The serine proteinase inhibitor antithrombin 111 (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 F'23-G'48, 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 L'30-G148 and K'36-G'48 di splayed attenuated heparin binding activities. In fact, K'36-G148 appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K'2'-A'34) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low pM concentrations of heparin induce significant a-helix conformation. K'2'-A'34 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-AT111 interactions.
Subspecies of heparin can be developed with significantly enhanced potency to inhibit vWF/platelet interactions. The vWF-inhibiting property of heparin can be dissociated from its antithrombin-binding activity. Based on a growing understanding of heparin/vWF interactions, combinations of affinity separations and chemical modifications could be designed to yield heparins uniquely suitable for prevention of arterial thrombosis.
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