Heparin cofactor II-dependent antithrombin activity of calcium spirulan.
Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, enhanced the antithrombin activity of heparin cofactor II (HC II) more than 10000-fold. The apparent second-order rate constant of thrombin inhibition by HC II was calculated to be 4.2 x 10(4) M-1 min-1 in the absence of Ca-SP, and it increased in the presence of 50 micrograms/ml Ca-SP to 4.5 x 10(8) M-1 min-1. Ca-SP effectively induced the formation of a thrombin-HC II complex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys173-->Leu and Arg 189-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHC II. This result indicates that the binding site of HC II for Ca-SP is different from the heparin- or dermatan sulfate-binding site. When we removed the calcium from the Ca-SP, the compound did not exert any antithrombin activity. Furthermore, Na-SP, which was prepared by replacement of the calcium in Ca-SP with sodium, accelerated the antithrombin activity of HC II as Ca-SP did. We therefore suggest that the molecular conformation maintained by Ca or Na is indispensable to the antithrombin activity of Ca-SP. The HC II-dependent antithrombin activity of Ca-SP was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, but not by treatment with chondroitinase ABC and chondroitinase AC II, suggesting that a heparin- or dermatan sulfate-like structure is not responsible for the activation of HC II by Ca-SP. Ca-SP is therefore thought to be a unique sulfated polysaccharide which shows a strong antithrombin effect in an exclusively HC II-dependent manner.
Hayakawa
Y
Y
Department of Clinical Laboratory Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Japan. hayakawa@ms.toyama-mpu.ac.jp
Hayashi
T
T
Hayashi
K
K
Hayashi
T
T
Ozawa
T
T
Niiya
K
K
Sakuragawa
N
N
D000818:Animals; D000990:Antithrombin III / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology*; D001777:Blood Coagulation / Q000187:drug effects*; D002236:Carbohydrate Conformation; D002417:Cattle; D018076:DNA, Complementary / Q000235:genetics; D006026:Glycoside Hydrolases / Q000494:pharmacology; D015844:Heparin Cofactor II / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013552:Swine
D000991:Antithrombins; D018076:DNA, Complementary; D011134:Polysaccharides; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C102617:spirulan; D015844:Heparin Cofactor II; D000990:Antithrombin III; D006026:Glycoside Hydrolases