α₂-Macroglobulin Is a Significant In Vivo Inhibitor of Activated Protein C and Low APC:α₂M Levels Are Associated with Venous Thromboembolism

Abstract

Background Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α₁-antitrypsin (α₁AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α₂-macroglobulin (α₂M) have been observed by immunoblotting. Here, we report an ELISA for APC:α₂M complexes in plasma.

Methods Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α₂M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α₂M, PCI and α₁AT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls.

Results In all the in vivo experiments, α₂M was a significant APC inhibitor. The VTE case–control study showed that VTE patients had significantly lower APC:α₂M and APC levels than the controls (p < 0.001). Individuals in the lowest quartile of APC:α₂M or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:α₂M or of APC. The risk increased for individuals with low levels of both parameters.

Conclusion The APC:α₂M assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:α₂M level is associated with increased VTE risk.

Keywords

activated protein C - α2-macroglobulin - ELISA - baboon - complexes

Authors' Contributions

S.N. performed the experiments, analysed the data and critically revised the manuscript. L.M. performed the experiments and analysed the data. L.A.R. performed the experiments. J.O. performed the experiments. A.F-P. performed the experiments. S.B.S. performed the plasma collection from VTE patients and controls and revised the manuscript. A.R.C. performed the plasma collection from VTE patients and controls and revised the manuscript. A.G. designed the experiments and revised the manuscript. J.H.G. designed the experiments and critically revised the manuscript. F.E. designed and performed the experiments, analysed the data and wrote the manuscript. P.M. designed and performed the experiments, analysed the data and wrote the manuscript.

^* These authors contributed equally to this study.

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