Proc. Natl. Acad. Sci. USAVol. 93, pp. 5899-5904, June 1996Medical Sciences

Urokinase-type plasminogen activator is effective in fibrinclearance in the absence of its receptor or tissue-typeplasminogen activator

(urokinase receptor/gene targeting/fibrinolysis/wound healing)

THOMAS H. BUGGE*t, MATTHEW J. FLICK*, MARY JO S. DANTON*, CYNTHIA C. DAUGHERTYt, JOHN R0MERt,KELD DANOt, PETER CARMELIET§, DESIRIE COLLEN§, AND JAY L. DEGEN*¶*Divisions of Developmental Biology and SPathology, Children's Hospital Research Foundation, Cincinnati, OH 45229; tFinsen Laboratory, Rigshospitalet,DK-2100 Copenhagen 0, Denmark; and §Center for Transgene Technology and Gene Therapy, Vlaams Interuniversitair Instituut Biotechnologie, KU Leuven,B-3000 Leuven, Belgium

Communicated by Earl W Davie, University of Washington, Seattle, WA, February 27, 1996 (received for review November 16, 1995)

ABSTRACT The availability of gene-targeted mice defi-cient in the urokinase-type plasminogen activator (uPA),urokinase receptor (uPAR), tissue-type plasminogen activator(tPA), and plasminogen permits a critical, genetic-basedanalysis of the physiological and pathological roles of the twomammalian plasminogen activators. We report a comparativestudy of animals with individual and combined deficits inuPAR and tPA and show that these proteins are complemen-tary fibrinolytic factors in mice. Sinusoidal fibrin deposits arefound within the livers of nearly all adult mice examined witha dual deficiency in uPAR and tPA, whereas fibrin depositsare never found in livers collected from animals lacking uPARand rarely detected in animals lacking tPA alone. This is thefirst demonstration that uPAR has a physiological role infibrinolysis. However, uPAR-/-/tPA-/- mice do not developthe pervasive, multi-organ fibrin deposits, severe tissue dam-age, reduced fertility, and high morbidity and mortalityobserved in mice with a combined deficiency in tPA and theuPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice donot exhibit the profound impairment in wound repair seen inuPA'-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasmin-ogen activation focused at the cell surface by uPAR is impor-tant in fibrin surveillance in the liver, but that uPA suppliessufficient fibrinolytic potential to clear fibrin deposits frommost tissues and support wound healing without the benefit ofeither uPAR or tPA.

Maintenance of vascular patency and the timely solubilizationof fibrin clots is a key function of the plasminogen (Plg)activation cascade and plasmin-mediated proteolysis (1). Ourfinding that Plg-deficient mice are predisposed to severethrombosis and secondary tissue damage but complete em-bryonic development, survive to adulthood, and are capable ofreproduction, suggests that fibrinolysis may be the only phys-iological process for which Pig is essential (2, 3). This conclu-sion is also supported by earlier studies of mice with combineddeficiencies in the two known Plg activators, tissue-type Pigactivator (tPA) and urokinase-type Pig activator (uPA) (4).Like Plg-/- mice, uPA-/-/tPA-/- mice suffer extensive spon-taneous fibrin deposition but develop to term, grow to adult-hood, and reproduce (4). Remarkably, despite the fact thattPA appears to be of primary importance in vascular fibrino-lysis via its fibrin binding property, mice lacking only tPAexperience essentially no thrombotic problems (4). Similarly,mice lacking only uPA are generally healthy, but these miceoccasionally develop hepatic fibrin deposits and ischemic

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rectal lesions (4). Together, these findings are consistent withthe simple hypothesis that Pig is fundamentally a fibrinolyticenzyme and both uPA and tPA are complementary proteasesthat are individually capable of effectively clearing spontane-ous vascular and extravascular fibrin.The value of two Plg activators in directing fibrinolysis might

be understood based on the unique biological properties oftPA and uPA. On the one hand, tPA, through its fibrin-bindingfunction (1), may be of primary importance in cell-independent fibrinolysis, such as the lysis of embolized clots(4). On the other hand, uPA, through its high-affinity cell-surface receptor (5-7), may primarily participate in cell-mediated fibrinolysis, such as fibrin solubilization in organizingwound fields. The uPA receptor (uPAR) provides a means ofprecisely focusing uPA-mediated proteolysis on the cell sur-face, and it is well-established that the rates of both pro-uPAand Pig activation in vitro are dramatically accelerated by thebinding of uPA to uPAR (6).

