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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2848-2854.)
© 1997 American Heart Association, Inc.

Articles

Induction of Vascular SMC Proliferation by Urokinase Indicates a Novel Mechanism of Action in Vasoproliferative Disorders

Sandip M. Kanse; Omar Benzakour; Chryso Kanthou; Christine Kost; H. Roger Lijnen; ; Klaus T. Preissner

From the Max-Planck-Institute, Kerckhoff-Klinik, Bad Nauheim, Germany (S.M.K., C. Kost, K.T.P.); the Thrombosis Research Institute, London, UK (O.B., C. Kanthou); and the Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Leuven, Belgium (H.R.J.).

	Abstract

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Abstract The urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c-fos/c-myc mRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor–independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.

Key Words: urokinase • proliferation • neointima • smooth muscle

	Introduction

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Vascular SMC proliferation is central to the process of restenosis after angioplasty and is also involved in the formation of atherosclerotic plaque. Platelets, macrophages, ECs, and SMCs secrete a range of growth factors that both initiate and sustain smooth muscle proliferation.¹ Upon injury to the vessel wall, there is immediate activation of the coagulation system, which is followed by fibrinolysis. Apart from regulating hemostasis, these systems are also involved in coordinating the cellular responses to injury that involve processes such as cellular adhesion, migration, and proliferation.² This is illustrated by the observation that vascular smooth muscle proliferation is stimulated by factors such as thrombin,³ factor Xa,⁴ protein S,⁵ factor XII,⁶ and TPA.⁷ Hence, the hemostatic system may contribute to vessel wall pathology through a number of different mechanisms.

Fibrinolytic activity is balanced by the levels of plasminogen activators and inhibitors that control the formation and action of plasmin. Though primarily mediating fibrin-clot lysis, these components are localized to the cell surface through specific receptors (or bound to specific extracellular molecules) and hence are also able to regulate pericellular proteolysis-related events.^{8 9 10} An accumulation of UPA on the leading edge of a migrating cell is thought to direct proteolysis of the matrix in relation to cell migration.^{11 12} The formation of a neointima involves remodeling of the extracellular matrix¹³ as well as activation of latent growth factors^{14 15} through the plasminogen activation system. UPA also induces cellular effects independent of its enzymatic activity, eg, stimulation of cell chemotaxis,¹⁶ adhesion of monocytes and neutrophils,^{17 18} release of tumor necrosis factor-,¹⁹ superoxide anion production,²⁰ and expression of matrix metalloproteinases,²¹ all of which contribute to neointimal formation.

The proliferative aspects of the components of the fibrinolytic system have been studied in considerable detail in nonvascular cells, and the involvement of UPA but not TPA has been demonstrated.^{15 22 23 24 25 26 27 28 29} UPA binding to cells leads to activation of a number of intracellular signaling events that include increased turnover of inositol phosphate, generation of diacylglycerol, phosphorylation of intracellular signaling proteins, and the induction of immediate-early genes such as c-fos.^{29 30 31 32 33 34}

Apart from its single-chain inactive precursor (ie, sc-UPA), there exist two other forms of UPA, a high- and a low-molecular-weight form (HMW-UPA and LMW-UPA, respectively). Internal cleavage at position 158 produces both of these two-chain forms, which are activators of plasminogen.^{35 36} The larger form includes the ATF (amino acids 1 to 131) that mediates binding to the cell-surface glycolipid–anchored UPA receptor.³⁷ Hence, the UPA receptor is a plausible signal transducing receptor for sc-UPA, HMW-UPA, or ATF but not LMW-UPA.³⁸

UPA, TPA, and PAI-1 activities are increased in the vessel wall in humans as well as in animal models of atherosclerosis.^{39 40 41 42 43} UPA expression particularly coincides with the proliferative phase of vascular SMCs, whereas TPA expression parallels the migratory phase in the balloon-injury model of neointimal hyperplasia.^{44 45} This together with the increased expression of the UPA receptor in atherosclerotic plaques⁴⁶ prompted us to further define the functional effects of UPA on vascular SMCs.

