Development of a screening method for vWF multimers, using collagen bead columns

用胶原珠柱评估的血小板留存率通过血小板通过胶原珠柱前后的数量变化来确定。该方法先前已被证明是一种用于床边血小板功能的简单且有用的方法。在这项研究中，我们试图开发新的胶原珠柱，这是最适合的筛选血管性血友病因子。使用珠直径为0.125 × 0.2mm的胶原珠柱，我们能够产生足够水平的剪切应力，其诱导与VWF/胶原相互作用相关的血小板活化，随后是VWF/GPIb相互作用。通过使用可从ISK Ltd.商购获得的注射泵，用1.5ml血液在40秒内的流速，可以评价与GPIb和GPIIb/IIIa有关的血小板活化。vWF抗原、vWF多聚体大小、瑞斯托卡林辅因子都可以用来评价vWF的活性，但性质略有不同。本研究发现这些因素与胶原微珠柱的血小板留存率密切相关。我们用SDS-PAGE、Western blotting和ELIZA法对VWF活性测定法和胶原珠柱法检测到高分子量VWF的病例进行了ADAMTS-13活性的测定。发现具有高VWF多聚体大小的几个病例具有低ADAMTS-13活性，再次证实了胶原珠柱方法用于筛选VWF多聚体大小和ADAMTS-13活性的有用性。

The platelet retention rate as assessed with collagen bead columns are determined by the change in the number of platelets before and after the passage through collagen bead columns. This method has been previously shown to be a simple and useful method for platelet function at bed side. In this study, we sought to develop new collagen bead columns which are most appropriate for the screening of von Willebrand factors. Using a collagen bead column with the bead diameter of 0.125〜0.2mm, we were able to generate sufficient levels of shear stress, which induces platelet activation related to VWF/collagen interaction, followed by VWF/GPIb interaction. With the use of an injection pump commercially available from ISK Ltd., the flow rate of 1.5ml blood within 40sec made it possible to evaluate platelet activation related to GPIb and GPIIb/IIIa.vWF antigen, vWF multimer size, ristocetin cofactor are all used to evaluate the activity of vWF with slightly different properties. We found in this study that all these factors closely correlate with the platelet retention rate of collagen bead columns. Thus, we suggest that the collagen bead column method can be a good screening method for vWF activity, especially that of vWF multimer size.The cases with which the VWF activity measuement and collagen bead columns detected the possibility of high VWF multimer size, ADAMTS-13 activity was measured, using SDS-PAGE and Western blotting, and also a recently developed ELIZA method. Several cases with high VWFmutlimer size were found to have low ADAMTS-13 activity, again confirming the usefulness of the collagen bead column method for screening for VWFmultimer size, and ADAMTS-13 activity.