Plasminogen activation in synovial tissues: differences between normal, osteoarthritis, and rheumatoid arthritis joints

OBJECTIVE: To analyse the functional activity of the plasminogen activators urokinase (uPA) and tissue type plasminogen activator (tPA) in human synovial membrane, and to compare the pattern of expression between normal, osteoarthritic, and rheumatoid synovium. The molecular mechanisms underlying differences in PA activities between normal and pathological synovial tissues have been further examined. METHODS: Synovial membranes from seven normal (N) subjects, 14 osteoarthritis (OA), and 10 rheumatoid arthritis (RA) patients were analysed for plasminogen activator activity by conventional zymography and in situ zymography on tissue sections. The tissue distribution of uPA, tPA, uPA receptor (uPAR), and plasminogen activator inhibitor type-1 (PAI-1) was studied by immunohistochemistry. uPA, tPA, uPAR, and PAI-1 mRNA values and mRNA distribution were assessed by northern blot and in situ hybridisations respectively. RESULTS: All normal and most OA synovial tissues expressed predominantly tPA catalysed proteolytic activity mainly associated to the synovial vasculature. In some OA, tPA activity was expressed together with variable amounts of uPA mediated activity. By contrast, most RA synovial tissues exhibited considerably increased uPA activity over the proliferative lining areas, while tPA activity was reduced when compared with N and OA synovial tissues. This increase in uPA activity was associated with increased levels of uPA antigen and its corresponding mRNA, which were localised over the synovial proliferative lining areas. In addition, in RA tissues, expression of the specific uPA receptor (uPAR) and of the plasminogen activator inhibitor-type 1     

Experimental study on efficacy of myocardial opacification owing to difference of echocardiographic contrast agents]

OBJECTIVE: Tissue plasminogen activator (tPA), a major regulator of fibrinolysis, is present in cerebrovascular endothelium. We have suggested that local regulation of tPA synthesis and release in brain microcirculation could be important determinants of the degree of damage after cerebral ischemia. In this study, the normal distribution of tPA antigen was determined in several stroke-prone regions in the rat brain often used to study the pathophysiological consequences of cerebral ischemia. METHODS: Immunohistochemistry and Western blot analysis were performed using an antibody that detects free tPA antigen and tPA complexed to its rapid inhibitor, plasminogen activator inhibitor-1 (PAI-1). Staining for von Willebrand factor, a brain endothelial cell marker, served as a positive control. RESULTS: Relative to von Willebrand factor, 8.6, 13, 11.4, and 20.4% of vessels in the parietal cortex, frontal cortex, striatum, and hippocampus, respectively, were tPA-positive. The majority of tPA-positive vessels (58-75%) were classified as precapillary arterioles and postcapillary venules (7-20 microm), whereas capillaries (4-7 microm) and small arterioles and venules (20-40 microm) accounted for 11 to 22% and 11 to 19%, respectively, of tPA-positive vessels. Western blot analysis of brain microvascular proteins confirmed the presence of free tPA (67 kDa) and a stronger band representing tPA-PAI-1 complexes. CONCLUSION: The tPA-containing cerebrovascular endothelium is distributed mainly in smaller vessels. In addition to the free pool of tPA, a large portion of tPA is complexed to PAI-1 and is therefore functionally inactive. The size of the free tPA cerebrovascular pool may be regulated by PAI-1, which in turn could suppress fibrinolysis in the cerebral microcirculation.     

Uremic medium increases cytokine-induced PAI-1 secretion by cultured endothelial cells

The overall fibrinolytic activity is depressed in patients with chronic renal failure where a prothrombotic state is described, thereby enhancing the risk of vascular occlusive events. The mechanism responsible for fibrinolysis derangement has not yet been elucidated. To evaluate the effect of the uremic environment on the fibrinolytic activity of endothelial cells, we studied plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) production by human umbilical vein endothelial cells (HUVEC) in culture, exposed either to uremic or normal sera, before and after cytokine stimulation. Twenty uremics were studied: 11 were on conservative dietary treatment and nine were on maintenance hemodialysis. Eight healthy subjects served as controls. Before cytokine stimulation, no difference in the HUVEC supernatant concentration of t-PA and PAI-1 was found among the groups studied. After stimulation with interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha, the HUVEC supernatant levels of PAI-1 in the uremics were higher than in the controls, whereas the supernatant levels of t-PA did not differ. Our data provide evidence that uremic serum, in concert with IL-1 or TNF-alpha, can enhance PAI-1 secretion by endothelial cells, thereby depressing the fibrinolytic system. This impaired endothelial fibrinolytic response to hypercoagulation could favor vascular events, which are the major cause of morbidity and mortality in patients with chronic uremia.     

