Partial amino acid sequence of purified von Willebrand factor-cleaving protease
Partial amino acid sequence of purified von Willebrand factor-cleaving protease
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DOI：
10.1182/blood.v98.6.1654
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发表时间：
2001-09-15
期刊：
影响因子：
20.3
通讯作者：
Furlan, M
中科院分区：
文献类型：
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作者：
Gerritsen, HE;Robles, R;Furlan, M
血管性血变因子切割蛋白酶（vWF-cp）负责内皮细胞释放的血浆vWF多聚体的持续降解。它在血栓性血小板减少性紫癜患者中是缺乏的，这些患者在血浆中表现出异常大的vWF多聚体。纯化的vWF-cp可用于这些患者的替代。现在接受血浆治疗。在这项研究中，vWF-cp通过对vWF-cp自身抗体患者的IgG部分进行亲和层析，并通过一系列进一步的层析程序，包括对蛋白G、IgG - therasorb、扁豆凝集素和肝素进行亲和层析，从正常人血浆中纯化出来。在非还原条件下，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离的4条单链蛋白条带显示M-r分别为150、140、130和110 kd，且具有相同的n端氨基酸序列，表明它们来源于同一条在羧基端部分降解的多肽链。建立了前15个残基的疏水序列（Ala-Ala-Gly-Gly-lie-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly）。蛋白酶在凝胶过滤中以高分子量复合物的形式与簇蛋白（一种具有伴侣蛋白活性的70 kd蛋白）一起迁移。结合在聚簇蛋白上的vWF-cp通过使用浓向乱盐解离。正常人血浆或血清中的vWF-cp与聚集素无关，这表明观察到的复合物是由于纯化过程中vWF-cp变性所致。在37℃的孵育期间，vWF-cp的活性异常稳定；其在柠檬酸人血浆、肝素血浆或血清中的体外半衰期超过1周。在孵育的前3天，蛋白酶活性甚至暂时增加。(C) 2001年由美国血液学会出版。
von Willebrand factor-cleaving protease (vWF-cp) is responsible for the continuous degradation of plasma vWF multimers released from endothelial cells. It is deficient in patients with thrombotic thrombocytopenic purpura, who show unusually large vWF multimers in plasma. Purified vWF-cp may be useful for replacement in these patients, who are. now treated by plasma therapy. In this study, vWF-cp was purified from normal human plasma by affinity chromatography on the IgG fraction from a patient with autoantibodies to vWF-cp and by a series of further chromatographic procedures, including affinity chromatography on Protein G, Ig-TheraSorb, lentil lectin, and heparin. Four single-chain protein bands, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, showed M-r of 150, 140, 130, and 110 kd and were found to share the same N-terminal amino acid sequence, suggesting that they were derived from the same polypeptide chain that had been partially degraded at the carboxy-terminal end. A hydrophobic sequence (Ala-Ala-Gly-Gly-lie-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly) of the first 15 residues was established. The protease migrates in gel filtration as a high-molecular-weight complex with clusterin, a 70-kd protein with chaperonelike activity. vWF-cp bound to clusterin is dissociated by the use of concentrated chaotropic salts. vWF-cp in normal human plasma or serum is not associated with clusterin, suggesting that the observed complex is due to vWF-cp denaturation during the purification procedure. Activity of vWF-cp is unusually stable during incubation at 37 degreesC; its in vitro half-life in citrated human plasma, heparin plasma, or serum is longer than 1 week. There was even a temporary increase in protease activity during the first 3 days of incubation. (C) 2001 by The American Society of Hematology.