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. The biological role of prothrombin in blood coagulation has been extensively studied while other biological roles remain to be elucidated. The role of prothrombin in mammalian development is unknown. Gene targeting provides an efficient way to address the biological roles of prothrombin. The applicants have recently generated mice with the prothrombin gene deleted in either a hemi- or homozygous state and now are able to address the biological properties of prothrombin.
In Specific Aim 1 the hemi- and homozygous prothrombin deficient mice will be characterized. Tissues and blood from mice or embryos will be characterized by histochemical, northern, western and hematological analyses including specific functional assays.
In Specific Aim 2 it will be determined whether liver-specific expression of prothrombin is sufficient for correction of the phenotype observed in prothrombin deficient mice. The liver is the major site of synthesis of prothrombin and therefore the albumin promoter/enhancer region will be used to express prothrombin in a liver-specific manner in transgenic mice. Mice generated from crosses with these transgenic mice and mice hemizygous for the knock-out allele will be used to test whether liver-specific expression is sufficient to correct the phenotype of null mice and for the development and survival of mice of adults. Experiments proposed in Specific Aim 3 involve correction of the phenotype of prothrombin deficient mice by the introduction of a knock-in vector containing the prothrombin cDNA under control of the endogenous prothrombin promoter and regulatory regions of the gene into ES cells containing one allele of the knock-out vector. For the structure-function studies proposed in Specific Aim 4, a vector is needed that will correct the phenotype of these mice in the event that the liver-specific transgene does not.
In Specific Aim 4 the mouse model of prothrombin deficiency will be used to test a mutant form of prothrombin. From in vitro studies of others, it has been determined that mutation of Gln344 results in a significant change in the ratio of clotting to anticoagulant activity of prothrombin. A mutagenized vector including the mutation at residue 344 will be electroporated into ES cells. After germ-line transmission is determined, hemi- and homozygous mice containing the mutation will be obtained and characterized.
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