Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Nov 3;54(43):6650-8.
doi: 10.1021/acs.biochem.5b00825. Epub 2015 Oct 26.

Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin

Lindsey D Handley  1 , Nicholas A Treuheit  2 , Varun J Venkatesh  1 , Elizabeth A Komives  1
Affiliations

Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin

Lindsey D Handley et al. Biochemistry. .

Abstract

Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the fourth, fifth, and most of the sixth EGF-like domain (TM456m) that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity versus those of previous TM456 constructs. In this work, we compare backbone amide exchange of human α-thrombin in three states: apo, D-Phe-Pro-Arg-chloromethylketone (PPACK)-bound, and TM456m-bound. Beyond causing a decreased level of amide exchange at their binding sites, TM and PPACK both cause a decreased level of amide exchange in other regions including the γ-loop and the adjacent N-terminus of the heavy chain. The decreased level of amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, hydrogen-deuterium exchange mass spectrometry results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(A) HDXMS coverage map for α-thrombin. Peptides are marked in grey bars underneath the amino acid sequence and sequential numbering of α-thrombin. Peptide masses are printed in white within each grey bar. The amino acid sequence is highlighted in different colors to mark important regions of α-thrombin: the light-chain is colored pink, the 30s loop is colored orange, the 60s loop is colored yellow, the 70s loop is colored blue, the 90s loop is colored red, the γ-loop is colored purple, the 170s loop is colored green, the 180s loop is colored light green, and the Na+-binding loop is colored brown. The chymotrypsin numbering is given for some important residues in bold over the amino acid sequence. (B) A crystal structure of PPACK-thrombin (1PPB) with loops colored, as identified in (A), and PPACK drawn in black sticks. A crystal structure of thrombin bound to TM456 (1DX5) was used to align TM456m with the PPACK-thrombin structure to indicate the TM binding site. TM456m is shown in cyan.
Figure 2
Figure 2
Relative deuterium uptake plots for peptides in PPACK-thrombin in black versus unbound α-thrombin in blue. (A) The 90s loop, which directly contacts PPACK, residues 117-132 (residues 85-99CT, MH+ mass of 2144.140), (B) the 170s loop, residues 202-221 (residues 161-180CT, MH+ mass of 2343.277), (C) the Na+-binding loop, residues 259-275 (residues 211-227CT, MH+ mass of 1845.822) which also directly contacts PPACK, and (D) the N-terminus of the heavy chain, residues 37-44 (residues 16-23CT, MH+ mass of 819.373).
Figure 3
Figure 3
Differences in deuterium exchange between (A) PPACK-thrombin versus α-thrombin and (B) TM456m-bound α-thrombin versus unbound α-thrombin displayed on the thrombin structure (1PPB). These differences in deuterium protection were calculated by comparing the deuterium uptake after 10 minute incubation with deuterium. Residues are colored with a gradient of white to red, with residues colored in red showing the highest degree of deuterium protection upon binding and residues colored in white showing no deuterium protection upon binding. Residues colored black were not covered in the experiment. In (A), PPACK is shown in blue sticks, and all residues, covered in the HDXMS experiment, which have amides within 5Å of PPACK are colored in yellow. In (B), residues constituting the TM binding site are colored in yellow.
Figure 4
Figure 4
Relative deuterium uptake plots for peptides in TM456m-bound α-thrombin in red versus unbound α-thrombin in blue. (A) The 30s loop, residues 54-61 (residues 33-39CT, MH+ mass of 1004.552), (B) the 70s loop in ABE1, residues 96-112 (residues 65-80CT, MH+ mass of 2127.189), (C) the Na+-binding site, residues 259-275 (211-227CT, MH+ mass of 1845.822), and (D) the N-terminus of the heavy chain, residues 37-44 (residues 16-23CT, MH+ mass of 819.373).
Figure 5
Figure 5
Comparison of the deuterium uptake in three regions covering the γ-loop in α-thrombin versus PPACK-thrombin (A-C) and α-thrombin versus TM456m-thrombin (D-F): (A, D) residues 166-177 (residues 130-141CT, MH+ mass of 1335.717), (B, E) residues 167-180 (residues 131-144CT, MH+ mass of 1506.781), and (C, F) residues 181-196 (residues 145-155CT, MH+ mass of 1714.912). (G) Structure of thrombin (1PPB) showing the interaction between the residues Gly178-Leu180 (residues 142-144CT) of the γ-loop (purple spheres) with residues Ile37-Gly40 (residues 16-19CT) of the N-terminus of the heavy chain (blue spheres). The same loop colorings from Figure 1B are used.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Furie B, Furie BC. The molecular basis of blood coagulation. Cell. 1988;53:505–518. - PubMed
    1. Bode W. Structure and interaction modes of thrombin. Blood Cells, Molecules, and Diseases. 2006;36:122–130. - PubMed
    1. Di Cera E. Thrombin. Mol Aspects Med. 2008;29:203–254. - PMC - PubMed
    1. Bode W. The transition of bovine trypsinogen to a trypsin-like state upon strong ligand binding: II. The binding of the pancreatic trypsin inhibitor and of isoleucine-valine and of sequentially related peptides to trypsinogen and to p-guanidinobenzoate trypsinogen. J Mol Biol. 1979;127:357–374. - PubMed
    1. Huber R, Bode W. Structural basis of the activation and action of trypsin. Acc Chem Res. 1978;11:114–122.

Publication types

MeSH terms

LinkOut - more resources