The general hypothesis this proposal addresses is that the cell membrane expression of the plasma kininogens [high molecular weight kininogen (HK) and low molecular weight kininogen (LK)] is essential for bradykinin (BK) delivery to cells. In order to describe how the kininogens deliver BK to cells the objectives of this proposal are to characterize the epitopes on HK and LK that bind to platelet membranes and to determine the platelet receptor(s) for the kininogens.
The specific aims of this proposal in order to achieve these objectives are as follows: (1) We will determine the epitopes on the kininogens that bind to platelets. Purified domains from each of the kininogens will be used to indirect and direct platelet binding experiments to determine the region(s) on these proteins involved in membrane binding. Monoclonal antibodies that inhibit kininogens' binding to platelets will be used to isolate kininogens' cell binding domains by determining the epitopes on the proteins will be used to isolate kininogens' cell binding domains by determining the epitopes on the proteins that these antibodies recognize and by characterizing peptides secreted from a lambdagt11 recombinant DNA kininogen expression library recognized by these antibodies. Synthetic peptides, corresponding to the deduced binding domains on the kininogens, will be produced to determine if they inhibit the parent molecules' ability to bind to platelets and can directly bind to the platelet surface. (2) Investigations will be performed to identify and characterize the platelet receptor(s) for the kininogens. Studies will be performed to identify a physiochemical receptor for the kininogens on unstimulated platelets and HEL cells by ligand blotting and chemical crosslinking agents. Purification of a physiochemical receptor for kininogen from unstimulated platelets and HEL cells will be performed by immuno- or ligand-affinity chromatography. Investigations will be performed to determine the mechanisms(s) by which platelet calpain becomes externalized on the activated platelet membrane and if its expression allows it to become an additional receptor for kininogen on thrombin-activated platelets. Characterization of the epitopes on the kininogens that bind to cells and determining the presence of a physiochemical receptor(s) for the kininogens on platelets and HEL cells should uncover mechanisms of importance for the regulation of BK availability to cells. Since both kininogens are the parent proteins for bradykinin, knowing how kininogens are expressed on cell membranes would be helpful to understand how bradykinin delivery to tissues can be modulated. Since modulation of bradykinin delivery to cells by captopril and enalapril has become very important for blood pressure management and treatment of heart failure, the proposed studies may provide for new approaches for the development of antagonists to bradykinin delivery to cells which could have therapeutic potential to modulate this peptide's availability.
Showing the most recent 10 out of 45 publications
