The interaction of thrombomodulin with Ca2+

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Structure and Function

Background:

• Thrombomodulin (TM) is an essential cofactor for thrombin-mediated protein C activation.
• Calcium ions (Ca2+) play a critical role in TM's cofactor activity, particularly at a consensus site within the sixth epidermal growth factor domain (EGF6).
• The precise role and number of Ca2+ binding sites in TM's extracellular domain require further elucidation.

Purpose of the Study:

• To investigate the presence and characteristics of additional Ca2+ binding sites in soluble thrombomodulin analogs.
• To determine the impact of Ca2+ binding on TM's structure, thrombin interaction, and cofactor activity.
• To localize Ca2+ binding sites within specific domains of thrombomodulin.

Main Methods:

• Preparation of soluble TM extracellular domain analogs (solulin, TME1-6, TMEi4-6).
• Equilibrium dialysis with 45Ca2+ to identify and quantify Ca2+ binding sites.
• Epitope mapping using Ca2+-dependent monoclonal antibodies.
• Surface plasmon resonance (BIAcore) to measure thrombin binding kinetics.
• Circular dichroism (CD) spectroscopy to assess Ca2+-induced conformational changes.
• Protease digestion assays to evaluate Ca2+-dependent structural stability.

Main Results:

• A high-affinity Ca2+ site (Kd ≈ 2 µM) was present in all analogs, removable only by EDTA.
• A second Ca2+ binding site (Kd = 30 ± 10 µM) was identified in solulin and TME1-6, but not TMEi4-6, attributed to EGF3.
• EDTA treatment of solulin reduced thrombin binding affinity by 75-fold.
• Ca2+ binding induced a more ordered structure in TM, as detected by CD spectroscopy.
• Bound Ca2+ stabilized TM against protease digestion at a specific site in EGF6.

Conclusions:

• Soluble thrombomodulin possesses at least two distinct Ca2+ binding sites, with one located in EGF3.
• Calcium ions are critical for maintaining TM's structural integrity and high-affinity interaction with thrombin.
• The identified Ca2+ binding sites significantly influence TM's cofactor function in protein C activation.