Occupancy of anion binding exosite 2 on thrombin determines Ca2+ dependence of protein C activation.
Thrombomodulin (TM) binds thrombin to form a complex that activates the plasma anticoagulant zymogen protein C. TM is an integral membrane glycoprotein that contains a chondroitin sulfate moiety. Interaction with thrombin involves both the protein component of TM, specifically the growth factor-like repeats 4-6 (TM 4-6), and chondroitin sulfate. Removal of chondroitin sulfate decreases the affinity for thrombin approximately 10-fold and shifts the Ca2+ dependence of protein C activation from simple saturation at > or = 500 microM Ca2+ to a distinct optimum at approximately 100 microM Ca2+. Thrombin possesses two regions of high positive charge, anion binding exosites 1 and 2. Anion binding exosite 1 interacts with the growth factor region of TM while exosite 2 is involved in binding prothrombin activation fragment 2 or heparin. We demonstrate that recombinant TM, truncated at the membrane-spanning domain, or TM 4-6 can bind thrombin when fragment 2 is present either covalently attached (meizothrombin des-fragment 1) or in reversible association. With meizothrombin des-fragment 1, the Ca2+ dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca2+, TM containing chondroitin sulfate binds thrombin (Kd(app) = 0.3 nM) approximately 45 times tighter than meizothrombin des-fragment 1 (Kd(app) = 14 nM). However, the chondroitin-free form binds thrombin (Kd(app) = 2.4 nM) only approximately 4 times tighter than meizothrombin des-fragment 1 (Kd(app) = 9.4 nM). These studies suggest that occupancy of anion binding exosite 2 by either chondroitin sulfate or fragment 2 alters thrombin conformation resulting in the altered Ca2+ dependence of protein C activation.
Liu
L W
LW
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City.
Rezaie
A R
AR
Carson
C W
CW
Esmon
N L
NL
Esmon
C T
CT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002809:Chondroitin Sulfates / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011486:Protein C; D018180:Thrombomodulin; D011516:Prothrombin; D002809:Chondroitin Sulfates; D013917:Thrombin; C026812:meizothrombin; D002118:Calcium