Activation of Heparin Cofactor II by Calcium Spirulan

Abstract

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 rH-CII) did. The second-order rate constant for the inhibition of ␣-thrombin by rHCII-⌬74 was 2.0 ؋ 10 8 M ؊1 min ؊1 in the presence of 50 g/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 anionbinding exosite I of thrombin not through the N-terminal acidic domain of HCII. The Arg 103 3 Leu mutant bound to Ca-SP-Toyopearl with normal affinity and inhibited ␣-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.Thrombin is considered to be the pivotal enzyme in the coagulation pathway (1). Besides its critical role in hemostasis, thrombin elicits cellular responses through the thrombin receptor that may contribute to inflammation, wound healing, and atherosclerosis (2). Heparin cofactor II (HCII) 1 is an important plasma protein that selectively inhibits thrombin (3). A number of sulfated polysaccharides in addition to heparin and dermatan sulfate catalyze HCII to accelerate markedly the reaction of HCII with thrombin (4 -6). Marine algae are a rich source of sulfated polysaccharides with novel structures (7).Calcium spirulan (Ca-SP) is a novel sulfated polysaccharide isolated from a blue-green alga Spirulina platensis by Hayashi et al. (8,9). They reported an inhibitory effect of Ca-SP on the replication of enveloped viruses. We have recently showed that Ca-SP enhances the rate of inhibition of thrombin by HCII and that the molecular conformation maintained by calcium ions is essential to exhibit antithrombin activity (10). However, the mechanism of activation of HCII by Ca-SP has not been clarified. Current evidence suggests that binding of either heparin or dermatan sulfate to HCII displaces the N-terminal acidic domain of the inhibitor, which normally occupies the glycosaminoglycan-binding site. The N-terminal acidic domain then binds to the anion-binding exosite I of thrombin, facilitating proteolytic attack on the reactive site peptide bond of HCII (11). Deletions within the N-te...

Proteoglycans Released from Cultured Bovine Aortic Endothelial Cell Layers by Sodium Spirulan Are Both Perlecan and Biglycan

“…The results support the hypothesis that Na-SP enhances the local anticoagulant activity in the liquid phase on the endothelium by two mechanisms: one is direct activation of heparin cofactor II 15,23) and the other is the promotion of the endothelial release of perlecan and biglycan that can activate antithrombin III 10) and heparin cofactor II, 12) respectively. Na-SP may be a beneficial agent for the prevention of atherosclerosis because of these anticoagulant properties of Na-SP, in addition to the enhancement of endothelial fibrinolytic activity 16) and the inhibition of vascular smooth muscle cell proliferation.…”

“…In addition, the present results suggest two possible mechanisms by which Na-SP exhibits anticoagulant activity in the liquid phase on the vascular endothelium. One is the direct activation of heparin cofactor II 15,32) in the blood, and the other is the indirect activation of antithrombin III and heparin cofactor II by promotion of endothelial perlecan and biglycan release.…”

“…13,14) On the other hand, Na-SP exhibits antithrombin activity in vitro in a fashion different from heparin, 15) suggesting that Na-SP may be an agent that can control anticoagulant activity in the blood. In addition, we found that Na-SP enhances the fibrinolytic activity of the cells through inhibition of plasminogen activator inhibitor-1 secretion.…”

Inhibition of Cultured Bovine Aortic Smooth Muscle Cell Proliferation by Colominic Acid

“…15,16) We hypothesized that CA or SCA may inhibits the proliferation of vascular smooth muscle cell proliferation, as does natural or semi-synthetic polysaccharides, including Na-SP, heparin, 17) pentosan polysulfate 18) and fucoidan.…”

“…12) In addition, Na-SP promotes the secretion of anticoagulant proteoglycans 13) such as perlecan and biglycan, 14) as well as exhibiting an antithrombin activity through activation of heparin cofactor II. 15,16) We hypothesized that CA or SCA may inhibits the proliferation of vascular smooth muscle cell proliferation, as does natural or semi-synthetic polysaccharides, including Na-SP, heparin, 17) pentosan polysulfate 18) and fucoidan. 19) The present study was undertaken to examine this hypothesis using a culture system of bovine aortic smooth muscle cells.…”