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

INTRODUCTION: Alpha-2-macroglobulin (α(2)M) is a broad specificity protease inhibitor which impacts several hemostatic pathways. Selective detection of various α(2)M 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 α(2)M in complex with a variety of hemostatic factors. MATERIALS AND METHODS: The assay makes use of the fact that α(2)M entraps proteases within a molecular "cage", leaving them inaccessible to macromolecular substrates while retaining functionality against small synthetic substrates. Wells coated with anti-α(2)M 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. RESULTS: Calibration curves constructed with purified protease-α(2)M 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 α(2)M-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 α(2)M. Thrombin-α(2)M 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. CONCLUSIONS: This assay provides a versatile platform method for quantification of multiple protease-α(2)M complexes. It may prove useful for mechanistic in vitro studies of hemostatic pathways, and potentially for clinical applications.