Ulrichts H, Vanhoorelbeke K, Girma J P, Lenting P J, Vauterin S, Deckmyn H
KU Leuven Campus Kortrijk, Kortrijk, Belgium.
J Thromb Haemost. 2005 Mar;3(3):552-61. doi: 10.1111/j.1538-7836.2005.01209.x.
Platelet adhesion and aggregation at sites of vascular injury exposed to rapid blood flow require von Willebrand factor (VWF). VWF becomes immobilized by binding to subendothelial components or by a self-association at the interface of soluble and surface-bound VWF.
As this self-association has been demonstrated only under shear conditions, our first goal was to determine whether the same interaction could be observed under static conditions. Furthermore, we wanted to identify VWF domain(s) important for this self-association.
Biotinylated VWF (b-VWF) interacted dose-dependently and specifically with immobilized VWF in an enzyme-linked immunosorbent assay (ELISA) assay, showing that shear is not necessary to induce the VWF self-association. Whereas anti-VWF monoclonal antibodies (mAbs) had no effect on the self-association, the proteolytic VWF-fragments SpII(1366-2050) and SpIII(1-1365) inhibited the b-VWF-VWF interaction by 70 and 80%, respectively. Moreover, a specific binding of b-VWF to immobilized Sp-fragments was demonstrated. Finally, both biotinylated SpII and SpIII were able to bind specifically to both immobilized SpII and SpIII. Similar results were observed under flow conditions, which confirmed the functional relevance of our ELISA system.
We have developed an ELISA binding assay in which a specific VWF self-association under static conditions can be demonstrated. Our results suggest a multiple domain interaction between immobilized and soluble VWF.
在暴露于快速血流的血管损伤部位，血小板的黏附和聚集需要血管性血友病因子（VWF）。VWF通过与内皮下成分结合或在可溶性和表面结合的VWF界面处进行自我缔合而固定下来。
由于这种自我缔合仅在剪切条件下得到证实，我们的首要目标是确定在静态条件下是否能观察到相同的相互作用。此外，我们希望确定对这种自我缔合重要的VWF结构域。
在酶联免疫吸附测定（ELISA）中，生物素化的VWF（b-VWF）与固定化的VWF呈剂量依赖性且特异性相互作用，表明剪切对于诱导VWF自我缔合并非必需。抗VWF单克隆抗体（mAb）对自我缔合没有影响，而蛋白水解的VWF片段SpII(1366 - 2050)和SpIII(1 - 1365)分别抑制b-VWF-VWF相互作用70%和80%。此外，还证实了b-VWF与固定化的Sp片段有特异性结合。最后，生物素化的SpII和SpIII都能够特异性结合固定化的SpII和SpIII。在流动条件下也观察到了类似结果，这证实了我们ELISA系统的功能相关性。
我们开发了一种ELISA结合测定法，可在静态条件下证明特定的VWF自我缔合。我们的结果表明固定化和可溶性VWF之间存在多结构域相互作用。