Modulation by endothelin-1 of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells: interaction of endothelin-1 with cytokines

The interaction of endothelin-1 (ET-1) with either interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on the release of tissue plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was investigated in a culture system of vascular endothelial cells derived from human umbilical vein. The t-PA:Ag release was significantly decreased by either IL-1 beta or TNF alpha; ET-1 intensified the suppressive effect of the cytokines. In contrast, PAI-1:Ag release was significantly increased by either IL-1 beta or TNF alpha; ET-1 significantly reduced the stimulatory effect of the cytokines. The data suggest that endothelial cell-mediated fibrinolysis may be modulated by ET-1.     

Urokinase plasminogen activator is elevated in human astrocytic gliomas relative to normal adjacent brain

We assayed urokinase plasminogen activator (uPA), tissue type plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1) in 43 human brain tumors (predominantly astrocytic gliomas) and in histologically disease-free brain tissue resected with 21 of the tumors. Levels of uPA, tPA, and PAI-1, measured by enzyme-linked immunosorbent assay, varied widely among individuals in neoplastic and in normal tissue but did not correlate with age or sex. Pairwise comparison of neoplastic and normal tissue from 21 individuals revealed that mean tumor uPA level was elevated 6-fold (P < 0.001). Mean tumor tPA and PAI-1 were 2.5-fold greater than those of normal brain, but these differences were not statistically significant. Tumor uPA was elevated 2- to 30-fold in 16/21 paired samples (76%). In contrast, tumor tPA was elevated 2- to 22-fold in 7/21 (33%) of pairs, whereas tumor PAI-1 was 2- to 13-fold greater in 10/21 (48%) of pairs. Our results demonstrate that elevation of uPA content is frequent in astroglial tumors, as is the case in other major human cancers.     

The mast cell as site of tissue-type plasminogen activator expression and fibrinolysis

Recent data suggest that mast cells (MC) and their products (heparin, proteases) are involved in the regulation of coagulation and fibrino(geno)lysis. The key enzyme of fibrinolysis, plasmin, derives from its inactive progenitor, plasminogen, through catalytic action of plasminogen activators (PAs). In most cell systems, however, PAs are neutralized by plasminogen activator inhibitors (PAIs). We report that human tissue MC as well as the MC line HMC-1 constitutively produce, express, and release tissue-type plasminogen activator (tPA) without producing inhibitory PAIs. As assessed by Northern blotting, highly enriched lung MC (>98% pure) as well as HMC-1 expressed tPA mRNA, but did not express mRNA for PAI-1, PAI-2, or PAI-3. The tPA protein was detectable in MC-conditioned medium by Western blotting and immunoassay, and the MC agonist stem cell factor (c-Kit ligand) was found to promote the release of tPA from MC. In addition, MC-conditioned medium induced fibrin-independent plasmin generation as well as clot lysis in vitro. These observations raise the possibility that MC play an important role in endogenous fibrinolysis.     

Expression of fibrinolytic antigens in redistributed cardiac mast cells in auricular thrombosis

Recent data suggest that auricular thrombosis is associated with an increase and accumulation of mast cells (MC) in the subendothelial region of the upper endocardium. However, the molecular basis and the functional role of MC in this process are not known. In the current study, expression of fibrinolytic and antifibrinolytic antigens in human cardiac MC was analyzed by immunohistochemistry. MC were found to react with antibodies against tissue-type plasminogen activator (tPA) and urokinase receptor (uPAR/CD87), but not with antibodies against urokinase (uPA) or plasminogen activator inhibitors (PAI-1, PAI-2). Significant changes were observed when the phenotype of accumulated MC in the upper endocardium in patients with auricular thrombosis was compared with the phenotype of myocardial MC in the same patients or with MC in normal hearts. These redistributed MC stained less intensely with antibodies against tPA and chymase but retained their staining for tryptase and uPAR. Together, these data indicate that cardiac MC are a source of fibrinolytic antigens and that accumulation of MC in auricular thrombosis is associated with phenotypic changes of MC and loss of cellular tPA. It is hypothesized that MC and their products may play a role in endogenous fibrinolysis in auricular thrombosis.     

