Vascular endothelial cells (EC) can regulate physiologic processes through the elaboration of secretory products and the expression of new cell surface molecules. Thrombin stimulates the rapid secretion of von Willebrand factor (vWF) and surface expression of P-selectin (GMP140/PADGEM) and is likely to be a major trigger for this event. The overall goal of this proposal is to elucidate the molecular mechanisms of thrombin-mediated secretion in vascular EC. In studies of both intact and saponin-permeabilized EC, we have identified Ca2+ and calmodulin (CaM) as predominant second messengers in this process. We propose to identify the intracellular targets of Ca2+/CaM which regulate vWF secretion. To examine the role of CaM-dependent kinases in this process, we will employ a series of well-characterized peptide inhibitors of these molecules utilizing our novel receptor-sensitive permeabilized cell model. These studies will be complemented by the transient expression of both wildtype and mutant homologs of two of these kinases: myosin light chain kinase (MLCK) and CaM kinase II (CaMKII). We will examine the role of CaM-regulated actin-binding proteins using an array of currently available inhibitory peptides and antibodies. We will determine if oscillatory [Ca2+]i transients, produced in response to low concentrations of thrombin, are capable of stimulating the CaM-dependent processes of secretion and cytoskeletal reorganization in individual EC. We will study MARCKS, an actin-binding protein and putative modulator of CaM-dependent processes, for its effects on vWF secretion. We hypothesize that among the targets of vWF regulation in EC are proteins associated with its secretory granule (Weibel-Palade body). We will use cell fractionation techniques previously developed in our laboratory to identify CaM-binding proteins associated with this organelle and establish their role in the secretory process. Our preliminary data demonstrate the feasibility of each of these approaches and provide evidence in partial support of the hypotheses which we have developed. the results of our proposed studies will provide a more detailed understanding of the molecular events of regulated secretion in EC. WE believe that regulated secretion in EC is of central importance to the early events of hemostasis, thrombosis and inflammation and that new knowledge derived from our studies will materially affect the future development of therapeutic approaches to control these processes.