The hypothesis that forms the basis of this proposal is that the endothelial cell receptor for the plasma kininogens is a multimeric structure that orders contact protein zymogen assembly and activation of prokallikreins for sequestered kinin liberation and other kallikrein- dependent biologic activities. Expression of kininogens, both high (HK) and low (LK) molecular mass kininogens on this receptor permits kinins [bradykinin (BK) and met-lys-bradykinin (kallidin)] to be liberated to influence local vascular biology. This hypothesis is based upon the finding that endothelial cells contain several membrane-expressed kininogen binding proteins. Second, both HK and LK themselves have multiple binding-domains for its particular orientation onto its endothelial cell binding site(s), putative receptor(s). Third, HK serves as the endothelial cell binding site for prekallikrein. Fourth, prekallikrein only bound to HK on endothelium is activated by an endothelial cell, calcium requiring metalloprotease for kinetically favorable pro-urokinase and subsequent plasminogen activation. To test the above hypothesis, the specific aims of this proposal are as follows: (1) The kininogen receptor(s) on endothelial cells will be determined. (2) The mechanism(s) regulating kinin liberation from cell- bound kininogens will be determined. (3) The fine mapping of sequences on kininogens that participate in its binding to endothelium will be characterized. These investigations will characterize how kinins are liberated from its parent proteins in the intravascular compartment to influence vascular biology. These studies will provide a new avenue for modulation of all bradykinin-dependent, endothelial cell activities such as tPA liberation, NO formation, elevation of cGMP, prostacyclin synthesis, superoxide formation, and smooth muscle hyperpolarization factor production. Modulation of bradykinin liberation is important in the regulation of local blood pressure in coronary, renal, pulmonary, and cerebral circulations.
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