Natriuretic peptides reduce plasminogen activator inhibitor-1 expression in human endothelial cells

Plasma concentrations of natriuretic peptides increase in some pathological conditions, but very little is known about the effect of these vasodilator peptides on the regulation of the blood coagulation system. The fundamental role in the regulation of fibrinolysis is played by plasminogen activator inhibitor type 1 (PAI-1). Recent studies demonstrate that natriuretic peptides can modulate PAI-1 expression in bovine aortic smooth muscle cells and rat aortic endothelial cells. In this report, we tested the effect of natriuretic peptides on PAI-1 expression in the human endothelial cell line (EA.hy 926). For this purpose, we treated the cell cultures with ANP, BNP and CNP, and modulation of PAI-1 synthesis was evaluated. We compared the effect of natriuretic peptides on synthesis and release of PAI-1 in unstimulated cells, and after activation with tumour necrosis factor alpha (TNFalpha). Natriuretic peptides abolished TNFalpha - induced upregulation of PAI-1 expression at both the PAI-1 mRNA and the antigen levels. The inhibitory efficiency was higher in the case of CNP when compared to that produced by ANP and BNP, particularly when TNFalpha-stimulated cells were used. We observed an inhibition of stimulatory effect of TNFalpha on PAI-1 expression also at the level of the PAI-1 promoter in cells transfected with a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was markedly inhibited by C-type natriuretic peptide, already at a very low (0.001 micro M) concentration of the peptide.     

Transmembrane signaling pathway mediates oxidized low-density lipoprotein-induced expression of plasminogen activator inhibitor-1 in vascular endothelial cells

Atherosclerotic cardiovascular disease is the number one cause of death for adults in Western society. Plasminogen activator inhibitor-1 (PAI-1), the major physiological inhibitor of plasminogen activators, has been implicated in both thrombogenesis and atherogenesis. Previous studies demonstrated that copper-oxidized low-density lipoprotein (C-oLDL) stimulated production of PAI-1 in vascular endothelial cells (EC). The present study examined the involvement of lectin-like oxidized LDL receptor-1 (LOX-1) and Ras/Raf-1/ERK1/2 pathway in the upregulation of PAI-1 in cultured EC induced by oxidized LDLs. The results demonstrated that C-oLDL or FeSO(4)-oxidized LDL (F-oLDL) increased the expression of PAI-1 or LOX-1 in human umbilical vein EC (HUVEC) or coronary artery EC (HCAEC). Treatment with C-oLDL significantly increased the levels of H-Ras mRNA, protein, and the translocation of H-Ras to membrane fraction in EC. LOX-1 blocking antibody, Ras farnesylation inhibitor (FTI-277), or small interference RNA against H-Ras significantly reduced C-oLDL or LDL-induced expression of H-Ras and PAI-1 in EC. Incubation with C-oLDL or F-oLDL increased the phosphorylation of Raf-1 and ERK1/2 in EC compared with LDL or vehicle. Treatment with Raf-1 inhibitor blocked Raf-1 phosphorylation and the elevation of PAI-1 mRNA level in EC induced by C-oLDL or LDL. Treatment with PD-98059, an ERK1/2 inhibitor, blocked C-oLDL or LDL-induced ERK1/2 phosphorylation or PAI-1 expression in EC. The results suggest that LOX-1, H-Ras, and Raf-1/ERK1/2 are implicated in PAI-1 expression induced by oxidized LDLs or LDL in cultured EC.     

Effect of retinoic acid on the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cells

The synthesis of plasminogen activators and inhibitors in endothelial cells is highly regulated by hormones, drugs and growth factors. The present study evaluates the effect of retinoic acid on the synthesis of tissue-type plasminogen activator (t-PA) and of plasminogen activator inhibitor-1 (PAI-1) by cultured human umbilical vein endothelial cells (HUVEC). Retinoic acid produced a time- and concentration-dependent increase in the secretion of t-PA-related antigen but not of PAI-1 related antigen into the culture medium. A maximal sevenfold increase of t-PA antigen after 24 h was observed with 10 microM and a half-maximal increase with 0.1 microM retinoic acid. Retinoic acid induced a time-dependent increase of the t-PA mRNA, with a maximum at 8 h and returning to normal at 24 h. The protein kinase inhibitor H7 decreased the t-PA antigen induced by both retinoic acid and phorbol 12-myristate 13-acetate. These results suggest that treatment of HUVEC with retinoic acid increases t-PA production by a pathway which, at some level, involves protein kinases. Thus, retinoic acid induces t-PA synthesis in the absence of altered PAI-1 synthesis, which may enhance the fibrinolytic potential of the endothelium.     

