Regulation of plasminogen activator inhibitor activity in endothelial cells by tissue-type plasminogen activator

Regulation of plasminogen activator inhibitor activity in endothelial cells by tissue-type plasminogen activator

Fibrinolysis(1996)10(3),183-191 © PearsonProfessionalLtd 1996 Regulation of plasminogen activator inhibitor activity in endothelial cells by tissue-t...

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Fibrinolysis(1996)10(3),183-191 © PearsonProfessionalLtd 1996

Regulation of plasminogen activator inhibitor activity in endothelial cells by tissue-type plasminogen act,vator G.-Y, Shi ~, C.-C. Hsu ~, B.-I. Chang 2, C.-E Tsai ~, H.-S. Han ~, M.-D. Lai ~, M. T, Lin ~, W,-C. Chang a, L,-Y. C, Wing 4, C. J, Jen 4, M.-J. Tang 4, H.-L. Wu 1 1Departments of Biochemistry, 2Medical Technology, 3Pharmacology, 4physiology, Medical College, National Cheng Kung University, Tainan, Taiwan, 70101, Republic of China

Summary Tissue-type plasminogen activator (t-PA) may stimulate the expression of plasminogen activator inhibitor type 1 (PAl-l) mRNA in cultured human umbilical vein endothelial cells. The PAl-1 antigen in the conditioned medium of cells, pretreated with t-PA, was less than the control, probably due to the formation and degradation of the t-PA.PAI-1 complex. However, the PAl activity of the t-PA-pretreated cells reached the same level as control group, 24 h after the residual t-PA activity was rapidly neutralized by the newly synthesized PAl-1. To test the release of PAl-1 from the substratum of the endothelial cells, the cultured cells were metabolically prelabeled with 35S-methionine for 3 h and then treated with t-PA. The 35S-PA1-1 of 46 kDa was found in the substratum and culture medium of cultured endothelial cells as analyzed by the SDS-PAGE after immunoprecipitation. During the treatment of endothelial cells with t-PA, the PAl-1 of 46 kDa in the cell substratum disappeared, and the 110 kDa t-PA.PAI-1 complex, the 81 kDa degraded t-PA.PAI-1, and the 44 kDa degraded PAl-1 products concomitantly appeared in the conditioned medium instead. In summary, t-PA can regulate the fibrinolytic activity of endothelial cells by enhancing PAl-1 mRNA biosynthesis and release PAl-1 from :the substratum to neutralize t-PA activity. The PAl-1 which released into the medium was immediately converted to the inactive latent form in the absence of active t-PA.

INTRODUCTION vascular endothelial cells play crucial roles in both haemostasis and fibrinolysis. They can synthesize von Willebrand factor, fibronectin and thrombospondin to promote platelet adhesion 1-3 and produce PGIE to inhibit the platelet aggregation.4 Anticoagulant factors, e.g. thrombomodulin and heparan sulfate are also produced by endothelial cells. 5'6 Endothelial cells are also capable of synthesizing several important fibrinolytic factors, e.g. tissue-type plasminogen activator (t-PA) and its specific inhibitor, plasminogen activator inhibitor, type 1 (PAIl). z-12 PAI-1 has been found in a variety of cell lines as

This study was supported by Grants NSC-83-0412-B006-044-M09, NSC-830412-B006-087, NSC-84-2331-B006-013-M09, and NSC-84-2331-B006-022 from the National Science Council, Republic of China. Received: 30 June 1995 Accepted after revision: 26 April 1996 Correspondence to: Dr Hua-Lin Wu, Tel. 06-2353535-5541; Fax. 06-2741694; E-mail: HALNWU @mail.ncku.edu.tw

well as platelets and plasma. 13-15 In cultured human umbilical vein endothelial cells (HUVECs), it is associated with the cytosol, membrane and growth substratum. 16 PAI-1 is also released into the culture medium of endothelial cells. 16-~8 The biosynthesis of PAI-1 can be induced in endothelial cells by a variety of substances, e.g. thrombin, phorbol ester, glucocorticoids, lipopolysaccharide, inflammatory cytokines, and growth modulators. 19-23 Thrombolytic agents such as t-PA, urokinase-type plasminogen activator, and streptokinase have been clinically used to treat patients with acute myocardial infarction, thereby decreasing their morbidity and mortality. However, the major limitations of thrombolytic therapy include the failure to achieve reperfusion, 24 haemorrhage side effects, 25'26 and the occurrence of subsequent reocclusion. 27'28 After thrombolysis with t-PA, the incidence of reocclusion ranges from 12% to 46%. 29,30 During thrombolytic therapy, the infusion of large dose plasminogen activators such as t-PA causes a transient increase of plasmin and t-PA in the circulation,26'3~ 183

