Activation of heparin cofactor II by calcium spirulan

Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose abil ity to inhibit alpha-thrombin is accelerated by a variety of sulfated polys accharides in addition to heparin and dermatan sulfate. Previous investigat ions have indicated that calcium spirulan (Ca-SP), a novel sulfated polysac charide, 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, I nterestingly, in the presence of Ca-SP, an N-terminal deletion mutant of HC II (rHCII-Delta 74) inhibited alpha-thrombin, as native recombinant HCII (n ative rH-CII) did. The second-order rate constant for the inhibition of alp ha-thrombin by rHCII-Delta 74 was 2.0 x 10(8) M-1 min(-1) in the presence o f 50 mu g/ml Ca-SP and 10,000-fold higher than in the absence of Ca-SP, The rates of native rHCII and rHCII-Delta 74 for the inhibition of gamma-throm bin were increased only 80- and 120-fold, respectively, Our results suggest ed that the anion-binding exosite I of cu-thrombin was essential for the ra pid inhibition reaction by HCII in the presence of Ca-SP and that the N-ter minal acidic domain of HCII was not required. Therefore, we proposed a mech anism by which HCII was activated allosterically by Ca-SP and could interac t with the anion-binding exosite I of thrombin not through the N-terminal a cidic 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 na tive rHCII, These results indicate that Arg(103) in HCII molecule is not cr itical for the interaction with Ca-SP.