The biochemistry, cell and molecular biology of high (HK) and low (LK) molecular weight kininogens will be studied to learn about structure-function correlates as they relate to binding to and effects on blood and vascular cells. D5 has been identified as the region that interacts with anionic surfaces and as a binding site for HKA to neutrophils. Deletion mutants as well as synthesized peptides should allow mapping of the sequence(s) required for binding to neutrophils and endothelial cells. Physical studies of D5 with and without Zn plus plus will be performed. A 31 amino acid sequence in D6 contains sufficient information to bind to two noncontinuous sites on prekallikrein. Using peptides and deletion mutagenesis of D6, the minimal sequences for binding and the topology of HK binding to prekallikrein will be determined. We use these peptides and cognate peptides of prekallikrein to down-regulate fibrinolysis on endothelial cells. D3 has previously been identified as one cell binding region for platelets, endothelial cells and neutrophils. Exons 7, 8, and 9 coding for D3 will be expressed to test the hypothesis that one of the exon products contains all of the information for neutrophil binding on the heavy chain, while another is responsible for inhibiting the binding of thrombin to platelets and endothelial cells. Synthesized, conformationally restrained peptides will be used for fine mapping based on surface-accessible regions of a molecular model of D3 of HK. HK binds to neutrophils on Mac-1, and we will further map the ligand and receptor to better define the interaction. We have recently shown that fibrinogen does not compete with HK binding to endothelial cells, and an antibody to alpha nu beta 3 does not inhibit HK binding, ruling out alpha nu beta 3 as the receptor for HK. However, we found that vitronectin and soluble urokinase receptor inhibit HK binding, suggesting that the UK receptor is the binding site for HK. We will further test this hypothesis by mapping the HK binding site on the UK receptor and map the binding site on HK to the receptor. The mechanism by which HK blocks thrombin binding to platelets by interaction with the thrombin receptor or GPIb will be explored. Kininogen-deficient rats will be used to verify whether HK or LK is an important anti-thrombotic protein. Peptides from kininogen domains will be tested in rats for their antiadhesive and antiplatelet potential. Such polypeptides could serve as templates for peptidomimetic compounds, which should be therapeutic in sepsis, arthritis, hereditary angioedema, and gingival disease in addition to reocclusion after thrombolytic therapy.
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