Abstract
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.
Highlights
Ca"+ andanother by Mg2+.The former was identified inhibitors of activatedprotein C (APC) in plasma include PC1 and alAT(13-16), as APC-a2-macroglobulin(APC-a2M)while the latter was not identified
Both of which inhibit APC relatively slowly compared with inhibition of other coagulation enzymes by plasma inhibitors
Inadditiontothe twopreviouslyidentified plasma inhibitors, here we report that a 2 M,a,AP, and possibly anand inhibition of APC by azAP wastimulated by Ca2+, other protein inhibit APC inwhole blood in a divalent metal
Summary
Kinetics for Inhibition of APC and OtherProteases-For determi- time for determination of APC anticoagulant activity, APC amidonation of the second order association rate consta(nk,t)for inhibition lytic activity, and APC antigen immunoblotting pattern asdescribed of APC by a2M,0.5 p~ APC was incubated at 37 "C with 2.6 p~ n,M above. Other additions as specified in TBS containing Identification of APC-Inhibitor Complexes-In orderto remove. Aliquots of 4 pl wereremoved over specific APC-inhibitor complexes from incubation mixtures of blood time, and the residual APC anticoagulant activity was tested by its and APC by immunoabsorption, the mixtures were incubated for a abilityto prolong theactivatedpartialthromboplastintime.The specified time and made mM in benzamidine usinga 500 mM control curvewas obtained using dilutionsof APC incubated without benzamidine solution and centrifuged1min in a Beckman Microfuge tr,M.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
