Antibody-mediated Targeting of the Urokinase-type Plasminogen Activator Proteolytic Function Neutralizes Fibrinolysis in Vivo

Abstract

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using genetargeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo. Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using genetargeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo. Tissue remodeling and cell migration are dependent on the confined degradation of proteins in the extracellular matrix and basement membranes, which is accomplished by the concerted action of several proteolytic enzymes, including those of the plasminogen activation and the matrix metalloproteinase systems (1Danø K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (398) Google Scholar, 2Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5244) Google Scholar). The two main proteases accomplishing plasminogen activation, i.e. conversion of plasminogen to the active broad spectrum serine protease plasmin, are urokinase-type plasminogen activator (uPA) 2The abbreviations used are: uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; uPAR, uPA receptor; mAb, monoclonal antibody; ATF, aminoterminal fragment of uPA; LMW-uPA, low molecular weight uPA; PrAg-U2, uPA-activable anthrax toxin-protective antigen-U2; FP59, fusion protein 59; PBS, phosphate-buffered saline; TNP, trinitrophenyl; SPR, surface plasmon resonance; ELISA, enzyme-linked immunosorbent assay; MSV, mouse sarcoma virus. and tissue-type plasminogen activator (tPA). At the molecular level, uPA is secreted as an inactive single-chain protein, pro-uPA, which becomes activated by proteolytic cleavage believed to be primarily performed by plasmin (3Ellis V. Scully M.F. Kakkar V.V. J. Biol. Chem. 1989; 264: 2185-2188Abstract Full Text PDF PubMed Google Scholar, 4Petersen L.C. Lund L.R. Nielsen L.S. Danø K. Skriver L. J. Biol. Chem. 1988; 263: 11189-11195Abstract Full Text PDF PubMed Google Scholar). The simultaneously occurring reciprocal zymogen activation reactions, composed of pro-uPA activation by plasmin and plasminogen activation by uPA, constitute a positive feedback mechanism, which is regulated at several levels. The naturally occurring inhibitors include serpins and in particular the plasminogen activator inhibitor-1 (PAI-1) for uPA and α2-antiplasmin for plasmin (1Danø K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (398) Google Scholar). Although the plasminogen activation processes can be focused to the cell surface by the uPA receptor (uPAR) and plasminogen-binding proteins (5Ellis V. Behrendt N. Danø K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar), additional functions of uPA seem to rely on a multifaceted array of interactions with cellular receptors, adhesion molecules, and extracellular matrix proteins (6Andreasen P.A. PubMed Scopus Google Scholar). role for uPA-mediated plasminogen activation in tissue remodeling processes and by studies of L.R. Ploug M. K. J. Nielsen Werb Z. Danø K. Rømer J. J. PubMed Scopus Google Scholar, Rømer J. Danø K. 93: PubMed Scopus Google Scholar, J. Lund L.R. Danø K. Nat. 2: PubMed Scopus Google Scholar), J. Danø K. Werb Z. Lund L.R. PubMed Scopus Google Scholar), and Lund L.R. 2005; PubMed Scopus Google in mice. The functions of the of uPA, tPA, have with fibrinolysis a of plasminogen activation of and plasminogen with Thromb. Haemostasis. 2005; 93: PubMed Scopus Google Scholar, L. L. J. PubMed Scopus Google Scholar). the in uPA and in physiological using mice. of of the plasminogen activation i.e. uPA L. L. J. PubMed Scopus Google Scholar), L. L. J. PubMed Scopus Google Scholar), and Rømer J. V. Danø K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. G. L. J. PubMed Scopus Google Scholar), by targeting not the of the as by and normal with to and In mice with in uPA and are by and L. L. J. PubMed Scopus Google Scholar). whereas a but in is in mice with in uPA, the of of the plasminogen activator the process L.R. Ploug M. K. J. Nielsen Werb Z. Danø K. Rømer J. J. PubMed Scopus Google Scholar, Rømer J. Danø K. 93: PubMed Scopus Google Scholar). studies on hepatic fibrinolysis in mice have in in uPA;tPA and double deficient mice with those in mice Rømer J. Danø K. 93: PubMed Scopus Google Scholar, L. L. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). the deficient mice, only the uPA mice developed hepatic and only Rømer J. Danø K. 93: PubMed Scopus Google Scholar, L. L. J. PubMed Scopus Google Scholar). that a of uPA and to the physiological of of and of Rømer J. Danø K. 93: PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar, L. M. J. Biol. PubMed Scopus Google Scholar). the of protease and the the plasminogen and with extracellular proteases as the matrix of the of uPA in mouse of including and have to normal physiological tissue remodeling and several of evidence the that plasminogen activation by uPA is a in these proteolytic (1Danø K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (398) Google Scholar, P.A. PubMed Scopus Google Scholar). the of the in vivo uPA have used directed against the of uPA to murine uPA a