Aspartic acid residues 72 and 75 and tyrosine-sulfate 73 of heparin cofactor II promote intramolecular interactions during glycosaminoglycan binding and thrombin inhibition.
We used site-directed mutagenesis to investigate the role of Glu(69), Asp(70), Asp(71), Asp(72), Tyr-sulfate(73), and Asp(75) in the second acidic region (AR2) of the serpin heparin cofactor II (HCII) during formation of the thrombin.HCII complex with and without glycosaminoglycans. E69Q/D70N/D71N recombinant (r)HCII, D72N/Y73F/D75N rHCII, and E69Q/D70N/D71N/D72N/Y73F/D75N rHCII were prepared to localize acidic residues important for thrombin inhibition. Interestingly, D72N/Y73F/D75N rHCII had significantly enhanced thrombin inhibition without glycosaminoglycan (4-fold greater) and with heparin (6-fold greater), showing maximal activity at 2 microg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) with maximal activity at 20 microg/ml heparin. The other rHCII mutants had lesser-enhanced activities, but they all eluted from heparin-Sepharose at significantly higher ionic strengths compared with wt-rHCII. Neutralizing and reversing the charge of Asp(72), Tyr-sulfate(73), and Asp(75) were done to characterize their individual contribution to HCII activity. Only Y73K rHCII and D75K rHCII have significantly increased heparin cofactor activity compared with wt-rHCII; however, all of the individual rHCII mutants required substantially less glycosaminoglycan at maximal inhibition than did wt-rHCII. Inhibition of either alpha-thrombin/hirugen or gamma(T)-thrombin (both with an altered anion-binding exosite-1) by the AR2 rHCII mutants was similar to wt-rHCII. D72N/Y73F/D75N rHCII and D75K rHCII were significantly more active than wt-rHCII in a plasma-based thrombin inhibition assay with glycosaminoglycans. These results indicate that improved thrombin inhibition in the AR2 HCII mutants is mediated by enhanced interactions between the acidic domain and anion-binding exosite-1 of thrombin and that AR2 may be a "molecular rheostat" to promote thrombin inhibition in the presence of glycosaminoglycans.
Mitchell
Jennifer W
JW
Department of Pathology, Center for Thrombosis and Hemostasis, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, North Carolina 27599-7035, USA.
Church
Frank C
FC
D000838:Anions; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D003871:Dermatan Sulfate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D015844:Heparin Cofactor II / Q000737:chemistry*; D006801:Humans; D007477:Ions; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*
D000838:Anions; D006025:Glycosaminoglycans; D007477:Ions; D011994:Recombinant Proteins; D003871:Dermatan Sulfate; C005662:tyrosine O-sulfate; D001224:Aspartic Acid; D014443:Tyrosine; D015844:Heparin Cofactor II; D006493:Heparin; D002918:Chymotrypsin; D013917:Thrombin