The aims of this proposal are the identification and characterization of the different regions on the light chain (LC) and heavy chain (HC), of high molecular weight kininogen (HK) responsible for the novel in- teraction with platelet thrombospondin (TSP), to define the primary structure and its functional role. Peptide fragments from the HK-LC and HK-HC will be generated by the use of a thiol protease enzyme from B. gingivalis, which cleaves efficiently both chains into small fragments: The proteolytic fragments will the be applied to a C-18 or C-4 column and purified by size exclusion or reverse phase HPLC. Peaks from HPLC will then be dot blotted onto a PVDF membrane. Radiolabeled TSP will serve as a probe to identify peaks containing binding domains, and a polyclonal antibody directed against either HK-LC or HK-HC will determine their chain of origin. The fraction(s) containing fragments reacting against both probes(labeled TSP and each of the two different antibodies) will be analyzed by SDS-PAGE electrophoresis to confirm its size and purity. The N-terminal sequence of each purified polypeptide will be determined, which will place them in the known amino acid sequence. The isolated peptides will be tested for their ability to compete for the binding of radiolabeled TSP to microtiter plates precoated with HK. To confirm the minimum sequence needed for the binding to TSP, a molecular biological approach will also be used to provide independent information. The HK-LC will be expressed in E. coli. After assessing the functional biological activity of the rHK-LC, its structure will be altered ( based on the results from the protein chemistry experiments) by site specific mutagenesis using polymerase chain reaction (PCR). The long-term goals are related to the functional role of the HK binding to TSP. TSP and HK are two multifunctional proteins playing an important role on the platelet function although their mechanism of action remains obscure. The correlation of structure-function will provide a better understanding of the interaction of TSP-HK during pathophysiological conditions.
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