Prothrombin plays a central role in both the processes of coagulation and anti-coagulation in the blood of mammals, is involved in the stimulation of platelets, is chemotactic for macrophages, and is involved in regulating the proliferation of endothelial cells and other cell types. Thus, prothrombin may play a crucial role in the development of cardiovascular disease due to its diverse functions as an important enzyme in the coagulation cascade and as a mitogen for specific cell types, especially endothelial cells. The biological role of prothrombin in blood coagulation has been extensively studied while other biological roles remain to be elucidated. We have recently generated mice with the prothrombin gene deleted in either a hemi- or homozygous state and we now are in a unique position to address the biological properties of prothrombin.
Specific Aims 1 and 2 will test the hypothesis that the expression of prothrombin in tissues other than the liver is required to maintain a normal phenotype in the mouse. This will be tested using two different approaches: generation of transgenic mice with liver-specific prothrombin expression (Specific Aim 1) and the generation of """"""""knock-in"""""""" mice to correct the prothrombin-deficient phenotype (Specific Aim 2).
Specific Aim 3 involves determination of the biological consequences of the tissue-specific deletion of prothrombin in the central nervous system. It is proposed to specifically ablate the expression of prothrombin in astrocytes and neurons. Due to the fact that prothrombin has a role in embryogenesis, the functional consequences of prothrombin loss at later stages of development and/or in the adult will be examined in mouse models using a conditional knockout strategy, which can be designed to bypass the embryonic lethality.
Specific Aim 4 addresses whether a change in the substrate specificity of thrombin results in thrombosis in mice. Prothrombin has been studied extensively by site-directed mutagenesis and by in vitro analysis of the resultant mutant proteins. Based on the high ratio of clotting to anticoagulant activity of a mutation at G1n344 as determined in vitro by others, this mutant form of prothrombin has been chosen for the studies proposed in this specific aim. Thus, a mutation in this region of prothrombin will be introduced into the mouse to study the in vivo consequences of this substitution in the whole animal. A better and more complete understanding of the coagulation process and the role that proteins involved in this process play in other biological functions is crucial for the treatment of patients with bleeding or thrombotic problems that result from physiological problems, surgery or from treatment with pharmaceuticals. Mouse model systems, such as those proposed in this application, may be useful for studying this complex system.
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