Open AccessThe interaction of thrombomodulin with Ca 2+
Author(s) -
Light David R.,
Glaser Charles B.,
Betts Melissa,
Blasko Eric,
Campbell Elizabeth,
Clarke Jeffrey H.,
McCaman Michael,
McLean Kirk,
Nagashima Mariko,
Parkinson John F.,
Rumennik Galina,
Young Tish,
Morser John
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00398.x
Subject(s) - chemistry , thrombomodulin , thrombin , binding site , conformational change , stereochemistry , trypsin , active site , protease , enzyme , biochemistry , biology , platelet , immunology
Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca 2+ site in the sixth epidermal growth factor domain (EGF6) is essential for this cofactor activity [Nagashima, M., Lundh, E., Leonard, J.C., Morser, J. & Parkinson, J.F. (1993) J. Biol. Chem. 268 , 2888–2892]. Three soluble analogs of the extracellular domain of TM, solulin (Glu4–Pro490), TM E 1–6 (Cys227–Cys462) and TM E i4–6 (Val345–Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca 2+ ‐depleted buffer. However, all three analogs still contained one tightly bound Ca 2+ ( K d ≈ 2 µ m ), which could only be removed by EDTA. Epitope mapping with Ca 2+ ‐dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca 2+ site. Equilibrium dialysis of the soluble TM analogs in [ 45 Ca 2+ ] between 10 and 200 µ m revealed a second Ca 2+ site ( K d = 30 ± 10 µ m ) in both solulin and TM E 1–6, but not in TM E i4–6. Ca 2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca 2+ site in EGF3. A 75‐fold decrease in the binding affinity of thrombin to TM was observed with immobilized solulin treated with EDTA to remove the high affinity Ca 2+ by measuring k assoc and k diss rates in a BIAcore™ instrument. Ca 2+ ‐dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca 2+ binding. Bound Ca 2+ stabilized soluble TM against protease digestion at a trypsin‐like protease‐sensitive site between Arg456 and His457 in EGF6 compared with protease treatment in EDTA. Finally, TM containing EGF domains 4–6, but lacking the interdomain loop between EGF3 and 4 (TM E 4–6), has an identical Ca 2+ dependence for the activation of protein C as found for TM E i4–6, indicating this interdomain loop is not involved in Ca 2+ binding.