血小板内血管性血友病因子裂解酶表达的鉴定  

Identification of von Willebrand factor-cleaving protease in peripheral platelets

作　　者：高维强[1] 李丽[2] 白霞[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所江苏苏州215006 [2]苏州市中心血站流式细胞仪室,江苏苏州215006

出　　处：《检验医学》2005年第2期130-133,共4页Laboratory Medicine

基　　金：国家自然科学基金资助项目(30470732)

摘　　要：目的　血管性血友病因子裂解酶(vWF cp)是一种新发现的血浆金属蛋白酶,他在人外周血细胞中的 表达尚待确定。本研究确定了该酶在血小板中的表达。方法　应用逆转录 聚合酶链反应(RT PCR)、流式细胞 术(FCM)和免疫共沉淀鉴定了血小板内vWF cpmRNA和蛋白的表达情况。同时应用FCM检测了白细胞、红细 胞内vWF cp蛋白的表达。结果　RT PCR显示,血小板中存在该蛋白酶mRNA的表达。同时,免疫沉淀及蛋白 印迹结果表明,血小板内存在相对分子质量约200000的vWF cp蛋白。FCM结果显示,经穿膜处理的血小板可 检测到vWF cp蛋白的明显表达,而不经穿膜处理以及经过穿膜处理的白细胞和红细胞中不存在该酶的表达。 结论　本研究不仅证实了外周血血小板中存在vWF cp,由于血小板亦为血管性血友病因子的一个主要合成/贮 存场所,这将有助于深入阐明血小板在血栓形成与止血中的作用。Objective von Willebrand factor-cleaving protease (vWF-cp) is a newly purified metalloproteinase in plasma. The expression of vWF-cp in blood cells remains to be characterized. The present study is to identify the protease in platelets. Methods The expression of vWF-cp in platelets was analyzed by reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM) and immunoprecipitation.Moreover, the expressions of vWF-cp in leukocytes and erythrocytes were detected by FCM. Results The data showed the vWF-cp mRNA was present in platelets, and a protein with 200 000 of the molecular size was observed by immunoprecipitation. The FCM analysis showed that the protein of vWF-cp could only be detected in platelets permeabilized with methanol, while the protease could not be detected in non-permeabilized platelets or permeabilized leukocytes and erythrocytes. Conclusions Since platelets is a main storage of vWF, and the multimeric sizes of vWF is regulated by vWF-cp, the data above not only further confirm that vWF-cp is present in platelets, but also will be helpful to study the role of platelets in thrombosis and haemostasis.

关 键 词：血管性血友病因子裂解酶 血小板 单克隆抗体 鉴定 

分 类 号：R446.63[医药卫生—诊断学]