Identification of divalent metal ion-dependent inhibition of activated protein C by α₂-macroglobulin and α₂-antiplasmin in blood and comparisons to inhibition of factor Xa, thrombin, and plasmin

The half-life of activated protein C (APC) was 31 min in citrated blood and 18 min in whole blood. Immunoblotting analysis of citrated blood identified APC-protein C inhibitor (APC-PCI) and APC-α₁-antitrypsin complexes. Whole blood contained two additional APC-inhibitor complexes, one stimulated by Ca²⁺ and another by Mg²⁺. The former was identified as APC-α₂-macroglobulin (APC-α₂M) while the latter was not identified. APC-α₂-antiplasmin complexes (APC-α₂AP) were identified, comigrating with APC-PCI complexes. Purified α₂M and α₂AP inhibited APC in the presence of Ca²⁺ (k₂ = 99 and 100 M^-1 s^-1, respectively. Inhibition of APC and Factor Xa by α₂M and inhibition of APC by α₂AP was stimulated by Ca²⁺, Mn²⁺, and Mg²⁺. Inhibition of thrombin by α₂M and of plasmin by α₂AP was not altered by EDTA or Ca²⁺, suggesting divalent metal ions affect APC and Factor Xa rather than the inhibitors. k₂ values for the APC inhibitors and their plasma concentrations suggest that PCI and α₁-antitrypsin are the more important APC inhibitors and that α₂M and α₂AP are metal ion-dependent auxiliary inhibitors. Inhibitors can account for the in vivo half-life of APC.