Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion
Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion
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DOI：
10.1182/blood.v87.10.4235.bloodjournal87104235
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发表时间：
1996-05-15
期刊：
影响因子：
20.3
通讯作者：
Tsai, HM
中科院分区：
文献类型：
--
作者：
Tsai, HM
血管性血友病因子（VWF）在循环中经历蛋白水解。在最近的一项研究中，我们报告说，正常血浆中含有蛋白酶的活性，切割vWF在剪切依赖性的方式，导致其多聚体的大小减少，同时产生的140 kD和176 kD片段的二聚体与正常血浆中发现的那些难以区分。在这项研究中，血浆蛋白酶已被部分纯化和表征和vWF的构象在其裂解的蛋白酶的作用进行了进一步的研究。盐酸胍引起展开的vWF在浓度依赖性的方式，导致其荧光发射最大值的位移到较长的波长。在1.1至1.2 mol/L盐酸胍浓度下，其蛋白水解敏感性急剧增加，vWF发射最大值仅发生3至4 nm的位移。虽然vWF分子重折叠盐酸胍通过透析去除，重折叠只伴随着部分恢复的蛋白水解抗性。通过Sephacryl S-300 HR凝胶过滤、Matrex凝胶橙子A染料亲和层析和Q Sepharose阴离子交换部分纯化约900倍的血浆蛋白酶具有约200 kD的分子量，并且被EDTA、EGTA或1，10-菲咯啉抑制。EGTA或EDTA的抑制作用可被Ca ~（2+）所逆转，但不被Mg ~（2+）所逆转。它不被一组合成和天然蛋白酶抑制剂抑制或被明胶-琼脂糖吸附，并且存在于缺乏凝血和抗凝蛋白的血浆中。由蛋白酶产生的vWF片段，如肽特异性抗体VP-1和LJ-7745所绘制的，与天然片段无法区分，但与纤溶酶产生的片段不同。暴露于盐酸胍或高剪切应力后，高分子量内皮vWF被蛋白酶切割成更小的形式。这些结果支持内皮分泌的vWF被一种新的血浆金属蛋白酶转化为多聚体的模型。虽然天然vWF以相对抗切割的构象存在，但剪切应力引起的构象改变可导致蛋白水解敏感性增强。该模型可以解释在各种临床条件下vWF多聚体大小的减少。(C)1996年，美国血液学会。
von Willebrand factor (VWF) in the circulation is subjected to proteolysis. In a recent study, we reported that normal plasma contains a protease activity that cleaves vWF in a shear-dependent manner, causing a decrease in its multimer size while generating dimers of the 140-kD and the 176-kD fragments indistinguishable from those found in normal plasma. In this study, the plasma protease has been partially purified and characterized and the role of vWF conformation in its cleavage by the protease has been further investigated. Guanidine HCl caused unfolding of vWF in a concentration-dependent manner, resulting in a shift in its fluorescence emission maxims to longer wavelengths. A dramatic increase in its proteolytic susceptibility was seen at 1.1 to 1.2 mol/L guanidine HCl, a concentration causing only a 3- to 4-nm shift in vWF emission maxima. Although vWF molecules refolded as guanidine HCl was removed by dialysis, the refolding was accompanied only by a partial recovery of the proteolytic resistance. The plasma protease, partially purified by approximately 900 folds by Sephacryl S-300 HR gel filtration, Matrex gel orange A dye affinity chromatography, and Q Sepharose anion exchange, had a molecular mass of approximately 200 kD and was inhibited by EDTA, EGTA, or 1,10-phenanthroline. The inhibition by EGTA or EDTA could be reversed by Ca2+ but not by Mg2+. It was not inhibited by a panel of synthetic and natural protease inhibitors or adsorbed by gelatin-agarose, and it was present in plasmas deficient in proteins involved in coagulation and anticoagulation. The vWF fragments generated by the protease, as mapped by peptide-specific antibodies VP-1 and LJ-7745, were indistinguishable from the natural fragments but distinct from those produced by plasmin. High molecular weight endothelial vWF, after exposure to guanidine HCl or high shear stress, was cleaved by the protease to smaller forms. These results support the model that endothelial secreted vWF is converted to multimers by a novel plasma metalloproteinase. Although native vWF exists in a conformation relatively resistant to cleavage, an alteration in the conformation by shear stress can lead to an enhanced proteolytic susceptibility. This model may explain the decrease in vWF multimer sizes in various clinical conditions. (C) 1996 by The American Society of Hematology.