Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells.

Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase.

controlling the biosynthesis of tPA, PAI-1, and their receptors. Impaired fibrinolytic activity has been observed in some patients with thrombotic disorders (14, 15), and a correlation between impaired fibrinolytic function and acute myocardial infarction has been reported (16,17). In patients with recurrent infarction, lower tPA activity and increased PAI-1 activity have been found (18).
Thrombolytic agents such as streptokinase, urokinase, and tPA have been used clinically to treat patients with acute myocardial infarction and deep vein thrombosis. However, reocclusion occurs in about 30% of the initially successful cases of myocardial infarction treated with thrombolytic agents (19). The risk factors for a reocclusion episode have not been well elucidated. During thrombolytic therapy, the infusion of plasminogen activators causes activation of a large amount of plasminogen and a transient increase of plasmin in the blood. The major side effects of a high concentration of plasmin in the blood include nonspecific hydrolysis of fibrinogen and inactivation of blood coagulation factors (20,21). As a result, bleeding complications are common, and the frequency of hemorrhagic events in large-scale clinical trials of thrombolytic therapy ranges from 0.3 to 19% (22). Califf et al. (22) have shown that catalytically active, fluid-phase plasmin can exist in the blood in large amounts either before it is inhibited by a,-antiplasmin or when the latter is totally exhausted by extensive plasmin generation following the administration of plasminogen activators. When this happens, the excess plasmin is capable of catalyzing fibrinogenolysis, which would lead to a significant drop in fibrinogen. The vascular endothelium is directly exposed to catalytically active plasmin, at least for a short time. Yet, to date, few studies have been reported on the possible effect of plasmin on the expression of fibrinolytic factors in endothelial cells. In our study the effects of plasmin on the production of tPA and PAI-1 in HUVEC were investigated. We demonstrated that tPA biosynthesis in the cells is down-regulated by plasmin but that the production of PAI-1 antigen is not affected.
In an attempt to elucidate the signal transduction pathways involved in the regulation of fibrinolytic activity of HUVEC, we also studied the effect of plasmin on protein phosphorylation in the cell. Protein kinase C activation has been portrayed as an important factor in the regulation of tPA and PAI-1 biosynthesis in endothelial cells (23, 24). Phorbol ester, which enhances protein kinase C activity, induces the biosynthesis of tPA in endothelial cells (25). Thrombin stimulates the biosynthesis of tPA antigen and protein kinase C activity of endothelial cells (26)(27)(28)(29). Treatment of HUVEC with thrombin results in receptor-and catalytic activity-dependent increases in protein kinase activity (28). In this report the effect of plasmin on the protein kinase activity of HUVEC was studied in comparison with other agonists.

MATERIALS AND METHODS
Cell Culture-Endothelial cells were isolated from human umbilical cord veins by the method previously described (27). Cells were grown to confluence in medium "199 containing 10% fetal calf serum, 25 pg/ml endothelial cell growth factor, 10 units/ml heparin, 100 units/ ml penicillin, and 100 units/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, at which time they were washed twice with "199 and incubated at 37 "C in 0.5 ml of "199 containing 0.5% BSA.
Cell Viability Evaluation-After treatment with protease and incubation in "199 containing 0.5% BSA for 24 h, the cell cultures were harvested by incubation for 10 min with 0.1% trypsin, and the viable cell numbers were counted by the trypan blue dye exclusion method. The amount of total protein of the attached cells, another parameter for viability, was determined with the Lowry method (30) after washing the cell cultures three times with phosphate-buffered saline.
Treatment of HUVEC with Proteases-Confluent cell cultures at passage three were washed twice with serum-free "199 containing 0.5% BSA. Cells were then treated with plasmin, plasmin derivatives, thrombin, trypsin, chymotrypsin, or collagenase at a determined concentration in 0.5 ml of "199 containing 0.5% BSA for defined time periods. Cultures were then washed three times with the same medium and incubated at 37 "C for various durations. Samples of the culture medium were collected at intervals, centrifuged at 15,000 X g, and made 0.01% with Tween 80. The lysates of the cells, from which medium had been removed at a predetermined interval, were obtained by adding 0.1% Triton X-100 to the washed cells at 4 "C for 1 h.
