Research Project
3471704
Factor IX is a vitamin K-dependent blood coagulation protein whose function is essential to normal hemostasis. lt participates in the middle phase of the coagulation cascade and its functional activity is mediated by the binding of calcium ions to the protein. The study of Factor IX provides an excellent model-for the role of calcium and the metal binding amino acid, Delta-carboxyglutamic acid, in the function of the vitamin K-dependent coagulation proteins. The purpose of this proposal is to characterize the role of metal ions in the function of Factor IX. Using immunochemical methods, I have demonstrated that Factor IX undergoes two metal- dependent conformation transition: FIX==greater than FIX'== greater than FIX*. The first transition (FIX==greater than FIX') is metal dependent but cation-nonselective. The second transition (FIX'==greater than FlX*) is metal-selective for Ca(II) or Sr(II). The second transition results in the expression of conformational determinants necessary for membrane binding and the Ca(II)- dependent activation of Factor IX by Factor XIa. Using these conformation specific antibodies 1 will isolate partially carboxylated Factor lX species from the blood of patients taking sodium warfarin which can undergo the first conformational transition (FlX== greater than FlX'), but not the second (FIX'=greater than FlX*). These antibodies can also be used to detect a mutant Factor lX protein from a patient with hemophilia B which is unable to undergo the second conformational transition. The structural and physicochemical characterization of the mutant and partially carboxylated Factor lX proteins will include protein sequencing, Gla content, protein metal binding studies, intrinsic fluorescence quenching and phospholipid binding. Functional characterization will include studies of the activation of the mutant and partially carboxylated Factor lX proteins by Factor XIa and Factor VIIa-Tissue Factor, enzymatic activity of activated mutant and partially carboxylated Factor lX proteins and studies of their interaction with Factor VIII-vWF. These studies will provide new information regarding the functional domains of Factor LX expressed on the metal dependent conformers.
