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

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.