The interaction of thrombomodulin with Ca2+

Thrombomodulin (TM) is a cofactor for protein C activation by thrombin and each residue of a consensus Ca2+ 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-Cys362) and TM(E)14-6 (Va134-Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+-depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (K-d approximate to 2 mu M), which could only be removed by EDTA. Epitope mapping with Ca2+-dependent monoclonal antibodies to EGF6 provided further localization of this tight Ca2+ site. Equilibrium dialysis of the soluble TM analogs in [Ca-45(2+)] between 10 and 200 mu M revealed a second Ca2+ site (K-d = 30 +/- 10 mu M) in both solulin and TM(E)1-6, but not in TM(E)i4-6. Ca2+ binding to this second site was unaffected by bound thrombin and we attribute it to the consensus Ca2+ 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 Ca2+ by measuring k(assoc) and k(diss) rates in a BIAcore(TM) instrument. Ca2+-dependent conformational transitions detected by CD spectroscopy in the far UV indicate a more ordered structure upon Ca2+ binding. Bound Ca2+ 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 Ca2+ dependence for the activation of protein C as found for TM(E)i4-6, indicating this interdomain loop is not involved in Ca2+ binding.