Urinary trypsin inhibitor efficiently inhibits urokinase production in tumor necrosis factor-stimulated cells

We demonstrated that urinary trypsin inhibitor (UTI) efficiently inhibits soluble and tumor cell-associated plasmin activity and subsequently inhibits tumor cell invasion and metastasis. The effect of UTI on tumor necrosis factor-alpha (TNF)-induced stimulation of urokinase-type plasminogen activator (uPA) in cultured human umbilical vein endothelial cells (HUVEC) and in the promyeloid leukemia U937 cells was studied. uPA antigen was evaluated in the cell lysate and in the conditioned media by enzyme-linked immunosorbent assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and Western blot. TNF can promote the production of uPA in HUVEC and in U937 cells. The PKC inhibitors (H7, calphostin C, and staurosporine) inhibited TNF-induced uPA expression and secretion in a dose-dependent manner. Analysis of the expression of cell surface receptor-bound uPA by flow cytometry using uPA-specific MAb indicates that induction of uPA expression by TNF was inhibited when these cells were incubated with UTI. On the other hand, treatment of the cells with UTI alone failed to alter uPA production. UTI also reduced the secretion of uPA in TNF-treated cells. UTI was as effective as PKC inhibitors in inhibiting uPA expression by TNF. Incubation of the cells with UTI, however, had no effect on the ability of PMA to stimulate cell-associated uPA expression. These data suggest that UTI may influence the PKC-dependent protein kinase pathway in uPA expression. The study on intracellular pathways involved in UTI modulation of uPA will enhance our understanding of the role that UTI plays in uPA-mediated cellular invasion.     

Lovastatin modulates in vivo and in vitro the plasminogen activator/plasmin system of rat proximal tubular cells: role of geranylgeranylation and Rho proteins

Interstitial fibrosis is one of the most deleterious events during the progression of renal deterioration after renal mass reduction. In vivo, hydroxymethylglutaryl CoA reductase inhibitors (HRI) were shown to reduce progression of glomerulosclerosis, but the mechanisms are still unclear. The present study investigates, in vivo, whether lovastatin, a potent HRI, was able to modulate the plasminogen-plasmin pathway, one of the most efficient systems involved in extracellular matrix remodeling, and characterizes in vitro the cellular mechanisms of these effects. Proximal tubules freshly isolated from rats treated for 2 d with lovastatin (4 mg/kg per d) showed increased tissue-type plasminogen activator (tPA) and urokinase (uPA) activities and antigens. Incubation with lovastatin (5 microM) of proximal tubules isolated from untreated rats induced an increase in tPA and uPA and a decrease in plasminogen activator inhibitor-1 (PAI-1) activities. In vitro, supernatants, cytosols, and membranes of renal proximal tubular cells in primary cultures had no detectable uPA activity, and lovastatin (0.1 to 10 microM) induced an increase in tPA and a decrease in PAI-1 activities and antigens. These effects were reversed by mevalonate and geranylgeranyl-pyrophosphate (GGPP) but not by farnesyl-pyrophosphate or LDL cholesterol. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and PAI- activity and blocked its reversion by GGPP. The effect of lovastatin was associated with a disruption of cellular actin stress fibers, which was reversed by GGPP and reproduced by C3 exoenzyme. In conclusion, HRI can modify the fibrinolytic potential of proximal tubules, most likely via inhibition of geranylgeranylated Rho protein and disruption of the cytoskeleton. The resulting increase of proteolytic activity of tubular cells may serve to prevent extracellular matrix deposition and renal interstitial fibrosis.     

