Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family
Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family
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DOI：
10.1182/blood.v98.6.1662
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发表时间：
2001-09-15
期刊：
影响因子：
20.3
通讯作者：
Chung, D
中科院分区：
文献类型：
--
作者：
Fujikawa, K;Suzuki, H;Chung, D
血管性血友病因子(von Willebrand factor，vWF)在巨核细胞和血管内皮细胞中以非常大的多聚体形式合成，但在血浆中以500~10000 kd的多聚体形式循环。大分子多聚体解聚的一个重要机制是血浆中存在的一种裂解vWF的蛋白水解酶。VWF裂解酶的缺失或失活会导致大分子多聚体的聚集，从而可能导致血栓性血小板减少性紫癜。VWF裂解酶是一种表观分子量约为300kd的钙依赖蛋白水解酶。然而，到目前为止，它还没有被孤立和表征。本文报道了用肝素-琼脂糖柱层析两步法从商业化的因子/vWF浓缩物中分离纯化人vWF裂解酶的方法。阴离子交换和凝胶过滤柱级分的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析表明，vWF裂解酶的活性对应于一条150kd的蛋白条带。还原后，其迁徙表观重量为190kd。该带的氨基末端序列为AAGGIL(H)LE(L)L-(D)AXG(P)X(V)XQ(单字母氨基酸编码)，初步残基显示在括号中。对人类基因组序列的搜索发现，vWF裂解酶是金属蛋白酶ADAMTS家族的一个新成员。HIGHSFGLEHE(单字母氨基酸编码)的活性位点序列位于该蛋白N端150个残基。(C)2001年，由美国血液病学会提供。
von Willebrand factor (vWF) is synthesized in megakaryocytes and endothelial cells as a very large multimer, but circulates in plasma as a group of multimers ranging from 500 to 10 000 kd. An important mechanism for depolymerization of the large multimers is the limited proteolysis by a vWF-cleaving protease present in plasma. The absence or inactivation of the vWF-cleaving protease results in the accumulation of large multimers, which may cause thrombotic thrombocytopenic purpura. The vWF-cleaving protease was first described as a Ca++-dependent proteinase with an apparent molecular weight of approximately 300 kd. Thus far, however, it has not been isolated and characterized. In this study, the purification of human vWF-cleaving protease from a commercial preparation of factor VIII/vWF concentrate by means of several column chromatographic steps, including 2 steps of heparin-Sepharose column, is reported. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the anion exchange and gel filtration column fractions showed that the vWF-cleaving protease activity corresponded to a protein band of 150 kd. After reduction, it migrated with an apparent weight of 190 kd. The amino terminal sequence of the 150-kd band was AAGGIL(H)LE(L)L-(D)AXG(P)X(V)XQ (single-letter amino acid codes), with the tentative residues shown in parentheses. A search of the human genome sequence identified the vWF-cleaving protease as a new member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motif) family of metalloproteinase. An active site sequence of HEIGHSFGLEHE (single-letter amino acid codes) was located at 150 residues from the N terminus of the protein. (C) 2001 by The American Society of Hematology.