Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation.
The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Olsen
P H
PH
Department of Biochemistry, University of New Hampshire, Durham 03824.
Esmon
N L
NL
Esmon
C T
CT
Laue
T M
TM
D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011486:Protein C / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000302:isolation & purification / Q000378:metabolism*
D023181:Antimicrobial Cationic Peptides; D010446:Peptide Fragments; D010455:Peptides; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C045866:peptide-Gly-Leu-amide; D004492:Edetic Acid; D010196:Pancreatic Elastase; D013917:Thrombin; D002118:Calcium