Structure and Function of Heparin Cofactor II

说明(申请人提供)：肝素辅因子II(HCII)是一种血浆中凝血酶的抑制物，可被血管平滑肌细胞和成纤维细胞上的硫酸皮肤素(DS)蛋白多糖激活。在HCII缺乏的小鼠模型中，我们证明了HCII调节对动脉损伤的反应。与野生型小鼠相比，HCHII-/-小鼠内皮损伤后在颈动脉形成血栓所需的时间更短，新生内膜平滑肌细胞的增殖程度更大。当高胆固醇血症时，HC-/-小鼠也会在主动脉中形成更多的动脉粥样硬化病变。为了建立在这些观察的基础上，我们提出了以下具体目标：(1)调查HCII缺陷129/SVJ小鼠新生儿死亡的原因以及具有HCII缺陷和其他亲血栓突变组合的小鼠的表型。虽然在C57BL/6背景下的HCII-/-小鼠产生了正常大小和存活能力的小鼠，但HCII-/-129/SVJ小鼠不能产生可存活的后代。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缺乏症有关，并将确定病理异常。C57BL/6小鼠的HCII缺陷合并因子V Leiden或蛋白Z缺陷将被观察为自发血栓形成或其他异常的证据。(2)在血管壁上获得与DS结合后激活HCII的证据，并鉴定其他组织中的HCII结合部位。HCII-/-小鼠将用在与肝素或DS结合方面存在特定缺陷的重组HCII变异体进行重组，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。将使用免疫组织化学方法来定位损伤后血管壁中的HCII和糖胺聚糖，并将确定其他组织中的HCII结合部位。(3)测定HCII+/+和HCII-/-小鼠血管内膜和动脉粥样硬化病变中凝血酶活性的位置和数量，以及血管内膜和巨噬细胞聚集的时间进程。水飞蓟素结合和发色底物分析将用于检查HCII-/-小鼠的动脉，以寻找在新的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。这些病变中纤维蛋白(原)、血小板、平滑肌细胞和巨噬细胞的积累率将在HCII-/-和野生型小鼠中确定。(4)比较猪皮肤和粘膜来源的HCII结合双链寡糖与血管平滑肌细胞和成纤维细胞的结构。大小分级的DS寡糖将经过HCII亲和层析，并将确定选定的高亲和力和低亲和力低聚糖的结构。代谢性标记的血管平滑肌细胞和成纤维细胞将被检测是否存在HCII结合的寡糖。这项工作将为深入了解血管氨基聚糖激活HCII的机制以及血栓形成和动脉粥样硬化等人类疾病的发病机制提供依据。

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.