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

用胶原蛋白珠柱评估的血小板保留率是通过通过胶原蛋白珠柱前后血小板数量的变化来确定的。该方法已被证明是床侧血小板功能的一种简单而有用的方法。在这项研究中，我们寻求开发新的胶原蛋白珠柱，最适合筛选血管性血友病因子。使用直径为0.125 ~ 0.2mm的胶原蛋白珠柱，我们能够产生足够水平的剪切应力，诱导血小板激活与VWF/胶原蛋白相互作用相关，然后是VWF/GPIb相互作用。使用ISK公司市售的注射泵，在40秒内1.5ml血液的流速使得评估与GPIb和GPIIb/IIIa相关的血小板活化成为可能。vWF抗原、vWF多聚体大小、里斯托霉素辅助因子均用于评价性质略有不同的vWF的活性。我们在本研究中发现，这些因素都与胶原蛋白珠柱的血小板保留率密切相关。因此，我们认为胶原珠柱法是一种很好的vWF活性筛选方法，特别是vWF多聚体的筛选方法。在VWF活性测定和胶原珠柱检测到高VWF多聚体大小可能性的情况下，使用SDS-PAGE和Western blotting以及最近开发的ELIZA方法测量ADAMTS-13活性。几例具有高vwf多聚体大小的病例被发现具有低ADAMTS-13活性，再次证实了胶原珠柱法筛选vwf多聚体大小和ADAMTS-13活性的有效性。

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.