Plasminogen Activator Inhibitor-1 (PAI-1) is the main physiological regulator of tissue-type plasminogen activator (tPA) in normal plasma. In addition to its critical function in fibrinolysis, PAI-1 has been implicated in other physiological and pathophysiological processes. Interestingly, both antifibrinolytic-related and non-antifibrinolytic-related roles of PAI-1 function have been implicated in the process of angiogenesis, which might account for some discrepancies observed in angiogenic models using PAI-1 deficient and over-expressing transgenic mice. To investigate the structure-function relationships of mouse PAI-1, the recombinant PAI-1 proteins were expressed in Escherichia coli and characterized. Our studies indicated that the complex interactions traditionally associated with different functions of human PAI-1 apply to the murine system, thus demonstrating a commonality of subtle functions among different species and evolutionary conservation of this protein. In an effort to separate these functions in vivo, knock-in targeting vector with targeted mutations to ablate the vitronectin binding ability was constructed. The composition of the targeting vector includes the 5' and 3' flanking sequences containing the mutation, a LoxP-FLT-Neo-FLT-LoxP cassette, and a CDA expression cassette. After homologous recombination with the WT allele, the "floxed" cassette may be removed by crossing with either Cre recombinase or flipase expressing transgenic mice. A cell culture-free system was developed to examine the cre-recombinase-mediated excision of the "floxed" element before the targeting vector was introduced into the embryonic stem cells. In order to bypass the lengthy backcrossing process from the 129 background into the C57BL/6 background, the homologous arms in the targeting vector were constructed using a C57BL/6 genetic background to facilitate the backcrossing. The targeting vectors were introduced into both C57 and C57/129 hybrid embryonic stem cells by electroporation, and the homologous recombination was screened by Southern Blot analysis. Positive ES cell clones with homologous recombination at both arms were identified. These PAI-1 knock-in mice would serve to further elucidate mechanisms associated with the observed phenotypes in PAI-1 deficient mice in a number of challenge models.