How are the thrombi that cause most heart attacks and strokes formed? How are normal hemostatic and inflammatory responses to tissue injury triggered? The coagulation cascade generates thrombin and related serine proteases upon disruption of vascular integrity, and thrombin is a potent activator of platelets, endothelial and other cells. How does a protease like thrombin behave like a hormone to regulate the cellular behaviors? We've characterized a family of protease-activated G protein-coupled receptors (PARs) that provide an answer. Thrombin cleaves PAR1's N-terminal exodomain to unmask a new amino terminus that then serves as a tethered peptide ligand, binding intramolecularly to the heptahelical segment of the receptor to cause transmembrane signaling. PAR1 is the prototype for a family of four receptors that appear to account for most cellular responses to coagulation and other trypsin-like proteases. Our laboratory currently focuses on understanding the roles of protease and PAR signaling and, more broadly, G protein-coupled receptors in cardiovascular biology:

PARs in physiology and disease. Using mice with individual and combined PAR deficiencies, we are exploring the importance of PAR signaling in platelets, endothelial cells and other cell types in mouse models of hemostasis and thrombosis, inflammation and other processes. A current emphasis is utilizing advanced light microscopy techniques to visualize the biochemical and cellular events that mediate various stages of thrombus assembly. Early studies show that PAR signaling is unnecessary for formation of an initial juxtamural platelet thrombus but required for enlargment and propagation of such thrombi. Thus different signaling mechanisms may be important at different points in the development of a thrombus, and exploiting such differences may permit the development of safer antithrombotic drugs.

PARs in embryonic development. PAR1 signaling in endothelial cells is important for normal vascular development in the mouse embryo. Efforts to identify the specific endothelial cell behaviors involved as well as the other targets of the coagulation cascade that are important for embryonic development are in progress. PARs, specifically PAR2, also appear to contribution to neurulation. Efforts to determine what PAR2 senses biochemically and physiologically and what it regulates in this context are ongoing. This line of research will reveal new roles for protease signaling.

Sphingosine kinases in development and disease. Sphingosine-1-phosphate acts through G protein-coupled EDG receptors to regulate heart and blood vessel formation in the embryo as well as leukocyte trafficking and other important processes in the adult. This pathway may modulate or mediate the effects of PARs, RTKs and other signaling systems. However, the exact sources of S1P and hence when and whether it functions primarily as a hormone or as a paracrine or autocrine factor are unknown. We have generated conditional alleles for the two sphingosine kinases in mice to explore these questions.

Novel roles for G protein signaling. The studies outlined above emphasize that G protein-coupled receptors can play important roles in embryonic development. The ~350 nonodorant G protein-coupled receptors in mice and humans couple through 4 main G protein families, G s , G q , G i , and G 12/13 . We are ablating G 12/13 and G i signaling in specific cell lineages to probe the roles of these pathways in embryonic development and other processes, then using a candidate approach to identify the receptors and ligands involved. Several novel phenotypes have been identified.