HEPARIN-BINDING AFFINITY OF NORMAL AND GENETICALLY-MODIFIED ANTITHROMBIN-III MEASURED USING A MONOCLONAL-ANTIBODY TO THE HEPARIN-BINDING SITE OF ANTITHROMBIN-III

The inhibitory activity of the plasma serine proteinase inhibitor anti thrombin III (AT III) is enhanced about 1000-fold upon binding to hepa rin. We have determined the dissociation constants, K(d), of 48.8 nM f or the heparin-AT III interaction, of 175 nM for the specific pentasac charide-AT III interaction, and of 13 muM for the low-affinity heparin -AT III interaction, using a binding assay based on a monoclonal antib ody (MAb) that recognizes an epitope at or close to the heparin bindin g site of AT III. The heparin binding affinities and proportions of no rmal and variant AT III in plasma from patients with mutations of AT I II have been quantitated for the first time using the binding assay. S ubstitution mutations in three regions of AT III have been investigate d: (i) mutations in the reactive site loop affecting Ala382, Arg393, a nd Ser394 have no discernible effect on heparin binding; (ii) mutation s in the previously identified N-terminal heparin binding region, affe cting Arg47, Leu99, and Arg129, produce variant AT III molecules with heparin affinities reduced 11-924-fold, the largest reduction being ob served for the substitution mutation Arg47-Cys in Padua 2, which has a n affinity of 65.6 muM; (iii) mutations in the hydrophobic regions aro und strand 1C of the C terminus, affecting Phe402, Ala404, Asn405, Pro 407, and Pro429, have pleiotropic effects that include the production of reduced amounts of low-affinity AT III with dissociation constants from 6 to 43 muM. The MAb-based method described is a simple means of determining the heparin binding affinity of variant AT III in complex solutions, and it will be useful in the study of novel natural mutatio ns or genetically engineered variants.