Protection of plasminogen activator inhibitor-1-deficient mice from nasal allergy

This study was performed to clarify the relationship between fibrinolytic components and the pathology of allergy, particularly that during the development of nasal allergy and nasal tissue changes. Intranasal OVA challenge after sensitization by i.p. administration of OVA induced a higher level of excess subepithelial collagen deposition in wild-type (WT) C57BL/6J mice than in plasminogen activator inhibitor (PAI)-1-deficient (PAI-1(-/-)) mice. The excess PAI-1 induction in the nasal mucosa and higher level of active PAI-1 in the nasal lavage fluid of WT-OVA mice compared with those in WT-control mice suggested that the decrease of proteolytic activity inhibits the removal of subepithelial collagen. The frequency of sneezing, nasal rubbing, nasal hyperresponsiveness, production of specific IgG1 and IgE in the serum, and production of IL-4 and IL-5 in splenocyte culture supernatant increased significantly in WT-OVA mice. In PAI-1(-/-) mice, these reactions were absent, and specific IgG2a in serum and IFN-gamma in splenocyte culture medium increased significantly. Histopathologically, there were marked goblet cell hyperplasia and eosinophil infiltration into the nasal mucosa in WT-OVA mice, but these were absent in PAI-1(-/-) mice. These results indicate that the immune response in WT-OVA mice can be classified as a dominant Th2 response, which would promote collagen deposition. In contrast, the Th2 response in PAI-1(-/-) mice was down-regulated, and the immune response shifted from Th2-dominant reaction to a Th1-dominant one. Taken together, these findings suggest that PAI-1 plays an important role not only in thrombolysis but also in immune response.     

Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells.

Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic factor, were assessed on PA activity and PA and PAI-1 mRNA levels in microvascular endothelial cells. u-PA and t-PA activity were increased by VEGF in a dose-dependent manner, with maximal induction at 30 ng/ml. u-PA and t-PA mRNAs were increased 7.5- and 8-fold respectively after 15 hours, and PAI-1 mRNA 4.5-fold after 4 hours exposure to VEGF. At equimolar concentrations (0.5 nM), VEGF was a more potent inducer of t-PA mRNA than bFGF, while bFGF was a more potent inducer of u-PA and PAI-1 mRNAs. In addition, VEGF induced u-PA and PAI-1 mRNAs with kinetics similar to those previously demonstrated for bFGF. These results demonstrate the regulation of PA and PAI-1 production by VEGF in microvascular endothelial cells and are in accord with the hypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesis.     

Oncostatin M induces tissue-type plasminogen activator and plasminogen activator inhibitor-1 in Calu-1 lung carcinoma cells

Oncostatin M (OSM) is a glycoprotein cytokine that is produced by activated T-lymphocytes, monocytes, and macrophages. In a DNA synthesis assay, OSM reduced tritiated thymidine incorporation by 53% in Calu-1 lung carcinoma cells. Radiolabeled cDNAs from untreated Calu-1 cells and 30-h OSM-treated cells were used to probe duplicate nylon membrane cDNA expression arrays. This study revealed OSM-mediated expression of mRNAs encoding tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Northern blot analysis showed that the steady-state level of tPA mRNA is nearly undetectable in Calu-1 cells. Exposure of these cells to OSM for 30 h increased tPA mRNA expression by 20-fold and PAI-1 mRNA expression by 5-fold. Exposure of these cells to other gp130 receptor family cytokines, including leukemia inhibitory factor (LIF), interleukin-6 (IL-6), and IL-11, do not significantly affect DNA synthesis or induction of tPA/PAI-1. Western blot studies demonstrated that OSM mediates a marked increase in secretion of the tPA protein. Secreted tPA was present in the conditioned medium almost exclusively as tPA/PAI-1 complexes. Inhibitor studies demonstrated that OSM-mediated induction of tPA and PAI-1 mRNAs is largely dependent upon activation of the MEK1/2 pathway. The JAK3/STAT3 pathway potentially serves a secondary role in these regulatory events.     

Polymerization of plasminogen activator inhibitor-1

The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into beta-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and beta-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-A-sheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving beta-strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet.     

A novel antithrombotic role for high molecular weight kininogen as inhibitor of plasminogen activator inhibitor-1 function

The adhesive glycoprotein vitronectin (VN) forms a function-stabilizing complex with plasminogen activator inhibitor-1 (PAI-1), the major fibrinolysis inhibitor in both plasma and vessel wall connective tissue. VN also interacts with two-chain high molecular weight kininogen (HKa), particularly its His-Gly-Lys-rich domain 5, and both HKa and PAI-1 are antiadhesive factors that have been shown to compete for binding to VN. In this study the influence of HKa and domain 5 on the antifibrinolytic function of PAI-1 was investigated. In a purified system, HKa and particularly domain 5 inhibited the binding of PAI-1 to VN and promoted PAI-1 displacement from both isolated VN as well as subendothelial extracellular matrix-associated VN. The sequence Gly(486)-Lys(502) of HKa domain 5 was identified as responsible for this inhibition. Although having no direct effect on PAI-1 activity itself, HKa domain 5 or the peptide Gly(486)-Lys(502) markedly destabilized the VN.PAI-1 complex interaction, resulting in a significant reduction of PAI-1 inhibitory function on plasminogen activators, resembling the effect of VN antibodies that prevent stabilization of PAI-1. Furthermore, high affinity fibrin binding of PAI-1 in the presence of VN as well as the VN-dependent fibrin clot stabilization by the inhibitor were abrogated in the presence of the kininogen forms mentioned. Taken together, our data indicate that the peptide Gly(486)-Lys(502) derived from domain 5 of HKa serves to interfere with PAI-1 function. Based on these observations potential low molecular weight PAI-1 inhibitors could be designed for the use in therapeutic interventions against thromboembolic complications.     

Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-1 from human endothelial cells

We have examined the effect of phorbol esters and cAMP elevating compounds on tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) secretion. Phorbol esters induce a time- and dose-dependent increase in tPA release from endothelial cells, while forskolin, isobutylmethylxanthine, dibutyryl cAMP, and 8-bromo-cAMP had no significant stimulatory effect on tPA secretion. However, elevation of cAMP simultaneously with phorbol ester treatment potentiated the phorbol ester-induced release of tPA 6 times from 22.2 ng/ml with phorbol myristate acetate (PMA) alone to 122.1 ng/ml (PMA and forskolin). Potentiation was dose-dependent (half-maximal potentiation = 4 microM forskolin), and tPA release was enhanced at all stimulatory concentrations of PMA with no change in the PMA concentrations causing half-maximal or maximum tPA release. The kinetics of release was also similar in PMA versus PMA-forskolin-treated cells. A 4-h delay was observed, enhanced release was transient, and was followed by the onset of a refractory period. In contrast, elevation of cAMP reduced constitutive secretion of PAI-1 by 30-40% and prevented the increase in PAI-1 secretion stimulated by PMA. Elevated cAMP also decreased the rate of PAI-1 deposition into the endothelial substratum. These studies indicate that activation of a cAMP-dependent pathway(s) in coordination with phorbol ester-induced responses plays a central role in modifying the tPA and PAI-1 secretion from endothelial cells, leading to a profibrinolytic state in the endothelial environment.     

