Occupancy of anion binding exosite 2 on thrombin determines Ca super(2+) dependence of protein C activation

• Published in: The Journal of biological chemistry Vol. 269; no. 16; pp. 11807 - 11812
• Main Authors: Liu, Le-Wen, Rezaie, A R, Carson, C W, Esmon, N L, Esmon, C T
• Format: Journal Article
• Language: English
• Published: 01-01-1994
• ISSN: 0021-9258

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 10-fold and shifts the Ca super(2+) dependence of protein C activation from simple saturation at greater than or equal to 500 mu M Ca super(2+) to a distinct optimum at 100 mu M Ca super(2+). 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 Ca super(2+) dependence of protein C activation is independent of the presence of the chondroitin sulfate on TM. At 0.27 mM Ca super(2+), TM containing chondroitin sulfate binds thrombin (K sub(d(app)) = 0.3 nM) 45 times tighter than meizothrombin des-fragment 1 (K sub(d(app)) = 14 nM).