The interaction of thrombomodulin with Ca2+

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Abstract
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), TME1–6 (Cys227–Cys462) and TMEi4–6 (Val345–Cys462) were prepared for equilibrium dialysis experiments by exhaustive dialysis against Ca2+‐depleted buffer. However, all three analogs still contained one tightly bound Ca2+ (Kd≈ 2 µm), 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 [45Ca2+] between 10 and 200 µm revealed a second Ca2+ site (Kd = 30 ± 10 µm) in both solulin and TME1–6, but not in TMEi4–6. 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 kassoc and kdiss rates in a BIAcore™ 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 (TME4–6), has an identical Ca2+ dependence for the activation of protein C as found for TMEi4–6, indicating this interdomain loop is not involved in Ca2+ binding.