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, solu
lin (Glu4-Pro490), TM(E)1-6 (Cys227-Cys362) and TM(E)14-6 (Va134-Cys462) we
re prepared for equilibrium dialysis experiments by exhaustive dialysis aga
inst Ca2+-depleted buffer. However, all three analogs still contained one t
ightly 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 immob
ilized solulin treated with EDTA to remove the high affinity Ca2+ by measur
ing k(assoc) and k(diss) rates in a BIAcore(TM) instrument. Ca2+-dependent
conformational transitions detected by CD spectroscopy in the far UV indica
te a more ordered structure upon Ca2+ binding. Bound Ca2+ stabilized solubl
e TM against protease digestion at a trypsin-like protease-sensitive site b
etween Arg456 and His457 in EGF6 compared with protease treatment in EDTA.
Finally, TM containing EGF domains 4-6, but lacking the interdomain loop be
tween EGF3 and 4 (TM(E)4-6), has an identical Ca2+ dependence for the activ
ation of protein C as found for TM(E)i4-6, indicating this interdomain loop
is not involved in Ca2+ binding.