Based on these findings, one might anticipate that thephenotypes of uPAR-/- and uPA-/- mice would be similar.Furthermore, one might anticipate that the phenotype of micewith a combined deficit in both uPAR and tPA would besimilar to that of mice with a combined deficit in uPA and tPA(i.e., predisposition to severe spontaneous thrombosis; ref. 4).However, we recently generated uPAR-/- mice (8, 9) andthese animals appear phenotypically distinct from uPA-/-animals (4). Specifically, uPAR-/- mice never develop thespontaneous hepatic fibrin deposits, ischemic rectal ulcer-ations, or rectal prolapses observed in uPA-/- mice. However,in view of the obvious functional overlap between uPA andtPA, a clear understanding ofuPAR function in uPA-mediatedfibrinolysis may be obscured in mice expressing tPA.To rigorously explore the role of uPAR in fibrin clearance,

mice with both individual and combined deficits in uPAR andtPA have now been generated and compared to mice lackinguPA and tPA. We report that the loss of both uPAR and tPAresults in the accumulation of hepatic fibrin deposits in themajority of animals, whereas spontaneous fibrin deposits arenever seen in uPAR-/- mice and are rarely seen in tPA-/-mice. However, the thrombotic consequences of combineduPAR and tPA deficiency are remarkably small when com-pared to those seen in mice with combined uPA and tPAdeficiency. uPAR-/-/tPA-/- mice do not exhibit the wide-spread fibrin deposition, extensive multi-organ tissue damage,and severe impairment of wound healing observed in uPA-/-/tPA-/- and Plg-/- mice (2-4, 10). Taken together, these

Abbreviations: Pig, plasminogen; uPA, urokinase-type Plg activator;uPAR, urokinase receptor; tPA, tissue-type Pig activator.1Towhom reprint requests should be addressed at: Children's HospitalResearch Foundation, Children's Hospital Medical Center, TCHRFRoom 2025, 3333 Burnet Avenue, Cincinnati, OH 45229-3039.

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findings demonstrate that receptor-bound uPA plays a role infibrinolysis, but that uPA is effective in general fibrin clearanceand can support wound healing in a uPAR- and tPA-independent manner.

MATERIALS AND METHODSGeneration of uPAR'-/tPA-/- Mice. Mice deficient in

both uPAR and tPA were generated by crossing tPA-/- mice(4) and uPAR-/- mice lacking either exon 3 (8) or exons 2-5(9) followed by interbreeding of the hemizygous offspring. Thegenotypes of mice raised were established by either PCR orSouthern blot analysis of tail biopsy DNA using hybridizationprobes and oligonucleotide primer sets complementary to thenormal and targeted uPAR and tPA alleles (4, 8, 9). Thewild-type tPA allele was detected by PCR with the exon 10primers, tPAEx10-1 (5'-ACCTGTGGCCTGAGGCAGTA-CAAACGGCCT-3') and tPAEx10-2 (5'-TGGGCAGCTGA-CAGCACCCAGCAGGAACTG-3'). These primers generatea 179-bp PCR product (11). The targeted tPA allele wasdetected using two primers complementary to the neomycinresistance gene, Neo-1 (5'-CATGAGAGCAGCCGATT-GTCTGTTGTGCCC-3') and Neo-2 (5'-TTGAACAAGAT-GGATTGCACGCAGGTTCTC-3') (12). Together theseprimers generate a 96-bp PCR product. Alternatively, thetargeted and wild-type tPA alleles were identified by Southernblot hybridization analysis of EcoRI-digested genomic DNAusing a 2.2-kb XbaI-EcoRI tPA probe (probe b in ref. 4). Thewild-type uPAR allele was detected by PCR with the exon 3primers, uPAR-E3-5' (5'-GATGATAGAGAGCTGGAG-GTGGTGAC-3') and uPAR-E3-3' (5'-CACCGGGTCT-GGGCCTGTTGCAGAGGT-3'), which generate a 148-bpproduct (8). The targeted uPAR allele was detected using theuPAR intron 3 primer, uPAR-I3 (5'-TCATCAGTCCTCCCT-GCTAAGGGC-3'), and the hypoxanthine phosphoribosyl-transferase (HPRT) primer, OligoHPRT-3 (5'-TATTAC-CAGTGAATCTTTGTCAGCAGTTCCC-3'), which gener-ate a 272-bp PCR product (8). With the offspring of micelacking uPAR exons 2-5, the mutant and wild-type uPARalleles were identified by Southern blot hybridization analysisof XbaI-digested genomic DNA using a 0.2-kb StuI-XbaIprobe complementary to a region immediately downstream ofthe uPAR gene (probe a in ref. 9).

