The molecular mechanisms of heparin action: II. Separation of functionally different heparins by affinity chromatography.

Abstract

Abstract Heparin was separated into functionally distinct fractions based on an affinity for bovine antithrombin III or bovine Factor X. Fractions with high affinity for antithrombin III (HA-ATIII) had high activity in the enhancement of antithrombin III inhibition of thrombin. These fractions functioned poorly in the anticoagulation of plasma and as inhibitors of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, HA-ATIII heparin stimulated antithrombin III inhibition of Factor Xa while in the presence of phospholipid HA-ATIII had little effect. Heparin fractions with a high affinity for antithrombin III were poor inhibitors of prothrombin activation and did not inhibit Factor Xa-phospholipid binding. Heparin fractions with high affinity for Factor X(HA-FX) were very effective in the inhibition of prothrombin activation by Factor Xa, calcium and phospholipid, as well as inhibition of Factor Xa binding to phospholipid vesicles. HA-FX fractions of heparin were poor anticoagulants in plasma and did not accelerate inhibition of thrombin by antithrombin III. HA-FX heparin accelerated antithrombin III inhibition of Factor Xa both in the presence and absence of phospholipid. When HA-FX heparin fractions were combined with HA-ATIII heparin fractions, the anticoagulant activity of the mixture was greater than the sum of the activities of the individual fractions. These results indicate that the interaction of heparin with both Factor Xa and antithrombin III is functionally important in the anticoagulation of blood.