	Methods

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Reagents
Recombinant, glycosylated sc-TPA was from Dr Karl Thomae (Biberach, Germany). Human urine–derived HMW-UPA, LMW-UPA, and ATF were from American Diagnostica. Active, recombinant PAI-1 was generously provided by Dr J. Deinum (Astra Hässle AB, Mölndal, Sweden). Recombinant Gly158–sc-UPA (a noncleavable and hence, nonactivatable mutant of HMW-UPA) and r32K–sc-UPA (containing only the catalytic domain; hence, the LMW form) were produced in Chinese hamster ovary cells as described before.^{35 36} Anti-UPA receptor antibodies R3 and R9 were kindly provided by Dr G. Hoyer-Hansen (Finsen Laboratory, Copenhagen, Denmark). PI-PLC was from Oxford Glycosystems. Plasminogen-free FCS was produced by passing serum over a lysine-Sepharose column five times and was subsequently checked and found to be negative for plasminogen activity.

HVSMC and HUVEC Cultures
Cultures of human aorta or saphenous vein SMCs (ie, HVSMCs) were established, characterized, and grown exactly as described before.⁴⁷ In brief, explant cultures were initiated from dissected sections of the media and, after 3 to 4 weeks, outgrowing cells were harvested by trypsinization. The cells were maintained in DMEM with 10% FCS (vol/vol) containing penicillin (100 U/mL) and streptomycin (100 µg/mL) and split every 10 to 14 days in a ratio of 1:3. The smooth muscle origin of the cells was confirmed by immunostaining for -smooth muscle–specific actin (Sigma). Early-passage cells (<10 population doublings) from two aortas and six veins were used for these investigations. ECs were isolated from normal-term umbilical vein (ie, HUVECs) and cultivated as described before,⁴⁸ together with the retina-derived growth factor extract provided by Dr B. Pötzsch (Kerckhoff Klinik, Bad Nauheim, Germany). Approval for the use of human tissue, which would have been discarded otherwise, was obtained from the local ethics committee.

DNA Synthesis and Proliferation Assays
Cells were plated in 96-well plates under normal culture conditions for 48 to 72 hours, after which their growth was arrested in serum-free medium (0.2% FCS for 48 hours for HVSMCs and 0.5% FCS for 18 hours for HUVECs). HVSMCs were then stimulated (triplicate wells) with the appropriate agonist for 48 hours in serum-free medium, and the cells were labeled with 1 µCi per well of [³H]thymidine (5 Ci/mmol; Amersham Buchler) for the last 24 hours of this stimulation period. HUVECs were stimulated for 24 hours, and the cells were labeled with [³H]thymidine for the last 6 hours. The cells were washed, precipitated with 10% (wt/vol) trichloroacetic acid, washed with methanol, and dissolved in 1N NaOH. The radioactivity incorporated into the DNA was quantified by scintillation counting.⁴⁷

For cell multiplication studies, HVSMCs were plated in 48-well plates and growth arrested as described above. The medium was then replaced with agonist-containing medium (0.2% FCS), and the cells were incubated for another 4 days. After this time the cells were washed, trypsinized, and quantified in a cell counter (Schärfe System).

Data Analysis
For proliferation and DNA synthesis assays, the raw data were converted to percentage of control values, which were the values for untreated wells with no test substance set at 100%. The proliferative or mitogenic index was calculated as follows: (counts from treated wells-counts from control wells)/(counts from 4% FCS–treated wells-counts from control wells). Untreated controls had an index of 0, whereas 4% FCS treatment yielded an index of 1. All data are expressed mean±SEM from a single experiment run in triplicate or from combined data of different experiments.

UPA Labeling and Receptor Binding
Gly158–sc-UPA and ATF were radiolabeled with [¹²⁵I]NaI (Amersham Buchler), and receptor binding analysis was carried out according to our previously described protocol.⁴⁸

MAPK Activation
Serum-starved cells were stimulated with the test substances, and cell extracts were prepared in SDS sample buffer according to standard protocols. Western blot analysis was performed with a monoclonal antibody specific for phosphorylated MAPK as described by the manufacturer (Promega). This antibody shows no cross-reactivity with nonphosphorylated MAPK. Blots were stained for total protein to check for equal loading of the wells.

