Shaun Coughlin - US grants

histocompatibility; immunomodulators; blood vessel transplantation; atherosclerosis; histocompatibility antigens; growth factor receptors; receptor expression; cytokine; leukocyte activation /transformation; coronary artery; growth factor; cardiovascular disorder epidemiology; vascular smooth muscle; cell type; platelet derived growth factor; disease /disorder model; myoepithelial cell; tissue /cell culture; natural gene amplification; in situ hybridization; laboratory rat; human tissue; human subject;

The long-term goal of this project is to define how thrombin triggers cellular responses and the roles of thrombin signaling in biology and disease. Thrombin's actions on platelets are critical for homeostasis and thrombosis, and its actions on leukocytes, endothelial, and mesenchymal cells may contribute to inflammatory and proliferative processes. Thrombin activates platelets and other cells at least in part via G protein- coupled protease-activated receptors (PARs). Further study of PARs promises to reveal new modes of receptor activation, interaction, and intracellular sorting. Defining the roles of PARs in vivo may provide new strategies for the prevention and treatment of thrombosis and other diseases. The following questions will be asked: 1) Does mPAR3 mediate transmembrane signaling on its own or, instead, act as a cofactor for mPAR4 activation? The paradigm of one receptor's binding a ligand that in turn activates a distinct receptor is unprecedented for GPCRs. PAR3 and PAR4 are the thrombin receptors known to participate in mouse platelet activation. Knockout for PAR3 attenuated activation of mouse platelets by low concentrations of thrombin, but expression of mPAR3 in heterologous systems has not conferred signaling. We hypothesize that mPAR3 functions by binding thrombin and promoting cleavage and activation of Mpar4. 2) Do cofactor mechanisms play a more general role in PAR activation? Results from Aim 1 will likely cast mPAR3 as a cofactor for ability of known protease cofactors to promote PAR activation will be assessed. 3) Do hPAR1 and Hpar4) have distinct roles and/or interact in human platelets? In contrast to mouse platelets, human platelets use PAR1 and PAR4 to mediate thrombin signaling, and activation of either receptor appears to be sufficient to trigger robust responses. Are hPAR1 and hPAR4 redundant or do they interact or serve at least partially distinct functions? Ability to activate distinct downstream signaling pathways and/or mediate signaling to proteases other than thrombin will be assessed. 4) How is activated PAR1 internalized and delivered to lysosomes? Internalization and sorting of activated receptors to lysosomes is important for the long-term down- regulation of signaling pathways. In multiple cell types, sorting of activated PAR1 to lysosomes is important for the long-term down- regulation of signaling pathways. In multiple cell types, sorting of activated PAR1 to lysosomes is rapid and robust. Thus PAR1 is a useful model system for the study of receptor down-regulation in mammalian cells. We will use complementation cloning to identify new molecules involved in PAR1 trafficking.

DESCRIPTION (provided by applicant): Cardiovascular disease remains a major cause of morbidity and mortality in the U.S. and is increasing in underdeveloped countries. Application of modern cell biology, genetics and genomic technologies is producing remarkable progress. The need for broadly trained scientists who can adopt innovative technologies, assemble tools from different disciplines, and bridge basic and clinical science is greater than ever. The overall goal of UCSF Training Program in the Molecular and Cellular Basis of Cardiovascular Disease is to train investigators who will be at the cutting edge of cardiovascular research. Toward this end we shall: (1) Capitalize on the strong multidisciplinary research environment of the CVRI and UCSF to provide outstanding training in areas of signal transduction, vascular biology, lipoprotein metabolism and obesity, muscle differentiation and function, developmental biology, and genetics, (2) Attract graduates of top Ph.D. and M.D.-Ph.D. programs to careers in cardiovascular research and (3) Provide opportunities for M.D.s in clinical fellowships at UCSF to obtain rigorous training in basic research. The Program brings together a diverse and outstanding group of mentors with a common interest in cardiovascular biology under auspices of the Cardiovascular Research Institute at UCSF, a multi-departmental and multi-disciplinary research organization. The Program places trainees in remarkably productive and interactive laboratories and provides a structured environment for acquisition of the knowledge and skills that will be required for success. The heart of the training program is execution of a substantial research project under the close supervision of a primary mentor. Multiple forums for scientific exchange and exposure to other laboratories are provided. Didactic programs are tailored to the individual's background and goals. Required practical skills and ethic courses are supplemented by an outstanding menu of seminars and an array of basic science courses.

