Liver regeneration is regulated by variety of growth factors. Release and activation of these growth factors are deeply related to degradation of extracellular matrix (ECM), which is regulated by plasminogen-activating system. Since plasminogen is assum ed to be essential in these functions in liver regeneration, partial 70% hepatectomy (PH) was performed on plasminogen deficient (Plg−/−) mice and wild-type (Plg+/+) mice. The sequential recovery of the liver weight after 70% PH gradually increased over 1 to 14 days in Plg+/+ mice. However, in Plg−/− mice, it increased over 1 to 7 days and thereafter increased no more. Thus, the recovery in Plg−/− mice was significantly impaired at 10 and 14 days compared with Plg+/+ mice. Plg+/+ mice exhibited an increase in DNA synthesis over5 days, and then a decrease thereafter; however, in Plg−/− mice, it increased over 3 days, and decreased thereafter, with the significant decrease at 14 days. The apoptotic nuclei labelling index slightly increased over 3 days, and decreased thereafter in Plg+/+ mice. In Plg−/− mice, it was few detected over 3 days, and gradually increased over 7 to 14 days. The apoptotic index in Plg−/− mice was higher than Plg+/+ mice at 14 days. In the histological examination of liver, focal area of cellular loss with fibrin deposition were detected in Plg−/− mice after PH, which were rarely detected in Plg+/+ mice. In Plg−/− mice, expressions of t-PA, u-PA and pro-MMP-9 were increased in the liver at 14 days after PH compared with Plg+/+ mice. These findings suggest that plasminogen plays an important role in liver regeneration after PH.
To assess the role of the fibrinolytic system in the pathogenesis of restenosis after percutaneous transluminal angioplasty (PTA) of peripheral arteries, 166 consecutive patients with peripheral atherosclerotic disease, ranging from 36 to 86 years old (median = 60), who had undergone successful PTA, were followed for 1 year. Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) antigens were determined before PTA and at 3, 6 and 12 months after, while t-PA activity, PAI-1 activi ty, fibrinogen, plasminogen, euglobulin clot lysis time anda2-antiplasmin were determined at 3 months after PTA. There were no significant differences in fibrinolytic parameters between patients with patent (121 patients – 73%) and those with restenosed (45 patients – 27%) arteries. Restenosis was more common in patients with poor outflow (17 out of 44 patients) than with good outflow (28 out of 122 patients;P< 0.05), with pre-existent occlusion (27 out of 93 patients) than with preexistent stenosis (17 out of 73 patients;P< 0.05), and with symptoms lasting longer than 6 weeks (35 out of 108 patients) than with symptoms lasting less (10 out of 58 patients;P< 0.05). We concluded that measured fibrinolytic parameters were not related to post-PTA restenosis, suggesting that the endogenous fibrinolytic system, estimated in peripheral blood, does not influence the development of restenosis in a peripheral artery.
Background and objective: Multivariate analyses on clusters of metabolic and hemostatic risk indicators implicitly assume good test-retest reliability of these variables, substantial covariance among the various indicators, stability of covariance structure over time, and comparable covariance structure in different subpopulations. The aim of the present study is to investigate these assumptions.Methods: Repeated samples were taken of fasting insulin, triglycerides (TG), high-density cholesterol (HDL-C), low-density cholesterol (LDL-C), fibrinogen, tissue-type plasminogen activator (t-PA) antigen, t-PA activity, plasminogen activator inhibitor-1 (PAI-1) antigen to address their intra-week reliability and covariance structure. In the same work-week blood was drawn three times from 125 sedentary males (age 45.2 +/- 5.3 years) and twice from 132 female nurses (age 33.7 +/- 8.0 years).Results: About half (44.8%) of these women were oral contraceptives (OC) users. Only minor intra-week changes in absolute levels were found. Intra-week test-retest correlations varied between 0.52 (t-PA activity) and 0.94 (HDL-C) with an average value of 0.81. In men, non-OC using women, and OC using women, the covariance matrices of the eight risk indicators were equal at day 1 and day 3, testifying the good stability of covariance structure over time. Differences in covariance structure of all three groups were observed, which remained after correction for BMI and age. In men and non-OC-using women, significant correlation was found on all days between insulin and the other risk indicators with exception of fibrinogen and LDL-C. In OC users, insulin was correlated with TG, LDL-C, and fibrinogen.Conclusion: The metabolic and hemostatic risk indicators showed good test-retest reliability, and their covariance is stable over time. Multivariate analyses of this cluster should be performed separately for men, non-OC-using women, and OC-using women. (C) 2001 Harcourt Publishers Ltd.
Markers of fibrinolytic activity and the effects of low-molecular-weight heparin treatment in unstable coronary artery disease.