Activation of the serine proteinase system in chronic kidney rejection

BACKGROUND: Activation of the serine proteinase system is an important mechanism that contributes to tissue remodeling. In the present study, we analyzed the expression of urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR), and plasminogen activator inhibitor type 1 (PAI-1) in samples of chronically rejected human kidneys. METHODS: Using Northern blot analysis, immunohistochemistry, and a uPA activity assay, specimens from 10 chronically rejected kidneys and 10 normal kidney samples were analyzed. RESULTS: By Northern blot analysis, the expression of uPAR and PAI-1 mRNA was 2.9-fold (P<0.05) and 2.3-fold (P<0.05) increased in chronically rejected kidney samples, respectively, compared with normal controls. In contrast, uPA mRNA levels in chronically rejected kidneys were comparable to those in the normal controls. Immunohistochemical analysis in normal kidneys showed weak immunostaining of uPA, moderate to intense uPAR and PAI-1 immunostaining in proximal tubules, and moderate immunostaining in distal tubules, but no signal in the glomeruli or cortical vessels. A similar staining pattern was found in the distal and proximal tubules in rejected kidney tissue samples. However, in the rejected kidneys, the number of tubules was markedly reduced. In addition, within the glomeruli of rejected kidney samples, there was positive immunostaining for uPA, uPAR, and PAI-1 in the mesangial cells, but negative staining in most of the endothelial cells, whereas the normal kidneys revealed no immunoreactivity in these structures. CONCLUSION: The demonstrated up-regulation of uPA/uPAR/PAI-1 in chronic renal rejection is consistent with the plasminogen/plasmin system contributing to tissue remodeling in this disorder. These factors might activate latent transforming growth factor-betas, which have been reported to be enhanced in this disorder, contributing to the generation of the extracellular matrix.     

Binding of tissue plasminogen activator to endothelial cells. The effect on functional properties. Localization of a ligand in the B-chain of tPA

The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8 x 10(5) and 1.2 x 10(5), respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.     

Small GTP-binding proteins in the brain-corpus cardiacum-corpus allatum complex of the silkworm, Bombyx mori: involvement in the secretion of prothoracicotropic hormone

BACKGROUND: Proteolysis, modulated in part by intramural fibrinolytic system proteins and their inhibitors, appears to influence vascular smooth muscle cell (SMC) migration and proliferation and remodeling of extracellular matrix (ECM). Alterations of fibrinolysis in circulating blood and of proteolysis within vessel walls in experimental animals and patients with diabetes have been associated with accelerated vascular disease. Hyaluronan, a prominent component of ECM in normal vessels, is increased in the tunica media of macroscopically normal arterial vessels from patients with type 2 diabetes. OBJECTIVE: To determine whether hyaluronan alters the expression of the fibrinolytic system protein, plasminogen activator inhibitor type-1 (PAI-1), in human vascular SMCs, thereby potentially accelerating vascular disease in patients with type 2 diabetes. METHODS: Urokinase-type and tissue-type plasminogen activators (uPA and tPA) and PAI-1 were assayed in vascular SMC conditioned media and in cell lysates, using enzyme-linked immunosorbent assay and western blotting. RESULTS: Hyaluronan increased the 24-h release of PAI-1 into conditioned media in a concentration-dependent and time-dependent manner (1.8-fold compared with control with 1 mg/ml hyaluronan; n = 9, P < 0.01). Although the accumulation of uPA in conditioned media tended to increase also, uPA content was reduced in cell lysates (64% of control with 0.1 mg/ml hyaluronan at 24 h; n = 9, P < 0.01) without any change in PAI-1. Concentrations of tPA in conditioned media and cell lysates were unchanged. Digestion of hyaluronan with hyaluronidase (50 turbidity reducing units (TRU)/ml) or exposure of the smooth muscle cells to antihuman CD44 antibody (1 microgram/ml) that binds to the hyaluronan cell surface receptor obviated the effects of hyaluronan. CONCLUSION: Our results indicate that increases in hyaluronan increase vascular SMC expression of PAI-1, a phenomenon that may alter the balance between proteolysis and its inhibition in vessels of patients with type 2 diabetes, thereby contributing to the acceleration of macroangiopathy.     

