Mechanism of activation of heparin cofactor II by calcium spirulan.
Calcium spirulan (Ca-SP), a novel sulfated polysaccharide, increases the rate of thrombin inhibition by heparin cofactor II (HCII) more than 1000-fold through a mechanism not requiring the amino-terminal acidic domain of HCII. Activation of HCII by Ca-SP was molecular-weight dependent. Furthermore, HD22, an aptamer that binds exosite II of thrombin, produced a concentration-dependent, 15-fold reduction at 5 microM in the rate of thrombin inhibition by HCII with Ca-SP, suggesting that Ca-SP interacts with exosite II of thrombin. Mutations of Lys173 to Leu (K173L) and Arg189 to Leu (R189L) in the HCII molecule resulted in large decreases in the rate of thrombin inhibition mediated by Ca-SP and in the NaCl concentration needed for elution from Ca-SP-Toyopearl. Mutations of Lys173 to Arg (K173R) and Arg189 to Lys (R189K) showed inhibition of thrombin similar to wild-type rHCII (wt-rHCII). These results indicate that Ca-SP binds to the positive charges of Lys173 and Arg189 on the HCII molecule. In the thrombin inhibitory process by HCII, Ca-SP appears to play as a template by binding to both thrombin and HCII.
Hayakawa
Yumiko
Y
Department of Neurosurgery, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, 930-0194, Toyama, Japan. hayakawa@ms.toyama-mpu.ac.jp <hayakawa@ms.toyama-mpu.ac.jp>
Hirashima
Yutaka
Y
Yamamoto
Hiromichi
H
Kurimoto
Masanori
M
Hayashi
Toshimitsu
T
Lee
Jung-Bum
JB
Endo
Shunro
S
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D002240:Carbohydrate Sequence; D015844:Heparin Cofactor II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism
D000596:Amino Acids; D011134:Polysaccharides; C102617:spirulan; D015844:Heparin Cofactor II; D013917:Thrombin