Structure and function studies on thrombin receptor activating peptide have revealed that certain residues in this peptide could be replaced with alanine. Attempts to prepare antagonist peptides by single amino acid modification of thrombin receptor activating peptide have not resulted in potent antagonist peptide. In the present study, we report an antagonist peptide with multiple alanine substitutions in both critical and non-critical residues. At a concentration of 32 μM, this peptide could completely block agonist-induced platelet aggregation. The magnitude of the antagonist effect of this peptide depends on the concentration of the antagonist and preincubation time. This peptide blocked the platelet aggregation induced by the agonist peptide and also by α-thrombin, but did not have any effect on adenosine diphosphate or collagen-induced platelet aggregation indicating that the antagonist affects of this peptide may be pertained to thrombin receptor mediated events only. This peptide may be useful for blocking thrombin-mediated events like thrombosis and restenosis or can be used as a template for developing more efficient thrombin receptor antagonists.
Abstract Circumstantial evidence suggests an important role of the fibrinolytic (plasminogen/plasmin) and matrix metalloproteinase (MMP) systems in biological processes involving (extra)cellular proteolysis and matrix degradation. The availability of mice with inactivation of main components of both systems has allowed to study directly the interactions between both systems and their biological role. In purified system, MMP-3 (stromelysin-1) specifically hydrolyzes plasminogen and urokinase-type plasminogen activator (u-PA), thereby removing the cellular binding domains from both proteins. In the presence of cells, MMP-3 may downregulate cell-associated plasmin activity by decreasing the amount of activatible plasminogen, without affecting cell-bound u-PA activity. During neointima formation after vascular injury in gene-deficient mice, expression of MMP-2 and MMP-9 (gelatinase A and B) is strongly enhanced, independently of the presence or absence of plasminogen or of the physiological plasminogen activators. Activation of proMMP-2 occurs independently of plasmin, whereas proMMP-9 activation occurs via plasmin-dependent as well as plasmin-independent mechanisms. The temporal and topographic expression patterns of MMP-2, MMP-3, MMP-9, MMP-12 (metalloelastase) and MMP-13 (collagenase) establish a potential role in neointima formation. This is further substantiated by the finding that neointima formation after vascular injury is significantly enhanced in mice with deficiency of TIMP-1, a main physiological MMP inhibitor. Atherosclerosis models in gene-deficient mice suggest an important role of u-PA in the structural integrity of the atherosclerotic vessel wall. u-PA-mediated plasmin generation may contribute to activation of latent MMPs (MMP-3, -9, -12, and -13) which degrade insoluble elastin and fibrillar collagen. Thus, studies with gene-deficient mice have allowed to establish novel interactions between the fibrinolytic and MMP systems, which may play a role in biological processes requiring cellular proteolytic activity and/or extracellular matrix degradation.
Objective: To study the role of alpha(2)-antiplasmin (alpha(2)-AP), the main physiological plasmin inhibitor, in controlling vascular and cellular proteolytic activity.Materials: Arteries, organs and cell cultures derived from alpha(2)-AP-deficient (alpha(2)-AP(-/-)) mice or from their wild-type littermates (alpha(2)-AP(+/+)).Results: In serum-free conditioned medium of alpha(2)-AP(+/+) or alpha(2)-AP(-/-) skin fibroblasts, the time course (0-72 h) of PAI-1 antigen and of t-PA or u-PA antigen and activity production was similar. Activation of proMMP-9 (gelatinase B) upon addition of plasmin(ogen) to serum-free conditioned medium of fibroblasts was consistently detectable with alpha(2)-AP(-/-) but not with alpha(2)-AP(+/+) cells. In aorta and femoral arterial extracts of alpha(2)-AP(+/+) or alpha(2)-AP(-/-) mice, t-PA and u-PA activity levels were comparable, and fibrin zymography with cryosections did not reveal significant differences in fibrinolytic activity. In liver or kidney extracts of alpha(2)-AP(+/+) or alpha(2)-AP(-/-) mice, t-PA, u-PA, PAI-1 and plasminogen antigen levels were comparable; t-PA or u-PA activity was not detected in liver extracts and was present at comparable levels in kidney extracts. Activation of plasminogen to plasmin in solution by cell-associated plasminogen activator, and activation of cell-bound plasminogen by tcu-PA was comparable for fibroblasts of both genotypes.Conclusions: alpha(2)-AP does not play a crucial role in controlling vascular or cellular proteolytic activity in mice. (C) 2000 Harcourt Publishers Ltd.
