Reactivity of heparin with the human plasma heparin-binding proteins thrombin, antithrombin III, and apolipoproteins E and B-100

Abstract

The heparin-binding properties of human plasma apolipoproteins B-100 and E (apoB-100 and E) of low density lipoproteins (LDL), thrombin, and antithrombin-III (AT-III) were investigated. A highly reactive heparin (HRH) to apoB-100 was isolated by chromatography of crude heparin on a column of LDL immobilized to Affi-Gel 10. This HRH showed a high, Ca 2+-dependent precipitating activity towards LDL; 1 μg HRH uronic acid precipitated 50–70 μg LDL-protein. HRH was fractionated further by chromatography on a column of AT-III bound to concanavalin A-Sepharose. The unretained fraction of heparin (HRH 1)had a low affinity for AT-III. The bound heparin (HRH 2) had a high affinity for AT-III and precipitated LDL in the presence of Ca 2+. To assess further their heparin-binding properties, the proteins were subjected to gradient-gel electrophoresis under denaturing conditions, transferred to nitrocellulose by electrophoresis, and then assayed for their ability to bind [ 125I]-labeled HRH 2. Autoradiographic analysis showed that thrombin, apolipoproteins E and B-100, and the AT-III-thrombin covalent complex bound HRH 2. Denatured AT-III did not bind HRH 2, indicating that its heparin recognition site may depend on conformation.