.) The long-term goal of this research is to understand the role of the coagulation system in development. Considerable evidence exists from knock-outs of components of that system that it has an important role in embryogenesis, including evidence from PAR-1 knock-out, which leads to the demise of approximately half of PAR1-deficient embryos at aboutE9.5. Death of these embryos was associated with bleeding but could not be attributed to defects in platelet function, implying that thrombin signaling may have a role in embryonic development unrelated to its role in hemostasis, at least in the usual sense. In situ hybridization revealed PAR1 mRNA expression in endocardial and endothelial cells at E9.5, suggesting that PAR1 signaling might be important for normal blood vessel development and vascular integrity. The Principal Investigator postulates that the coagulation cascade functions in part as a """"""""leak detector"""""""" that monitors and regulates the integrity of developing blood vessels. To test these hypotheses he proposes the following specific aims.
Aim 1 will ask the question: In which cell type(s) is PAR1 signaling important for embryonic development? To address this question they will: a) Use a lacZ knock-in to identify the cell types that express PAR1 and determine their fate in PAR1-deficient embryos; b) Characterize vascular development in PAR1-deficient embryos; c) determine by transgenic rescue whether lack of PAR1 function in endothelial cells is the primary defect in PAR1-deficient embryos; d) Use knock-in of gain-of-function mutations to identify the cells in which PAR1 activation normally occurs during embryonic development; e) Test the hypothesis that disinhibited thrombin production and PAR1 signaling are responsible for the death of thrombomodulin-deficient embryos.
In Aim 2, they ask the question: Beyond PAR1, what are the other effectors of the coagulation cascade during embryonic development? They will: a) Test the hypothesis that fibrinogen becomes important in the absence of PAR1, b) Determine whether PAR4 and/or as yet unidentified receptors provide """"""""back-up"""""""" thrombin signaling in embryonic development, c) Determine the effect of PAR2 deficiency in combination with other PAR-deficiencies on embryonic development.
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