Northern Blot Analysis of c-fos/c-myc mRNAs
Serum-starved cells were prepared as described above, and isolation of RNA from appropriately stimulated cells, electrophoresis through agarose, and transfer to Hybond membrane filters were performed as described previously.^{3 49} The filters were hybridized with c-fos cDNA probe and after stripping, with the c-myc cDNA probe followed by the glucose-6-phosphate dehydrogenase cDNA probe as a control to check for equal loading of the wells.^{3 49}

	Results

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Regulation of HVSMC Growth by UPA
The effect of different forms of UPA (and for comparison, TPA) was tested in DNA synthesis and cell proliferation assays in several HVSMC cell strains. To account for differences between individual experiments, the data were combined to calculate the growth percentage (with respect to untreated cells) and the growth index (relative to stimulation with 4% FCS; the Table). Highly purified HMW-UPA from human urine as well as mutant HMW-UPA (Gly158–sc-UPA; uncleavable and hence, with no plasminogen activation properties) was found to stimulate cell proliferation and DNA synthesis in HVSMC. In contrast, the LMW forms of UPA (containing only the catalytic domain without the receptor-binding fragment), either isolated from human urine or recombinant, had no significant effects, as did the diluents used for each UPA preparation. Both ATF and TPA were also ineffective in stimulating HVSMC growth. These data indicate that the complete UPA molecule is necessary to elaborate the proliferative effect on HVSMCs. As a positive control, a twofold induction with 4% FCS and 25 ng/mL of PDGF-BB was obtained in every HVSMC growth assay.

View this table: [in this window] [in a new window]   Table 1. Effect of Different Test Substances on DNA Synthesis and Cell Proliferation in HVSMCs

The most effective forms of UPA, HMW-UPA and Gly158–sc-UPA, were found to induce DNA synthesis in a dose-dependent manner in the concentration range from 12 to 200 nmol/L (Fig 1), although maximal stimulation was probably not reached at the highest concentration used. In ECs, none of the tested UPA isoforms stimulated DNA synthesis, whereas the positive controls (FCS and RDGF) induced a robust mitogenic response, which indicated that the effect of UPA is SMC specific (Fig 2).

View larger version (17K): [in this window] [in a new window]   Figure 1. Semilog plot of dose-dependent effect of UPA on DNA synthesis in HVSMCs. Quiescent HVSMCs were treated with various concentrations (as indicated) of HMW-UPA () or Gly158–sc-UPA () for 48 hours. [3H]Thymidine incorporation into DNA was measured between 24 and 48 hours. Data are expressed as percentage of control (mean±SEM) from a typical experiment. Similar results were obtained in 2 other separate experiments.

View larger version (31K): [in this window] [in a new window]   Figure 2. Effect of UPA on DNA synthesis in HUVECs. Quiescent HUVECs were treated with different isoforms of UPA, TPA (each 100 nmol/L), 1% retinal growth factor extract (RDGF), or 4% FCS. [3H]Thymidine incorporation into DNA was measured between 18 and 24 hours. Data are expressed as percentage of control (mean±SEM) from 5 or 6 different experiments.

Enzymatic Activity of UPA Is Not Required for Mitogenesis
A possible mechanism for the stimulation of proliferation may be via the cleavage of plasminogen by exogenous UPA that, in turn, leads to activation of latent growth factors or their release from the matrix. This possibility seems unlikely, since the mutant form of UPA with no plasminogen activation properties (Gly158–sc-UPA) also stimulated HVSMC growth. To strengthen this point, further experiments were performed in the presence of plasminogen-free FCS and excess PAI-1, and in both cases, the growth-stimulatory effect of UPA was unaltered (Fig 3). The plasminogen-free FCS was as effective as "normal" FCS in stimulating cell proliferation, and coadministration of UPA with plasminogen-free FCS gave an additive effect. Hence, plasminogen activation plays no role in transducing the mitogenic effect of UPA.