Samples of conditioned medium and cell lysate were frozen at -40 "C until assayed.
Caseinolytic Activity of Various Proteases-The proteolytic activity of plasmin, trypsin, chymotrypsin, and collagenase was determined by using a-casein as the substrate (35)(36)(37). To 1 ml of 4% casein solution were added 0.8 ml of 0.067 M phosphate buffer and 0.2 ml of protease sample. The mixture was incubated at 37 "C for 30 min, and then 3.0 ml of 15% trichloroacetic acid were added to stop the reaction, and the sample was cooled on ice for 10 min and filtered through Whatman No. 40 filter paper. The absorbance at 280 nm of the filtrate was taken using casein only as the blank. One CTA unit of protease catalyzed the casein hydrolysis and caused a release of 0.1 peq of tyrosine/min as defined (35).
Quantitation of tPA and PAI-1 Antigen Levels-tPA and PAI-1 were assayed by procedures recommended by the manufacturer of Imubind-5 tPA and PAI-1 enzyme-linked immunosorbent assay kits (American Diagnostica).
Assay of PAI Actiuity-PA1 activity was measured by titrating samples with increasing amounts of tPA into a fixed volume of endothelial cell-conditioned medium or control medium (38,39). The excess tPA activity was quantitatively assayed as described by Verheijen et al. (39). PA1 activity was calculated from the intersection of the asymptote of the titration curve with the x axis and expressed in international units of tPA inhibited.
Phosphate Labeling of Endothelial Cells-The method described by Levin and Santell (28) was followed. Briefly, confluent cells in 12well tissue culture dishes were washed with phosphate-free RPMI 1640 medium and incubated in the same medium containing 50 pCi/ ml [32P]orthophosphate for 1 h; the test compounds were then added, and the reaction was allowed to take place for the defined period of time. The reactions were stopped by removing the medium and by adding 500 pl of 10% trichloroacetic acid to the cells. After washing twice with 1 ml of cooled water, the protein was solubilized with 160 pllwell of 62.5 mM Tris-HC1, pH 6.8, and 1% SDS.
Electrophoresis-SDS-PAGE was performed according to the procedure of Laemmli (40), using a 12.5% separating gel and a 5% stacking gel. A final protein content of 20 pg/sample was reduced with 5% P-mercaptoethanol and boiled for 3 min prior to application to the gel. All gels were stained with Coomassie Brilliant Blue and dried before autoradiography.
Reagents-Medium "199, fetal calf serum, penicillin, and streptomycin were purchased from GIBCO. Goat anti-PAI-1 antibody (at the 50% inhibition point, 1 mg inhibits about 1000 IU of PAI-1) was purchased from American Diagnostica; the endothelial cell growth factor was from Collaborative Research; heparin, S-2251 (D-Val-Leu-Lys-p-nitroanilide), trypsin, chymotrypsin, collagenase, and BSA were purchased from Sigma; and bovine thrombin was from Miles. All other chemicals were of the highest grade available commercially. FIG. 1. Inhibition of tPA antigen production and stimulation of PA1 activity by pretreatment with plasmin: dose titration. Increasing concentrations of plasmin were added to confluent cultures of endothelial cells, which were prewashed and incubated in medium "199 containing 0.5% BSA. After treatment with plasmin at 37 "C for 1 h, the cultures were washed and incubated in "199 containing 0.5% BSA for 24 h. The supernatant media and cell lysates were collected, and the amounts of tPA antigen (A) were determined by enzyme-linked immunosorbent assay; PA1 activity ( B ) was determined by titration with excess tPA as described under "Materials and Methods." Values are the mean & S.D. of five experiments