title: Inhibition of a thrombin anion-binding exosite-2 mutant by the glycosaminoglycan-dependent serpins protein C inhibitor and heparin cofactor II.
abstract: Antithrombin (ATIII), heparin cofactor II (HCII) and protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) are serine protease inhibitors (serpins) whose thrombin inhibition activity is accelerated in the presence of glycosaminoglycans. We compared the inhibition properties of PCI and HCII to ATIII using R93A/R97A/R101A thrombin, an anion-binding exosite-2 (exosite-2) mutant that has greatly reduced heparin-binding properties. Heparin-enhanced PCI inhibition of R93A/R97A/R101A thrombin was only approximately 2-fold compared to 40-fold enhancement with wild-type recombinant thrombin. Thrombomodulin (TM) (with or without the chondroitin sulfate moiety) accelerated PCI inhibition of both wild-type and R93A/R97A/R101A thrombins. HCII achieved the same maximum activity in the presence of heparin with both wild-type and R93A/R97A/R101A thrombins; however, the optimum heparin concentration was 20 times greater than the reaction with wild-type thrombin, indicative of a decrease in heparin affinity. Dermatan sulfate (DSO4)-catalyzed HCII thrombin inhibition was unchanged in R93A/R97A/R101A thrombin compared to wild-type recombinant thrombin. These results suggest that PCI is similar to ATIII and depends upon ternary complex formation with heparin and these specific thrombin exosite-2 residues to accelerate thrombin inhibition. In contrast, HCII does not require Arg(93), Arg(97) and Arg(101) of thrombin exosite-2 and further supports the hypothesis that HCII uses an allosteric process following glycosaminoglycan binding to inhibit thrombin.
authors.affiliation: Department of Pathology, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC 27599, USA.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Cooper
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
authors.forename: Charles T
authors.initials: CT
authors.lastname: Esmon
authors.forename: Frank C
authors.initials: FC
authors.lastname: Church
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites; D006025:Glycosaminoglycans / Q000378:metabolism; D015844:Heparin Cofactor II / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D009154:Mutation; D018046:Protein C Inhibitor / Q000737:chemistry / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics
chemical_list: D006025:Glycosaminoglycans; D018046:Protein C Inhibitor; D015844:Heparin Cofactor II; D013917:Thrombin