Histological Examination of uPAR-/-/tPA-/- mice. Micewere sacrificed under CO2 narcosis and tissues were fixed for24 h at 4°C in zinc-formalin fixative (United States Biotex,Webbville, KY). Fixed tissues were processed into paraffin andsectioned. Sections were either stained with hematoxylin/eosin or immunostained with anti-mouse fibrinogen antiserumusing the Vectastain Elite ABC system (Vector Laboratories)and nickel-enhanced diaminobenzidine as described (2).

Skin Wound Healing. Adult mice were anaesthetized byinhalation of 2% Isoflurane (USP, Ohmeda PPD, LibertyCorner, NJ) before the surgical incision. Full-thicknesswounds, 20 mm long, were made in the mid-dorsal skin.Immediately after surgery, the wounds were spindle-shapedwith well-separated incision edges. The wounds were neitherdressed nor sutured, and by the second day were covered by adehydrated wound crust. The mice were caged individually andthe progress of wound healing was determined by visualinspection. Healed wounds were defined as those with mac-roscopic closure of the incision interface and restoration ofepithelial covering. Both the surgery and evaluation of woundhealing was done by a blinded investigator.

Hematological Analysis. Mice were anaesthetized with 0.1ml per 30 g of body weight of ketamine/xylazine/aceproma-zine (4:1:1) and blood was collected from the inferior vena cavainto one-tenth vol of 0.129M sodium citrate. Blood cell counts,hematocrit and hemoglobin, were determined as described (2).Plasma was obtained from whole blood by centrifugation at

2500 x g for 10 min at 4°C. Thrombin-stimulated clotting timeof plasma in reaction mixtures containing equal volumes ofplasma and 20 units/ml of bovine thrombin in PBS wasdetermined as described (2). Casein-Plg zymography fordetection of plasma uPA and tPA activity was performed asdescribed (2) using 5 ,ul of plasma. Pig and fibrinogen weredetected by Western blot analysis using sheep anti-rat Pig anti-serum (2) and rabbit anti-mouse fibrinogen antiserum (13).

RESULTS

Viability and Growth of Mice Lacking Both uPAR and tPA.Mice with combined uPAR and tPA deficiency (uPAR-/-/tPA-/-) were born and appeared outwardly normal regardlessof whether the uPAR allele was disrupted by deletion of exon3 (8) or exons 2-5 (9). Like mice with individual deficits inuPAR-/- (8, 9) and tPA-/- (4), uPAR-/-/tPA-/- miceexhibited normal weight gain. The body weights of control,tPA-/-, uPAR-/-, and uPAR-/-/tPA-/- mice measured at8-11 weeks of age were 24.5 ± 4.1 g (n = 4), 26.0 ± 4.0 g (n =

32), 25.6 ± 1.7 g (n = 5), and 24.4 ± 3.4 g (n = 26), respectively.In contrast to uPA-/-/tPA-/- mice, which on average weigh lessthan one-third of control animals at 6 months of age (4), the bodyweights of uPAR-/-/tPA-/- mice older than 6 months wereessentially indistinguishable from control mice [29.2 ± 3.6 g (n =13) versus 30.8 ± 5.0 g (n = 10) for control animals].

It was shown earlier that mice with a single deficit in uPAR(8, 9) and tPA (4) transmit these mutant alleles to theiroffspring in a Mendelian inheritance pattern with no apparentloss of uPAR-/- or tPA-/- mice during development. Breed-ing data from mice with various combinations of mutant uPARand tPA alleles indicated that the transmission of the targeteduPAR and tPA alleles uniformly followed an independentMendelian inheritance pattern. For example, of 85 progenygenerated from uPAR+/-/tPA-/- breeding pairs, 21 (24.7%)were found to have a uPAR-/-/tPA-/- genotype, 40 (47.1%)were found to have a uPAR+/-/tPA-/- genotype, and 24(28.2%) were found to have a uPAR+/+/tPA-/- genotype.Thus, neither the individual nor combined loss of uPAR andtPA results in a failure to develop to term. If it is assumed thatthe principal role of uPAR is facilitating uPA-mediated Pigactivation, then these data are consistent with the earlierfinding that there is little or no fetal loss of either uPA-/-/tPA-/- or Plg-/- mice (2-4).Combined uPAR and tPA Deficiency Does Not Impair