DESCRIPTION: Thrombin is a multifunctional serine protease generated at sites of vascular injury. Its in vitro actions on platelets, leukocytes, endothelial and mesenchymal cells suggest that thrombin may mediate not only hemostasis and thrombosis but also inflammatory and proliferative responses to vascular damage in vivo. The overall goal of the application is to understand thrombin signaling, thereby providing strategies to prevent unwanted responses to vascular injury such as formation of platelet thrombi overlying ruptured atherosclerotic plaques, the usual cause of unstable angina and myocardial infarction. Knockout of par1, the first thrombin receptor cloned and characterized by the investigator s group, provided definitive evidence for the existence of second thrombin receptor in mouse platelets and for tissue specific roles for distinct thrombin receptors. Dr. Coughlin recently cloned and characterized par3, a second thrombin receptor that is expressed in human bone marrow and mouse megakaryocytes. The proposal seeks to define the role of par3 in vivo and the signaling mechanisms that it utilizes. The investigator seeks to answer the following questions: What are the roles of par1 and par3 in human platelets and other cells? Does par3 account for thrombin signaling in par1 -/- mouse platelets or do still other thrombin receptors exist? Are the signaling mechanisms utilized by par3 distinct from those used by par1, and what is the signaling pathway from par3 activation to platelet secretion and aggregation?

DESCRIPTION (Adapted from abstract) This is one of three collaborative ROls submitted in response to RFA HL-99-015 on Arterial Thrombosis. The applicants proposal's overall goal is to define the signaling mechanisms by which platelets are activated, thereby providing a framework for therapeutic development and risk factor identification. The goal of this individual project is to define the role of thrombin signaling in hemostasis and thrombosis. Thrombin activates platelets via proteaseactivated G protein-coupled receptors (PARs). The applicants shall use mice deficient in these receptors to ask: 1) Is thrombin signaling in platelets necessary for normal hemostasis? PAR3 and PAR4 are the thrombin receptors known to participate in mouse platelet activation. It appears that mPAR3 does not mediate transmembrane signaling but instead acts as a cofactor for mPAR4 cleavage and activation by low concentrations o f thrombin. Thus the applicants expect PAR4 to be required for thrombin signaling in mouse platelets. The applicants will use PAR4-deficient mice to test the importance of thrombin signaling in hemostasis. 2) Does attenuation or ablation of thrombin signaling in platelets inhibit thrombosis? Human platelets use PAR1 and PAR4 for thrombin signaling, and it is unknown whether inhibition of PAR1 and/or PAR4 would be useful for preventing or treating thrombosis. PAR3- deficient mouse platelets are analogous to PARl-inhibited human platelets (both rely on PAR4 for thrombin signaling). PAR4-deficient mouse platelets will likely prove analogous to human platelets in which all thrombin signaling has been blocked. The applicants will use PAR3- and PAR4-deficient mice to determine whether partial or complete inhibition of thrombin signaling in platelets protects against arterial and microvascular thrombosis. 3) Is endothelial cell activation by thrombin or other proteases important in hemostasis and thrombosis? The applicants hypothesize that activation of endothelial PAR1 and PAR2 promotes thrombosis and inflammation by promoting platelet and leukocyte rolling and adhesion. The applicants will determine whether mice deficient in PAR1 and/or PAR2 are protected in models of inflammation and microvascular and arterial thrombosis. 4) Do gain-of-function mutations in platelet G protein-coupled receptors (GPCRs) promote thrombosis? The discovery of prothrombotic mutations in genes that regulate cellular behaviors has lagged that in genes encoding the plasma proteins. Several human diseases are mediated by gain-of-function mutations in GPCRs. The applicants will use mice bearing gain-of-function mutations in PAR4 to determine if such GPCR mutations, alone or in combination with mutations in other genes, might be a basis for thrombophilia.