Sphingomyelinase and cell-permeable ceramide analogs increase the release of plasminogen activator inhibitor-1 from cultured endothelial cells

We investigated the effect of exogenous staphylococcal sphingomyelinase (SMase) on the release of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) from cultured human umbilical vein endothelial cells (HUVEC). Addition of SMase (2 units/ml) to the culture medium induced an approx. 15-fold increase in the extracellular level of PAI-1 antigen at 3 h. No significant increase in the level of t-PA antigen was detected. Treatment of HUVEC with SMASE (2 units/ml) for 3 h resulted in a significant decrease in the cellular sphingomyelin (SM) level, accompanied by a corresponding increase in the ceramide level. Cell-permeable ceramide analogs also enhanced the release of PAI-1 from cultured HUVEC in concentration- and time-dependent manners. A 6-fold increase in PAI-1 antigen level was observed after incubation for 3 h with 10 microM N-acetylsphingosine. Similar effect was noted as early as 2 h with 10 microM N-hexyanoylsphingosine. Addition of sphingosine failed to affect the release of PAI-1 from cultured HUVEC, indicating that the effects of ceramide analogs were independent of sphingosine generation. Pretreatment with cycloheximide or actinomycin D abated the response of HUVEC to N-acetylsphingosine in the increased levels of both extracellular and intracellular PAI-1. These results suggest that ceramide, generated via "SM cycle", acts as a lipid mediator of PAI-1 release from vascular endothelial cells, and may contribute to a better understanding of the pathogenesis of the PAI-1-associated thrombotic disorders.     

Proteases and their inhibitors are indicative in gestational disease

OBJECTIVE: To assess whether various proteolytic factors which are involved in trophoblast invasion show different concentrations in plasma and placenta of patients with HELLP syndrome, pre-/eclampsia and highly pathological Doppler flow measurements but without additional complications (hpD). DESIGN: Case control and observational study; 18 women with HELLP syndrome, 21 with pre-/eclampsia, 13 with hpD, as well as healthy pregnant women (matched pairs); statistical analysis: sign test and Wilcoxon test. RESULTS: Urokinase-type plasminogen activator (uPA), uPA receptor, tissue-type plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), matrix metalloproteinases MMP-8, MMP-9 and tissue inhibitor of metalloproteinases TIMP-1 were measured by ELISA. PAI-1 plasma levels are significantly elevated in all three groups studied. In HELLP syndrome, tPA and TIMP-1 are also elevated, and in patients with hpD, MMP-8 is increased, whereas MMP-9, and TIMP-1 are lower. In placenta extract, only pre-/eclampsia shows reduced MMP-9 concentrations. CONCLUSIONS: The increased frequency of small-for-gestational-age infants observed in all three study groups is an expression of impaired placental implantation and remodelling processes. These disturbances manifest themselves in the form of changes in some of the factors in plasma and placenta extract that are involved in these processes.     

Concentration of endogenous tPA antigen in coronary artery disease: relation to thrombotic events, aspirin treatment, hyperlipidemia, and multivessel disease

Tissue plasminogen activator (tPA) is the major plasminogen activator responsible for dissolving blood clots found in blood vessels. However, elevated concentrations of tPA antigen were found to be related to adverse events in patients with coronary artery disease (CAD). Considerable controversy about the significance of these results exists. The goal of this cross-sectional study was to identify independent determinants for tPA antigen concentrations in patients with CAD, to possibly clarify the above paradoxical relationship. The baseline tPA antigen concentrations of 366 patients with angiographic evidence of coronary sclerosis were determined. Univariate analysis showed that age (P=0.013), angiographic extent of disease (P<0.001), presence of angina at rest (P<0.001), diabetes mellitus (P=0.004), hypercholesterolemia (P=0. 045), hypertriglyceridemia (P=0.015), and chronic intake of nitrates (P<0.001) were significantly and positively related to tPA antigen concentration, while the chronic intake of aspirin was inversely related to tPA antigen (P<0.001). In addition, plasminogen activator inhibitor type 1 (PAI-1) activity was found to be significantly and positively associated with tPA antigen concentration (P<0.001). A multivariate analysis identified chronic low-dose aspirin therapy (P<0.001), PAI-1 activity (P<0.001), hypertriglyceridemia (P=0.005), the type of angina (P=0.026), multivessel disease (P=0.041), and hypercholesterolemia (P=0.043) as significant and independent determinants of tPA antigen. While hypertriglyceridemia and hypercholesterolemia both are related to the underlying disease, the type of angina and the number of involved vessels are linked to the severity and extent of disease, and all of them are indicators of a prothrombotic state found during the progression of CAD. In contrary, low-dose aspirin rather would decrease the likelihood of thrombotic events. The relation of tPA antigen to PAI-1 activity furthermore underlines the relation between tPA antigen concentration and a prothrombotic state. Therefore, the positive or-in case of aspirin therapy-negative correlation of these parameters with tPA antigen concentration would indicate that thrombus formation and simultaneous endothelial cell activation might be major determinants for tPA antigen concentration in CAD.     

