动物模型与人类重大疾病机理重点实验室知识库

动脉粥样硬化是一种与脂质代谢异常及血管壁病变相关的动脉疾病，被广泛地认知为一种复杂的慢性炎症性疾病。动脉粥样硬化斑块的破裂是导致动脉粥样硬化血栓形成疾病，例如，心肌梗死、缺血性脑中风及外周血管病等的主要原因，给人类健康及社会发展带来严重的负担。临床上主流的治疗动脉粥样硬化药物有很多种类，这些药物主要针对动脉粥样硬化发病的重要的几个过程或者重要的致病因子，主要分为调节胆固醇水平类药物，例如，他汀类药物、胆固醇吸收类抑制剂；免疫抑制剂类药物，例如，糖皮质激素；调节血管机能和促进血管再生类药物；抗氧化剂，例如，维生素E；抗血小板聚集类药物及多烯脂肪酸类药物等。关于对动脉粥样硬化发病机制的研究一直是科学界的研究热点，纵观当今科学界对动脉粥样硬化发病机制的研究，多种学说占据着研究的关键位置，例如，炎症反应学说、氧化应激学说、脂质浸润学说甚至血栓形成学说等。尽管各种学派对动脉粥样硬化的研究众说纷纭，但是由于其发病机制确实过于复杂，导致我们对动脉粥样硬化的理解其实仍停留在一个模糊的阶段。将凝血系统与动脉粥样硬化的发病联系起来的原因是由于在动脉粥样硬化斑块中发现了特定的凝血系统相关蛋白，例如，组织因子、凝血酶、凝血因子FX和凝血因子FXII等。动脉粥样硬化与高促凝状态相关而在动脉粥样硬化斑块中发现的上述凝血因子可能直接诱导了高凝倾向及动脉粥样硬化的发生，但是动脉粥样硬化的高凝倾向形成原因及与动脉粥样硬化发病的因果关系的机制研究仍不清楚。在本研究中我们首先着重对铁代谢相关转铁蛋白诱导高凝倾向及动脉粥样硬化发病的机制进行探索，提供全新的动脉粥样硬化干预靶点。在另一部分延伸研究中我们侧重从吸血生物菲牛蛭中筛选靶向于动脉粥样硬化发病相关凝血因子的具有抗凝血功能的多肽抑制剂，挖掘潜在的抗动脉粥样硬化多肽药物分子。第一部分研究我们筛选并鉴定了转铁蛋白是动脉粥样硬化病人血浆中重要的促凝血分子，且动脉粥样硬化病人血浆的高凝倾向与转铁蛋白上调具有非常紧密的联系。调查发现转铁蛋白含量在动脉粥样硬化病人血浆及动脉粥样硬化斑块中显著上调。通过高脂饲料诱导载脂蛋白E敲除小鼠成功构建小鼠动脉粥样硬化模型，并同样发现该模型小鼠血浆及动脉粥样硬化斑块中转铁蛋白的含量显著上调。深入研究发现转铁蛋白与凝血酶和凝血因子FXIIa具有明显的相互作用且其平衡解离常数为7-13 nM。分子对接及突变实验证明转铁蛋白是通过作用于凝血酶和凝血因子FXIIa的外结合位点I功能域从而增强上述两种酶的酶催化活性。动脉粥样硬化病人血浆及动脉粥样硬化斑块中转铁蛋白-凝血酶/凝血因子FXIIa复合物含量明显上调并直接导致了动脉粥样硬化病人血浆和斑块中凝血酶和凝血因子FXIIa的酶催化活性的增高。在载脂蛋白E敲除小鼠动脉粥样硬化模型中转铁蛋白的过表达可以显著加重动脉粥样硬化斑块的形成而转铁蛋白的敲降和转铁蛋白抗体干预可以明显减轻动脉粥样斑块的形成。更重要的是，根据转铁蛋白结合凝血酶和凝血因子FXIIa的外结合位点I功能域的结合模式而设计合成的分别干预转铁蛋白与凝血酶或者凝血因子FXIIa结合的干预多肽TH16和FX18可以显著的抑制高凝倾向的发生及动脉粥样硬化的发病。综上所述，转铁蛋白结合并能增强凝血酶和凝血因子FXIIa的酶催化活性并是诱导高凝倾向及动脉粥样硬化发病的重要血浆因子。因此，转铁蛋白可以作为检测动脉粥样硬化疾病的候选标志物分子，且转铁蛋白可以作为优秀的动脉粥样硬化治疗靶点。以凝血酶和凝血因子FXIIa外结合位点I功能域为基础设计的干预多肽可以作为临床上潜在的抗动脉粥样硬化候选药物。在接下来的一部分研究中，我们从吸血生物菲牛蛭中筛选分离能作用于与动脉粥样硬化发病相关凝血因子的多肽抑制剂，以期筛选到优秀的抗动脉粥样硬化候选多肽药物。吸血动物为了顺利的从宿主获得血餐会分泌类型丰富的抗凝血成分以抑制吸血微环境的凝血过程。利用吸血生物这一生存策略我们从菲牛蛭中分离得到一个全新的antistasin家族丝氨酸蛋白酶抑制剂poecistasin。对该抑制剂的深入研究发现，多肽poecistasin能够抑制内源性凝血通路的激肽释放酶及凝血因子FXIIa并显著延长活化部分凝血活酶时间以抑制凝血进程。同时，在FeCl3诱导的小鼠颈动脉血栓模型上，poecistasin也能够与阳性对照肝素钠一样明显的抑制血栓形成，这也充分证明poecistasin在抑制凝血进程上发挥的重要作用。更重要的是，在小鼠动脉粥样硬化模型上poecistasin表现出显著的抗动脉粥样硬化效果，可以作为优秀的抗动脉粥样硬化候选多肽药物。综合上述两部分研究，凝血系统调控与动脉粥样硬化的发病具有直接的重要的联系，针对动脉粥样硬化相关凝血因子的干预可以显著的起到治疗动脉粥样硬化的效果。我们的研究首次阐明转铁蛋白通过调控凝血因子酶催化活性从而诱导高凝倾向的发生及动脉粥样硬化的发病，部分解释了动脉粥样硬化高凝倾向的重要分子机制。特异性的干预转铁蛋白或者转铁蛋白与其靶点凝血酶和凝血因子FXIIa作用的过程可以起到显著的抗动脉粥样硬化作用，而且针对该病理过程可以开发制备多种抗动脉粥样硬化候选药物。另外，从吸血菲牛蛭中分离的抗凝多肽poecistasin在动物模型上具有明显的抗动脉粥样硬化功能，也是临床潜在的治疗动脉粥样硬化候选药物分子。

Atherosclerosis (AS) is a kind of artery disease characterized by abnormal lipid metabolism and blood vessel wall lesions, and widely perceived as a complicated chronic inflammatory disease. Atherosclerotic plaque rupture is a predominant underlying cause of acute atherothrombotic events such as myocardial infarction, ischemic stroke, and peripheral vascular disease, etc, which poses serious burden to human health and social development. Several kinds of clinical mainstream drugs to treat AS are available and mainly target several important atherosclerotic disease processes or important pathogenic factors, and mainly include cholesterol regulation drugs, for example, statins, cholesterol absorption inhibitor; Immunosuppressive drugs, for example, sugar cortical hormone drugs; Blood vessel function adjusting and angiogenesis promoting drugs; Antioxidants, for example, vitamin E; Anti-platelet aggregation drugs and polyene fatty acid drugs, etc. Research on the pathogenesis of AS has been the prime focus of the scientific community. A variety of theories occupy the key position of the AS pathogenesis study, for example, inflammatory reaction theory, oxidative stress theory, lipid infiltration theory and even thrombosis formation theory, etc. Despite the extensive research on AS by various academic schools, our understanding of AS still remain obscure because of its complicated pathogenesis. The first reported significant correlation between blood coagulation system and pathogenesis of AS is due to the detection of particular proteins involved in blood clotting system in AS plaque, for example, tissue factor, thrombin, FX and FXII, etc. AS