DESCRIPTION (provided by applicant): In the adult, protease-activated receptors (PARs) respond to coagulation proteases to help orchestrate cellular responses involved in hemostasis, inflammation and repair. In the previous project period, we showed that PARs also play distinct and important roles in embryonic development. Indeed, PAR1 signaling in endothelial cells is required for proper remodeling and/or integrity of developing blood vessels in mouse embryos. What biochemical and physiological processes does PAR1 monitor during blood vessel formation? Which endothelial cell responses to PAR1 activation are important for proper vessel development? We hypothesize that the coagulation cascade and PARs together provide a system for monitoring and regulating the formation, remodeling, and integrity of developing blood vessels. Toward testing this hypothesis we shall first determine whether PARs sense coagulation proteases during mouse embryonic development, and whether activation of PARs account for the roles of coagulation factors in this context. Specific questions include a) Does combined deficiency of PARs phenocopy tissue factor deficiency? b) Is tissue factor expression in or around blood vessels necessary and sufficient to support embryonic development? and c) Can embryos bearing prothrombin mutations be rescued by complementary mutations in PAR1? We shall next determine which signaling pathways are important for the function of PAR1 and other GPCRs in endothelial cells during vascular development by ablating the function of specific G protein pathways in endothelial cells in the mouse embryo. We expect these studies to illuminate a novel role for the coagulation cascade and PARs and to provide new insights regarding the mechanisms governing vascular development and perhaps new blood vessel formation in other settings.

DESCRIPTION (provided by applicant): Cardiovascular disease remains a major cause of morbidity and mortality in the U.S. and is increasing in underdeveloped countries. Application of modem cell biology, genetics and genomic technologies is producing remarkable progress. The need for broadly trained scientists who can adopt innovative technologies, assemble tools from different disciplines, and bridge basic and clinical science is greater than ever. The overall goal of UCSF Training Program in the Molecular and Cellular Basis of Cardiovascular Disease is to train investigators who will be at the cutting edge of cardiovascular research. Toward this end we shall: (1) Capitalize on the strong multidisciplinary research environment of the CVRI and UCSF to provide outstanding training in areas of signal transduction, vascular biology, lipoprotein metabolism and obesity, muscle differentiation and function, developmental biology, and genetics, (2) Attract graduates of top Ph.D. and M.D. Ph.D. programs to careers in cardiovascular research and (3) Provide opportunities for M.D.s in clinical fellowships at UCSF to obtain rigorous training in basic research. The Program brings together a diverse and outstanding group of mentors with a common interest in cardiovascular biology under auspices of the Cardiovascular Research Institute at UCSF, a multidepartmental and multi-disciplinary research organization. The Program places trainees in remarkably productive and interactive laboratories and provides a structured environment for acquisition of the knowledge and skills that will be required for success. The heart of the training program is execution of a substantial research project under the close supervision of a primary mentor. Multiple forums for scientific exchange and exposure to other laboratories are provided. Didactic programs are tailored to the individuals background and goals. Required practical skills and ethic courses are supplemented by an outstanding menu of seminars and an array of basic science courses.

DESCRIPTION (provided by applicant): The long term goal of this proposal is to define the role of protease-activated receptors (PARs) in inflammatory responses. PARs mediate cellular responses triggered by coagulation proteases and other proteases that are generated or released at sites of tissue injury. The effects of PAR activation in different cell types, largely defined in culture, suggest that PARs may help orchestrate a coordinated response to tissue injury that includes hemostasis, inflammation, and perhaps even regulation of the adaptive immune response. Local PAR activation by proteases generated at sites of local bacterial inoculation may protect against spread of bacteria by promoting both recruitment of leukocytes and microvascular thrombosis. However, more regional or systemic activation of PARs as may occur in the setting of tissue ischemia or sepsis may promote tissue damage by the same mechanisms. Toward testing these hypotheses, we have generated knockout mice for the known PARs as well as relevant double knockouts. Par4 -/- mice, for example, have no platelet responses to thrombin. Mice lacking both PAR1 and PAR4 may have lost thrombin signaling in all cell types. Using such mice and cells derived from them, we shall determine which PARs are responsible for mediating responses to coagulation proteases and other proteases in endothelial and other cell types, the extent to which different PARs in the same cell serve redundant functions, and the potential roles for endothelial PAR activation in vivo. We shall then go on to determine the effects of loss of PAR function in models of a) local bacterial infection and dissemination of same, b) systemic inflammatory response syndromes induced by endotoxin and by pancreatitis, and c) ischemia/infarction. Endpoints will include survival; markers of platelet and fibrin deposition and leukocyte accumulation in tissues; vascular permeability and edema; cytokine production; organ histology; and in a hind-limb ischemia-reperfusion model, reflow and infarct size. Lastly, because PARs sense tissue injury and hence "immunological danger," we shall ask whether PARs influence the decision to mount an adaptive immune response. These studies may point to new strategies for modulating injurious inflammatory responses in man.

