Structure and Function of Heparin Cofactor II

肝素辅因子 II 的结构和功能

• 批准号: 7869359
• 负责人: DOUGLAS M TOLLEFSEN
• 金额: $ 57.17万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7869359
• 关键词: Affinity; Affinity Chromatography; Aorta; Arterial Fatty Streak; Arterial Injury; Arteries; Atherosclerosis; Binding; Binding Sites; Biological Assay; Blood Platelets; Blood Vessels; Breeding; C57BL/6 Mouse; Carotid Arteries; Cell Proliferation; Chromogenic Substrates; Defect; Dermatan Sulfate; Development; Family suidae; Fibrin; Fibroblasts; Glycosaminoglycans; Heparin; Heparin Cofactor II; Hirudin; Hirudins; Injury; Label; Lesion; Location; Methods; Modeling; Mucous Membrane; Mus; Mutation; Neonatal Mortality; Oligosaccharides; Pathogenesis; Pathologic; Pharmacia brand of estropipate; Phenotype; Plasma; Recombinants; Skin; Smooth Muscle Myocytes; Structure; Thrombin; Thrombosis; Thrombus; Time; Tissues; Variant; Venous Thrombosis; Wild Type Mouse; Work; dermatan sulfate proteoglycan; factor V Leiden; fetal; human disease; inhibitor/antagonist; insight; macrophage; neointima formation; offspring; plasma protein Z; reconstitution; response

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.

描述（申请人提供）：肝素辅因子 II (HCII) 是血浆中凝血酶的抑制剂，可被血管平滑肌细胞和成纤维细胞上的硫酸皮肤素 (DS) 蛋白聚糖激活。在 HCII 缺乏的小鼠模型中，我们发现 HCII 调节对动脉损伤的反应。与野生型小鼠相比，HCHII-/-小鼠在内皮损伤后颈动脉中形成血栓所需的时间更短，并且具有更大程度的新内膜平滑肌细胞增殖。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，我们提出以下具体目标：（1）研究 HCII 缺陷型 129/SvJ 小鼠新生儿死亡的原因以及 HCII 缺陷型和其他易血栓突变组合的小鼠的表型。虽然 C57BL/6 背景中的 HCII-/- 小鼠产生正常大小和活力的窝，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 缺乏有关，并确定病理异常。将观察具有 HCII 缺陷并伴有因子 V Leiden 或蛋白质 Z 缺陷的 C57BL/6 小鼠，以寻找自发血栓形成或其他异常的证据。 (2) 获得HCII与血管壁中的DS结合后激活的证据，并确定其他组织中的HCII结合位点。将使用在与肝素或 DS 结合方面具有特定缺陷的重组 HCII 变体重建 HCII-/- 小鼠，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。免疫组织化学方法将用于定位损伤后血管壁中的 HCII 和糖胺聚糖，并鉴定其他组织中的 HCII 结合位点。 (3)测定HCII+/+和HCII-/-小鼠新生内膜和动脉粥样硬化病变中凝血酶活性的位置和量以及平滑肌细胞和巨噬细胞聚集的时间过程。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。将在 HCII-/- 和野生型小鼠中测定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。 (4)比较来自猪皮肤和粘膜的HCII结合DS寡糖与血管平滑肌细胞和成纤维细胞的结构。对尺寸分级的 DS 寡糖进行 HCII 亲和层析，并确定所选高亲和力和低亲和力寡糖的结构。将检测代谢标记的血管平滑肌细胞和成纤维细胞是否存在 HCII 结合寡糖。这项工作将深入了解血管糖胺聚糖激活 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。

项目成果

Amino acid residues of heparin cofactor II required for stimulation of thrombin inhibition by sulphated polyanions.

硫酸化聚阴离子刺激凝血酶抑制所需的肝素辅因子 II 的氨基酸残基。

• DOI: 10.1016/s0167-4838(99)00051-5
• 发表时间: 1999
• 期刊: Biochimica et biophysica acta
• 作者: Colwell,NS;Grupe,MJ;Tollefsen,DM
• 通讯作者: Tollefsen,DM

COX-1(+/-)COX-2(-/-) genotype in mice is associated with shortened time to carotid artery occlusion through increased PAI-1.

小鼠中的 COX-1(/-)COX-2(-/-) 基因型与 PAI-1 增加导致颈动脉闭塞时间缩短相关。

• DOI: 10.1111/j.1538-7836.2010.04156.x
• 发表时间: 2011
• 期刊: Journal of thrombosis and haemostasis : JTH
• 作者: Riehl,TE;He,L;Zheng,L;Greco,S;Tollefsen,DM;Stenson,WF
• 通讯作者: Stenson,WF

Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces.

• DOI: 10.1088/0957-4484/25/39/395101
• 发表时间: 2014-10-03
• 期刊: Nanotechnology
• 影响因子: 3.5
• 作者: Myerson JW;He L;Allen JS;Williams T;Lanza G;Tollefsen D;Caruthers S;Wickline S
• 通讯作者: Wickline S

Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli.

大肠杆菌中表达的人肝素辅因子 II 的糖胺聚糖结合特性和动力学特征。

• DOI: 10.1016/j.ab.2010.07.024
• 发表时间: 2010
• 期刊: Analytical biochemistry
• 影响因子: 2.9
• 作者: Sarilla,Suryakala;Habib,SallyY;Tollefsen,DouglasM;Friedman,DavidB;Arnett,DianaR;Verhamme,IngridM
• 通讯作者: Verhamme,IngridM

Dermatan sulfate and bone marrow mononuclear cells used as a new therapeutic strategy after arterial injury in mice.

硫酸皮肤素和骨髓单核细胞用作小鼠动脉损伤后的新治疗策略。

• DOI: 10.3109/14653249.2010.548378
• 发表时间: 2011
• 期刊: Cytotherapy
• 影响因子: 4.5
• 作者: Godoy,JulianaAP;Block,DanielB;Tollefsen,DouglasM;Werneck,ClaudioC;Vicente,CristinaP
• 通讯作者: Vicente,CristinaP