REQUIREMENT OF LYSINE RESIDUES OUTSIDE OF THE PROPOSED PENTASACCHARIDE BINDING REGION FOR HIGH-AFFINITY HEPARIN-BINDING AND ACTIVATION OF HUMAN ANTITHROMBIN-III

Variant forms of human antithrombin III with glutamine or threonine su bstitutions at Lys(114), Lys(125), Lys(133), Lys(136), and Lys(139) we re expressed in insect cells to evaluate their roles in heparin bindin g and activation. Recombinant native ATIII and all of the variants had very similar second order rate constants for thrombin inhibition in t he absence of heparin, ranging from 1.13 x 10(5) M(-1)min(-1) to 1.66 x 10(5) M(-1)min(-1). Direct binding studies using I-125-fluoresceinam ine-heparin yielded a K-d of 6 nM for the recombinant native ATIII and K136T, whereas R114Q and K139Q bound heparin so poorly that a K-d cou ld not be determined. K125Q had a moderately reduced affinity. Heparin binding affinity correlated directly with heparin cofactor activity. Recombinant native ATIII was nearly identical to plasma-purified ATIII , whereas K114Q and K139Q were severely impaired in heparin cofactor a ctivity. K125Q and K136T were only slightly impaired. Based on these d ata, Lys(114) and Lys(139) which are outside of the putative pentasacc haride binding site, play pivotal roles in the high affinity binding o f heparin to ATIII and the activation of thrombin inhibitory activity.