title: Altering heparin cofactor II at VAL439 (P6) either impairs inhibition of thrombin or confers elastase resistance.
abstract: Heparin cofactor II (HCII) is plasma glycoprotein and thrombin inhibitor of the serpin type previously shown to inhibit thrombin in the absence of its N-terminal 74 amino acids, and to be cleaved by neutrophil elastase (NE) at two sites: I66-F67 and V439-G440, the P6-P5 bond of the reactive center loop. We examined the contribution of Val439 to the reaction of HCII with thrombin and NE. Hexahistidine-tagged HCII proteins lacking residues 1-66 (H6delta66HCII) containing either the wild-type Val 439 or one of six substitutions were-expressed in E. coli. The rates of heparin-catalyzed thrombin inhibition of the V439L, C, or R variants were reduced at least 80-fold compared to wild-type H6delta66HCII, while those of the F, S, or W variants were largely unchanged. Following controlled exposure to NE in the presence of heparin, these latter variants retained 3.5- to 4.5-fold more residual anti-thrombin activity than wild-type H6delta66HCII treated in the same manner. This resistance arose due to deflection of NE attack from V439-G440 to secondary sites. The F, S, or W V439 variants exhibited a similar or greater degree of NE resistance when re-expressed as full-length hexahistidine-tagged HCII proteins, suggesting that the I66-F67 NE site is not well recognized in non-glycosylated HCII. Of these full-length variants, the V439F was the most active, exhibiting only a 2-fold reduction in its heparin-catalyzed rate of thrombin inhibition. HCII can therefore be made NE-resistant without severely compromising its capacity to inhibit thrombin.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cunningham
authors.forename: Varsha
authors.initials: V
authors.lastname: Bhakta
authors.forename: William P
authors.initials: WP
authors.lastname: Sheffield
mesh_terms: D015844:Heparin Cofactor II / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors*
chemical_list: D015844:Heparin Cofactor II; D019272:Leukocyte Elastase; D013917:Thrombin