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thereby directly exposing the endothelium to catalytically active plasmin and t-PA, at least for a short time interval. The understanding of effect of plasmin or t-PA on the expression of fibrinolytic factors becomes very important. In our recent study, plasmin could inhibit the biosynthesis of t-PA antigen in HUVECs. is However, such treatment led to an increase in PAI activity, whereas the amount of PAI-1 antigen was unchanged. ~s Previous studies have demonstrated that exogenous t-PA can form complex with extracellular matrix-associated PAI-1 in Hep G2 cells to form an SDSstable t-PA-PAI-1 complex. And the t-PA.PAI-1 complex is rapidly released and specifically binds, with a high affinity to a cell-surface receptor which mediates endocytosis and degradation of the bound ligand. 32-35 A PAI-l-independent endothelial cell surface binding site or receptor for t-PA was also demonstrated and purified. 36'37 In this study, the direct effect of t-PA on the biosynthesis of PAI-1 in endothelial cells is investigated. Since PAI-1 is largely stored in the substratum of endothelial cells,16 the effect of exogenously added t-PA on the PAI-1 released from the substratum is also investigated. Furthermore, the formation and degradation of t-PA.PAI-1 complex in h u m a n endothelial cells are also analyzed.

in our laboratory from mice. All other chemicals were of the highest grade commercially available.

MATERIALS AND METHODS

Treatment of endothelial cells with t-PA

Reagents

Medium M-199, endothelial-SFM (serum-free media), methionine-free DMEM, fetal calf serum, penicillin, and streptomycin were purchased from GIBCO. Goat antiPAI-1 antibody was from American Diagnostica; the endothelial cell growth factors was from Collaborative Research; heparin, aprotinin, pepstatin, phenylmethanesulfonyl fluoride cq-antitrypsin and BSA were purchased from Sigma. Recombinant human one-chain t-PA (Actilyse) was purchased from Boehringer Ingelheim, Germany. The t-PA sample used to treat endothelial cells was further purified by passing through a Centricon-30 membrane (Amicon) to remove the ingredients that have molecular weight less than 30 000. The t-PA sample was dissolved and diluted in phosphate-buffered saline and concentrated in a Centricon-30 tube by centrifugation. The buffer exchange procedures were performed repeatedly for three times. Some insoluble fraction could be observed and was removed by centrifugation. The soluble fraction retained 70% of the t-PA activity of the original sample and was diluted in the culture medium. The tPA sample after dilution in the culture medium was soluble through the incubation period. Unless indicated, the purified one chain t-PA from Boehringer Ingelheim was used in all experiments, t-PA antibody was prepared Fibrinolysis (1996) 10(3), 183-191

Cell culture

Endothelial cells were isolated from human umbilical veins as previously described. 3s Cells were grown to confluence in medium M-199 containing: 10% fetal calf serum; 25 gg/ml endothelial cell growth factor; 10 u/ml heparin; 100 u/ml penicillin, and 100 u/ml streptomycin. Passaged cells were subcultured in 24-well dishes and allowed to grow for 3 days to confluence under the same conditions used for primary cultures. Cells of the third passage were used in all experiments. Cell viability evaluation

After being treated with t-PA and incubated in medium for various periods, the cells were harvested by incubation for 10 min with 0.1% trypsin, and numbers of viable cells were counted by the trypan blue dye exclusion method. The amount of total protein of the attached cells, another parameter for viability, was determined via the Lowry method 39 after washing the cell cultures three times with phosphate-buffered saline.

