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

使用胶原珠柱开发 vWF 多聚体筛选方法

基本信息

批准号：
16590450
负责人：
OZAKI Yukio
金额：
$ 2.18万
依托单位：
University of Yamanashi
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

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.

用胶原珠柱评估的血小板保留率是通过通过胶原珠柱前后血小板数量的变化来确定的。这种方法已经被证明是一种简单而有效的床边血小板功能检测方法。在这项研究中，我们试图开发最适合筛选von Willebrand因子的新型胶原珠柱。使用直径为0.125~0.2 mm的胶原珠柱，我们能够产生足够水平的剪应力，诱导与VWF/胶原相互作用相关的血小板激活，然后是VWF/GPIB相互作用。使用ISK公司的注射泵，40秒内1.5ml血液的流速使评估与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、瑞斯托西汀辅因子均可用于评估vWF的活性，但性质略有不同。我们在本研究中发现，所有这些因素都与胶原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筛选vWF活性的方法，尤其是vWF多聚体大小的筛选。针对VWF活性测定和胶原珠柱检测到vWF多聚体大小的可能性的情况，用SDS-PAGE和Western blotting以及最近发展的Eliza方法测定了ADAMTS-13的活性。几个VWF多聚体大小较高的病例发现ADAMTS-13活性较低，再次证实了胶原珠柱法在筛选VWF多聚体大小和ADAMTS-13活性方面的有效性。