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.

Fig. 1

Calibration of the ELISA for APC:α₂M complex. APC:α₂M complex was diluted with pre-treated casein-EDTA buffer as indicated in section ‘Materials and Methods’ to reach the final concentrations of APC complexed to α₂M indicated, and samples were assayed for APC:α₂M as indicated in section ‘Materials and Methods’.

Fig. 2

Time course for complexation of APC with α₂M during APC infusion into baboons. 0.25 (●–●) or 1.0 (○–○) mg APC/kg in 1 hour (30% initial bolus; remainder by continuous infusion) was infused into two different baboons on separate days. Blood samples were withdrawn at different time intervals and anticoagulated with 1/9 volume citrate containing 0.3 M benzamidine before, during and after APC infusion, and APC:α₂M complexes were measured as indicated in section ‘Materials and Methods’.

Fig. 3

Time course of complexation of APC during i.v. APC infusion in humans. Forty-four adult men and postmenopausal or surgically sterile females were infused i.v. with recombinant human APC (XIGRIS) in two dosing periods, 6 (A, C, E) and 24 hours (B, D, F) separated by at least 6 days, at 12 (A and B; n = 12), 24 (C and D; n = 18) and 36 (E and F; n = 14) µg/kg/hour. Blood samples were withdrawn at different time intervals as indicated in section ‘Materials and Methods’, before and during or after APC infusion, and APC (Δ–Δ) and its complexes with PCI (●–●), α1AT (○–○) and α2M (▲–▲) were measured as indicated in section ‘Materials and Methods’.