HEPARIN-COFACTOR II-DEPENDENT ANTITHROMBIN ACTIVITY OF CALCIUM SPIRULAN

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated fro m the blue-green alga Spirulina platensis, enhanced the antithrombin a ctivity of heparin cofactor II (HC II) more than 10 000-fold. The appa rent second-order rate constant of thrombin inhibition by HC II was ca lculated 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 mu g/ml Ca-SP to 4.5 x 10(8) M(-1) min(-1). Ca-SP effectively induced the formation of a thrombin-HC II c omplex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys(173) --> Leu and Arg(189) --> His, which are defective i n interactions with heparin and dermatan sulfate, respectively, inhibi ted thrombin in a manner similar to native rHC II. This result indicat es that the binding site of HC II for Ca-SP is different from the hepa rin- or dermatan sulfate-binding site. When me removed the calcium fro m the Ca-SP, the compound did not exert any antithrombin activity. Fur thermore, Na-SP, which was prepared by replacement of the calcium in C a-SP with sodium, accelerated the antithrombin activity of HC II as Ca -SP did. We therefore suggest that the molecular conformation maintain ed 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 hepari tinase, but not by treatment with chondroitinase ABC and chondroitinas e 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 the refore thought to be a unique sulfated polysaccharide which shows a st rong antithrombin effect in an exclusively HC II-dependent manner.