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of enzymatic activity of UPA on mitogenesis. Quiescent HVSMCs were treated with buffer (dotted bars) or Gly158–sc-UPA (100 nmol/L) (hatched bars) in the absence (CON) or presence of 5% plasminogen (Plsg)-free FCS or 200 nmol/L PAI-1, respectively. [3H]Thymidine incorporation into DNA was measured between 24 and 48 hours. Data are expressed as percentage of control (mean±SEM) from 1 of 2 representative experiments.

Effect of UPA on Intracellular Proliferation-Related Events in HVSMCs
Recent studies have demonstrated that p44^MAPK and p42^MAPK (together referred to as MAPK) each undergo rapid tyrosine and threonine phosphorylation as well as nuclear translocation in response to growth factors, thereby leading to stimulation of their intrinsic kinase activities.⁵⁰ Western blot analysis with an antibody specific for dually phosphorylated MAPK indicated that both forms undergo rapid phosphorylation (40-fold) in HMW-UPA–stimulated HVSMCs (Fig 4A). The effect of recombinant UPA isoforms was dose dependent, and Gly158–sc-UPA was twice as effective as r32K–sc-UPA (data not shown). Since most proliferative responses lead to rapid induction of c-fos and c-myc mRNAs in HVSMCs,^{3 49} the effect of UPA as a possible inducer of these immediate-early genes was also tested. At 30 minutes there was a rapid increase (30-fold) in c-fos induction that declined after 2 hours, whereas there was sustained stimulation of c-myc (2-fold at 30 minutes) for as long as 4 hours after stimulation of cells with HMW-UPA (Fig 4B). There was marginal stimulation (1.2-fold) of inositol phosphate turnover in these cells after addition of UPA, but the cAMP levels remained unaltered. These data indicate a rapid and specific stimulation by UPA of proliferation-related signaling events in HVSMCs.

View larger version (101K): [in this window] [in a new window]   Figure 4. Induction of intracellular signaling events in HVSMCs. A, Quiescent HVSMCs were stimulated for 15 minutes to 2 hours with 100 nmol/L HMW-UPA, and the cell extracts were analyzed for phosphorylated MAPK by Western blot analysis. B, Cells were stimulated with 100 nmol/L HMW-UPA for 30 minutes to 4 hours, and the RNA samples were processed for Northern blot analysis. Expression of c-fos, c-myc, and the "housekeeping" enzyme glucose 6-phosphate dehydrogenase (G6PD) was determined from the same blot. These experiment were performed 3 times with identical results.

Role of the UPA Receptor in Inducing Mitogenicity
To determine whether UPA mediates its cellular effects on HVSMCs through the "classic" UPA-receptor, the interference induced by blocking monoclonal antibodies to the UPA receptor was tested. Antibodies (25 µg/mL) were added 15 minutes before the addition of either HMW-UPA or Gly158–sc-UPA. Both monoclonal antibodies R3 and R9 (each directed against domain I of the UPA receptor) were found to inhibit UPA binding to its receptor; however, neither antibody inhibited UPA-stimulated mitogenesis (Fig 5A).

View larger version (34K): [in this window] [in a new window]   Figure 5. Role of the UPA receptor in mediating the mitogenic effect of UPA on HVSMCs. A, Cells were pretreated with 25 µg/mL each of the UPA receptor–blocking antibodies R3 or R9 or no antibody (CON), and the HMW-UPA (50 nmol/L)-stimulated DNA synthesis was determined (dotted bars; UPA-stimulated DNA synthesis in the absence of antibody is 100%), or specific UPA binding was measured (hatched bars; binding in the absence of antibody is 100%). B, Cells were not treated (hatched bars) or pretreated with PI-PLC (0.5 U/mL) for 2 hours in 0.2% FCS (dotted bars) followed by stimulation with buffer (CON), 50 nmol/L HMW-UPA, or 25 ng/mL PDGF-BB, respectively, and DNA synthesis was determined. For both panels, results from 1 representative experiment of 3 are shown (mean±SEM as the percentage of control).

