Structure and Function of Heparin Cofactor II

项目描述（由申请人提供）：本项目的长期目标是阐明肝素辅助因子H （HCII）的作用机制和生理功能。HCII是一种凝血酶抑制剂，存在于血浆中浓度为-1 μ m。肝素或硫酸皮聚糖可显著提高HCII的抗凝血活性，成纤维细胞和平滑肌细胞表面的硫酸皮聚糖可刺激HCH。在此之前，我们分离了人类和小鼠HCII的cDNA和基因组克隆，在大肠杆菌中表达了重组HCII，通过定点诱变鉴定了与凝血酶和糖胺聚糖相互作用的重要氨基酸残基，并鉴定了猪皮肤硫酸盐中与HCII结合的高亲和力六糖的结构。虽然HCII在体内的功能尚不清楚，但间接证据表明，HCII的活性在怀孕期间增加，可能有助于抑制胎盘中母体血液的凝固。此外，HCII在正常人动脉内膜中的存在以及动脉粥样硬化病变中皮肤硫酸酯成分的改变（刺激HCII活性降低）为HCII在动脉粥样硬化中的作用提供了间接证据。通过对胚胎干细胞进行靶向基因破坏，建立了小鼠HCII缺陷模型。提出以下具体目的：(1)确定HCII缺乏对小鼠止血、动脉粥样硬化、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HCH在人和小鼠组织中的位置，并在组织切片中确定外源性HCII的结合位点。(3)表征来自胎盘和其他组织的糖胺聚糖中与HCII相互作用所需的低聚糖结构。(4)确定动脉粥样硬化患者HCII缺乏的患病率。关于HCII在体内的结构、作用机制和功能的新信息将有助于更好地理解正常和病理条件下凝血酶活性的调节。

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.