Confluent cell cultures at passage three in 24-well dishes were washed twice with serum-free M-199 containing 25 gg/ml endothelial cell growth factor, 10 u/ml hepafin, and 0.5% BSA and treated with the Centricon-30 purified t-PA in the same medium for 30 min. Cultures were then washed three times and incubated in 0.5 ml of serum-free M-199 containing: 10% endothelial-SFM (GIBCO BRL); 25 gg/ml endothelial cell growth factor; 10 u/ml heparin, and 0.5% BSA at 37°C for various durations. Approximately 0.3% of the added t-PA still remained bound to the cells as determined by 125I-labeled t-PA. Samples of the culture medium were collected at intervals, centrifuged at 15 000xg, and made to 0.01% with Tween 80. Cell lysates were obtained by adding 0.5 ml of 0.1% Triton X-100 to the washed cells and incubating at 4°C for 1 h. Samples of conditioned medium and cell lysates were frozen at -40°C until assayed for PAI-1 antigen. Quantitation of PAl-1 antigen levels

PAI-1 antigen was assayed by procedures recommended by the manufacturer of Imubind PAI-1 enzyme-linked immunosorbent assay (ELISA)kits (American Diagnostica). The kit is suitable for measuring free PAI-1 antigen as well as PAI- 1 antigen in t-PA.PAI- 1 complex as suggested by the manufacturer. © Pearson Professional L td 1996

PAl activity and t-PA

Assay of PAl activity PAI activity was measured by titrating samples with increasing amounts of t-PA into fixed volume of endothelial cell-conditioned medium or control medium. 4°'41 The excess t-PA activity was quantitatively assayed as described by Verheijen et al. 41 PAI activity was calculated from the intersection of the asymptote of the titration curve with the x axis and expressed in international units (IU) of t-PA inhibited, t-PA standard was purchased from Boehringer Mannheim, Germany. One IU t-PA corresponds to 1.66 ng t-PA antigen.

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bated at 37°C for various durations. Next, samples of the culture medium were collected at each interval by centrifugation at 15 000xg, and made to a final concentration of 0.01% Tween 80, I mM phenylmethanesulfonyl fluoride, 10 mM pepstatin, and 10 gg/ml aprotinin. Cell lysates were obtained by adding 0.1% Triton X- 100 to the washed cells at 4°C for 1 h. For growth substratum isolation, cells were first removed by treatment with 0.1% EDTA and 0.2 M urea in phosphate-buffered saline at 37°C for 10 rain and substratum was then isolated from the culture dish by adding 0.1% SDS and 0.5% Triton X100 (0.4 ml twice per well in a 12-well dish) and scraping with a rubber policeman. 16

Preparation of RNA and northern hybridization analysis HUVECs in 6-cm plates were treated with increasing concentrations of t-PA for various durations. Total cellular RNA from each 6-cm plate of t-PA-treated and control cultures was isolated at intervals according to the method of Chomczynski and S a c c h i . 42 10gg of RNA was subjected to gel electrophoresis in formaldehyde-agarose gels. 43 After electrophoresis, the RNA was transferred to nylon filter (Hybond N, Amersham, UK) and crosslinked to the membrane by UV irradiation. Hybridization was performed with eDNA probes of PAI-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) labeled by the random primer method. 44 Autoradiography was performed by exposing the nylon filter to imaging plates for 15 h and visualization and quantitation of the autoradiographic signals was conducted on a Fuji Bio-Imaging Analyzer. All autoradiographic values for PAI-1 mRNA levels were normalized relative to the level of GAPDH mRNA to correct for possible differences in the amount of RNA loaded. The probes used were a 2-kb EcoR1 PAI-1 eDNA fragment 4s (a kind gift from Dr D. Ginsburg, Howard Hughes Medical Institute, Departments of Medicine and Human Genetics, University of Michigan, USA) and a 1.25-kb PstI eDNA fragment of human GAPDH. Radioactive labeling of cell cultures, preparation of conditioned medium, and isolation of the growth substratum Confluent endothelial cells were washed three times with methionine-free DMEM (GIBCO BILL) and labeled with 50gCi/ml L-BS-methionine (Amersham) in the same medium for 3 h. After labeling, cells were washed with serum-free M-199 containing: 25gg/ml endothelial cell growth factor; 10u/ml heparin, and 0.5% BSA and treated with t-PA for up to 30 min. Samples of the conditioned medium and growth substratum were obtained at various intervals and analyzed for 35S-PAI-1. Changes of PAI-1 after incubation were analyzed by washing cells three times after a 30 min treatment with t-PA and incu© Pearson Professional Ltd 1996

