Inhibition of dysthrombins Quick I and II by heparin cofactor II and antithrombin

• Published in: The Journal of biological chemistry 1993-02, Vol.268 (5), p.3321-3327
• Main Authors: PHILLIPS, J. E, SHIRK, R. A, WHINNA, H. C, HENRIKSEN, R. A, CHURCH, F. C
• Format: Article
• Language: English
• Subjects: active sites; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; antithrombin; Antithrombins - pharmacology; Biological and medical sciences; Chromatography, Affinity; dysthrombins; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Heparin - metabolism; heparin cofactor II; Heparin Cofactor II - pharmacology; Humans; Hydrolases; inhibition; Kinetics; Models, Molecular; mutants; Mutation; Protein Conformation; Protein Structure, Secondary; Thrombin - antagonists & inhibitors; Thrombin - chemistry; Thrombin - isolation & purification; Thrombin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)53696-6

Heparin cofactor II and antithrombin are plasma serine proteinase inhibitors whose ability to inhibit alpha-thrombin is accelerated by glycosaminoglycans. Dysfunctional thrombin mutants Quick I (Arg67-->Cys) and Quick II (Gly226-->Val) were used to further compare heparin cofactor II and antithrombin interactions. Quick I, Quick II, and alpha-thrombin were eluted at the same salt concentration from heparin-Sepharose suggesting that the putative heparin-binding site (also termed anion binding exosite-II) is functional. Antithrombin yielded similar inhibition rates for Quick I and alpha-thrombin in the absence or presence of various amounts of heparin. Also, Quick I was inhibited similarly to alpha-thrombin by heparin cofactor II in the absence of glycosaminoglycan. In contrast, glycosaminoglycan-accelerated Quick I inhibition by heparin cofactor II was greatly reduced indicating that anion binding exosite-I (where the mutation occurs in Quick I) is critical for increased inhibition by heparin cofactor II. We also found that heparin cofactor II formed a SDS-resistant bimolecular complex with Quick II and alpha-thrombin at similar rates and the rate of complex formation was accelerated in the presence of glycosaminoglycans. A three-dimensional molecular model of the Quick II active site compared to alpha-thrombin suggested that the heparin cofactor II Leu-Ser-reactive site sequence (P1-P1') is a compatible "pseudosubstrate" in contrast to the Arg-Ser sequence found in antithrombin. The importance of heparin cofactor II as a thrombin regulator will depend upon its ability to interact with glycosaminoglycans and the functional availability of thrombin exosites.