A major aim is the identification of the cause(s) of the antithrombin III (ATIII) abnormalities observed in hemophilic patients' plasma following intravenous treatment with various therapeutic concentrates of the vitamin-K dependent proteins (factor IX concentrates or prothrombin complex concentrates). The consistent finding of a cathodal shift in electrophoretic mobility of ATIII antigen suggested that ATIII complexes were present in the post-infusion plasmas or that a new or modified ATIII fraction had appeared. Attempts to characterize this putative variant and/or complexed ATIII will employ several complementary biochemical and immunologic techniques to study ATIII in plasma, including analytic isoelectric focusing in agarose, crossed immunoelectrofocusing, and immunoblotting following SDS-polyacrylamide gel electrophoresis. Other studies will be directed at isolating ATIII from plasma by either immunoprecipitation with anti-ATIII or affinity chromatography on heparin-agarose, followed by elution and analysis. A newly described normal variant of ATIII (ATIII-Beta) will be purified and used for comparison with ATIII fractions in post-infusion plasmas. ATIII-Beta, protein C, and heparin cofactor II in pre- and post-infusion plasmas will also be measured. A second major aim is the study of the biologic activity and fate of infused factors VII and VIIa following factor IX concentrate therapy, since infused factors VII/VIIa may have thrombogenic or hemostatic potential. Studies will include radioimmunoassay of factor VII in hemophilic plasmas, inactivation studies of factor VIIa with ATIII-Alpha, ATIII-Beta and heparin cofactor II in vitro, and quantitation of the rates of thrombin generation and inactivation in hemophilic plasma, after addition of factor VIIa and/or tissue factor. The observation that patients with hyperlipidemia and increased risk of arteriosclerotic disease have elevated factor VII levels is the basis for proposed studies to test whether factor VIIa is increased in such patients.
A third aim i s the characterization of the contents and effects of the new heat-treated factor IX concentrates on post-infusion AT III activity and antigen characteristics, factor VIIa recovery and survival, and immunologic suppression associated with concentrate therapy.
The aims of this proposal are directly pertinent to the clinical management of patients with hemophilia and may enhance the understanding of thrombotic disorders and transfusion-related immunosuppression.