The effect of removal of the glycolipid-anchored UPA receptor from the cell surface was also tested. Treatment of cells with PI-PLC removed 60% to 70% of the cell-surface UPA receptors, as checked in a subsequent binding assay (data not shown). The same enzyme preparation under identical conditions reduced UPA binding to HUVECs by 95%.⁴⁸ The proliferative response of PI-PLC–treated cells to HMW-UPA and PDGF-BB was unchanged (Fig 5B). Similarly, induction of MAPK phosphorylation by UPA in HVSMCs was also unaffected by PI-PLC–mediated removal of the cell-surface UPA receptor (Fig 6). Thus, the above results indicate that UPA mediates its effects, at least partly, independently of the UPA-receptor.

View larger version (40K): [in this window] [in a new window]   Figure 6. Role of the UPA receptor in mediating MAPK phosphorylation. Quiescent HVSMCs were not treated (lanes 5-8) or pretreated with PI-PLC (lanes 1-4) followed by stimulation with buffer (lanes 1 and 5), 100 nmol/L Gly158–sc-UPA (lanes 2 and 6), 20 ng/mL PDGF-BB (lanes 3 and 7), or 10% FCS (lanes 4 and 8) respectively, and the phosphorylation status of MAPK was determined by Western blotting. Results from 1 representative experiment of 2 are shown.

	Discussion

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The present study clearly demonstrates the potential of UPA to stimulate growth of vascular SMCs and to activate those intracellular mechanisms related to cell proliferation. This mitogenic effect of UPA was independent of its ability to activate plasminogen and to bind to the classic UPA-receptor, suggesting a novel mechanism of action. These results, together with the well described effect of UPA on cell migration and its role in remodeling of the extracellular matrix, provide additional evidence for the involvement of UPA in vasoproliferative disorders.

The specificity of the growth-stimulatory effect of UPA was consolidated by the following observations: (1) highly purified HMW-UPA from urine or its recombinant analogue (Gly158–sc-UPA) were effective, whereas urinary LMW-UPA or its recombinant version (r32 k–sc-UPA) were not; (2) the mitogenic effect of UPA was dose dependent and cell specific, in that ECs showed no similar responses; (3) the structurally and functionally similar molecule TPA showed no growth-stimulatory activity; and (4) UPA stimulated proliferation-related intracellular signaling mechanisms in HVSMCs.

The ability of UPA to activate MAPK phosphorylation as well as the transient transcription of the immediate-early genes c-fos and c-myc within minutes makes it likely that UPA stimulates growth by a rapidly acting direct mechanism. This is also supported by time-course studies of DNA synthesis, which showed that the peak of DNA synthesis induced by UPA occurred at the same time as that induced by FCS (24 to 36 hours after stimulation).

Since the enzymatically inactive (noncleavable) mutant Gly158–sc-UPA was effective in stimulating cell proliferation, a role for the enzymatic activity of UPA in this process can clearly be excluded. Further validation of this point can be deduced from our observation that neither the enzymatically active forms of LMW-UPA nor TPA induced growth. Furthermore, no modulation of UPA mitogenic activity with PAI-1 or in the presence of plasminogen-depleted serum was observed, again confirming a plasminogen activation–independent effect. In a previous analysis of the growth-stimulatory effect of plasminogen activators on HVSMCs, it was observed that UPA was ineffective whereas TPA induced cell proliferation.⁷ In this study, the mitogenicity of TPA was completely attributed to its enzymatic activity but was not related to the activation of plasminogen, because plasmin by itself did not stimulate growth. In this case, it could be hypothesized that TPA activates/mobilizes growth factors directly. These divergent results could be related to the source of plasminogen activators, the cell systems, and conditions of the growth assays.

Structure-function studies indicated that the HMW forms of UPA were most effective in inducing proliferation, whereas little or no activity was observed for the LMW forms (carboxy-terminal-fragment of UPA) or ATF-UPA. Binding of the ATF-UPA fragment but not LMW-UPA to HVSMCs was observed, which confirms earlier observations that the binding region of UPA for its receptor lies exclusively within the ATF.³⁷ Thus, binding of ATF-UPA to the UPA receptor may not be sufficient to induce the necessary conformational change to drive intracellular signaling events, whereas HMW-UPA recruits other receptors in addition to the UPA receptor. Similar observations have been made in other cell systems, where the mitogenic effect of UPA could not be reproduced with ATF-UPA.^{19 20 25 29} Taken together, the whole UPA molecule seems to be necessary to elaborate its proliferative effect on HVSMCs.

