How Thrombomodulin Enables W215A/E217A Thrombin to Cleave Protein C but Not Fibrinogen

Biochemistry. 2022 Jan 18;61(2):77-84. doi: 10.1021/acs.biochem.1c00635. Epub 2022 Jan 3.


The W215A/E217A mutant thrombin is called "anticoagulant thrombin" because its activity toward its procoagulant substrate, fibrinogen, is reduced more than 500-fold whereas in the presence of thrombomodulin (TM) its activity toward its anticoagulant substrate, protein C, is reduced less than 10-fold. To understand how these mutations so dramatically alter one activity over the other, we compared the backbone dynamics of wild type thrombin to those of the W215A/E217A mutant thrombin by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS). Our results show that the mutations cause the 170s, 180s, and 220s C-terminal β-barrel loops near the sites of mutation to exchange more, suggesting that the structure of this region is disrupted. Far from the mutation sites, residues at the N-terminus of the heavy chain, which need to be buried in the Ile pocket for correct structuring of the catalytic triad, also exchange much more than in wild type thrombin. TM binding causes reduced H/D exchange in these regions and also alters the dynamics of the β-strand that links the TM binding site to the catalytic Asp 102 in both wild type thrombin and in the W215A/E217A mutant thrombin. In contrast, whereas TM binding reduces the dynamics the 170, 180 and 220 s C-terminal β-barrel loops in WT thrombin, this region remains disordered in the W215A/E217A mutant thrombin. Thus, TM partially restores the catalytic activity of W215A/E217A mutant thrombin by allosterically altering its dynamics in a manner similar to that of wild type thrombin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Fibrinogen / metabolism*
  • Humans
  • Models, Molecular
  • Point Mutation
  • Protein Binding
  • Protein C / metabolism*
  • Protein Conformation, beta-Strand
  • Proteolysis
  • Thrombin / chemistry
  • Thrombin / genetics
  • Thrombin / metabolism*
  • Thrombomodulin / metabolism*


  • Protein C
  • Thrombomodulin
  • Fibrinogen
  • Thrombin