performed in duplicate. changed (Fig. L4). This effect of plasmin on the endothelial cells took place very rapidly, reaching a maximum after a 10min treatment. However, no further decrease in tPA biosynthesis was observed when the duration of treatment of HU-VEC with plasmin was extended beyond 10 min and up to 2 h. Experiments in which the cell medium, treated or untreated with plasmin, was sequentially sampled in the ensuing 24 h showed that tPA antigen in the conditioned medium of the control group increased linearly with the duration of incubation, whereas in the cells pretreated with 100 nM plasmin for 1 h, the amount of tPA dropped to as low as 30% of that produced in a control group after a 24-h incubation (Fig. 2.4). The inhibitory effect of plasmin on tPA biosynthesis was blocked by 10 mM t-aminocaproic acid, which is known to interfere with the binding of plasmin to HUVEC (Table I). t-Aminocaproic acid alone at the same concentration exerted no significant effect on the biosynthesis of tPA in HUVEC (Table I) I Effect of c-aminocaproic acid (EACA), plasmin derivatives, and various proteases on production of tPA and PAI-1 by endothelial ce1l.s HUVEC were treated with various compounds at the indicated concentrations for 1 h, washed, and incubated for 24 h as described in Fig. 1 kringles 1-3, and kringle 4 did not affect the production of tPA antigen in HUVEC (Table I). Microplasmin, a plasmin derivative with no kringle domains, reduced the production of tPA antigen by 50% (Table I). Cells treated with other proteases (0.2 CTA unit/ml) such as trypsin, chymotrypsin, and collagenase under the same conditions had no effect on tPA biosynthesis in the cell culture (Table I). Thrombin caused a 1.7-fold increase in the tPA antigen biosynthesis (Table I). After a 24-h incubation there was no change in cell viability of the cultures treated with proteases for 1 h.

Endothelial cells of the
Treatment with plasmin at 100 nM for up to 2 h did not have any significant effect on the biosynthesis of PAI-1 antigen of the cells. The amount of PAI-1 antigen in c6nditioned media of both plasmin-treated and control HUVEC increased linearly with incubation time (Fig. 2B). However, treatment with plasmin resulted in a dose-dependent increase of PA1 activity in conditioned medium, while PA1 activity in cell lysates of HUVEC remained unchanged (Fig. 1B). Like the inhibitory effect of plasmin on tPA antigen biosynthesis, the enhancing effect of plasmin on PA1 activity also developed very rapidly. The maximum amount of PA1 activity was obtained in conditioned medium of cells treated with plasmin for 10 min, beyond which no further increase could be observed even when the duration of treatment was increased to as long as 2 h. The PA1 activity in conditioned medium of HUVEC pretreated with 100 nM plasmin increased with duration of incubation (Fig. 2C). In the control group PA1 activity in conditioned medium also increased but reached a plateau of 2 IU/ml after a 4-h incubation, and no further increase was observed (Fig. 2C). Kringle domains of plasmin, such as kringles 1-5, kringles 1-3, and kringle 4 did not have a significant effect on PA1 activity in conditioned medium of HUVEC (Table I). Microplasmin caused a 2-fold increase in PA1 activity compared with the control (Table I). Trypsin, chymotrypsin, and collagenase did not change PA1 activity in conditioned medium of HUVEC (Table I). Thrombin caused a 2-fold increase in PA1 activity (Table I). PA1 activity in all these conditioned media could be specifically inhibited by a PAI-1 specific antibody (Fig. 3), proving that PA1 activity in these media was due to PAI-1.