Fertility. To examine the breeding behavior and fertility ofuPAR-/-/tPA-/- mice, 11 uPAR-/-/tPA-/- breeding pairswere monitored daily. Nine of 11 pairs mated within 6 daysbased on the finding of a vaginal plug and all of those females(separated from their mates at the time vaginal plugs wereobserved) delivered litters within the standard 3-week gesta-tion period. These data indicate that both male and femaleuPAR-/-/tPA-/- mice have excellent reproductive drive andsuccess, with little or no failures of pregnancy. The averagelitter size produced by uPAR-/-/tPA-/- breeding pairs (8.1 ±2.6; n = 13) was also similar to that observed with the controlbreeding pairs (8.4 ± 3.6; n = 25). Finally, no generalimpairment was found in uPAR-/-/tPA-/- females in gener-ating and raising multiple litters (data not shown). Thus,neither the individual loss of uPAR nor the combined loss ofuPAR and tPA appears to significantly diminish reproductivepotential or fertility. In contrast, the combined loss ofuPA andtPA results in a significant reduction in fertility (4), with aboutone-third of the breeding pairs never producing litters evenafter several months. Therefore, whatever the basis for thereduced fertility in Pig activator-deficient mice, it appears thatuPA alone, without the contribution of either uPAR or tPA,can sustain reproductive potential.

Pathological Consequences of the Loss of Both uPAR andtPA. Of 89 uPAR-/-/tPA-/- mice that could be monitored for

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 5901

more than 12 weeks, none developed overt lesions of any kindand only two died of unknown causes. Furthermore, nodisorders or additional fatalities were observed among 54 micethat could be monitored beyond 5 months of age, or among 17mice that could be monitored beyond 8 months. In contrast, ahigh incidence of visible rectal lesions or rectal prolapse wasfound in mice with a combined deficiency of uPA and tPA (7of 10 monitored to 19 weeks of age; P < 0.001) and micelacking uPA alone (4 of 9 mice monitored to 20 weeks of age;P < 0.001) when housed in parallel with the uPAR-/-/tPA-/-colony.A microscopic survey of tissues collected from 20 apparently

healthy uPAR-'-/tPA-/- mice ranging in age from 8 to 32weeks revealed the presence of multiple small fibrin depositsin the hepatic sinusoidal space of 16 (80%) of the animals; the4 mice that were apparently free of hepatic fibrin deposits wereamong the youngest analyzed (8-9 weeks old). These depositswere similar to those documented previously in uPA'-/tPA-/- and Plg-/- mice (refs. 2 and 4 and see below) and wererecognized as a fibrillar material in hematoxylin/eosin-stainedsections that stained strongly using a polyclonal antibodyraised against mouse fibrinogen (Fig. 1). In contrast, hepaticfibrin deposits were virtually never observed in uPAR-/- miceand were absent in 12 of 16 8-34-week-old tPA-/- miceexamined in parallel (Fig. 1). In the few tPA-/- mice wherefibrin deposits were encountered, they were extremely rare(e.g., one or two clusters). The fibrin deposits in uPAR-/-/tPA-/- and uPA-/-/tPA-/- mice were not associated withsignificant necrosis of adjacent tissue, but local hepatocytedropout was often apparent and the lesions occasionallycontained spindle-shaped cells, possibly infiltrating macro-phages (Kupffer cells) (for examples, see Fig. 1B). Thesedeposits were generally scattered but tended to be morefrequent near the liver capsule, implying that mechanicalsurface trauma may initiate the deposition of some fibrin. Theuneven distribution of fibrin deposits within the liver and thearbitrary nature of tissue sections and fields surveyed made aformal quantitative analysis complex, but as a semiquantitativemeans to compare the frequency of fibrin deposits in age-matched uPAR-/-/tPA-/- mice and uPA-/-/tPA-/- mice,the deposits were counted within randomly-selected surveyareas ('90 mm2; 60 high-powered fields) of two 3-month-oldmice of each genotype. Counts of 8 and 14 were obtained in thetwo uPAR-/-/tPA-/- animals, whereas counts of 60 and 93were established in the two uPA-/-/tPA-/- animals. There-fore, hepatic fibrin deposits are common in both uPA-/-/tPA-/- and uPAR-/-/tPA-/- animals but there appears to befewer deposits in the latter.

