们发现HCII调节对动脉损伤的反应。与野生型小鼠相比，HCHII-/-小鼠在内皮损伤后需要更少的时间在颈动脉中形成血栓，并且具有更大程度的新生内膜平滑肌细胞增殖。当高胆固醇血症时，HC-/-小鼠也在主动脉中发展更多的动脉粥样硬化病变。基于这些观察结果，我们提出了以下具体目标：（1）研究HCII缺陷129/SvJ小鼠新生儿死亡的原因以及HCII缺陷和其他嗜血栓性突变组合的小鼠的表型。虽然HCII-/-小鼠在C57 BL/6背景下产生正常大小和活力的窝，HCII-/-129/SvJ小鼠不产生可行的后代。将进行育种研究，以确定新生儿死亡率是否与母亲，父亲，或胎儿HCII缺乏症，并将确定病理异常。将观察HCII缺乏联合因子V Leiden或蛋白Z缺乏的C57 BL/6小鼠是否存在自发性血栓形成或其他异常的证据。(2)获得HCII与血管壁中DS结合后激活的证据，并确定其他组织中的HCII结合位点。HCII-/-小鼠将用在与肝素或DS结合方面具有特异性缺陷的重组HCII变体重建，以确定HCII-糖胺聚糖相互作用在动脉和静脉血栓形成模型中的重要性。将使用免疫组织化学方法定位损伤后血管壁中的HCII和糖胺聚糖，并鉴定其他组织中的HCII结合位点。(3)测定HCII +/+和HCII-/-小鼠新生内膜和动脉粥样硬化病变中凝血酶活性的位置和量以及平滑肌细胞和巨噬细胞蓄积的时间过程。水蛭素结合和显色底物试验将用于检查HCII-/-小鼠的动脉，以寻找新生内膜形成或动脉粥样硬化病变发展期间凝血酶活性增加的证据。将在HCII-/-和野生型小鼠中测定这些病变中纤维蛋白（原）、血小板、平滑肌细胞和巨噬细胞的蓄积速率。(4)比较来自猪皮肤和粘膜与血管平滑肌细胞和成纤维细胞的HCII结合DS寡糖的结构。将对大小分级DS寡糖进行HCII亲和层析，并测定选定的高亲和力和低亲和力寡糖的结构。将测定代谢标记的血管平滑肌细胞和成纤维细胞中是否存在HCII结合寡糖。这项工作将提供深入了解血管糖胺聚糖激活HCII的机制和人类疾病，如血栓形成和动脉粥样硬化的发病机制。