The effect of plasmin on protein phosphorylation in HU-VEC was studied in comparison with that of phorbol ester PMA and H-7. Plasmin (100 nM), PMA (100 nM), H-7 (90 p~) , or forskolin (100 p~) was added to the culture for defined periods of time as indicated after incorporation of ["Plorthophosphate. The phosphoproteins were fractionated and detected by one-dimensional 12.5% SDS-PAGE and autoradiography (Fig. 4). The pattern of bands stained with Coomassie Brilliant Blue in each sample was essentially identical. However, on autoradiography, it was clear that PMA induces a rapid increase in phosphorylation of proteins in HUVEC (Fig.   4). Phosphorylation of protein(s) of M, 30,000 was observed in cells treated with PMA but not in the control cells. PMA had the most profound effect on the enhancement of the phosphorylation of proteins of M, 80,000, 66,000, 60,000, 43,000,40,000, 30,000, and 19,000. No further increase of the extent of protein phosphorylation was observed when forsko- lin and PMA were added to the cells at the same time. On the other hand, a significant decrease of basal phosphorylation of various proteins in HUVEC was observed when treated with plasmin or H-7 for 20 min (Fig. 4). In experiments in which plasmin was added to cultures 20 min before PMA or PMA/ forskolin, protein kinase activity dropped significantly compared with control experiments with no plasmin pretreatment.

DISCUSSION
Endothelial cells can synthesize both tPA and its specific inhibitor PAI-1, which are two of several important factors involved in the regulation of fibrinolytic function in the vascular system (3,41,42), and many studies have been reported on the regulation of their biosynthesis (43)(44)(45). Since vascular endothelial cells line the surface of the system in which blood flows, any changes in its character will promptly affect the dynamics of blood circulation. Therefore, regulation of the fibrinolytic tendency of endothelial cells, if it can be achieved, will be of enormous significance in the prevention and treatment of thrombotic incidents. Several physiological agents such as histamine and thrombin have been shown to stimulate the biosynthesis of tPA in these cells (25)(26)(27)46). Tumor-promoting phorbol esters stimulate tPA release from HUVEC, and the elevation of CAMP resulting from forskolin treatment has been shown to further potentiate this effect (47). These agonists also induced the biosynthesis of PAI-1, although the effect was not as pronounced as that on the biosynthesis of tPA (29,47). Thrombin is believed to play essential roles in the regulation of both blood coagulation and the fibrinolytic character of the endothelium (27). Incubation with thrombin causes an increase in tPA biosynthesis in endothelial cells (Table I) (26,27). Previous studies suggested that protein kinase C activation could be one of the factors responsible for increased production of tPA in response to treatment with thrombin and possibly with other physiological agonists (24).
Plasmin is the major enzyme directly responsible for fibrinolysis. Specific binding sites on the endothelium for plasmin have been reported (12). In thrombolytic therapy, infusion of large doses of plasminogen activators would result in a transient increase of plasmin concentration in the blood circulation. A situation similar to the experimental conditions reported here is then created. Under these circumstances, plasmin may have an important effect on the functions of the endothelium, and the consequences may be critical to the outcome of thrombolytic therapy. As shown in this study, plasmin may act as a feedback regulator of the fibrinolytic system. It may signal the endothelial cells to turn off the biosynthesis of tPA (Fig. LA). On the other hand, the biosynthesis of PAI-1 antigen was not affected (Fig. 2B). The increased PA1 activity in conditioned medium may be in part because less tPA antigen was synthesized and released. As a result the endothelial cells can attenuate fibrinolytic activity in response to stimulation by plasmin (Fig. 2C).
The reaction of plasmin with endothelial cells is bindingand catalytic activity-dependent (Table I). Plasmin containing both kringle and active-site domains was more potent than microplasmin in reducing tPA biosynthesis (Table I). The experiments in which plasmin, thrombin, and other proteases were compared indicated that the influence of plasmin and thrombin on the biosynthesis of fPA did not result simply from the proteolytic perturbation of endothelial cells in culture. Specific binding of catalytically active plasmin or thrombin to receptors on the surface of endothelial cells is required for their unique effects. Protein synthesis was re-quired for tPA and PAI-1 to be present in the medium, since cycloheximide completely inhibited their production (data not shown). The PA1 activity in the medium was mostly from PAI-1-related inhibitors, since the tPA inhibitor activity diminished when PAI-1-specific antibody was added to the conditioned medium of HUVEC with or without plasmin pretreatment (Fig. 3). The HUVEC were shifted to a less fibrinolytic state since a higher residual activity of PAI-1 was found in conditioned medium after treatment with plasmin. In plasma, plasmin may easily reach a concentration ranging from 10 to 100 nM during thrombolytic therapy with plasminogen activators or under some uncontrolled fibrinolytic pathological conditions in the local area, since the normal concentration of plasminogen is as high as 2.2 PM (48). The elevated concentration of plasmin may down-regulate the biosynthesis of tPA in endothelial cells. Plasmin, therefore, could act as one of the physiological regulators in the feedback control of the fibrinolytic system.
