Structure-function studies of the epidermal growth factor domains of human thrombomodulin

Structure-function relationships in the 6 epidermal growth factor-like domains of human thrombomodulin (TME, residues 227–462) were studied by deletion mutagenesis. Purified and characterised proteins were used for kinetic studies. Deletion of EGF1, EGF2 and residues 310–332 in EGF3 had no effect on thrombin binding (Kd) or on kcatKM for protein C activation by the thrombin-thrombomodulin complex. Deletion of the rest of EGF3 and the interdomain loop between EGF3 and EGF4 had no effect on Kd but decreased kcatKM to 10% of TME. Deletion of residues 447–462 of EGF6 had no effect on kcatKM but increased Kd for thrombin ∼6-fold. Thus, the region 333–350 in EGF3-4 is critical for protein C activation by the thrombin-thrombomodulin complex and the region 447–462 in EGF6 is critical for thrombin binding.