Modification of human thrombin: effect on thrombomodulin binding.
Thrombomodulin, an endothelial cell protein, binds thrombin with high affinity and alters thrombin from a procoagulant to an anticoagulant molecule. In this study, chemical and/or proteolytic modification of thrombin was carried out to identify the essential components required for its interaction with thrombomodulin. Modification of thrombin at the catalytic site serine and histidine residues, with Diisopropylfluorophosphate and Tosyl-L-lysine chloromethyl ketone, resulted in loss of clotting and amidolytic activity. Both Diisopropyl phosphoryl-thrombin and Tosyl-L-chloromethyl ketone-thrombin inhibited native-thrombin: thrombomodulin catalyzed protein C activation with Ki values of 5 nM and 6 nM respectively indicating no loss of affinity for thrombomodulin. Oxidation of tryptophan residues with N-bromosuccinimide or iodination of tyrosine residues of thrombin led to reduced clotting and amidolytic activity as well as a reduced ability to interact with thrombomodulin. Modification of arginine residues with Phenylglyoxal and 2,3,Butanedione led to loss of thrombomodulin binding affinity. Limited proteolysis of thrombin by trypsin yielded the derivative beta-thrombin which had also lost its ability to interact with thrombomodulin. Deglycosylation of thrombin did not alter its binding affinity for thrombomodulin. These results indicate that one or more tryptophan, arginine and tyrosine residues are essential for the recognition of thrombin by thrombomodulin whilst the carbohydrate side chain and the active site residues of the thrombin molecule are not involved in thrombomodulin binding.
Thompson
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EA
Department of Medicine, Monash Medical School, Prahran, Victoria, Australia.
Salem
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HH
D001974:Bromosuccinimide; D055598:Chemical Phenomena; D002621:Chemistry; D003931:Diacetyl; D006801:Humans; D007531:Isoflurophate; D010658:Phenylglyoxal; D011193:Potassium Iodide; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism*; D014107:Tosyllysine Chloromethyl Ketone; D014357:Trypsin / Q000378:metabolism
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D007531:Isoflurophate; D011193:Potassium Iodide; D014107:Tosyllysine Chloromethyl Ketone; D014357:Trypsin; D013917:Thrombin; D003931:Diacetyl; D001974:Bromosuccinimide; D010658:Phenylglyoxal