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J Biol Chem, Vol. 275, Issue 15, 11379-11382, April 14, 2000

Activation of Heparin Cofactor II by Calcium Spirulan^*

Yumiko Hayakawa, Toshimitsu Hayashi^§, Jung-Bum Lee^§, Tetsuo Ozawa, and Nobuo Sakuragawa

From the Department of Clinical Laboratory Medicine, Faculty of Medicine and the ^§ Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan

Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose ability to inhibit -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 -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-74) inhibited -thrombin, as native recombinant HCII (native rHCII) did. The second-order rate constant for the inhibition of -thrombin by rHCII-74 was 2.0 × 10⁸ M¹ min¹ in the presence of 50 µg/ml Ca-SP and 10,000-fold higher than in the absence of Ca-SP. The rates of native rHCII and rHCII-74 for the inhibition of -thrombin were increased only 80- and 120-fold, respectively. Our results suggested that the anion-binding exosite I of -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 Arg¹⁰³  Leu mutant bound to Ca-SP-Toyopearl with normal affinity and inhibited -thrombin in a manner similar to native rHCII. These results indicate that Arg¹⁰³ in HCII molecule is not critical for the interaction with Ca-SP.

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