Outside of the liver, uPA-/-/tPA-/- and uPAR-/-/tPA-/-mice differed dramatically with regard to both fibrin deposi-tion and secondary organ damage. Among the 20 uPAR-/-/tPA-/- mice analyzed microscopically, extra-hepatic fibrindeposits were encountered in only 3. These were small fibrindeposits detected at the tips of lung lobes in 29-32-week-oldmice. No thrombotic lesions were detected in any other tissue,including stomach, colon, spleen, pancreas, thymus, heart, andkidney (data not shown). In contrast, as expected from earlierfindings (2-4), extensive fibrin deposition was found in thegastrointestinal tract, lung, and other tissues of all of six6-13-week-old uPA-/-/tPA-/- mice analyzed in parallel (datanot shown). Four of these uPA-/-/tPA-/- mice had gastriculcers and one had suffered a rectal prolapse, apparently as asecondary consequence of vaso-occlusion and ischemic necro-sis. Taken together, the above findings suggest that uPAR,uPA, and tPA are complementary factors in fibrinolysis butuPA alone supplies sufficient fibrinolytic potential to maintainvascular patency and prevent ischemic necrosis in most tissues,including tissues that are particularly susceptible to occlusivetissue damage, such as rectal tissue.

FIG. 1. Hepatic fibrin deposits in mice with combined deficits inuPAR, uPA, and tPA. (A) Hematoxylin/eosin-stained liver section froman 88-day-old uPAR-/-/tPA-/- mouse showing a typical fibrin deposit(arrowheads). (Bar = 35 pm.) (B) Hematoxylin/eosin-stained liversection from a 45-day-old uPA-/-/tPA-/- mousewith two fibrin deposits(arrowheads) with infiltrating spindle cells, probably macrophages orfibroblasts. (Bar = 35 pm.) (C) Immunoperoxidase-stained sinusoidalfibrin deposits (asterisk and arrowhead) in another liver section preparedfrom the same 88-day-old uPAR-/-/tPA-/- mouse highlighted inA. Asshown in this representative field, the fibrin depositswere generally sparseand modest in size. (Bar = 100 pum.) (D) Immunoperoxidase-stainedsinusoidal fibrin deposits (arrowheads) in a liver section from a 45-day-olduPA-/-/tPA-/- mouse. (Bar = 100 pm.) Note the higher frequency ofthese deposits relative to the much older uPAR-/-/tPA-/- mouse shownin C. (E and F) Higher magnification of representative sinusoidal fibrindeposits of the uPAR-/-/tPA-/- mouse shown in C. The fibrin depositshown inE is the same as that labeled with an asterisk in C. (Bars inE andF = 2.2 pm.) (G andH) Higher magnification ofrepresentative sinusoidalfibrin deposits found in the uPA-/-/tPA-/- mouse shown in D. (Bars inG andH = 1.4 and 2.2 pm, respectively.) (/) Representative immunoper-oxidase-stained liver section from a 66-day-old tPA-/- mouse showingnormal fibrinogen staining in the sinusoidal space but no deposits. (Bar =100 pm.) (J) Liver section from a uPAR-/-/tPA-/- mouse processed forimmunostaining in parallel with those samples shown in C-H except thatthe primary anti-fibrin(ogen) antiserum was omitted. (Bar = 100 pm.)

Impact of uPAR- and tPA-Deficiency on HematologicalProfile. The development of hepatic fibrin deposits inuPAR-/-/tPA-/- mice, as well as the absence of hepatic fibrin

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deposits in uPAR-/- and tPA-/- mice, does not appear to berelated to secondary alterations in the general hematologicalprofile of these mice. No differences were found betweenanimals with each of these genotypes with regard to platelet,red cell and white cell counts, hematocrit and thrombinclotting time (Table 1). Furthermore, the plasma concentra-tions of the key coagulation and fibrinolytic system compo-nents, fibrinogen, uPA, and Pig, were unaffected by eitheruPAR, tPA, or combined uPAR and tPA deficiency (Fig. 2).Although a more extensive survey of hemostatic factors may bewarranted, no evidence has been found for compensatorychanges in the plasma level of one fibrinolytic factor inresponse to the wholesale loss of another.Normal Wound Healing in the Skin of uPAR-/-/tPA-/-