Immunoprecipitation of PAl-1 Radiolabeled samples of culture medium, cell lysate and substratum were immunoprecipitated with goat anti-PAIl polyclonal antibody as describedY 400 gl of sample was incubated with 2 gl of antibody (1 mg/ml) at 4°C for overnight. Protein A-Sepharose beads (40 gl of a 1:1 slurry in phosphate-buffered saline) were added, and the samples were incubated at 4°C for 2 h. The beads were then washed three times each in a 0.5 ml of ice-cold washing solution (0.1% (v/v) Triton X-100, 0.02°/0 (w/v) SDS, 150mM NaC1, 50mM Tris-HC1, pH 7.4, 5mM EDTA, and 10 gg/ml aprotinin) and then resuspended in a sample buffer for SDS-PAGE. The supernatants of the above Protein A-Sepharose beads were subjected to SDSPAGE. SDS-PAGE and autoradiography SDS-PAGE was performed according to the procedure of Laemmli, 4z using a 10% separating gel and a 3% stacking gel. All samples were reduced with [}-mercaptoethanol (5% v/v) and boiled for 3 min prior to application to the gel. After electrophoresis, proteins were transferred to nitrocellulose membranes (0.45 gm, Bio-Rad) for I h at 4°C and 100V using a Bio-Rad Trans-Blot apparatus. Autoradiography was performed by exposing the nitrocellulose filter to imaging plates for 24 h and visualization of the autoradiographic signals was conducted on a Fuji No-Imaging Analyzer. RESULTS To determine whether the expression of PM-1 mRNA was affected by t-PA, endothelial cells were treated with 0.5 and 1 gM t-PA for 8 and 12 h. The cells were then harvested and the PAI-1 mRNA was measured at each interval. The PAI-1 gene is generally known to be transcribed into two distinct PAI-1 mRNA species with lengths of 3.4 and 2.4kb in human endothelial cells. 45,48 In cells Fibrinolysis (1996) 10(3), 183-191

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treated with 0.5 or 1 btM t-PA, the two PAI-1 mRNA species increased about 2- or 3-fold at 12 h incubation as their ratios to the GAPDH mRNA (Fig. 1A). czl-antitrypsin, t-PA antibody aboIished the stimulatory effect of t-PA on PAI-1 mRNA expression (Fig. 1B). The heated sample oftPA also did not have the stimulatory effect, suggesting that the catalytic activity of t-PA was required for this effect (Fig. 1B). An incubation with a different source oftPA {two-chain t-PA, purchased from American Diagnostica Inc., USA) is as efficient as one-chain t-PA in stimulating endothelial cell PAI-1 mRNA expression (Fig. 1B). The viability of the ceils was unaffected by treatment with t-PA as determined by trypan blue staining, cell counting and total protein determination. The amounts of PAI-1 antigen released in the conditioned medium and in cell lysate of endothelial cells were measured by ELISA method. The PAI-1 antigen here represents the net amount of PAI-1 antigen released by the cells, which is the balance between its synthesis and degradation. In both the control and t-PA pretreated cells, the PAI-1 antigen in conditioned medium increased linearly with the incubation time (Fig. 2A). The net PAI-1 antigen in conditioned medium of cells pretreated with tPA was lower than the untreated cells, probably due to the formation and degradation of t-PA.PAI-1 complex. The PAI activity in conditioned medium of control cells increased slowly and reached a plateau in 6 h (Fig. 2B). Residual t-PA activity was recovered in the culture medium of cells which were pretreated with 0.5 or 1 gM t-

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Fig. 1 Northern hybridization analysis of PAl-1 mRNA of t-PAtreated endothelial cells. (A) HUVECs were treated with 0.5 or 1 gM t-PA for 8 and 12 h in serum-free M-199 containing 10% endotheliaI-SFM (GIBCO BRL), 25 gg/ml endothelial cell growth factor, 10 u/ml heparin, and 0.5% BSA, and the RNA was extracted at the indicated times. Total cell RNA (10 gg/lane) was fractionated b2Ypelectrophoresis,transferred to nylon paper, and hybridized with -labeled cDNA fragments of human PAl-1 and glyceraldehyde-3phosphate dehydrogenase (GAPDH). The stabilizer in the t-PA sample (purchased from Boehringer Ingelheim) was removed by centrifugation in a Centricon-30 tube in phosphate-buffered saline and the purified t-PA retained 70% catalytic activity of the original sample. The signal intensity of PAl-1 mRNA 3.4 kb was quantitated by a Fuji Bio-lmaging Analyzer. The difference in loading was corrected by comparison to the intensity of GAPDH. (B) The levels of PAl-1 mRNA of t-PA-treated endothelial cells in the presence of a~-antitrypsin and anti-t-PA antibody. HUVECs were treated with tPA for 12 h in the presence of oh-antitrypsin or t-PA antibody, or treated with heated t-PA, and the effects on the amount of PAl-1 mRNA were measured, a" control, no treatment; b: t-PA 1 gM; c: o~lantitrypsin 10 gM only; d: t-PA 1 gM+(z~-antitrypsin 10 gM preincubated at 4°C for 4 h before adding to cells; e: t-PA polyclonal antibody (10-fold dilution) only; f." t-PA 1 pM+t-PA polyclonal antibody preincubated at 37°C for 2 h before adding to the cells; g: t-PA 1 gM boiled for 30 min; h: two-chain t-PA 1 gM (purchased from American Diagnostica Inc. without further purification). Each data point represents the mean+_S.D,of three independent experiments.

