Identification of divalent metal ion-dependent inhibition of activated protein C by alpha 2-macroglobulin and alpha 2-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-alpha 1-antitrypsin complexes. Whole blood contained two additional APC-inhibitor complexes, one stimulated by Ca2+ and another by Mg2+. The former was identified as APC-alpha 2-macroglobulin (APC-alpha 2M) while the latter was not identified. APC-alpha 2-antiplasmin complexes (APC-alpha 2AP) were identified, comigrating with APC-PCI complexes. Purified alpha 2M and alpha 2AP inhibited APC in the presence of Ca2+ (k2 = 99 and 100 M-1 S-1, respectively. Inhibition of APC and Factor Xa by alpha 2M and inhibition of APC by alpha 2AP was stimulated by Ca2+, Mn2+, and Mg2+. Inhibition of thrombin by alpha 2M and of plasmin by alpha 2AP 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 alpha 1-antitrypsin are the more important APC inhibitors and that alpha 2M and alpha 2AP are metal ion-dependent auxiliary inhibitors. Inhibitors can account for the in vivo half-life of APC.
Heeb
M J
MJ
Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, California 92037.
Gruber
A
A
Griffin
J H
JH
D004789:Enzyme Activation; D065427:Factor Xa Inhibitors; D005341:Fibrinolysin / Q000037:antagonists & inhibitors*; D006207:Half-Life; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D011486:Protein C / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors*; D000979:alpha-2-Antiplasmin / Q000502:physiology*; D000511:alpha-Macroglobulins / Q000502:physiology*
D065427:Factor Xa Inhibitors; D008670:Metals; D011486:Protein C; D000979:alpha-2-Antiplasmin; D000511:alpha-Macroglobulins; D013917:Thrombin; D005341:Fibrinolysin