Anti–UPA receptor antibodies were effective in inhibiting the binding of UPA to HVSMCs, as was the PI-PLC–mediated removal of the glycolipid-anchored UPA receptor, yet neither of these treatments inhibited the growth-stimulatory activity of UPA. It is possible to assume that not all of the receptors were blocked or removed from the cell surface or that de novo synthesis of (unoccupied) receptors may have occurred after these treatments. Alternatively, there may be a receptor subpopulation resistant to these manipulations. An attractive possibility that needs further experimental validation is that the effect of UPA is transduced independently of the classic UPA receptor. There is ample evidence for novel, cell-surface UPA binding proteins on platelets⁵¹ and liver parenchymal cells⁵² that seem to be distinct from the classic UPA receptor. Moreover, UPA can also interact with extracellular matrix proteins, such as vitronectin, in the presence of soluble UPA receptor⁵³ and bind to lipoprotein receptor–related protein in the presence of PAI-1.⁵⁴ These reports provide additional evidence that UPA could function through another fast-acting UPA receptor–independent mechanism. Although other mitogens such as thrombin⁴⁹ act on HVSMCs via the release of PDGF, a PDGF-AA blocking antibody had no effect of UPA-mediated cell proliferation (data not shown) in the present study.

Recently, transgenic mouse strains deficient in individual components of the fibrinolytic system have been generated that should provide ideal models to test their influences in proliferative vascular diseases. Following vascular trauma, TPA or UPA receptor deficiency did not influence the formation of a neointima, whereas a UPA deficiency substantially reduced neointimal cell accumulation.¹³ In such complex animal models, there could be direct or indirect effects that combine to influence cell migration, proliferation, and matrix remodeling in the vasculature. Nevertheless, these in vivo observations are paralleled by deductions from our in vitro experiments, wherein no effect of TPA and a stimulatory effect of UPA (independent of UPA receptors) on smooth muscle proliferation was observed. In conclusion, induction of SMC proliferation is a novel function of UPA and may be a key mechanism leading to the formation of neointimal hyperplasia in restenosis and atherosclerosis.

	Selected Abbreviations and Acronyms

 

ATF = amino-terminal fragment of UPA (amino acids 1-131) FCS = fetal calf serum HMW-UPA = high-molecular-weight UPA HUVEC = human umbilical vein endothelial cell HVSMC = human vascular smooth muscle cell LMW-UPA = low-molecular-weight UPA MAPK = mitogen-activated protein kinase PAI-1 = plasminogen activator inhibitor-1 PDGF = platelet-derived growth factor PI-PLC = phoshatidylinositol phospholipase C TPA = tissue-type plasminogen activator UPA = urinary-type plasminogen activator/urokinase

	Acknowledgments

 
H.R.L. was supported by a grant from the Belgian National Fund for Scientific Research (G.0126.96). O.B. and C. Kanthou acknowledge the support of Professor V.V. Kakkar, the British Heart Foundation and the Thrombosis Research Trust. This work was performed under grant DFG No. Pr 327/1-3 (to K.T.P.) from the Deutsche Forschungsgemeinschaft, Bonn, Germany. The excellent technical assistance of T. Chavakis and B. Yutzy is greatly appreciated. We thank Dr G. Hoyer-Hansen (Copenhagen, Denmark) for the anti–UPA receptor monoclonal antibodies and Dr J. Deinum (Astra Hässle AB, Mölndal, Sweden) for the active PAI-1.

	Footnotes

 
Reprint requests to Dr Sandip Kanse, Max-Planck-Institute, Kerckhoff-Klinik, Sprudelhof 11, D-61231 Bad Nauheim, Germany.

Received February 7, 1997; accepted June 18, 1997.

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