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PA and washed three times with culture medium (Fig. 2B). In the t-PA pretreated cells, the t-PA activity was neutralized by newly synthesized PAI-1 in a 9 h incubation (Fig. 2B). After t-PA activity was neutralized, the PAI activity increased rapidly and reached a similar steady state level as cells without t-PA treatment (Fig. 2B). Comparing the results of Figure 2A and 2B, it might be concluded that most of the PAI-1 in the 24-h conditioned medium was in a latent form, and about 1% of total PAI-1 antigen was in an active form. The amount of PAI-1 antigen in cell lysate was 3 to 10% of that in the conditioned © Pearson Professional Ltd 1996

PAl activity and t-PA

medium, and no difference was observed between the control and t-PA-pretreated cells (Fig. 2C). In the presence of cycloheximide PAI-1 antigen synthesis and release into culture medium was inhibited (Fig. 3), suggesting that de novo protein synthesis was required for the t-PA-induced increase of PAI-1 antigen in the medium. To study whether exogenously added t-PA could affect the level of substratum-associated PAI-1 and whether this PAI-1 was susceptible to form complex with t-PA, cells were prelabeled with 35S-methionine for 3 h prior to t-PA treatment. The substratum and conditioned media were

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Fig. 2 Time-course of t-PA effect on PAl-1 antigen (A) and PAl activity (B) in the conditioned medium and PAl-1 antigen in the cell lysate (C) of endothelial cells. HUVECs in 24-well dishes were treated with 0 (©), 0.5 (.) or 1 (.) gM t-PA in 0.5 ml of serum-free medium M'199 c°ntaining 25 gg/ml end°thelial cell gr°wth fact°r' 10 u/ml heparin, and 0.5% BSA for 30 min. The cultures were washed and incubated for specified time periods. Samples of the conditioned medium were collected at intervals and cell lysates were obtained by adding 0.5 ml of 0.1% Triton X-100 to the washed cells as described. The amounts of PAl_l antigen in the conditioned medium (A) and in the cell lysate (C) were determined by enzymelinked immunosorbent assay. PAl activity in the conditioned medium(B) was determined by titration with excess t-PA as described. Negative values of PAl activity represent a net t-PA activity. Values are the means+S.D, of three experiments performed in duplicate. Key for Figure 2B: [] 2 h, [] 6 h, ~ 9 h, [] 12 h, [] 24 h.

harvested after treatment with 0.1 and 1 gM t-PA for2 and 30 min. 35S-PAI-1 was immunoprecipitated with antiPAI-1 antibody, adsorbed with Protein A-Sepharose, and analyzed by SDS-PAGE. PAI-1 with molecular mass of 46 kDa was detected both in the substratum (Fig. 4A) and in the conditioned medium (Fig. 4B) of the control cells. However, no PAI-1 of 46 kDa was detected in the substraturn and conditioned medium of endothelial cells treated with 0.1 and I gM t-PA (Fig. 4A, B). Instead bands of 110, 81 and 44 kDa, which represent a t-PA-PAI-1 complex, a possible degraded product of t-PA-PAI-1 complex and a possible degraded product of PAI-1, respectively, were detected in the conditioned medium of cells treated with either 0.1 or I gM t-PA for 2 and 30 rain (Fig. 4B). No complex or degradation products of PAI-1 were observed in the substratum of the cells treated with t-PA (Fig. 4A). This result suggested that addition of t-PA to the endothelial cells caused a rapid depletion of PAI-1 from the substratum to form a t-PA.PAI-1 complex in the Fibrinolysis (1996) 10(3), 183-191

