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

我们发现一位66岁的日本患者，患有I型先天性肝素辅因子(HC)II缺乏症，表现为多处动脉粥样硬化病变。扩增所有5个外显子及其侧翼区的测序结果显示，外显子5的12,854位核苷酸发生了单一的C-T转换，使Pro^&lt；443&gt；密码子(CCG)变为亮氨酸密码子(CTG)。瞬时转染、代谢标记和脉冲追逐实验及免疫沉淀分析表明，与野生型相比，COS-1细胞分泌的突变型HC II分子数量减少，并且突变型分子与CHO-K1细胞内伴侣GRP78/Bip的结合增强。此外，免疫组织化学研究显示，切除的狭窄动脉中对硫酸皮肤素和HC II的免疫反应性分别增加和降低。我们还发现甲亢患者血浆中HC II的水平显著降低，体外研究表明T_3治疗可剂量依赖性地增强培养的人肝细胞中HC II的mRNA水平。这一结果可能揭示了甲状腺功能减退症患者易患动脉粥样硬化性疾病的可能机制。对接受普通老式球囊血管成形术或支架(支架)治疗的冠心病患者的血浆HC II活性进行分析发现，血浆HC II活性超过110%的患者支架植入术后再狭窄的发生率显著低于那些HC II活性低于110%的患者(p&lt；0.0469)。关于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，G418抗性克隆现在通过Southern blotting进行筛选。

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.