title: Activation of heparin cofactor II by calcium spirulan.
abstract: Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose ability to inhibit alpha-thrombin is accelerated by a variety of sulfated polysaccharides in addition to heparin and dermatan sulfate. Previous investigations have indicated that calcium spirulan (Ca-SP), a novel sulfated polysaccharide, enhanced the rate of inhibition of alpha-thrombin by HCII. In this study, we investigated the mechanism of the activation of HCII by Ca-SP. Interestingly, in the presence of Ca-SP, an N-terminal deletion mutant of HCII (rHCII-Delta74) inhibited alpha-thrombin, as native recombinant HCII (native rHCII) did. The second-order rate constant for the inhibition of alpha-thrombin by rHCII-Delta74 was 2.0 x 10(8) M(-1) min(-1) in the presence of 50 microgram/ml Ca-SP and 10, 000-fold higher than in the absence of Ca-SP. The rates of native rHCII and rHCII-Delta74 for the inhibition of gamma-thrombin were increased only 80- and 120-fold, respectively. Our results suggested that the anion-binding exosite I of alpha-thrombin was essential for the rapid inhibition reaction by HCII in the presence of Ca-SP and that the N-terminal acidic domain of HCII was not required. Therefore, we proposed a mechanism by which HCII was activated allosterically by Ca-SP and could interact with the anion-binding exosite I of thrombin not through the N-terminal acidic domain of HCII. The Arg(103) --> Leu mutant bound to Ca-SP-Toyopearl with normal affinity and inhibited alpha-thrombin in a manner similar to native rHCII. These results indicate that Arg(103) in HCII molecule is not critical for the interaction with Ca-SP.
authors.affiliation: Department of Clinical Laboratory Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. hayakawa@ms.toyama-mpu.ac.jp
authors.forename: Y
authors.initials: Y
authors.lastname: Hayakawa
authors.forename: T
authors.initials: T
authors.lastname: Hayashi
authors.forename: J B
authors.initials: JB
authors.lastname: Lee
authors.forename: T
authors.initials: T
authors.lastname: Ozawa
authors.forename: N
authors.initials: N
authors.lastname: Sakuragawa
mesh_terms: D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D015844:Heparin Cofactor II / Q000378:metabolism*; D006801:Humans; D011134:Polysaccharides / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D011134:Polysaccharides; C102617:spirulan; D015844:Heparin Cofactor II; D013917:Thrombin