An experimental model to study the in vivo survival of von Willebrand factor -: Basic aspects and application to the R1205H mutation
An experimental model to study the in vivo survival of von Willebrand factor -: Basic aspects and application to the R1205H mutation
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DOI：
10.1074/jbc.m310436200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Denis, CV
中科院分区：
文献类型：
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作者：
Lenting, PJ;Westein, E;Denis, CV
为探讨血管性血友病因子（VWF）清除的分子基础，建立了VWF缺陷小鼠的实验模型。通过注射放射性标记的VWF来检测生物分布，VWF主要定向到肝脏，在其他器官中有少量。VWF从血浆中消失的特征为快速初始期（t(1/2) α = 13 min）和缓慢次级期（t(1/2) α = 3 h），平均停留时间（MRT）为2.8 h。仅由高分子量或低分子量多聚体组成的VWF也观察到类似的清除，表明在我们的实验模型中，清除与多聚体分布无关。这使我们能够比较全长VWF与截断变体的存活率。氨基末端D'-D3和羧基末端D4-CK结构域的缺失导致片段具有与野生型VWF相似的清除率。仅删除D'-D3区域与几乎2倍的恢复率降低和清除率增加相关（MRT = 1.6 h），而仅删除D4-CK区域导致清除率显著降低（MRT = 4.5 h, p < 0.02）。这些结果表明D'-D3区域在阻止VWF清除中的作用。此外，His取代D3结构域残基Arg-1205导致清除率显著增加（MRT = 0.3 h; p = 0.004）。因此，这种突变似乎取消了D'-D3区域的保护作用。对该突变体的体外分析还显示，在低pH下，Arg-1205对VWF前肽的亲和力降低了2倍，这表明Arg-1205突变不仅导致清除率增加，而且还与VWF前肽的pH依赖性相互作用受损有关。
To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase ( t(1/2) alpha = 13 min) and a slow secondary phase (t(1/2) alpha = 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of full-length VWF to truncated variants. Deletion of both the amino-terminal D'-D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D'-D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT = 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance ( MRT = 4.5 h, p < 0.02). These results point to a role of the D'-D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance ( MRT = 0.3 h; p = 0.004). Therefore, this mutation seems to abrogate the protective effect of the D'-D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide.