Conditional Disruption of the Murine Prothrombin Gene.

Thrombin is the core protease in the hemostatic system. Thrombin directs thrombus formation through the proteolytic conversion of fibrinogen to fibrin and the local activation of protease-activated receptors on platelets and other cells. In addition, thrombin controls the coagulation system through the activation of fXI, protein C and other key hemostatic factors. Interestingly, the biological role of thrombin is seemingly not limited to the maintenance of vascular integrity. There is appreciable evidence that thrombin-mediated proteolysis plays an important role in development, the inflammatory response, tissue repair, tumor cell metastasis and other physiological and pathological processes. Unfortunately, the embryonic and perinatal lethal phenotype previously described in prothrombin null (fII^−/−) mice limited the utility of those knockout animals in better defining the larger role of fII in vivo. In order to develop the means to explore the importance of thrombin in disease processes within adult animals, a mouse line was generated carrying a conditional (“floxed”) fII knockout allele (fIIfx mice). Homozygous fIIf^x/f^x mice and compound heterozygous mice carrying one fII floxed allele and one fII null allele (fII^fx/− mice) developed to term, were present in offspring in the expected Mendelian frequencies, survived to adulthood and retained normal reproductive success. In the absence of Cre-mediated recombination, fII^fx/− mice maintained circulating fII levels that were low (approximately 10% of normal), but spontaneous bleeding events were never encountered in these animals. Studies of fII^fx/− mice carrying a Cre recombinase transgene known to be constitutively expressed in the liver showed that prothrombin levels can be reduced to levels incompatible with post-natal survival. More sophisticated studies using the polyI:C-inducible Mx-Cre system revealed that unchallenged Mx-Cre⁺/fII^fx/− mice consistently survived to adulthood. However, induction of Cre under conditions that result in near-complete recombination of target floxed alleles within the liver resulted in the development of spontaneous bleeding events and death within 7 days. Multiple sites of hemorrhage were evident in these challenged adults, including lower gastrointestinal and intracranial sites of bleeding. Immunological analysis of plasma collected from these animals revealed that they carried levels of fII below current detection limits (< 1% of normal). Studies are underway to establish both the lowest level of plasma fII compatible with long-term survival and define the effects of extremely low fII levels on disease processes in vivo.