Heparin cofactor II-dependent antithrombin activity of calcium spirulan.
Hayakawa Y, Hayashi T, Hayashi K, Hayashi T, Ozawa T, Niiya K, Sakuragawa N.
Department of Clinical Laboratory Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Japan.
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.
PUB: Blood Coagul Fibrinolysis. 1996 Jul;7(5):554-60.