Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis
Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis
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DOI：
10.1182/blood.v87.10.4223.bloodjournal87104223
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发表时间：
1996-05-15
期刊：
影响因子：
20.3
通讯作者：
Lammle, B
中科院分区：
文献类型：
--
作者：
Furlan, M;Robles, R;Lammle, B
血管性血友病因子（vWF）的蛋白水解裂解发生在健康受试者的循环血液中，并在一些2A型血管性血友病患者中增加。具有止血活性的大vWF多聚体降解为较小的活性较低的形式。已经表明vWF的多肽亚基在842 Tyr-843 Met的肽键处被切割。我们纯化（约10，000倍）从人血浆vWF降解蛋白酶，使用螯合琼脂糖凝胶，疏水相互作用色谱，和凝胶过滤。该酶被发现在通过反复冻融获得的血小板裂解物中几乎不存在。通过凝胶过滤和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断，蛋白水解活性与高分子量蛋白（约300 kD）相关。在中性缓冲液中，VWF在生理离子强度下对蛋白酶具有抗性，但在低盐浓度或1 mol/L尿素存在下被降解。在相同的实验条件下，没有发现人纤维蛋白原、牛血清白蛋白或小牛皮肤胶原被纯化的蛋白酶降解。蛋白水解活性表现出最佳pH值为8至9，并强烈抑制螯合剂，而只有缓慢的抑制与N-乙基马来酰亚胺观察。碘乙酰胺、亮抑酶肽或丝氨酸蛋白酶抑制剂均无抑制作用。最佳的肽基重氮甲基酮抑制剂是Z-Phe-Phe-CHN 2。二价金属离子的活化程度依次为：Zn（2+）> Cu（2+）> Cd（2+）> Ni（2+）> Co（2 +
Proteolytic cleavage of von Willebrand factor (vWF) takes place in the circulating blood of healthy subjects and is increased in some patients with von Willebrand disease type 2A. The hemostatically active large vWF multimers are degraded to smaller less active forms. It has been suggested that the polypeptide subunit of vWF is cleaved at the peptide bond 842Tyr-843Met. We purified (approximate to 10,000-fold) from human plasma a vWF-degrading protease, using chelating Sepharose, hydrophobic interaction chromatography, and gel filtration. The enzyme was found to be virtually absent in the platelet lysates obtained by repeated freezing and thawing. The proteolytic activity was associated with a high molecular weight protein (approximate to 300 kD) as judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. VWF was resistant against the protease in a neutral buffer at physiological ionic strength but became degraded at low salt concentration or in the presence of 1 mol/L urea. No degradation of human fibrinogen, bovine serum albumin, or calf skin collagen by the purified protease was noted under the same experimental conditions. Proteolytic activity showed a pH optimum at 8 to 9 and was strongly inhibited by chelating agents, whereas only slow inhibition was observed with N-ethylmaleimide. There was no inhibition by iodoacetamide, leupeptin, or serine protease inhibitors. The best peptidyl diazomethyl ketone inhibitor was Z-Phe-Phe-CHN2. Activation by divalent metal ions was found to increase in the following order: Zn(2+)approximate to Cu(2+)approximate to Cd(2+)approximate to Ni2+ approximate to Co2+