Allosteric activation of human α-thrombin through exosite 2 by suramin analogs

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Abstract

Thrombin is a serine protease that plays fundamental roles in hemostasis. We have recently elucidated the crystal structure of thrombin in complex with suramin, evidencing the interaction through the anion binding exosite 2. Here, we show that the activity of thrombin toward natural and synthetic substrates is enhanced by suramin as well as analogs of suramin at a low micromolar range prior to an inhibitory component at higher concentrations. Suramin analogs substituted by phenyl and chlorine instead of methyl were the most efficient in promoting allosteric activation, with an enhancement of enzymatic activity of 250% and 630% respectively. We discuss the importance of exosite 2 as a regulatory site for ligands in both the procoagulant and inhibitory scenarios.

Highlights

► Suramin binds to thrombin through exosite 2. ► Suramin activates thrombin at low micromolar range. ► At higher suramin concentration thrombin is inhibited. ► A series of suramin analogs reveals the mechanism for activation and inhibition.

Abbreviations used

FPRCK
D-Phe-Pro-Arg-chloromethylketone
Suramin
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-(4-methylphenylene)carbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF037
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-phenylenecarbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF127
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-(4-ethylphenylene)carbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF151
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-(4-isopropylphenylene)carbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF198
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-(4-phenylphenylene)carbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF157
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-(4-fluorophenylene)carbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF258
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-(4-chlorophenylene)carbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF260
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-(4-(methoxyphenylene) carbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF222
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino-3,1-(4-(methoxymethyl)phenylene)carbonylimino]]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
NF023
8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino]bis[naphthalene-1,3,5-trisulfonic acid] hexasodium salt
MK012
4,4′-[carbonylbis[imino-3,1-phenylenecarbonylimino]]bis[benzene-1,3-disulfonic acid] tetrasodium salt
S-2238
H–D-Phe-Pip-Arg-p-nitroanilide
TBS
20 mM Tris–HCl, 150 mM NaCl, pH 7.5

Keywords

Thrombin
Fibrinogen
Suramin
Exosite-2
Allosteric activation

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1

These authors contributed equally to this work.