M. N. V. K. Thromb. Haemostasis. PubMed Scopus Google Scholar), is a for inhibitors of mouse uPA with in vivo. To be to uPA-mediated in physiological we have now murine inhibitory monoclonal antibodies (mAbs) against mouse murine uPA for of the in vivo on uPA functionality in a of murine In the we performed a and of two murine uPA mAbs epitopes located on the B-chain of uPA and of action on uPA The in vivo of was by systemic administration to mice treated with a anthrax uPA-mediated activation, and the potent was used for of the physiological of of uPA activity in mice. proteins by of a The of and and have in Ploug M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google for the murine pro-uPA pro-uPA the murine fragment of uPA and murine (i) active murine uPA (ii) murine with the proteolytic cleavage i.e. and low molecular weight by of from a of mouse sarcoma as K. V. L. Nielsen L.S. PubMed Scopus Google Scholar). The proteins from the and plasmin the and murine tPA, PAI-1, uPA, and The and from The uPA-activable anthrax toxin-protective and the fusion protein of the anthrax to the of from and of of mice of and including to the and to and double deficient mice as well as performed as Rømer J. Danø K. 93: PubMed Scopus Google Scholar, L. L. J. PubMed Scopus Google Scholar, Rømer J. V. Danø K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). at the The by of L.R. Ploug M. K. J. Nielsen Werb Z. Danø K. Rømer J. J. PubMed Scopus Google Scholar). The in this to and by the mice to be of murine in with the of for of used for and at the of the tissue and performed by of the mouse and mice deficient in uPA L. L. J. PubMed Scopus Google used for at with by of to the of the cell of recombinant mouse pro-uPA was in in of with for in of phosphate-buffered for The for of from mice and of from the mice as well as the fusion and murine performed as J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google Scholar), that in the we used uPA-deficient mice. antibodies specific for mouse uPA by The of the mAbs by the mouse and of mAbs from the as J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google Scholar), that of mAbs for in vivo was performed using in which in J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google additionally with and The of mU1, mU3, and the monoclonal J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google for using the to the and to be level of mouse The for antibodies against mouse uPA was performed by as J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google Scholar), that the with recombinant mouse pro-uPA in of in i.e. and from two mice was used as positive as a for was at in a with at on a used to (i) a of the for antibodies uPA, (ii) the of of the anti-uPA mAbs on mouse uPA, and the and for of the of the these the proteins in and on by to the of was performed by of from which cell by a at and as J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google that the proteins mouse and pro-uPA and a of uPA and of on uPA of the mAbs performed by of the in on the and of pro-uPA by an of an of of the anti-uPA was at a low mU3, and a of murine pro-uPA uPA was at a of at from a cell as well as as of the using the The and by of the to a to of the of the The used in Western pro-uPA and the murine proteins pro-uPA, uPA, LMW-uPA, ATF, tPA, and by uPA with a of at as well as the uPA ATF, and by was performed not and Western was performed as J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google Scholar), that the with anti-uPA and uPA was was a positive a of from two mice, in was of the on the surface of murine was performed as J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google with was with pro-uPA, mU1, mU3, and to for at to the for a at The are from at used to on mouse (i) uPA-mediated plasminogen activation, (ii) plasmin-mediated pro-uPA activation, and pro-uPA and plasminogen The kinetic assays performed and to Behrendt N. K. P.A. Danø K. J. PubMed Scopus Google for the The in with of The reactions, performed at by of and as the and conversion was by the at for to in a activity with the activity of the protease in the of at the by in and using uPA-mediated mouse uPA, and plasmin in the of the of the antibodies mU3, and inhibitors as mouse pro-uPA, plasmin, and uPA in the of mU1, mU3, inhibitors as The was used in a of this the of the antibodies against the activity of performed by mouse uPA with in the of mU1, mU3, and mouse pro-uPA, and in the of the of mU1, mU3, The uPA inhibitors and as well as the plasmin inhibitors α2-antiplasmin and as in the assays mice mU1 mU3, in PBS, by from which was as well as of the The of the mAbs by as that was to of mouse The of the mAbs from the of the the assays in performed using the murine and as J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. 