Mice. Healthy, well-maintained mice may be less likely to showthe adverse consequences of uPAR and tPA deficiency thanare animals subjected to a fibrinolytic challenge such asinfection or injury. To explore the importance of uPA anduPAR in tissue repair, uPAR-/-/tPA-/-, uPA-/-/tPA-/- andcontrol mice were challenged with a 2-cm full-thickness skinincision wound and the progress ofwound repair followed (Fig.3). In control mice, the dehydrated crust or scab covering thewound fields was lost 10-16 days after incision and the woundswere uniformly healed as defined by macroscopic closure ofthe incision interface and restoration of the epithelial coveringby day 26 (Fig. 3J). In contrast, uPA'J-/tPA-/- mice displayedsubstantially slower wound healing, with only one of eightjudged healed by day 30 and only three healed after 5 weeks(Fig. 3J). These data are consistent with our earlier report ofseverely impaired skin healing in Plg-/- mice (13) and high-light the importance of plasmin generation in tissue remod-eling within fibrin-rich wound fields. Remarkably, the com-bined loss of both uPAR and tPA did not significantly impairwound healing in the same model system. Like control mice,half the uPAR-/-/tPA-/- mice were healed by day 19 and theywere uniformly healed by day 27 (Fig. 3). This shows that uPAin the absence of uPAR and tPA directs sufficient Pig activa-tion to support the prompt resolution of fibrin-rich woundfields.

DISCUSSIONThis study provides strong genetic evidence that uPAR andtPA are complementary fibrinolytic factors in mice-hepaticfibrin deposits are frequently detected in mice with combineddeficits in uPAR and tPA and rarely found in animals withsingle deficits in either uPAR or tPA. However, a moreremarkable finding drawn from this work is that uPA isgenerally quite effective in fibrin clearance, even in the ab-sence of both uPAR and tPA. The overall thrombotic risk inuPAR-/-/tPA-/- mice is tiny when compared to the devas-tating spontaneous thrombosis, multi-organ tissue damage,and early morbidity and mortality seen in uPA-/-/tPA-/- andPlg-/- mice (2-4). This finding is particularly unexpected inlight of the importance of uPAR in the potentiation ofuPA-mediated Pig activation in vitro (see below) and the impor-tance generally assigned to tPA in physiological fibrinolysis.The extraordinarily mild consequences of combined uPAR

and tPA deficiency raises the question of whether there are

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FIG. 2. uPA, tPA, Pig, and fibrinogen in the plasma of uPAR- andtPA-deficient mice. (Upper) Zymographic detection of uPA and tPAactivity in 5-,l plasma samples electrophoretically-fractionated on

SDS/polyacrylamide gels cast with Plg and casein. Immunoblot anal-ysis of Pig (Middle) and fibrinogen (Lower) in 1.0-uLI and 0.1-Al plasmasamples, respectively. Plasma samples were collected from individualuPAR+/+/tPA+/+ (lanes 1-3), uPAR-/-/tPA+/+ (lanes 4-6),uPAR+/+/tPA-/- (lanes 7-9), and uPAR-/-/tPA-/- mice (lanes10-12). The control plasma in the upper panel (lane 13) was collectedfrom a mouse lacking both uPA and tPA (4) and demonstrates thespecificity of the zymographic assay for Pig activator. The controlplasma in the middle panel (lane 13) was collected from a Plg-/-mouse (2) and demonstrates the specificity of the immunological assayfor Pig. The control plasma in the lower panel (lane 13) was collectedfrom a fibrinogen-/- mouse (13) and demonstrates the specificity ofthe immunological assay for fibrinogen. The positions of tPA, uPA,Pig, and the fibrinogen Aa, B3, and y chains are indicated at left. Thepositions of standard molecular mass markers are indicated at right.other high-affinity receptors for uPA that might facilitate Pigactivation. Whereas this possibility has not been formallyexcluded, no definitive evidence has been reported for anotherspecific high-affinity receptor in the decade since uPAR wasfirst identified (for review see ref. 7). Furthermore, specificuPA binding activity is undetectable in either cultured mac-

rophages or tissue extracts prepared from uPAR-/- mice,whereas uPA binding activity is easily detected in parallelsamples prepared from control mice (8). Nevertheless, thesefindings do not rigorously dismiss the existence of otherhigh-affinity receptors that cannot be detected in standardbinding assays, or the existence of an unidentified low-affinityreceptor for uPA that partially rescues uPAR-/-/tPA-/- micefrom thrombosis.The importance of uPAR in the potentiation of Pig activa-

tion in macrophages (8) may provide a clue as to why the liveris uniquely susceptible to fibrin deposition in uPAR-/-/tPA-/- mice. A key function of the macrophages stationedwithin the hepatic sinusoids (i.e., Kupffer cells) is the clearanceof foreign material and general debris that might otherwisecompromise hepatic vascular patency or function. Chance

