The gla domain of the vitamin-K dependent proteins allows thee proteins to bind to metal ions, an interaction which results in a change in the three dimensional structure. This conformational transition allows these proteins to bind to membrane surfaces and is necessary for zymogen activation of these proteins,f or their participation as enzymes, and for their assembly into protein complexes with cofactors. This project will evaluate whether the gla domains of factor IX, factor X and prothrombin help to determine the unique functional characteristics of each of these proteins or whether the homologous gla domains in each of these proteins is interchangeable. The cDNA encoding the gla domain of factor IX, factor X and prothrombin will be shuffled to create chimeric cDNAs coding for chimeric vitamin K-dependent proteins: factor IX with its native gla domain replaced by the gla domain of factor X or prothrombin, factor X with its native gla domain replaced with the gla domain of factor IX or factor X. The cDNA for these chimeric proteins will be introduced into Chinese hamster ovary cells, expressed and the corresponding protein purified. The activation of these chimeric proteins by activated coagulation factors will be assessed. The chimeric proteins will be activated and used as enzymes to assess their ability to activate the appropriate native zymogens within the coagulation cascade. These recombinant chimeric proteins will be evaluated in assays to see if their interaction with known cofactors such as factor V and factor VIII has remained the same or has been altered. The interaction of the recombinant chimeric proteins with phospholipids, endothelial cells and platelets will be examined. The immunochemical and metal binding characteristics of the recombinant proteins will be compared to the native proteins. The gross characteristics of the recombinant proteins will be evaluated in standard coagulant assays. These results will demonstrate whether the gla domain defines the unique interactions of each of the vitamin K-dependent proteins within the coagulation cascade.
| Deitcher, S R; Erban, J K; Limentani, S A (1996) Acquired free protein S deficiency associated with multiple myeloma: a case report. Am J Hematol 51:319-23 |
| Limentani, S A; Gowell, K P; Deitcher, S R (1995) In vitro characterization of high purity factor IX concentrates for the treatment of hemophilia B. Thromb Haemost 73:584-91 |
