Human alpha-thrombin binding to nonpolymerized fibrin-Sepharose: evidence for an anionic binding region.
In order to investigate ligand binding sites in alpha-thrombin that interact with nonpolymerized fibrin, fibrinogen was conjugated (with CNBr) to Sepharose 4B and converted to the nonpolymerized fibrin resin with alpha-thrombin. Human alpha-thrombin was bound to the resin at 22 degrees C and eluted with a linear NaCl gradient [50-300 mM in 50 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6] with midpeak elution occurring at an ionic strength that corresponds to 170 +/- 5 mM NaCl. Among various ligands examined, ATP and its analogues caused alpha-thrombin to elute with 125 mM or less salt. Apparent dissociation constants were estimated by the dependence of elution volume on ligand concentration. The most potent ligands for desorption from the column were anionic (e.g., adenine nucleotides), which also inhibit thrombin esterolytic/amidolytic and clotting activity [Conery, B. G., & Berliner, L. J. (1983) Biochemistry 22, 369-375]. The desorption series was at 10 mM concentrations: ATP = ADP greater than pyrophosphate greater than citrate greater than oxalate greater than PO4(3-). Contrastingly, serotonin and related apolar compounds did not cause dissociation of alpha-thrombin from the fibrin resin, even though several of these substances inhibit fibrinogen clotting and esterolytic/amidolytic activities of the enzyme. These data imply that independent sites for apolar and anionic binding in alpha-thrombin are required for converting fibrinogen into clottable fibrin and that alpha-thrombin-fibrin binding involves an anionic site.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002846:Chromatography, Affinity; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009994:Osmolar Concentration; D011485:Protein Binding; D012685:Sepharose / Q000378:metabolism; D013917:Thrombin / Q000302:isolation & purification / Q000378:metabolism*
D004800:Enzymes, Immobilized; D008024:Ligands; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005337:Fibrin; D012685:Sepharose; D013917:Thrombin