α ₂ -Macroglobulin Is a Significant in Vivo Inhibitor of Activated Protein C and Low APC:α ₂ M Levels Are Associated with Venous Thromboembolism

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 α ₁ -antitrypsin (α ₁ AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α ₂ -macroglobulin (α ₂ M) have been observed by immunoblotting. Here, we report an ELISA for APC:α ₂ M complexes in plasma. Methods Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α ₂ M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α ₂ M, PCI and α ₁ AT 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, α ₂ M was a significant APC inhibitor. The VTE case-control study showed that VTE patients had significantly lower APC:α ₂ M and APC levels than the controls (p < 0.001). Individuals in the lowest quartile of APC:α ₂ M or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:α ₂ M or of APC. The risk increased for individuals with low levels of both parameters. Conclusion The APC:α ₂ M 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:α ₂ M level is associated with increased VTE risk.