DESCRIPTION (provided by applicant): Protease-activated receptors (PARs) were discovered in the context of an effort to understand how thrombin and other proteases regulate cellular behaviors. Four PARs are found in mammals. PARs 1,3, and 4 are thrombin receptors that mediate activation of platelets and other cells by thrombin and play key roles in hemostasis and perhaps inflammatory and other responses to tissue injury. By contrast, physiological activators of PAR2 have not been clearly identified, and its roles in vivo are uncertain. Our recent work suggests that PARs play roles in contexts other than response to injury. PAR1 function in endothelial cells is important for proper vascular development, and we now find that PAR2 also plays distinctive roles in the embryo. In one strain background, Par2-/- embryos develop profound anemia. Moreover, Par1 -/-Par2 -/- embryos show a strong phenotype not seen in either single knockout: exencephaly, a hallmark of failed neural tube closure. PAR2 is expressed in the epidermal ectoderm overlying the neuroepithelium before and during neural tube closure, that is, at the right place and time to play a direct role. We will better define these interesting phenotypes and identify the exact cell types involved by transgene rescue and cell type-specific knockout. What does PAR2 regulate and what does it sense in these contexts? We have developed mouse lines that will be used to ablate specific G protein signaling pathways in the cell types in which PAR2 function is important for development. Phenocopies of PAR-deficiencies will help identify which of the several G proteins regulated by PARs is important for the phenotypes described above, and novel phenotypes will point to new roles for G protein-coupled receptors in embryonic development. We also plan systematic candidate-driven approach to identify PAR2-activating proteases that might function in the embryo. These studies will provide new insights into basic mechanisms underlying hematopoiesis, vascular development and/or neurulation.

A central goal of my laboratory has been to determine how thrombin regulates cellular behaviors involved in hemostasis, thrombosis, inflammation and other processes, and to elucidate the roles of thrombin signaling in vivo. This grant has focused on hemostasis and thrombosis. Our previous work showed that protease- activated receptors (PARs) are necessary for platelet activation by thrombin and important for thrombosis and hemostasis in mouse models. These and other studies support exploration of PAR antagonism for the prevention or treatment of thrombosis in humans. We now propose studies to define the roles of PARs in more detail and to determine how thrombin signaling integrates with other platelet activation and coagulation mechanisms in vivo. We shall ask:1) Are vWF, collagen and thrombin the major initiators of platelet activation in vivo? How do these pathways interact? Using sophisticated mouse models, we will test the hypothesis that GPIb and GP-VI signaling is necessary and sufficient to drive platelet adhesion and juxtamural thrombus formation at a site of injury but that PAR signalingis necessary for propagation of the thrombus away from the vessel wall. Genetic and pharmacological approaches will be used. PAR interactions with P2Y12 and Tbxa2r (TP) will also be probed. 2) How do thrombin-inducedplatelet activation and fibrin formation interact in hemostasis and thrombosis? Does their relative importance change when thrombin generation or activity is reduced or inhibited? We shall determine whether platelet activation by thrombin is important for propagation of thrombin generation and fibrin formation away from the vessel wall. We shall also ask whether disruption of PAR signalingin the setting of low thrombin generation has synergistic effects on thrombosis or hemostasis. 3) What is the role of tissue factor expression in endothelial and hematopoietic cells in hemostasis and thrombosis? In endotoxemia? Can roles for such tissue factor be uncovered or amplified by knockout of alternative mechanisms for propagation of coagulation? Tissue factor expression will be ablated in endothelial and hematopoietic tissues to probe the source and roles of "circulatingtissue factor". These studies will illuminate how key effectors of hemostasis and thrombosis interact.

