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

IntroductionAlpha-2-macroglobulin (α2M) is a broad specificity protease inhibitor which impacts several hemostatic pathways. Selective detection of various α2M 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 α2M in complex with a variety of hemostatic factors.Materials and MethodsThe assay makes use of the fact that α2M entraps proteases within a molecular “cage”, leaving them inaccessible to macromolecular substrates while retaining functionality against small synthetic substrates. Wells coated with anti-α2M 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.ResultsCalibration curves constructed with purified protease-α2M 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 α2M-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 α2M. Thrombin-α2M 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.ConclusionsThis assay provides a versatile platform method for quantification of multiple protease-α2M complexes. It may prove useful for mechanistic in vitro studies of hemostatic pathways, and potentially for clinical applications.