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The three-domain GPI-anchored urokinase receptor (uPAR) plays a key role in the focalization of pericellular plasminogen activation through its binding of urokinase (uPA). To enable in vivo studies investigating the effect of inhibiting the uPA-uPAR interaction in murine models, we have generated murine mAbs directed against murine uPAR (muPAR) by immunizing uPAR deficient mice with recombinant muPAR. We have selected four mAbs (mR1-mR4), of which mR1 and mR3 recognize epitopes in the N-terminal domain I of muPAR, while mR2 and mR4 recognize epitopes in domain II-III of muPAR. mR1, mR2, and mR4 cross-react with the human form of uPAR, thus recognizing epitopes conserved between species. In cell binding experiments, mR1 and mR4 antagonize the binding of 125I-mATF (amino-terminal fragment of muPA) to muPAR expressing cells with IC50 values of 0.67 nM for mR1 and 0.40 nM for mR4, compared to 0.14 nM for unlabeled mATF. mR3 binds domain I without interfering with uPA binding. Interestingly, mR1, mR3, and mR4 did not prevent vitronectin binding to muPAR. Thus, in immunocytochemistry experiments, mR3 stained intact muPAR independent of mATF binding, while mR1 could not stain intact muPAR after preincubation of the cells with mATF. The N-terminal domain I in muPAR can be liberated and mR4 only recognizes this form if the cells has been preincubated with mATF. Hence, our murine anti-muPAR mAbs have the ability to selectively recognize different muPAR populations on the surface of murine cells. In vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of an uPA-activatable anthrax toxin. Another in vivo approach demonstrated that mR1 treated tPA deficient mice mimicked the hepatic fibrin deposits of uPAR;tPA double deficient mice. Thus, mR1 is an efficient in vivo antagonist of the muPA-muPAR interaction and well suited for therapeutic intervention.