DESCRIPTION (provided by applicant): We will characterize two signaling systems that regulate the permeability and integrity of blood vessels: coagulation proteases and protease-activated receptors, and sphingosine-1-phosphate and S1P receptors. We will test the hypothesis that both systems sense extravasation of plasma and trigger appropriate endothelial cell responses, and we will explore parallels and possible connections between these systems. We shall ask: 1) How is S1P important for regulation of vascular permeability and integrity? We generated adult mice that fail to supply S1P to plasma and found striking abnormalities in vascular permeability and integrity. We shall determine a) whether altered barrier function in these "pS1Pless" mice is due to failure to metabolize sphingosine with consequent endothelial cell-autonomous metabolic/toxic effects or to failure to supply S1P to plasma and S1P receptor activation on endothelial and other cells, b) the anatomic basis for their increased vascular leak and whether it shows tissue or vessel-type specificity, c) whether endothelial cells are the main target of plasma S1P signaling in this context, and if so, whether such signaling is continuous or whether plasma S1P provides a dynamic signal that enables endothelial cells to sense and help terminate leaks, and d) the long-term effects of lack of plasma S1P and whether they are due to dysregulated barrier function. 2) How are PARs important for regulation of vascular permeability and integrity? We generated knockout mice for all the PARs and relevant transgenics and will use these to determine a) the effects of activation of different endothelial PARs on vascular permeability and integrity in vivo and whether pS1Pless mice provide a sensitized system for uncovering such roles for PARs, b) whether PAR signaling is parallel to, partially redundant with, or dependent upon S1P signaling. 3) Do differences in apical and basal S1P and PAR function contribute to their roles in barrier regulation? Our preliminary studies suggest a model that would permit S1P signaling to serve a dynamic leak detector function and raise new questions regarding PARs by analogy. We will determine a) whether endothelial cell S1P1 receptors display apical-basal polarity in vitro and in vivo to enable the dynamic leak detecting function we posit, and b) whether apical and basal differences, such as EPCR ligation, modulate the effects of PAR activation on either surface. Complementary genetic and pharmacological approaches will be used in mouse models and in cell culture. Preliminary studies reveal an important role for plasma S1P in regulating endothelial barrier function in vivo, distinct barrier responses to activation of different PARs, dramatic effects of manipulation of S1P and PAR signaling on survival in models of anaphylaxis, and long-term effects of altered barrier function in vivo. The proposed studies will provide new information regarding vascular physiology and pathophysiology. PUBLIC HEALTH RELEVANCE: Our studies will reveal new molecular and cellular mechanisms by which the endothelial cells that line blood vessels sense and regulate leakiness. Alterations in endothelial permeability play an important role in allergic reactions, new blood vessel growth in cancer, heart attacks and strokes, atherosclerosis, and inflammatory states including sepsis. Thus, understanding of how to manipulate the endothelial leakiness might benefit patients with a range of disorders.

DESCRIPTION (provided by applicant): Cardiovascular diseases remain a leading cause of morbidity and mortality. Many of these diseases originate at least in part from genetic defects that impact the development and maturation of the cardiovascular system. Despite substantial progress in our understanding of cardiovascular development and its role in disease susceptibility, much remains to be learned. The focus of this proposal is on cardiac trabeculation, a fundamental aspect of cardiac development that modulates cardiomyocyte mass and cardiac function. Interestingly, trabeculae initially form only in the ventricle but not the atrium, and within the ventricle, they only form in the outer but not the inner curvature. These observations indicate that cardiac trabeculation is regulated by genetic as well as epigenetic factors, such as flow. We will continue to take advantage of the zebrafish system to investigate several aspects of cardiac trabeculation. Our data indicate that 1) cardiac trabeculation initiates by the directed migration/delamination of cardiomyocytes from the compact layer, 2) in zebrafish, as in mouse, Erbb2 signaling is absolutely required for cardiac trabeculation (but importantly why or how it is required remains unclear), and 3) cardiac function is required for cardiac trabeculation. These and other data lead us to propose to test the following three hypotheses: 1) cardiac trabeculation initiates via the directed migration/ delamination of cardiomyocytes. We will analyze at single cell resolution a) the distribution and behavior of small clones of labeled cardiomyocytes as trabeculae form, b) the patterns of cardiomyocyte division during the initiation and expansion of trabeculae, and c) apicobasal polarity of cardiomyocytes in the compact and trabecular layers. 2) Erbb2 signaling regulates cardiac trabeculation via its role in modulating cardiomyocyte epithelial to mesenchymal transformation (EMT) including the loss of apicobasal polarity. Experiments are designed to a) test the cell autonomy of Erbb2 function in the initiation of cardiac trabeculation, b) investigate the regulation of Neuregulin (Nrg) in the initiation of cardiac trabeculation, c) investigate the mechanism of action of Erbb2 signaling in the initiation of cardiac trabeculation, and d) investigate the potential link between Erbb2 signaling and cardiomyocyte EMT, loss of apicobasal polarity and apical constriction. 3) cardiomyocyte contractility and shear stress regulate cardiac trabeculation. Experiments are designed to a) test the role of cardiomyocyte contractility in the initiation of cardiac trabeculation, and b) test the role of flow in the initiation of cardiac trabeculation. These studies will help understand mechanisms of cardiac trabeculation. They should also shed new light on the function of Erbb signaling in cardiomyocyte biology beyond trabeculation as Erbb signaling has been implicated in differentiated cardiomyocyte proliferation and repair of heart injury. Furthermore, they should help elucidate the critical, yet poorly understood, role of cardiac function in cardiac development.

