Structure and Function of Heparin Cofactor II

描述（由申请方提供）：本项目的长期目标是阐明肝素辅因子H（HCII）的作用机制和生理功能。HCII是凝血酶的抑制剂，其以约1 μ M的浓度存在于血浆中。HCII的抗凝活性被肝素或硫酸皮肤素大大增加，成纤维细胞和平滑肌细胞表面的硫酸皮肤素蛋白聚糖刺激HCH。以前，人和鼠HCII的cDNA和基因组克隆被分离，重组HCII在E.大肠杆菌中，进行定点诱变以鉴定与凝血酶和糖胺聚糖相互作用的重要氨基酸残基，并鉴定了与猪皮硫酸皮肤素中HCII结合的高亲和力六糖的结构。虽然HCII在体内的功能仍然未知，间接证据表明，HCII活性增加，在怀孕期间，可能有助于抑制母体血液在胎盘中的凝固。此外，HCII在正常人动脉内膜中的存在和在动脉粥样硬化病变中硫酸皮肤素的改变的组成（具有降低的HCII刺激活性）提供了HCII在动脉粥样硬化形成中的作用的间接证据。通过在胚胎干细胞中靶向基因破坏，已经开发了HCII缺陷的小鼠模型。具体目的如下：（1）确定HCII缺乏对小鼠止血、动脉粥样硬化形成、伤口修复和炎症的影响。(2)通过免疫组织化学方法确定内源性六氯环己烷在人类和小鼠组织中的位置，并确定组织切片中外源性HCII的结合位点。(3)表征胎盘和其他组织中与HCII相互作用所需的糖胺聚糖中的寡糖结构。(4)确定动脉粥样硬化患者中HCII缺乏症的患病率。关于HCII在体内的结构、作用机制和功能的新信息将提供对正常和病理条件下凝血酶活性调节的更好理解。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the mechanism of action and physiologic function of heparin cofactor H (HCII). HCII is an inhibitor of thrombin that is present in plasma at a concentration of -1 uM. The anticoagulant activity of HCII is greatly increased by heparin or dermatan sulfate, and dermatan sulfate proteoglycans on the surface of fibroblasts and smooth muscle cells stimulate HCH. Previously, cDNA and genomic clones for human and murine HCII were isolated, recombinant HCII was expressed in E. coli, site-directed mutagenesis was performed to identify important amino acid residues that interact with thrombin and glycosaminoglycans, and the structure of a high-affinity hexasaccharide that binds HCII in porcine skin dermatan sulfate was identified. Although the function of HCII in vivo remains unknown, indirect evidence suggests that HCII activity increases during pregnancy and perhaps serves to inhibit coagulation of maternal blood in the placenta. In addition, the presence of HCII in the intima of normal human arteries and the altered composition of dermatan sulfate (with reduced HCII-stimulating activity) in atherosclerotic lesions provide circumstantial evidence of a role for HCII in atherogenesis. By means of targeted gene disruption in embryonic stem cells, a murine model of HCII deficiency has been developed. The following specific aims are proposed: (1) Determine the effects of HCII deficiency on hemostasis, atherogenesis, wound repair, and inflammation in the mouse. (2) Determine the location of endogenous HCH in human and murine tissues by immunohistochemical methods and identify binding Sites for exogenous HCII in tissue sections. (3) Characterize the oligosaccharide structures in glycosaminoglycans derived from placenta and other tissues that are required for interaction with HCII. (4) Determine the prevalence of HCII deficiency in patients with atherosclerosis. New information about the structure, mechanism of action, and function of HCII in vivo will provide a better understanding of the regulation of thrombin activity under normal and pathologic conditions.