Calcium ions are directly involved in the regulation of blood coagulation in two distinct, but related, ways. Ca2+ binding to individual proteins induces conformational changes that are required for the activation of the proteins and for their function. Ca2+ is also required for the assembly of the multicomponent macromolecular complexes which mediate clotting in vivo. We propose to investigate the interaction of Ca2+ with (i) the high affinity binding site in protein C and in factor IX that lacks Gamma-carboxyglutamic acid residues, and (ii) the single binding site in factor Va. In addition, we propose to examine the structure of the prothrombinase complex and the interactions between the individual components of the enzyme-substrate complex. Fluorescence spectroscopy will be used to monitor, at physiological concentrations, these macromolecular interactions, the metal ion-induced conformational changes in protein C and factor IX and their derivatives, and the kinetics of Ca2+ dependent factor Va subunit association. The fluorescence-detected structural changes will be analyzed both kinetically and thermodynamically, and will be correlated with changes in function whenever possible. Various proteins, each with a single fluorescent dye covalently attached to a single site, will be prepared and used as probes when the modification does not interfere with function. Singlet-singlet energy transfer experiments will provide information about the molecular architecture of the prothrombinase complex by measuring the distance between two appropriate protein-bound dyes, or between a protein-bound dye and either fluorophores trapped inside phospholipid vesicles or dyes in the phospholipid bilayer. Lipophilic photoreactive reagents will be used to determine whether or not factor Va and other proteins are embedded in the phospholipid bilayer.
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