Thrombin and histamine are known to activate the phosphoinositide pathway and to generate diacylglycerol, which is a physiological activator of protein kinase C (28, 49). Activation of protein kinase C in endothelial cells has also been demonstrated when tPA synthesis is stimulated by thrombin and histamine, as well as by phorbol esters (25-28). It has also been suggested that H-7-sensitive protein kinase regulates the secretion of tPA (24), and H-7 and staurosporine, the protein kinase inhibitors, causes depression of the basal level of tPA, although they have no effect on PAI-1 secretion (24). In this regard, since the effect of plasmin is in many ways very similar to that of H-7, we became interested in whether the plasmin-induced decline in tPA synthesis resulted through its action on the protein kinase activity of HUVEC. Incubation of HUVEC with plasmin caused a decrease in the basal phosphorylation of proteins in HUVEC, as in the case of H-7 (Fig. 4). Plasmin also had the same effect on HUVEC as H-7 in inhibiting tPA synthesis. Parallel effects of plasmin and H-7 on protein phosphorylation and tPA and PAI-1 synthesis of HUVEC are consistent with the idea that protein kinases serve as mediators in the regulation of tPA secretion in HUVEC stimulated by phorbol ester, thrombin, histamine, and other physiological agonists. Forskolin caused an increase in cellular cAMP by stimulating adenyl cyclase activity. However, forskolin did not have a significant effect either on the increase of protein phosphorylation induced by PMA or on the decrease of protein phosphorylation caused by plasmin. The results indicated that cAMP is not involved in the effects of plasmin in decreasing protein kinase activity of HUVEC.
Thrombin is a potent agonist for a number of biological responses in platelets and endothelial cells (50-52). It is still uncertain whether thrombin activates its receptor by a simple occupancy mechanism or by a novel mechanism involving proteolytic cleavage of the receptor, as proposed by the recent studies of Vu et al. (53) and Huang et al. (54). However, the activating effect of thrombin on platelet phospholipase C and the accumulation of 3-phosphorylated phosphoinositides could be accounted for by specific thrombin proteolytic activity. Although the responses of endothelial cells to plasmin in tPA and PAI-1 release are not the same as that of thrombin (Table I), some similarities in the actions of these two agonists have been observed. Endothelial cells have binding sites for the kringle domains of plasmin and thrombin, and the effect of these two agonists depends on their proteolytic activity. However, the binding sites for plasmin and thrombin in endothelial cells are obviously different, since the two enzymes are not competitive in binding (12). Whether the same mechanisms of thrombin acting on its endothelial cell receptors could be invoked to explain the action of plasmin on its specific receptor requires further investigation.
In most previously reported studies of tPA induction in endothelial cells, protease was left in contact with endothelial cells for many hours. However, one would expect that a protease would be inactivated rapidly in vivo, and therefore, an extrapolation of these findings in vitro to the situation in vivo requires considerable caution (38). In our study, treatment of endothelial cells with plasmin at physiological concentration for 20 min was sufficient to induce a decrease in protein kinase activity and to cause a decrease in tPA production in endothelial cells. Taken together, these results on the effects of plasmin on the fibrinolytic tendency of endothelial cells may be clinically important and should be considered during fibrinolytic therapy. Our study also provides several important clues for further research on molecular mechanisms involved in the interaction of plasmin and endothelial cells in the regulation of thrombosis and fibrinolysis.