is associated with pro-coagulant state and these clotting factors may directly induce the occurrence of hypercoagulability of early AS, but the mechanism of hypercoagulability of early AS and its relationship with the onset of AS remain elusive. In this study, we firstly clarified that the iron metabolism related protein transferrin (Tf) is an important plasma factor that induces hypercoagulability and AS and provided a new intervention target for AS. In another extended study we tend to screen for peptide inhibitors which can inhibit coagulation factors related to AS and develop clinical anti-AS peptide drugs from blood sucking leech Poecilobdella manillensis.In the first part of the research, we screened and identified that Tf is an important pro-coagulant factor in AS plasma and the hypercoagulability of AS patients’ plasma is strongly related to the elevated Tf level. A remarkable elevated Tf level was found in plasma and plaque of atherosclerotic patients. Atherosclerotic model was constructed successfully in apolipoprotein E-deficient (Apoe-/-) mice and elevated Tf levels in plasma and plaque were also detected. Tf directly interacts with thrombin and FXIIa with high affinity (equilibrium dissociation constant: 7-13 nM). Molecular docking and mutation experiment proved that Tf significantly potentiates the activity of these enzymes via binding their exosite I motifs. Elevated Tf and Tf-prothrombin/FXII complexes are found in atherosclerotic plasma and plaques and are associated with the ability of AS plasma and plaque to potentiate activities of these enzymes. Tf-overexpression aggravates atherosclerotic lesion while Tf-knockdown or anti-Tf antibody alleviates the lesion in mouse models of AS. More importantly, small peptides TH16 and FX18 designed based on the exosite I-binding motifs of Tf interferes with Tf interaction with thrombin or FXIIa and inhibits AS progression. In conclusion, Tf is an important plasma factor promoting hypercoagulability and AS progression by interacting and potentiating thrombin and FXIIa. Thus, Tf can be a candidate marker for detection of atherosclerotic disease, and be an excellent therapeutic target of AS. Exosite I motif-based peptides have potentials as a novel avenue for clinical anti-AS medicine.In the next part of the study, we purified inhibitors which can inhibit coagulation factors related to AS to screen for excellent candidate anti-AS drugs. Blood-sucking animal can secrete anti-coagulant factors to get blood meal by overcoming host’s blood coagulation. In light of this survival strategy, we purified a novel antistasin-type serine protease inhibitor from Poecilobdella manillensis, and named it poecistasin. Poecistasin can inhibit kallikrein and FXIIa of the intrinsic coagulation pathway and prolong activated partial thromboplastin time then inhibit the coagulation process. Poecistasin showed anti-thrombosis activity with a similar effect of heparin sodium in FeCl3-induced carotid artery thrombosis model, this proves the important role of poecistasin in coagulation inhibition. More importantly, poecistasin exerts anti-AS effect in mice AS model, and might be an excellent candidate for the development of clinical anti-AS peptide drug.In all, regulation of coagulation system tightly related to AS, and intervention of AS-related coagulation factors can significantly treat AS. Our research firstly clarified that Tf can induce hypercoagulability and AS by regulating enzyme catalytic activity of coagulation factors and partly explained the mechanism of hypercoagulability in AS. Tf and Tf-thrombin/FXIIa interaction intervention exert a significant anti-AS effect, and multiple anti-AS drugs can be developed based on the pathlogy process. In addition, anti-coagulation peptide poecistasin exerts significant anti-AS effect and can be an ideal candidate for clinical anti-AS drug.