adhesive glycoprotein present in the circulation and in different tissues. It has been postulated to regulate intra- and extravascular proteolytic processes including blood coagulation and fibrinolysis through its interaction with thrombin-antithrombin III (TAT)-complexes, type 1 and 3 plasminogen activator inhibitor (PAI-1 and PAI-3), fibrin, and vimentin-type intermediate filaments (Preliminary Studies). However, our understanding of the function(s) of Vn in vivo is somewhat obscured by its conformational lability. Vn exists in vivo in at least two different conformational states, the native and the modified or activated form, and these forms have different ligand binding properties. The long term objectives of this proposal are to define the domains in native and modified Vn that account for the different ligand binding functions, and to delineate the structural basis for the conformational lability of Vn. More specifically, in a series of structure/function studies, the interaction of native and modified Vn with TAT-complexes, PAI-3, fibrin, and vimentin will be analyzed and compared to results obtained with PAI- 1. Binding experiments will be performed not only to establish kinetic parameters for the interactions of these molecules with native and modified Vn, but also to localize the binding domains in Vn for these molecules. These latter studies will employ defined Vn fragments, inhibitory monoclonal antibodies, and recombinant Vn polypeptides. Results will be confirmed using genetically engineered Vn molecules that contain altered putative binding sites. In a second series of experiments, MABs will be developed to native and modified Vn and employed to define epitopes preferentially expressed in each molecule. The binding sites for these conformational sensitive MABs will be identified in immunoprecipitation experiments using defined Vn fragments. These MABs will also be used to develop assays systems to quantitate changes in the relative amount of native and modified Vn under various conditions. Finally, the regions contributing to the conformational lability of the Vn molecule will be identified using genetically engineered, truncated Vn molecules and mutant Vn molecules in which, for example, cysteines and hydrophobic amino acids have been replaced. Conformational lability will be assessed using the conformation specific immunoassays, and by the formation of multimers. This detailed analysis of the conformational states of Vn and their effect on its ligand binding properties, together with the elucidation of the structural basis for the conformational lability of this molecule, should lead to a better understanding of the structure of Vn and the role of conformational modulations in its function in vivo.
