Deficiencies in several different components of the hemostatic mechanisms are commonly found in newborn infants, particularly those born prematurely, and are associated with a number of serious and often fatal disorders. The condition disseminated intravascular coagulation (DIC) constitutes part of the underlying pathology of many bleeding abnormalities, apparently because blood clotting proteins are consumed and are not adequately replaced. In order to develop specific therapies for treating these coagulopathies, it is essential to understand the regulation of synthesis of procoagulant and anticoagulant proteins, almost all of which are made in the liver. Fibrinogen is particularly important to study because it is the precursor of fibrin, which is absolutely required for forming the structural network of a clot, and it is depleted from the plasma in DIC. The research program described in this proposal is directed toward characterizing the induction of fibrinogen synthesis by glucocorticoid hormones, which act directly on the liver to increase the mRNA levels for the three subunits of fibrinogen. These investigations utilize the frog Xenopus laevis because in this organism it is possible to induce fibrinogen synthesis from low basal levels in purified liver parenchymal cells maintained under fully-defined culture conditions. Work in progress will determine whether the hormone increases steady state mRNA levels by enhancing transcription or messenger stability. The experiments outlined here will determine the underlying molecular mechanisms by defining the specific regions of the fibrinogen genes necessary for hormonal stimulation. This will be accomplished by developing a gene transfection system in which regulated expression of exogenously- added cloned fibrinogen genes can be achieved. This powerful technology will allow functional regulatory elements to be identified and subsequently mapped in fine detail by site-directed mutagenesis. Complementary studies will be carried out to locate specific glucocorticoid receptor binding sites near the fibrinogen genes, in order to correlate them with the activation of the genes. One of the major goals is to ascertain whether the coordinate induction of the three subunits is due to the hormone acting by precisely the same mechanism on the separate genes. The Xenopus system also has potential for future investigations into liver differentiation and the acquisition of competence to express the fibrinogen genes. Experimentation in this system has relevance to development, maturation, and hormonal regulation of hemostatis.
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