Isolation and Partial Characterization of Equine Antithrombin III.
A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.