Identification of tissue-type plasminogen activator-specific plasminogen activator inhibitor-1 mutants. Evidence that second sites of interaction contribute to target specificity

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the P1 and P1' positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at P1 including Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P1'. Kinetic analyses of selected variants identified P1Tyr and P1His as the most efficient tPA-specific inhibitors, with second-order rate constants (ki) of 4.0 x 10(5) M-1s-1 and 3.6 x 10(5) M-1s-1, respectively. Additional PA-specific PAI-1 variants containing substitutions at P3 through P1' were constructed. P3Tyr-P2Ser-P1Lys-P1'Trp and P3Tyr-P2Ser-P1Tyr-P1'Met had ki values of 1.7 x 10(6) M-1s-1 and 2.5 x 10(6) M-1s-1 against tPA, respectively, but both were inactive against uPA. In contrast, P2Arg-P1Lys-P1'Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several P1Arg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.     

Activated protein C resistance, factor V Leiden and peripheral vascular disease

AIM: To study cerebrospinal fluid (CSF) concentrations of plasminogen activator inhibitor type-1 (PAI-1) in patients with neurological disease. METHODS: CSF PAI-1 concentrations were measured in 51 patients with neurological disease and 20 reference subjects using an ELISA. The patient group comprised three patients with viral meningitis, 20 with encephalitis, nine with acute lymphoblastic (n = 7) and myeloid (n = 2) leukaemia (with central nervous system involvement), and 19 with multiple sclerosis. RESULTS: Raised PAI-1 concentrations were observed in patients with leukaemia, encephalitis and multiple sclerosis. There was no difference in the mean concentrations of PAI-1 in patients with meningitis when compared with the reference subjects. The highest mean (SEM) PAI-1 concentration was found in patients with leukaemia (1.28 (0.36) ng/ml), and the next highest in those with encephalitis (1.19 (0.20) ng/ml). these values were much higher than those in patients with viral meningitis. In a previous report, raised CSF tissue-type plasminogen activator (tPA) activities were detected in patients with multiple sclerosis, leukaemia and encephalitis, with mean activities in decreasing order. PAI-1 concentrations in the same patients were the reverse of their corresponding tPA activities, being higher in those with leukaemia and encephalitis, than in patients with multiple sclerosis. There was no association between CSF PAI-1 concentrations and age in either patients or controls. Similarly, there was no association between CSF PAI-1 concentrations and urokinase-type plasminogen activator (uPA). CONCLUSIONS: Raised CSF PAI-1 concentrations may be used as a non-specific marker of neurological disease. Moreover, PAI-1 may play an important role in regulating the functions tPA, and probably uPA, in CSF.     

Immunohistochemical characterization of the plasminogen activator system in psoriatic epidermis

The relative topographical distribution of urokinase-type plasminogen activator (uPA), tissue-type PA (tPA), PA-inhibitor-1 (PAI-1), PA-inhibitor-2 (PAI-2), plasmin(ogen), alpha 2-antiplasmin, and alpha 2-macroglobulin was studied in lesional epidermis of psoriasis vulgaris, and in normal epidermis, by immunohistochemistry. In psoriatic epidermis, tPA predominated, although uPA was found in some biopsies. PAs were not detected in normal epidermis. PAI-1 was not detected in normal epidermis and was only present in a proportion of biopsies of psoriatic lesions. PAI-2 was found in normal and psoriatic epidermis. Plasmin(ogen) was confined to the basal cell layer of normal epidermis, whereas in lesional psoriatic skin it was scattered throughout the epidermis. Alpha 2-antiplasmin and alpha 2-macroglobulin were not found in the epidermis of normal skin. In psoriatic epidermis alpha 2-antiplasmin was confined to the subcorneal layer, whereas staining for alpha 2-macroglobulin was found only in a proportion of biopsies, in the upper epidermis. Our immunohistological findings indicate that colocalization of tPA and its substrate plasminogen may allow efficient generation of plasmin, and that the focal absence of plasmin inhibitors may then favour the persistence of plasmin activity.     