Table 1. Hematological parameters in uPAR and tPA double-deficient mice

Control uPAR-/- tPA-/- uPAR-/-/tPA-/-Plasma thrombin time(s) 15.1 ± 1.7 (3) 17.0 + 3.6 (3) 15.7 ± 2.3 (3) 17.0 ± 3.6 (3)WBC (x 109/liter) 5.9 ± 3.0 (4) 2.4 ± 0.5 (3) 2.9 ± 1.4 (4) 4.0 ± 3.6 (4)RBC (x 10'2/liter) 8.24 ± 0.47 (4) 8.23 ± 0.39 (3) 7.97 ± 0.59 (4) 6.49 ± 1.14 (3)Hemoglobin (gram %) 13.2 ± 0.6 (4) 14.4 + 0.9 (3) 12.9 ± 0.4 (4) 11.7 ± 2.8 (4)Hematocrit (%) 41.1 ± 2.0 (4) 44.6 ± 2.8 (3) 40.2 + 1.6 (4) 39.7 ± 5.8 (3)Platelets (x 109/liter) 1004 ± 98 (4) 802 + 216 (3) 762 + 274 (4) 795 ± 64 (4)Data are presented as the mean ± SEM with the number of mice analyzed in parentheses. Mice were 6-18 weeks old.

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FIG. 3. The time course of wound healing following full-thicknessskin incision in uPAR-'-/tPA-/- and uPA-/-/tPA-/- mice. Repre-sentative examples of the progression of tissue repair in a controlmouse (A, D, and G), a uPAR-/-/tPA-/- mouse (B, E, and H) anda uPA-/-/tPA-/- mouse (C, F, and 1) at day 2 (A-C), day 13 (D andE), day 14 (F), day 22 (G and H), and day 26 (I). (J) Rate of woundhealing in control mice O (n = 16), uPAR-/-/tPA-/- miceo (n = 20)and uPA-/-/tPA-/- mice A (n = 8). The percentage of mice healed,defined as loss of the wound scab and complete covering of the woundwith epidermis, is plotted versus time after the incision.

fibrin deposits might be a common form of "debris" confront-ing Kupffer cells and the loss of uPAR may partially compro-mise their ability to remove fibrin within the sinusoidal space.Macrophages and many macrophage/monocyte cell lines are

normally well-equipped for fibrin dissolution; these cells ex-

press both uPA and uPAR, and this expression is dramaticallyincreased by inflammatory agents (14-17). It is also notewor-thy that in the human disease, paroxysmal nocturnal hemo-

globinuria (PNH), in which glycolipid-anchored proteins suchas uPAR are lacking on the surface of circulating cells, thereis an increased tendency for thrombosis, with thromboembo-lism being the major cause of death (18, 19). A significant roleof uPA/uPAR-producing inflammatory cells in fibrin surveil-lance would provide an explanation for the generally unex-pected earlier finding that uPA-/- mice are more likely todevelop hepatic fibrin deposits and occlusive rectal lesionsthan tPA-/- mice (4). This concept would also provide anexplanation for the paradoxical susceptibility of the liver tofibrin deposits in uPAR-/-/tPA-/- and uPA-/- mice eventhough uPAR, tPA, and uPA are each present in only vanishingquantities in unchallenged liver (20-23); the overwhelmingcontribution of hepatocytes to liver mass relative to Kupffercells is likely to obscure the expression of these factors by thefar less abundant macrophages. A detailed characterization ofthe fibrinolytic potential of inflammatory cells in vivo would beuseful in clarifying the proposed role of these cells in fibrinsurveillance.uPAR has been shown to focus uPA to the leading edge of

