Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin.
Thrombomodulin (TM), or its epidermal growth factor-like domains 456 (TM456), enhances the catalytic efficiency of thrombin toward both protein C and protein C inhibitor (PCI) by 2-3 orders of magnitude. Structural and mutagenesis data have indicated that the interaction of basic residues of the heparin-binding exosite of protein C with the acidic residues of TM4 is partially responsible for the efficient activation of the substrate by the thrombin-TM456 complex. Similar to protein C, PCI has a basic exosite (H-helix) that constitutes the heparin-binding site of the serpin. To determine whether TM accelerates the reactivity of thrombin with PCI by providing a binding site for the H-helix of the serpin, an antithrombin (AT) mutant was constructed in which the H-helix of the serpin was replaced with the same region of PCI (AT-PCIH-helix). Unlike PCI, the H-helix of AT is negatively charged. It was discovered that TM456 slightly (<2-fold) impaired the reactivity of AT with thrombin; however, it enhanced the reactivity of AT-PCIH-helix with the protease by an order of magnitude. Further studies revealed that the substitution of Arg35 of thrombin with an Ala also resulted in an order of magnitude enhancement in reactivity of the protease with both PCI and AT-PCIH-helix independent of TM. We conclude that TM enhances the reactivity of PCI with thrombin by providing both a binding site for the serpin and a conformational modulation of the extended binding pocket of thrombin.
Yang
Likui
L
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Manithody
Chandrashekhara
C
Walston
Timothy D
TD
Cooper
Scott T
ST
Rezaie
Alireza R
AR
D000494:Allosteric Regulation; D001665:Binding Sites; D006801:Humans; D018046:Protein C Inhibitor / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D015843:Serpins / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018180:Thrombomodulin / Q000502:physiology*
D018046:Protein C Inhibitor; D011994:Recombinant Proteins; D015843:Serpins; D018180:Thrombomodulin; D013917:Thrombin