Thrombomodulin (TM) has been defined in the context of the blood coagulation system as an endothelial thrombin receptor that suppresses the activation of the blood clotting mechanism. Formation of a thrombin- TM complex alters the substrate specificity of thrombin to generate the natural anticoagulant, activated protein C. In humans, genetic deficiencies leading to the reduced function of the TM-protein C pathway are commonly inherited risk factors for thrombotic disease. The complete ablation of TM function in mice via gene targeting results in early embryonic lethality, before the establishment of a functional cardiovascular system. Biochemical and genetic evidence indicates that the receptor's survival function in development does not involve the known anticoagulant mechanisms of protein C activation or thrombin binding, but a potentially novel activity that is absolutely required for a normal interaction between the developing fetus and the maternal environment. These findings suggest that TM has additional functions not related to blood coagulation that may fulfil important roles in yet other physiological processes, as suggested by the highly regulated expression pattern of the receptor in the developing lung, bone forming tissue, and in other non-endothelial cell types of adult mice. The objective of the proposed studies is to describe in precise molecular terms the mechanism underlying TM function in development and to delineate the physiological consequences of complete receptor deficiency in different stages of embryogenesis and in adult mice. Specifically, the survival of TM deficient mice will be examined in the complete absence of fibrin or fibrinolytic mechanisms to ascertain whether the death of TM null mice involves the formation or dissolution of blood clots. Critical structural domains of TM required for survival and interaction with intracellular pathways will be delineated by testing receptor mutants for their ability to support embryonic viability. Components interacting with these domains will eventually be identified with biochemical methods. The suspected role of TM in later stages of development will be determined by prolonging the survival of TM knock- out mice via a stage- and cell type-restricted restoration of TM expression in selected embryonic tissues. The cell type restricted ablation of the receptor gene will be employed to evaluate the proposed role of TM in lung morphogenesis and to examine in vivo the consequences of complete receptor deficiency in vascular endothelium and vascular smooth muscle cells.
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