Plasminogen activator activity during decidualization of human endometrial stromal cells is regulated by plasminogen activator inhibitor 1

Progesterone acts on the estradiol (E2)-conditioned human endometrium to induce decidualization of stromal cells. Consistent with these differential hormone actions in vivo, progestins regulate several end points of decidualization in human endometrial stromal cell monolayers, and E2 augments the effects of progestin. This study shows that in vitro decidualization of the stromal cells is accompanied by diminished plasminogen activator (PA) expression. Polyacrylamide gel electrophoretic separation after immunoprecipitation of biosynthetically labeled PAs revealed that medroxyprogesterone acetate (MPA) lowered levels of secreted tissue type PA (tPA) at 67 kilodaltons and urokinase type PA (uPA) at 55 kilodaltons. These levels were reduced further by E2 plus MPA despite a lack of response to E2 alone. Although tPA activity was readily measured by a chromogenic assay, detection of uPA activity required prior activation, indicating that uPA is released as the pro-uPA zymogen. Comparisons of levels of immunogenic PAs, as measured by specific enzyme-linked immunosorbent assays, with the corresponding catalytic activities revealed selective progestational inhibition of PA activity vs. antigen after 3 days of experimental incubation. Thus, 10(-7) mol/L MPA produced about a 2-fold greater reduction of levels of PA activity than that of its corresponding antigen. More strikingly, 10(-8) mol/L E2 plus 10(-7) mol/L MPA virtually eliminated both tPA activity (99% inhibition; P < 0.005) and uPA activity (93% inhibition; P < 0.005); the reductions in levels of the corresponding antigens were only about 50% of the control levels and did not attain statistical significance. Only after 3-6 days of incubation with E2 plus MPA was statistically significant inhibition achieved for immunogenic levels of both tPA (P < 0.05) and uPA (P < 0.005). Preferential inhibition of levels of PA activities compared with those of the corresponding PA antigens reflects the action of the potent PA inhibitor PAI-1. Thus, the concentration of PAI-1 in the stromal cell-conditioned medium at the end of 0-3 days exceeded those of tPA and uPA, respectively, by 28- and 12-fold in response to MPA and by 52- and 25-fold in response to E2 plus MPA. Extrapolation of these in vitro results to the events of the luteal phase, whose steroidal milieu is mimicked by E2 plus MPA, indicates that decidual cell-derived PAI-1 is a key regulator of proteolytic degradation of extracellular matrix and fibrinolysis during implantation and menstruation.     

Human endothelial cell migration is stimulated by urokinase plasminogen activator:plasminogen activator inhibitor 1 complex released from endometrial stromal cells stimulated with transforming growth factor beta1; possible mechanism for paracrine stimulation of endometrial angiogenesis

Human endometrial stromal cell cultures, stimulated for two days with recombinant transforming growth factor beta1 (TGFbeta1; 10 ng/ml), contained conditioned medium concentrations of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI1), and uPA:PAI1 complex. Since a number of cellular effects have been reported to follow a binding of enzymatically inactive uPA to the receptor in different cell types, we studied the influence of uPA:PAI1 complex on human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1). Increasing concentrations of uPA:PAI1 complex as well as free uPA resulted in a dose-dependent stimulation of endothelial cell migration. Stimulation by the complex was of the same magnitude as that of free uPA on a molar basis and reached its maximum at 1 nM in both cell types. PAI1 by itself, however, had no effect on cell migration. The migratory response to both uPA and the uPA:PAI1 complex was inhibited by antibody adhesion to the cell surface receptor for uPA. In addition, we found that TGFss1 had a direct stimulatory effect on migration in both HUVEC and HMEC-1. This response did not, however, involve the binding of uPA to the uPA receptor. Since TGFbetas are expressed in endometrial tissue and reportedly stimulate angiogenesis in other tissues in vivo, though not endothelial cell proliferation in vitro, they may engage in the regeneration of endometrial vasculature indirectly via perivascular cells. We found that the uPA:PAI1 complex, when released from endometrial stromal cells in response to TGFbeta1, stimulated endothelial cell migration. This suggests a possible mechanism for paracrine stimulation of endometrial angiogenesis.