Heparin binding domain peptides of antithrombin III: Analysis by isothermal titration calorimetry and circular dichroism spectroscopy

Abstract

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F¹²³-G¹⁴⁸, which encompasses both the purported high-affinity pentasaccharide binding region and an adjacent, C-terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino-terminal truncations of this sequence, including L¹³⁰-G¹⁴⁸ and K¹³⁶-G¹⁴⁸ displayed attenuated heparin binding activities. In fact, K¹³⁶-G¹⁴⁸ appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K¹²¹-A¹³⁴) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low μM concentrations of heparin induce significant α-helix conformation. K¹²¹-A¹³⁴ also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure-function relations of heparin-ATIII interactions.