IL-8 induces imbalances between nitric oxide and endothelin-1, and also between plasminogen activator inhibitor-1 and tissue-type plasminogen activator in cultured endothelial cells

Interleukin-8 (IL-8), a member of the CXC chemokine family, plays an important role in the modulation of multiple biological functions in endothelial cells containing the receptors CXCR1 and CXCR2. It has previously been shown that IL-8 directly enhances endothelial cell survival, and stimulates the production of matrix metalloproteinases, which in turn regulates angiogenesis. However, its role in the regulation of the production of vasoactive substances in endothelial cells is less well defined. In this study, we investigate the effects of IL-8 on the proliferation of human umbilical vein endothelial cells (HUVECs). In addition, we also study the effects of IL-8 on the production of vasodilator, vasoconstrictor and fibrinolytic factors in these cells. The results show that recombinant IL-8 (50-200ng/ml) induces neither HUVEC proliferation nor nitric oxide (NO) release. However, it significantly increases the production of endothelin-1 (ET-1) in a concentration-dependent manner. Furthermore, incubation of endothelial cells with IL-8 (200ng/ml) up-regulates the plasminogen activator inhibitor-1 (PAI-1) in HUVECs, while it down-regulates the tissue plasminogen activator (t-PA). These findings suggest that IL-8 offsets the balance between endothelial vasoconstrictors and vasodilators. Furthermore, IL-8 also leads to an imbalance between PAI-1 and t-PA, which causes the ECs to become procoagulative and hypofibrinolytic.     

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.     

Longistatin, a novel plasminogen activator from vector ticks, is resistant to plasminogen activator inhibitor-1

Thrombo-occlusive diseases are major causes of morbidity and mortality, and tissue-type plasminogen activator (t-PA) is recommended for the treatment of the maladies. However, both t-PA and u-PA are rapidly inactivated by plasminogen activator inhibitor-1 (PAI-1). Here, we show that longistatin, a novel plasminogen activator isolated from the ixodid tick, Haemaphysalis longicornis is resistant to PAI-1. Longistatin was relatively less susceptible to the inhibitory effect of SDS-treated platelet lysate than physiologic PAs. Platelet lysate inhibited t-PA and tcu-PA with the IC₅₀ of 7.7 and 9.1 μg/ml, respectively, whereas for longistatin inhibition IC₅₀ was 20.1 μg/ml (p < 0.01). Similarly, activated PAI-1 (20 nM) inhibited only 21.47% activity of longistatin but almost completely inhibited t-PA (99.17%) and tcu-PA (96.84%). Interestingly, longistatin retained 76.73% initial activity even after 3 h of incubation with 20 nM of PAI-1. IC₅₀ of PAI-1 during longistatin inhibition was 88.3 nM while it was 3.9 and 3.2 nM in t-PA and tcu-PA inhibition, respectively. Longistatin completely hydrolyzed fibrin clot by activating plasminogen efficiently in the presence of 20 nM of PAI-1. Importantly, unlike t-PA, longistatin did not form complex with PAI-1. Collectively, our results suggest that longistatin is resistant to PAI-1 and maybe an interesting tool for the development of a PAI-1 resistant effective thrombolytic agent.     

Daunorubicin attenuates tumor necrosis factor-alpha-induced biosynthesis of plasminogen activator inhibitor-1 in human umbilical vein endothelial cells

The anthracycline antibiotic daunorubicin is reported to induce apoptosis in cells by triggering ceramide generation through de novo synthesis or sphingomyelin hydrolysis. Treatment of human umbilical vein endothelial cells (HUVEC) with daunorubicin markedly decreased the mRNA expression and protein release of plasminogen activator inhibitor-1 (PAI-1). This cellular event was accompanied by a significant increase in the total ceramide content in HUVEC. On the other hand, tumor necrosis factor (TNF)-alpha treatment of HUVEC led to an increase in both PAI-1 mRNA expression and protein release, and an enhancement of total ceramide content was also observed. The stimulating effect of TNF-alpha on PAI-1 synthesis was attenuated by the pretreatment of HUVEC with daunorubicin. Interestingly, the daunorubicin-induced increase in ceramide content was blocked by addition of the potent ceramide synthase inhibitor fumonisin B(1), while the TNF-alpha-induced ceramide increase was not affected by this drug. Fumonisin B(1) treatment restored the daunorubicin-induced decrease in PAI-1 release to approximately 70% of the control, but did not affect the TNF-alpha-induced increase in PAI-1 release. Thus, these data imply the possibility that the subcellular topology of ceramide production determines its lipid mediator function in the regulation of PAI-1 synthesis in HUVEC, because both TNF-alpha and daunorubicin could increase the ceramide levels.     

The prognostic value of vascular endothelial growth factor, urokinase plasminogen activator and plasminogen activator inhibitor-1 in node-negative breast cancer

The vascular endothelial growth factor (VEGF) and the plasminogen activator system play an essential role in solid tumor angiogenesis and in tumor invasion and metastasis. In the present study we investigated the relationship between patient outcome and levels of VEGF, urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in tumor cytosols of 196 node-negative primary invasive breast cancer patients who did not receive any adjuvant therapy. The median follow-up was 65 months. VEGF, uPA and PAI-1 were measured by commercially available enzyme-linked immunosorbent assays. Cox's univariate analysis showed that pT (p = 0.0007), uPA (p = 0.0156) and PAI-1 (p = 0.0015) had a significant impact on relapse-free survival, whereas VEGF did not have any prognostic value (p = 0.18). Bivariate analysis showed significant interactions between uPA and PAI-1 (p = 0.0035) and between VEGF and PAI-1 (p = 0.006). Our study confirms that uPA and PAI-1 cytosol levels can be considered as prognostic factors for relapse-free survival in node-negative breast cancer. Moreover, the interaction between VEGF and PAI-1 warrants further investigation into the relationship between the biomarkers of angiogenesis and those of the protease cascade.     

Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator.

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.     

Protein movement during complex-formation between tissue plasminogen activator and plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1) rapidly inactivates tissue plasminogen activator (tPA). After initial binding and cleavage of the reactive-centre loop of PAI-1, this complex is believed to undergo a major rearrangement. Using surface plasmon resonance and SDS-PAGE, we have studied the influence of a panel of monoclonal antibodies on the reaction leading to the final covalent complex. On the basis of these data, we suggest the mechanisms for the action of different classes of inhibitory antibodies. We propose that the antibodies which convert PAI-1 into a substrate for tPA do this by means of preventing the conversion of the initial PAI-1/tPA complex into the final complex by sterical intervention. Moreover, the localisation of the binding epitopes on free PAI-1, as well as on the PAI-1/tPA complex, suggests that tPA in the final complex cannot be located near helices E and F, as has previously been proposed.     

Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells

The site of the reaction between plasminogen activators and plasminogen activator inhibitor 1 (PAI-1) was investigated in cultures of human umbilical vein endothelial cells. In conditioned medium from endothelial cells, two forms of a plasminogen activator-specific inhibitor can be demonstrated: an active form that readily binds to and inhibits plasminogen activators and an immunologically related quiescent form which has no anti-activator activity but which can be activated by denaturation. In conditioned medium, only a few percent of PAI-1 is the active form. However, the addition of increasing concentrations of tissue-type plasminogen activator (t-PA) or urokinase to confluent endothelial cells produced a saturable (3.0 pmol/5 x 10(5) cells), dose-dependent increase of the activator-PAI-1 complex in the conditioned medium even in the presence of actinomycin D or cycloheximide. This resulted also in a dose-dependent decrease of the residual PAI activity measured by reverse fibrin autography both in the conditioned medium and cell extracts. Short-time exposure of endothelial cells to a large amount of t-PA caused almost complete depletion of all cell-associated PAI activity. Although there was no detectable PAI activity even after activation of PAI by denaturants or antigen in the culture medium at 4 degrees C without the addition of t-PA, the addition of t-PA at 4 degrees C not only resulted in the formation of 70% of the amount of the t-PA.PAI complex in conditioned medium at 37 degrees C, but also induced PAI-1 antigen in a time and dose-dependent manner in the conditioned medium. Moreover, 125I-labeled t-PA immobilized on Sepharose added directly to endothelial cells formed a complex with PAI-1 in a dose-dependent manner. On the other hand, no detectable complex was formed with PAI-1 when Sepharose-immobilized 125I-labeled t-PA was added to endothelial cells under conditions in which the added t-PA could not contact the cells directly but other proteins could pass freely by the use of a Transwell. All these results suggest that a "storage pool" on the surface of endothelial cells or the extracellular matrix produced by endothelial cells contains almost all the active PAI-1, and reaction between PA and PAI-1 mainly occurs on the endothelial cell membranes, resulting in a decrease of the conversion of active PAI-1 to the quiescent form.