moving cells (24) and to significantly potentiate Pig activationin vitro by: (i) increasing the rate of pro-uPA activation byplasmin; (ii) decreasing the apparent Km of uPA for Pig tobelow 2 ,tM, the concentration of Pig in plasma; and (iii)increasing the apparent kcat/Km of uPA for Pig (6). Based onthis, and the anticipated unfocused and inefficient pro-uPAand Pig activation in uPAR-/-/tPA-/- mice, the apparenteffectiveness of "free" uPA in general fibrin surveillance andthe resolution of fibrin-rich wounds is surprising. However, theimpact of uPAR on pro-uPA activation rates in vivo has not yetbeen explored, and it should be noted that pro-uPA activationin at least one organ system, the urogenital tract, does notdepend on either uPAR (8) or Pig (2). One model that mightexplain the minimal fibrin accumulation in uPAR-/-/tPA-/-mice is that the local secretion ofuPA by cells in direct contactwith fibrin matrices may effectively promote local fibrinolysisdespite somewhat inefficient and unfocused Pig activation inthe absence of uPAR. Under this general model, the impor-tance of uPAR in fibrin surveillance may be minimized if thecells participating in fibrin clearance are equipped with recep-tors to bind fibrin and are copious uPA producers. Notably,macrophages, which stand out as potential participants infibrin clearance in tissues, are known to bind fibrin through themacrophage-specific integrin, Mac-l(CDllb/CD18, aM32)(25, 26), and produce both uPA and uPAR (8, 9, 14-17).Similarly, keratinocytes navigating within fibrin-rich woundfields produce both uPAR and uPA (27, 28) and are capableof both binding and solubilizing fibrin (29).

It was shown previously that wound healing in the skin isseverely impaired in Plg-deficient mice and the delay in woundrepair is associated with a major impediment in keratinocytemigration and re-epithelialization of the wound field (10). Wehave now shown that wound healing is impaired in a similarway in uPA-/-/tPA-/- mice but not in uPAR-/-/tPA-/-mice. Thus, it appears that uPA alone can supply sufficientfibrinolytic potential for prompt re-epithelialization, even un-der conditions that directly challenge the fibrinolytic system.An important extension of this work, which will clearly illu-minate the linkage between uPA-mediated fibrinolysis andwound repair, will be to establish whether the rate of woundhealing is restored to normal in PA- and Plg-deficient micewhen they also lack fibrin(ogen). Fibrinogen-deficient mice areavailable (13) and these animals are presently being raised.

Several features of normal wound repair have also beenrecognized in the context of common life-threatening diseasessuch as atherosclerosis and cancer (30-32). For example, localfibrin deposition, secretion of Pig activators, and invasive cellmigration are all well-established features of both tissue repairand the pathological progression of vessel wall disease andsolid tumors (33-38). Although there is substantial evidence

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5904 Medical Sciences: Bugge et al.

available to suggest that Pig activation is important in thepathogenesis of these diseases (36-41), it is unknown whetherdisease progression in vivo is served by plasmin-mediatedsolubilization of fibrin, activation of proteases, activation oflatent growth factors, proteolysis of general extracellular ma-trix proteins, or cleavage of some combination of substrates(42, 43). We anticipate that the availability of mice withindividual and combined deficits in tPA, uPA, uPAR, Pig andfibrinogen will be valuable assets in defining the role of Pigactivation in cancer and atherosclerosis since these diseases areeasily modeled in mice (44, 45).The data now available on uPA, uPAR, tPA, and Pig-

deficient mice (2-4, 8, 9), strongly argue that uPA and tPA arecomplementary factors in fibrinolysis with a high functionaloverlap. Our current working hypothesis for the evolutionaryadvantage of two Pig activators in mammals is that: (i) uPAprovides a mechanism for efficient cell-mediated fibrinolysisthat is driven by local secretion (receptor-independent fibri-nolysis) and/or cell-surface binding (receptor-dependent fi-brinolysis) and (ii) tPA provides a mechanism for efficientcell-independent fibrinolysis via its fibrin-binding capability.Consistent with this general hypothesis, tPA appears be ofprimary importance in the dissolution of embolized fibrin clots(4), whereas uPA-mediated Pig activation appears to be ofprimary importance in the resolution of fibrin-rich woundfields in the skin.

We thank Ed Reich for the kind gift of antibodies against Pig, KennHolmback and Bhavani Pathak for critically reading the manuscript,and Barbara O'Toole and Keith Kombrinck for their excellent tech-nical assistance. P.C. and D.C. thank M. Dewerchin for generating theuPAR- and uPAR/tPA-deficient mice used in the experiments carriedout in Leuven, Belgium. This work was supported by grants to J.L.D.from the National Institutes of Health (HL47826) and the NationalAmerican Heart Association (with funds contributed by the AmericanHeart Association Ohio Affiliate) (92-1103) and to T.H.B. from theDanish Medical Research Council. This study was done during thetenure of an Established Investigatorship (J.L.D.) from the AmericanHeart Association (93002570). T.H.B. was supported by fellowshipsfrom the Danish Medical Research Foundation, the Danish CancerResearch Foundation, and the Danish Plasmid Foundation.

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