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extracellular matrix and also to other PAI-1 independent binding sites of HUVECs. 9,51 A mannose receptor on the liver endothelial cells which can bind t-PA and function as a mechanism to concentrate circulating t-PA was also identified. 5° Thus, liver endothelial cells m a y also participate in t-PA clearance, although to a lesser extent than liver parenchymal cells, s° In this study, part of the t-PA added to endothelial cells was associated with PAI-1 as demonstrated from the 35S-methionine prelabeling experiment. Approximately 0.3% of the added t-PA still

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t-PA Concentration (ILM) Fig. 3 Effect of cycloheximide on the release of PAl-1 antigen by tPA-pretreated endothelial cells. HUVECs were preincubated with and without cycloheximide (1 gg/ml) for 10 rain at room temperature. Cells were then treated with t-PA for 30 min at 37°C, washed, and incubated with and without cycloheximide (1 gg/ml) for 24h. The supernatant media were collected and the amounts of PAl-1 antigen were determined. Values are the means_+S.D, of three experiments performed in duplicate.

supernatant. To analyze changes of PAI-1 during a further incubation, cultures were washed three times after treatment with t-PA for 30 min and reincubated at 37°C for various durations. Samples of the culture medium and cell lysate were collected at 3 and 5 h of incubation and the 35S_PAI_1 was analyzed as previously described. PAI- 1 of 46 kDa was observed in the conditioned medium of the control cultures but not in the t-PA-pretreated cultures (Fig. 4C). On the other hand, PAI-1 degraded products of lower molecular mass were observed in the conditioned medium of t-PA-pretreated culture (Fig. 4C). The result indicated that the 35S-PAI-1 was degraded in the conditioned medium of cells after treatment with t-PA for 30 min and wash with culture medium. DISCUSSION This study has demonstrated that treatment of endothelial cells with t-PA led to an increase in the expression of PAI-1 mRNA. The stimulatory effect of PAI-1 mRNA expression b y t-PA is dependent on the catalytic activity of t-PA since the active-site-inhibited sample of t-PA failed to have this effect. It has been shown that t-PA specifically binds to HUVECs with binding sites of both high and low affinity. 49,5° Moreover, it was further demonstrated that t-PA binds to PAI-1 expressed on the Fibrinolysis (1996) 10(3), 183-191

© Pearson Professional Ltd 1996

PAl activity and t-PA

Fig. 4 SDS-PAGE and autoradiography of metabolically radiolabeled PAl-1 synthesized by endothelial cells. Depletion of PAl-1 from the substratum by exogenously added t-PA (A), the formation of t-PA.PAI-1 complex in the conditioned medium of the tPA-treated endothelial cells (B), and the degradation of the t-PA-PAI1 complex in the culture medium after incubation (C). Endothelial cell cultures were labeled with 50 gCi/ml a5S-methionine for 3 h, washed, treated with 0.1 and 1 gM t-PA for 2 and 30 min. The substratum (A) and conditioned medium (B) were harvested as described and 35SPAl-1 was detected on a Fuji Bio-lmaging Analyzer after immunoprecipitationwith anti-PAl-1 antibody, adsorption with Protein A-Sepharose, and analysis by SDS-PAGE. In a parallel experiment, the cultures were treated with 1 gM t-PA for 30 min, washed with M199 three times, the conditioned medium was collected at 3 and 5 h of incubation, and 35S-PA1-1 was detected as described (C).

remained bound to the endothelial cells even after the cultures were washed three times as monitored by the lasI-t-PA. A similarly small amount of residual plasminogen activator activity could be detected in the conditioned medium of HUVECs 2 and 6 h after treatment with t-PA (Fig. 2B). The active t-PA which is bound to its low affinity binding sites might be reversibly dissociated from the cells. 52 The binding of plasminogen activators to their unique membrane binding sites, other than PAI-1 binding sites, on endothelial cells may contribute to the regulation of fibrinolysis. 36'52 However, the exact mechanism is unclear. Complex formation and subsequent internalization and degradation of endogenous PM-1 in substratum of different cell cultures in interaction with t-PA and urokinase have been studied. Addition of t-PA to the isolated metabolically labeled substratum of HUVECs led to the formation and release of t-PA.PAI-1 complex as well as an inactive PAI-1 of 44 kDa. 16 Incubation of confluent cul© Pearson Professional Ltd 1996