DESCRIPTION (provided by applicant): Protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that permit thrombin and other extracellular proteases to regulate cellular behaviors. Together with the coagulation cascade, PARs link tissue injury to cellular responses that help orchestrate hemostasis and thrombosis, inflammation, cytoprotection, repair, and pain perception. Given these and other roles, PARs are potential drug targets. Available evidence supports a model in which thrombin activates the prototypical PAR, PAR1, by cleaving the N-terminal exodomain of the receptor at a specific site to generate a new N-terminus that then functions as a tethered peptide agonist, binding intramolecularly to the receptor's heptahelical bundle to effect receptor activation. Structures that test this model and reveal how the tethered ligand binds and how such binding drives transmembrane domain (TM) movement and G protein activation are lacking, as are structures to support development of pharmaceuticals targeting PARs. Building on our recent crystal structure of inactive-state PAR1 complexed with the antagonist vorapaxar, we propose studies to illuminate the mechanism of PAR activation, signaling and antagonism at a structural level. We will 1) Solve crystal structures of thrombin- activated PAR1 in complex with Gi and either Gq or G12/13. 2) Determine the basis for vorapaxar's specificity for PAR1 over closely related receptors and the route of vorapaxar entry (from the plasma membrane or the extracellular space), and 3) Solve the crystal structure of a PAR2-antagonist complex. Cutting edge crystallographic approaches, including use of stabilizing nanobodies and single particle EM to assess complexes, will be employed. Molecular dynamics simulations will aid design and interpretation of mutational studies. Our studies of PAR1 will reveal the mechanism by which the PAR1 tethered agonist binds and triggers TM movement and G protein activation, the structural basis for PAR1's promiscuous coupling to multiple G protein subtypes (Gi, Gq, and G12/13), a novel route of antagonist entry and the importance of entry route for specificity. Detailed studies of the PAR1-vorapaxar structure and the PAR2 crystal structure will provide an entry to structure-based discovery and optimization of better PAR antagonists needed to explore the role of these receptors in human disease. Our studies will provide the first structure of a peptide agonist- GPCR-G protein complex and the first structural insight into whether distinct conformers of individual GPCRs recognize different G proteins.

Project Summary/Abstract My laboratory seeks to understand how proteases, key biological regulators that act by cleaving other molecules, govern cellular behaviors and the roles of such protease signaling in embryonic development, adult physiology and disease. We discovered and characterized Protease-Activated Receptors (PARs). These molecules span the cell membrane, sense extracellular protease activity and transmit this information inside the cell to trigger responses. Proteases trigger signaling by cleaving PARs at a specific site to unmask an activator that is part of the receptor but hidden until uncovered by the protease. This work revealed how thrombin, a key blood clotting enzyme, activates blood platelets, the small cells that plug broken blood vessels to stop bleeding or diseased arteries to cause heart attacks and strokes. These discoveries led to a new type of antithrombotic drug (vorapaxar/Zontivity). The work also uncovered unexpected roles for protease signaling in endothelial, smooth muscle, and epithelial cells, neurons and other cell types, many of which remain largely unexplored. We seek to build on this foundation to better understand how PARs function as signaling machines and their roles in normal biology and disease. Our future work will focus on three important areas: 1) The structural basis of how PAR1 becomes active upon cleavage by thrombin, transmits information across the cell membrane, and chooses the signaling proteins that couple to the inside of the activated receptor and transmit information to the cell's interior. 2) The role of PAR signaling in the vessel wall in the formation and maintenance of blood vessels in the embryo and adult. 3) The role of protease signaling in regulating the behavior of epithelia, organized sheets of cells that separate compartments and play critical roles for the formation and function of the heart, lungs, kidneys, gut, liver, endocrine and exocrine glands, brain, skin and other organs. Success in our studies will advance our understanding of PARs themselves and of the large family of receptors to which they belong (G protein-coupled receptors - key regulators and drug targets). It will also advance our understanding of the mechanisms that govern the formation and function of blood vessels and epithelia, with potential broad impact.