Interaction of antithrombin with sulfated, low molecular weight lignins: opportunities for potent, selective modulation of antithrombin function.
Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891-31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80-87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.
Henry
Brian L
BL
Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Connell
Justin
J
Liang
Aiye
A
Krishnasamy
Chandravel
C
Desai
Umesh R
UR
D000991:Antithrombins / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002392:Catechin / Q000031:analogs & derivatives / Q000378:metabolism; D019075:Electrophoresis, Capillary; D015949:Factor IXa / Q000037:antagonists & inhibitors / Q000378:metabolism; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006865:Hydrogenation; D007700:Kinetics; D008024:Ligands; D008031:Lignin / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D013431:Sulfates / Q000737:chemistry / Q000378:metabolism*; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D000991:Antithrombins; D008024:Ligands; D013431:Sulfates; D013463:Sulfuric Acid Esters; C452422:epicatechin sulfate; D002392:Catechin; D006493:Heparin; D008031:Lignin; D015942:Factor VIIa; D015949:Factor IXa