Structure and Function of Heparin Cofactor II

描述（由申请人提供）：肝素辅助因子II （HCII）是血浆中凝血酶的抑制剂，可被皮肤硫酸酯（DS）蛋白聚糖在血管平滑肌细胞和成纤维细胞上激活。在HCII缺乏的小鼠模型中，我们发现HCII可以调节对动脉损伤的反应。与野生型小鼠相比，HCHII-/-小鼠内皮损伤后颈动脉血栓形成所需时间更短，新内膜平滑肌细胞增殖程度更高。当高胆固醇血症时，HC-/-小鼠的主动脉也会出现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缺陷和其他血栓性突变组合小鼠的表型。虽然C57BL/6背景下的HCII-/-小鼠能产生正常大小和存活率的胎仔，但HCII-/- 129/SvJ小鼠不能产生存活率的后代。将进行育种研究，以确定新生儿死亡率是否与母体、父亲或胎儿HCII缺乏有关，并确定病理异常。C57BL/6小鼠HCII缺乏合并Leiden因子V或蛋白Z缺乏将观察自发性血栓形成或其他异常的证据。(2)获得HCII与血管壁DS结合后激活的证据，并在其他组织中确定HCII的结合位点。HCII-/-小鼠将通过重组HCII变体与肝素或DS结合的特异性缺陷进行重组，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。使用免疫组织化学方法定位损伤后血管壁上的HCII和糖胺聚糖，并确定其他组织中的HCII结合位点。(3)测定HCII+/+和HCII-/-小鼠新内膜和动脉粥样硬化病变中凝血酶活性的位置和数量以及平滑肌细胞和巨噬细胞积聚的时间过程。水蛭素结合和显色底物测定将用于检查HCII-/-小鼠的动脉，以寻找在新内膜形成或动脉粥样硬化病变发展过程中凝血酶活性增加的证据。纤维蛋白（原）、血小板、平滑肌细胞和巨噬细胞在这些病变中的积累率将在HCII-/-和野生型小鼠中进行测定。(4)比较了从猪皮肤和粘膜中提取的与hcii结合的DS寡糖与血管平滑肌细胞和成纤维细胞的结构。通过HCII亲和层析对分级后的DS寡糖进行分析，确定高亲和寡糖和低亲和寡糖的结构。代谢标记的血管平滑肌细胞和成纤维细胞将被检测hci结合寡糖的存在。这项工作将为血管糖胺聚糖激活HCII的机制以及血栓和动脉粥样硬化等人类疾病的发病机制提供新的见解。

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.