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tures of HT-1080 cells with urokinase also resulted in the release of PM-1 from the substratum. 53 Studies with Hep G2 cells also showed that exogenous t-PA was able to form a complex with endogenous PAI-1 in the extracellular matrix. In addition, the t-PA.PAI-1 complex bound to a specific high-affinity receptor of Hep G2 cells and was internalized and degraded. 32 t-PA also binds to smooth muscle cell-associated PM-1 with subsequent t-PA.PM-1 internalization and degradation. 54 In the incubation of HUVECs with t-PA, a 81 kDa degraded t-PA.PAI-1 complex and a 45kDa degradation product of PAId were found in the 24-h conditioned medium. 17 In this study, treatment of HUVECs with t-PA also induced a rapid PAIl release from the substratum and the formation and release of intact 110 kDa and degraded 81 kDa t-PA.PAI-1 complexes, as well as degraded PM-1 of 44 kDa in the culture medium (Fig. 4A, B). Further incubation of the tPA-treated cells for 3 and 5 h resulted in the degradation oft-PA.PM-1 complex or PM-1 (Fig. 4C). In this study, HUVECs in serum-free medium were treated with 0.5 or I gM t-PA, and the elevation of PM-1 mRNA biosynthesis was observed. A large dose of t-PA (150 mg) may be infused in a few hours during the treatment of patients with acute myocardial infarction. A rapid initial clearance of t-PA from the circulation, followed b y a slower second phase of elimination, were observed. 55 These findings are compatible with the observation that the active t-PA may be concentrated on the surface of endothelial cells because of specific binding, followed by slow release from the low affinity binding sites (Fig. 2B). 52 The experimental conditions reported here may mimic the situation as thrombolytic therapy. Incubation of HUVECs with 2 gg/ml t-PA in the presence of 0.1% fetal calf serum for up to 2 4 h had no obvious effect on the steady state PAI-1 mRNA levels. 17 Under such experimental conditions, relatively low concentration of t-PA was used, which may not be capable of neutralizing and degrading all the substratum-associated PAI-1. Thus, there may not be enough t-PA to induce the biological responses including PAI-1 synthesis. In summary, the fibrinolytic activity of endothelial cells is subjected to a very complex regulatory mechanism. The cells may facilitate regulation of fibrinolysis through the balance in the expression of PAI-1 and low affinity t-PA binding sites on the cell surface. 52 Our previous investigation has indicated that plasmin inhibited the biosynthesis of t-PA in the endothelial cells, is In the present study, it is demonstrated that PAI-1 was depleted from the substratum by t-PA to form t-PA-PAI-1 complex in the culture medium in a very short reaction time. In the presence of excess t-PA, the biosynthesis of PAI-1 mRNA was significantly stimulated. The excess t-PA was neutralized by newly synthesized PM-1 which was most likely to be in active form. Once the excess t-PA activity Fibrinolysis (1996) 10(3), 183-191

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was c o m p l e t e l y n e u t r a l i z e d b y PAI-1, t h e PAI-1 r e l e a s e d in t h e m e d i u m was c o n v e r t e d to i n a c t i v e l a t e n t form. T h u s , H U V E C c a n a u t o r e g u l a t e t h e f i b r i n o l y t i c a c t i v i t y in t h e c u l t u r e m e d i u m b y i n c r e a s e t h e p r o d u c t i o n o f PAI-1 in r e s p o n s e to e x c e s s t-PA. It r e m a i n s to b e e l u c i d a t e d w h e t h e r o u r o b s e r v a t i o n w o u l d o c c u r in v i v o d u r i n g t h r o m b o l y t i c i n f u s i o n o f t-PA, a n d a n y c o r r e l a t i o n b e t w e e n t h e e l e v a t i o n of PAI-1 a n d r e o c c l u s i o n . Studies to a n s w e r t h e s e q u e s t i o n s are c u r r e n t l y in p r o g r e s s in o u r laboratory.

ACKNOWLEDGEMENTS T h e a u t h o r s w o u l d like to t h a n k H s i a n g - H u e y Y e n a n d LiC h i n g C h a n g for t h e i r o u t s t a n d i n g t e c h n i c a l assistance.

Abbreviations PAI-1: p l a s m i n o g e n a c t i v a t o r i n h i b i t o r t y p e 1; t-PA: r e c o m b i n a n t tissue-type p l a s m i n o g e n activator; SDS-PAGE: s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e sis; GAPDH: g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e ; HUVECs: h u m a n u m b i l i c a l v e i n e n d o t h e l i a l cells.

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