abstract
- We have produced a molecule comprising of permanently-activated covalently linked antithrombin and heparin (ATH). This study was designed to elucidate the covalent linkage point(s) for heparin on antithrombin and conformational properties of the ATH molecule. ATH was produced using Schiff base/Amadori rearrangement by incubating antithrombin with unfractionated heparin for 14 d at 40 degrees C. ATH was then digested using Proteinase K, and the heparin-peptide was reacted with NaIO4/NaBH4/mild acid to degrade the heparin moiety. Sequencing of the remaining peptide was performed by Edman degradation with linkage point confirmation by LC-MS. The degree of insertion of the reactive center loop (RCL) of antithrombin into the A-sheet of ATH was examined using synthesized antithrombin RCL peptides. Binding between the peptides and ATH, and the formation of ATH in the presence of the peptides were tested. CD was used to further examine the secondary and tertiary structures of ATH. The results suggest that heparin is conjugated to the amino terminal of antithrombin in the majority of ATH molecules, proximal to the previously determined heparin binding domain of antithrombin. From the linkage data, a model is proposed for the structure of ATH. Studies using the RCL peptides and CD analysis of ATH support this model.