钩端螺旋体vwa基因产物竞争性抑制vWF介导血小板聚集引发出血分子机制

Hemorrhage is the typical pathological change in leptospirosis, but is mechanism remains unknown. We found that both the products of vwa-I and vwa-II gens of Leptospira interrogans strain Lai caused the serious pulmonary hemorrhage in Syrian hamsters, and the product of vwa-I gene had an ability to bind to the GPIbα receptor of human platelets. Moreover, our bioinformatic analysis indicated that the two leptospiral genes contain the functional domains and motifs similar to those A functional region of human von Willebrand factor (vWF) that mediates platelet aggregation. Therefore, we presume that the two leptospiral gene products cause hemorrhage by competitive inhibition of vWA-mediated platelet aggregation. In this project, we first determine the role of vwa-I and vwa-II gene products of L. interrogans strain Lai binding platelets as well as inhibiting vWF to bind to platelets or mediate platelet aggregation. Subsequently, we determine the activities of the two leptospiral gene products to bind to the GPIbα on platelets, vWF-A1 region and collagens as well as trigger platelet aggregation-associated PI3K/Akt-cGMP-ERK and PLC/PKC signaling pathways, up-regulate the Ca2+, TXA2 and ADP levels, activate the talin and αIIbβ3 integrin in platelets. Finally, we determine the role and diversity of the two leptospiral gene products as well as either vwa-I or vwa-II gene-deleted mutants and the wild-type leptospiral strain to cause the hemorrhage in Syrian hamsters. The research results of this project will reveal the molecular mechanism of hemorrhage during leptospirosis with high originality and medical significance.

钩端螺旋体（简称钩体）感染时脏器出血是钩体病典型病变，但机制不明。我们发现钩体vwa-I和vwa-II基因产物引起金地鼠肺组织严重出血，vwa-I基因产物能结合人血小板GPIbα受体，生物信息学分析显示上述基因均含介导血小板聚集的人血管性血友病因子（vWF）A功能区相似结构域或基序，推测上述基因产物竞争性抑制vWF介导血小板聚集导致出血。本项目中我们首先确定问号钩体赖株vwa-I和vwa-II基因产物结合血小板及抑制vWF结合或介导血小板聚集作用，然后确定上述基因产物与血小板GPIbα、vWF-A1区或COL结合活性与基序以及激活血小板聚集PI3K/Akt-cGMP-ERK和PLC/PKC通路、上调Ca2+及TXA2和ADP水平、活化踝蛋白和αIIbβ3整合素活性，采用金地鼠了解上述基因产物及其敲除突变株与野生株引起体内出血作用及差异，以期阐明钩体病出血分子机制，有较高创新性和医学意义。

致病性钩端螺旋体（简称钩体）感染引起的钩体病是全球流行的人兽共患传染病。肺等脏器出血是钩体病典型病变，但其发病机制不明。本项目历时四年，取得了如下研究成果：.1．问号钩体是全球流行最广的致病性钩体，问号钩体黄疸出血群赖型是我国最主要流行的致病性钩体血清群型，本项目中采用问号钩体黄疸出血群赖型赖株和人血小板（遵照医学伦理规范）为研究对象。.2．通过E. coli-pET42a原核表达系统表达了问号钩体黄疸出血群赖型赖株vwa-I和vwa-II基因编码蛋白， Ni-NTA亲和层析法获得了大量vwa-I和vwa-II基因重组表达产物（rLep-vWA-I和rLep-vWA-II）。.3．证实了rLep-vWA-I和rLep-vWA-II能与血小板以及血小板膜表面GPIbα呈高亲和力结合，表明血小板GPIbα是问号钩体赖株rLep-vWA-I和rLep-vWA-II受体。.4．以重组人vWF为阳性对照，确定rLep-vWA-I和rLep-vWA-II与血小板GPIbα结合后，不能激活血小板聚集信号通路关键激酶（AKT、ERK1/2、PLCγ2和PKC）、不能上调上述血小板聚集关键活性分子和游离Ca2+水平、血小板不出现聚集现象。.5．确定了G13/G47/R36和G76/Q126分别是Lep-vWA-I和Lep-vWA-II结合血小板GPIbα位点。.6．rLep-vWA-I和rLep-vWA-II注入C3H/HeJ和C57BL/6小鼠后，其外周血凝集时间（CT）、凝血酶原时间（PT）、凝血酶时间（TT）、部分活化凝血活酶时间（APTT）、凝血酶生成时间（TGT）均显著延长，小鼠出现弥漫性肺出血和局灶性肾出血。.本研究已发表SCI论文4篇（其中3篇IF>5）、国家核心期刊论文5篇；主编了高等教育出版社出版的普通高等教育十二五国家规划教材《医学微生物学》第三版；在2017年11.28-12.03新西兰北帕市第10届国际钩体病学术会议上做了两个大会报告并再次当选为国际钩体病学会理事会理事（1/13）；全日制6名博士生、3名硕士生毕业；另有1篇SCI论文已投稿、2篇SCI论文正在编辑或撰写中。对照本项目合同指标，我们全面完成了本项目研究任务并取得了优异成绩。