The short loop between epidermal growth factor-like domains 4 and 5 is critical for human thrombomodulin function

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Thrombomodulin (TM) is a crucial cofactor for thrombin-mediated protein C activation.
• Oxidation of methionine 388 (Met388) in TM significantly reduces its cofactor activity.
• The interdomain loop between EGF-like domains 4 and 5 is critical for TM function.

Purpose of the Study:

• To investigate the role of the interdomain loop in TM cofactor activity.
• To identify specific mutations that enhance TM anticoagulant properties.
• To elucidate the mechanisms underlying TM activity loss upon Met388 oxidation.

Main Methods:

• Site-directed mutagenesis of the TM interdomain loop, including substitutions, deletions, and alanine insertions.
• Expression and purification of wild-type and mutant TM analogs in various cell systems (bacteria, insect, mammalian).
• Kinetic analysis (kcat/Km, Kd) of TM-thrombin-protein C interactions, with a focus on calcium dependence.

Main Results:

• Most loop mutations (>50%) decreased TM cofactor activity by over 50%.
• Mutations altering loop length resulted in >90% activity loss.
• The Met388 to Leucine (Met388-->Leu) substitution yielded a 2-fold increase in cofactor activity.
• Met388-->Leu TM analogs exhibited enhanced catalytic efficiency (kcat/Km), particularly at physiological calcium levels.
• Met388 oxidation impairs TM function by reducing thrombin binding (Kd) and TM-thrombin complex interaction with protein C (Km).

Conclusions:

• The interdomain loop, particularly Met388, is essential for TM's anticoagulant function.
• The Met388-->Leu mutation significantly enhances TM cofactor activity through improved catalytic efficiency.
• Targeted mutagenesis of the TM interdomain loop offers a strategy to improve anticoagulant properties.