Activation of heparin cofactor II by calcium spirulan

Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose ability to inhibit α-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 α-thrombin by HCII. In this study, we investigated the mechanism of the activation of HCII by CaSP. Interestingly, in the presence of Ca-SP, an N-terminal deletion mutant of HCII (rHCII-Δ74) inhibited α-thrombin, as native recombinant HCII (native rHCII) did. The second-order rate constant for the inhibition of α-thrombin by rHCII-Δ74 was 2.0 x 10⁸ M^-1 min^{- 1} 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-Δ74 for the inhibition of γ-thrombin were increased only 80- and 120-fold, respectively. Our results suggested that the anion-binding exosite I of α-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 α-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.