Determination of alpha-2-macroglobulin complexes by a new immuno-activity assay

详细信息	查看全文 | 推荐本文 |

• 作者：Helen M. Atkinson^a ; Nagina Parmar^b ; Leslie R. Berry^a ; Anthony K.C. Chan^a ; ^{akchan@mcmaster.ca}
• 关键词：伪2M ; alpha-2-macroglobulin ; H ; heparin ; 伪2-AP ; alpha-2-antiplasmin ; APC ; activated protein C ; AT ; antithrombin ; PCI ; protein C inhibitor ; IIa ; thrombin ; Xa ; factor Xa ; PPACK ; D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone
• 刊名：Thrombosis Research
• 出版年：2012
• 期刊代码：247_00493848
• 类别：med
• 出版时间：May, 2012
• 卷：129
• 期：5
• 页码：635-640
• 文件大小：395 K

摘要

class="h4">Introduction

Alpha-2-macroglobulin (伪₂M) is a broad specificity protease inhibitor which impacts several hemostatic pathways. Selective detection of various 伪₂M complexes may be useful to define markers for the status of different hemostatic components. We present proof of principle for a novel assay to quantitatively measure 伪₂M in complex with a variety of hemostatic factors.

class="h4">Materials and Methods

The assay makes use of the fact that 伪₂M entraps proteases within a molecular 鈥渃age鈥? leaving them inaccessible to macromolecular substrates while retaining functionality against small synthetic substrates. Wells coated with anti-伪₂M antibodies were used to isolate the complexes from buffer or plasma, followed by detection of specific proteases with chromogenic substrates. Macromolecular inhibitors were added to eliminate signal from any unbound proteases.

class="h4">Results

Calibration curves constructed with purified protease-伪₂M complexes were sigmoidal in nature, as is typical with immuno-assays. The specificity of signal production was confirmed with inhibitors that target either free protease, or both free and 伪₂M-bound protease. The detection range of the assay was dependent on the protease being measured, and the surrounding matrix. Interference in detection of complexes in plasma was found to be caused, in part, by free 伪₂M. Thrombin-伪₂M complexes were quantified in adult and newborn plasma following induction of thrombin generation and found to be significantly higher in adults, likely due to higher prothrombin levels.

class="h4">Conclusions

This assay provides a versatile platform method for quantification of multiple protease-伪₂M complexes. It may prove useful for mechanistic in vitro studies of hemostatic pathways, and potentially for clinical applications.