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The was in a cellular as mU1 mU3 blocked the of to on the surface of murine the an in of was in the of resulting from and To a of the of mU1 and mU3 for pro-uPA and uPA, we using Both antibodies to for pro-uPA and uPA, i.e. in the low with pro-uPA and active uPA, mU3 a for the of the anti-uPA mAbs to pro-uPA and uPA for the the mAbs and mouse pro-uPA mouse uPA by from used to the and i.e. for mU1, pro-uPA and uPA, and for mU3, pro-uPA and and the and from at and used to the The are that the of pro-uPA and active uPA derived from cellular and be with to that the of pro-uPA and active uPA derived from cellular and be with to that the of pro-uPA and active uPA derived from cellular and be with to in a of an enzyme kinetic to the conversion of a plasmin we first the of mU1 and mU3 on uPA-mediated plasminogen in and antibodies uPA-catalyzed plasminogen activation in a mU1 at a of whereas mU3 was with and The was by of the not with naturally occurring inhibitors as a of uPA be in the with whereas the plasmin activity be by of the two plasmin α2-antiplasmin and not a low molecular weight uPA a of was to be to uPA activity the not activation of pro-uPA is an in the plasminogen activation an of this be a highly for of the in a we the of the mAbs against plasmin-mediated pro-uPA activation using a uPA In this mU1 to be of the activation of pro-uPA in a as low as mU3 in in this with as that be with and directed against the activity of the uPA, whereas a was with of α2-antiplasmin and to plasmin not In a we used a with pro-uPA, and the plasmin including of the two activation N. K. P.A. Danø K. J. PubMed Scopus Google Scholar). In this mAbs of the and with the these the that mU1 was against activation for activity against uPA to In the assay, of α2-antiplasmin and the plasmin activity not whereas of the resulting uPA as an of and we to a of the catalytic of uPA was in the this we the of the mAbs against the activity of uPA with the uPA was that mU3 on this whereas of mU1 was only in the the of mU1 to include to a blocked with plasminogen at the active and plasmin-mediated pro-uPA activation, whereas the of mU3 is only directed against the uPA-mediated plasminogen mU1 with uPA-activable anthrax in which is dependent on a specific cleavage of an i.e. PrAg-U2, by uPA J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google was used to the of mU1 and mU3 in and in vivo. on uPA functionality activation, catalytic and with be in a cellular In the of of murine to and the recombinant FP59, which to becomes to cell on the of mU1 and mU3, we that these mAbs the of uPA in this cellular a of treated with the uPA-activable anthrax protoxin was of the with of the antibodies The of functions by mU1 the level of the positive mouse ATF, which proteolytic and to with uPA for to on the this a inhibitory of mU1 on uPA catalytic activity in this in vitro, whereas the with mU3 was have uPA mice to be to with of PubMed Scopus Google Scholar), uPA activity as an for activation of and of in vivo. To the in vivo of mU1 and mU3, we administration of the in to with an of FP59, the by this in mice. was by an with administration of mU1 and mU3 to mice the of the two mAbs in to be and not of the resulting in two mU1 the at to the mice the anthrax was of of the mice treated with mU1 by an anthrax whereas only of of of the two and and not to the of mU3 and including mU1 as that was administration of mU3 to the mice of mice of uPA-deficient mice anthrax to the uPA of the of these mice by the not with PubMed Scopus Google Scholar). we additional i.e. mice only mU1 mice and that administration of mU1 and mU3 not that mU1 in vivo can the by the uPA anthrax by activation of and of mU1 of in targeting of uPA and in of in the and this is in uPA deficient mice Rømer J. Danø K. 93: PubMed Scopus Google Scholar, L. L. J. PubMed Scopus Google Scholar). To the of mU1 on a uPA process in we the on hepatic by systemic of mice. in a we the of hepatic in mice, including i.e. uPA and and uPA;tPA double deficient mice. In the of uPA;tPA double deficient mice, we as for the double deficient mice in the Rømer J. Danø K. 93: PubMed Scopus Google Scholar). with the and that in and and uPA deficient mice, the and of in uPA;tPA double deficient mice to was to the activity of uPA functions by mU1 mice treated with mU1 To of the i.e. in with the a of mU1 by two of mU1 was to and mice the of was to the mice. the of the by mU1 in at the of on weight of the mice not systemic administration of mU1 in a in hepatic in mice with as by of the of The in mice treated with mU1 was of The level of in the mice was only and not from the level in uPA;tPA double deficient mice of mice treated with mU1 not in hepatic with mice deficient mice. that the in the of mice by a specific of uPA-dependent plasminogen activation and not a of to plasminogen activator In with this studies have the of in a of as well as mice with an on hepatic fibrinolysis J. Høyer-Hansen G. Lund L.R. Nielsen Danø K. Rømer J. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). At the these the role of uPA and tPA, which specific uPA targeting in a The of the was to the of targeting the proteolytic activity of uPA in mice. was using murine monoclonal antibodies directed against murine uPA and of uPA-mediated plasminogen activation with uPA receptor monoclonal mU1, a inhibitory on uPA functionality in and in an that of the plasminogen activation i.e. uPA-catalyzed plasminogen activation and activation of the uPA Although a of the have for of inhibitory against the of uPA, only a have of murine uPA a M. N. V. K. Thromb. Haemostasis. PubMed Scopus Google the that the are for studies in murine antibodies against uPA be to this the of murine antibodies is by of against the murine whereas antibodies in to (1Danø K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (398) Google Scholar, K. Lund L.R. J. Nielsen Danø K. Rømer J. Johnsen M. J. Cancer. 2005; PubMed Scopus Google Scholar). our using immunization of uPA-deficient mice with murine uPA, was to The of this was by a by M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google on of mouse that the of murine mAbs with using mice. The in the extracellular proteases and inhibitors is a for the and of tissue In a of physiological including K. PubMed Scopus Google Scholar), L.R. Ploug M. K. J. Nielsen Werb Z. Danø K. Rømer J. J. PubMed Scopus Google Scholar, Rømer J. Danø K. 93: PubMed Scopus Google Scholar, L. M. J. Biol. PubMed Scopus Google Scholar), and hepatic Rømer J. Danø K. 93: PubMed Scopus Google Scholar), in uPA to and of these physiological processes to rely on with those of proteases as and the matrix In the that systemic administration of mU1 a of in the in mice, a physiological that of the of uPA;tPA double deficient mice Rømer J. Danø K. 93: PubMed Scopus Google Scholar, L. L. J. PubMed Scopus Google Scholar). our that a monoclonal that uPA catalytic activity is of the in mice. we have that a murine mAb, directed against the mouse uPA receptor and to J. Lund Lund L.R. Ploug M. Rømer J. Danø K. Høyer-Hansen G. Thromb. Haemostasis. PubMed Scopus Google can be used for systemic of mice to a phenotype mimicking that of double deficient mice Rømer J. Danø K. 93: PubMed Scopus Google Scholar, J. Høyer-Hansen G. Lund L.R. Nielsen Danø K. Rømer J. J. Thromb. Haemostasis. PubMed Scopus Google Scholar). an by N. 2: PubMed Scopus Google that mice, treated with a directed against murine by as with with our the inhibitory was of the N. 2: PubMed Scopus Google Scholar). The by these on of the resulting in activity of the protein. a the in our is that to the of the uPA and proteolytic systems in the mice, the of uPA activity in these mice. Hence, can be of of models, a of the functionality of the protein as a of as and of of functions with the protein. of extracellular proteases is an to (1Danø K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (398) Google Scholar, P.A. PubMed Scopus Google Scholar). of studies K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (398) Google Scholar, M. N. V. K. Thromb. Haemostasis. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, and N. K. M. V. M. Thromb. Haemostasis. PubMed Scopus Google have in particular focused on uPA as a and that in several of (i) uPA is (ii) uPA is a and and uPA, with PAI-1, is a for to the of uPA by of the uPA in mice by administration of against uPA to in K. Lund L.R. J. Nielsen Danø K. Rømer J. Johnsen M. J. Cancer. 2005; PubMed Scopus Google Scholar, J. M. V. M. Thromb. Haemostasis. 2005; 93: PubMed Scopus Google Scholar, 2002; Google Scholar, K. M. PubMed Scopus Google Scholar, J. PubMed Google Scholar). uPA to a role and and with the of an phenotype of uPA-deficient mice, this as an with to be evidence demonstrating uPA and receptor to be by of at the of the (1Danø K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (398) Google Scholar). the of as antibodies against uPA in is by of against mouse uPA derived from the cell of the and (1Danø K. Behrendt N. Høyer-Hansen G. Johnsen M. Lund L.R. Ploug M. Rømer J. Thromb. Haemostasis. 2005; 93: 676-681Crossref PubMed Scopus (398) Google Scholar). The of with monoclonal antibodies on antibodies directed against cell proteins the receptor of a PubMed Scopus Google Scholar). In only a studies have the of mAbs targeting the catalytic activity of an this is highly to mU1 murine for of in a of murine The used in this can be by administration of of inhibitors directed against two several of the plasminogen and the matrix metalloproteinase In the administration of an be to the of mice. whereas the the of of the protein in the of specific mAbs for the of of several functions of the protein. mU1 blocked the activity of uPA the which is for the of the uPA (6Andreasen P.A. PubMed Scopus Google Scholar). Although be used to a these are highly and antibodies can be in studies in which two several are to double deficient the is for of the of the protein normal physiological tissue remodeling processes as well as using administration of the as well as in with the of the of the mAbs is In this is highly that the administration in our in mice which the are at In we have in highly potent murine inhibitors of mouse uPA and simultaneously we have a of in vivo of of in mouse systems in which uPA plays a We that inhibitory murine anti-uPA mAbs can be as to uPA functions in vivo normal and tissue remodeling processes. The of L. M. M. K. L. and M. is We and of for with and as well as J. for with and J. for with the of the with