Role and clinical significance of heparin cofactor II as an anti-atherosclerotic factor

肝素辅因子II作为抗动脉粥样硬化因子的作用及临床意义

• 批准号: 11838011
• 负责人: AZUMA Hiroyuki
• 金额: $ 1.92万
• 依托单位: University of Tokushima
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11838011/
• 关键词: heparin cofactor II; atherosclerosis; thrombin; vascular smooth muscle cell; dermatan sulfate; knockout mouse; トロンビン

We found a 66-year-old Japanese patient with type I congenital heparin cofactor (HC) II deficiency manifesting multiple atherosclerotic lesions. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T transition at nucleotide position 12,854 in exon 5, which changed a Pro^<443> codon (CCG) to Leu codon (CTG). Transient transfection, metabolic labeling and pulse-chase experiments followed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared with the wild-type, and that an enhanced intracellular association of the mutant molecules with a chaperon, GRP78/BiP, was observed in CHO-K1 cells. In addition, immunohistochemical study showed that the immunoreactivities against dermatan sulfate and HC II in resected aortic artery with stenosis were increased and decreased, respectively.We also found that the plasma levels of HC II in patients with hyperthyroidism significantly decreased by treatment, and in vitro study showed that T_3 treatment enhanced dose-dependently the mRNA levels of HC II in cultured human hepatocytes. This result may indicate a possible mechanism whereby patients with hypothyroidism are prone to atherosclerotic disease.Analysis of plasma HC II activity in patients with coronary heart disease treated with plain old balloon angioplasty (POBA) or stenting (stent) revealed that patients with more than 110% of plasma HC II activity showed significantly less frequent (p<0.0469) in restenosis after stent grafting than those with less than 110% of HC II activity.Regarding HC II-knockout mouse, since targeting vector for desrupting mouse HC II gene has been prepared, G418-resintant clones are now screening by Southern blotting.

我们发现了一位66岁的日本患者，患有I型先天性肝素辅助因子（HC） II缺乏症，表现为多发性动脉粥样硬化病变。对所有5个外显子及其侧翼区域进行扩增后的测序分析显示，在第5外显子12,854核苷酸位置有一个单C到T的转变，将Pro^<443>密码子（CCG）转变为Leu密码子（CTG）。瞬时转染、代谢标记和脉冲追踪实验以及免疫沉淀分析表明，与野生型相比，重组突变体HC II分子在COS-1细胞中分泌量减少，并且在CHO-K1细胞中观察到突变体分子与伴侣子GRP78/BiP的细胞内关联增强。此外，免疫组化研究显示，主动脉狭窄切除后，对硫酸皮聚糖和HCⅱ的免疫反应活性分别升高和降低。我们还发现，治疗后甲亢患者血浆HC II水平显著降低，体外研究表明，T_3治疗可剂量依赖性地提高培养的人肝细胞中HC II mRNA水平。这一结果可能提示甲状腺功能减退患者容易发生动脉粥样硬化性疾病的可能机制。对接受普通旧球囊血管成形术（POBA）或支架（支架）治疗的冠心病患者血浆HC II活性的分析显示，血浆HC II活性大于110%的患者在支架移植后再狭窄的发生率明显低于HC II活性小于110%的患者（p<0.0469）。对于HC - II基因敲除小鼠，由于已经制备了破坏小鼠HC - II基因的靶向载体，目前正通过Southern blotting筛选g418抗性克隆。

项目成果

Kanagawa et al.: "Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at reactive P2 site: HC II Tokushima"Thrombosis and Haemostasis. 85. 101-107 (2001)

神奈川等人：“由反应性 P2 位点错义突变引起的 I 型先天性肝素辅因子 (HC) II 缺乏症的分子机制：HC II 德岛”血栓形成和止血。

Kanagawa Y et al: "Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at P2 reactive site : HC II Tokushima"Thrombosis and Haemostasis. 85(1). 101-107 (2001)

Kanakawa Y等人：“P2反应位点错义突变引起的I型先天性肝素辅因子（HC）II缺乏症的分子机制：HC II德岛”血栓形成和止血。

Kanagawa et al.: "Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at reactive P2 site : HC II Tokushima"Thrombosis and Haemostasis. 85・1. 101-107 (2001)

神奈川等人：“由反应性 P2 位点错义突变引起的 I 型先天性肝素辅因子 (HC) II 缺乏的分子机制：HC II 德岛”血栓形成和止血 85・1。