Structure and Function of Heparin Cofactor II

描述(申请人提供)：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是一种凝血酶抑制物，以-1微米的浓度存在于血浆中。肝素或硫酸皮肤素可显著增强HCII的抗凝血活性，而成纤维细胞和平滑肌细胞表面的硫酸皮肤素蛋白多糖刺激HCH。在此之前，我们分离了人和小鼠HCII的cDNA和基因组克隆，在大肠杆菌中表达了重组的HCII，通过定点突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皮肤素中与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活性在怀孕期间增加，可能有助于抑制母体血液在胎盘中的凝结。此外，正常人动脉内膜中HCII的存在和动脉粥样硬化病变中硫酸皮肤素成分的改变(HCII刺激活性降低)提供了HCII在动脉粥样硬化形成中作用的间接证据。通过在胚胎干细胞中进行靶向基因破坏，已经建立了小鼠HCII缺乏症模型。提出了以下具体目标：(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)用免疫组织化学方法确定内源性六氯环己烷在人和小鼠组织中的定位，并在组织切片中确定外源性六氯环己烷的结合部位。(3)鉴定胎盘和其他组织中与HCII相互作用所需的糖胺聚糖的寡糖结构。(4)测定动脉粥样硬化患者HCII缺乏症的患病率。有关体内HCII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.