In plasma all plasminogen activator inhibitor type 1 (PAI-1) is found in complex with vitronectin (VN). We have investigated the interaction of human PAI-1 with monomeric native human VN by size exclusion chromatography with VN in solution and using the surface plasmon resonance technique (SPR) with immobilised VN. With these techniques PAI-1 is found to bind in a tight one-to-one complex. VN in solution competed with amine coupled VN in binding free active PAI-1 with aKD of 20 nM. A KDof 0.1 nM at 25°C was instead found for PAI-1 binding to immobilised VN. The extremely rapid binding of PAI-1 to immobilised VN had an association rate constant, κonof 20μ M–1s–1. By the immobilisation of VN the affinity for PAI-1 thus increased manifold. The stable substrate mutant PAI-1-Ala335Glu had similar binding characteristics for immobilised VN as active PAI-1. Upon injection of human α-thrombin onto PAI-1 captured on VN the ternary complex was formed and PAI-1 rapidly dissociated from immobilised VN, indicating that VN was not converted into the denatured form by amine coupling. Monoclonal antibodies against PAI-1, belonging to five different classes did all bind to PAI-1 captured on immobilised VN. There is thus no direct overlap of either of these five antibody binding epitopes on PAI-1 with the binding site for VN. PAI-1 was released from immobilised VN by addition of tissue plasminogen activator (tPA) (E.C. 3.4.21.68), with a dissociation rate depending on the tPA concentration. The preformed complex of tPA and PAI-1 did not bind to immobilised VN. An intermediate ternary complex with tPA and active PAI-1 captured on VN is not detected with SPR. On the contrary, a transient ternary complex was observed with tPA and PAI-1-Ala335Glu captured on VN. In conclusion, these results reject that VN is bound to any of the five antibody binding epitopes, and support the proposal that the binding surface for VN on PAI-1 is located near the epitope of tPA in the final complex, including parts of α-helices C, E and β-strand 1A.
Matrix metalloproteinase-2 (MMP-2) degrades basement membrane collagen and its abnormal expression is associated with glomerulonephritis and glomerulosclerosis. All-trans retinoic acid (ATRA) has been indicated as preventing age-related glomerulosclerosis. The results of our study showed that ATRA upregulated expression of MMP-2 mRNA and secretion of MMP-2 proteins, and increased enzymatic activity of MMP-2 in cultured mesangial cell. In addition, ATRA also caused a dose-dependent increase in the secretion of transforming growth factor-β (TGF-β1) peptides. Since TGF-β1 regulated the expression of MMP-2 in mesangial cell, it is possible that TGF-β1 is involved in ATRA-induced MMP-2 expression. Using antisense TGF-β1 RNA strategy, antisense TGF-β1 construct almost completely blocked secretion of active TGF-β1 peptides in the antisense-bearing transfectants. Northern blot analysis showed that the transfectants treated with 10–6M ATRA for 72 h neither increased nor decreased the levels of MMP-2 messenger ribonucleic acid (mRNA). Moreover, addition of anti-TGF-β1 antibodies to mesangial cell in the presence of 10–6M ATRA reduced ATRA-induced MMP-2 expression dose dependently. These data suggest that TGF-β1 might be involved in ATRA-induced MMP-2 expression.
Fibrinolytic genes are involved in many biological processes, such as fibrinolysis, wound healing, inflammation and tumor metastasis, some of which rely on non-catalytic properties of the gene products. Reflecting the broad biological functions, expression of these genes is controlled by several mechanisms. Since the identification of fibrinolytic genes 2 decades ago, a vast amount of information has accumulated about the many signals, signaling pathways and mechanisms inducing their transcription. Our knowledge in this field is still expanding, and in this article we discuss two further emerging mechanisms by which expression of these genes is regulated: cell cycle-mediation and mRNA stability.
Objective: Plasminogen binds with apparent low affinity to cell-surface receptors via its lysine binding sites. This enhances/stabilizes the activation-susceptible conformation. However, it is not known whether this lysine-mediated conformational change of plasminogen may affect its subsequent dissociation rate and hence its stability at the cell surface. Therefore, we sought to determine the relationship between the lysine-dependent conformation of plasminogen and its dissociation rate from its receptor.Design: BIACORE experiments were used to determine the kinetics of the interaction of glu-plasminogen with its receptor alpha -enolase. Intrinsic and extrinsic fluorescence spectroscopy were utilized to confirm if alpha -enolase induced a conformational change to glu-plasminogen as predicted by analyses of the BIACORE data.Results: The dissociation of glu-plasminogen from alpha -enolase was mediated by at least two components with apparent dissociation rate constants of k(d1) = 4.7 x 10(-2)s(-1) and k(d2) = 1.6 x 10(-3)s(-1). This second slower dissociation event reflects an increase in the stability of the complex. Global analysis of the interaction suggested a two-state conformational change reaction, mediated by a concentration-dependent increase in the initial association rate constant. The apparent K-d predicted by this analysis was 1 muM. Fluorescence spectroscopy confirmed that alpha -enolase induced a more open conformation of glu-plasminogen.Conclusions: These results provide direct evidence that the binding of glu-plasminogen to alpha -enolase is not simply a low-affinity interaction, but involves a multivalent, competition binding reaction that is associated with a glu-plasminogen conformational change. This mechanism is compatible with the structure of glu-plasminogen. This has implications for the stability of binding and activation of glu-plasminogen at the cell surface. (C) 2000 Harcourt Publishers Ltd.
Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily because of its conformational and functional flexibility. Different systems have been used to express PAI-1, e.g. Chinese hamster overy (CHO) cells, Escherichia coli cells and HT 1080 cells. Although glycosylation may influence the biochemical properties of proteins, to date minor differences have been observed between glycosylated and non-glycosylated PAI-1. In the present study, we have investigated the effect of glycosylation on the inactivation of PAI-1 by Triton X-100 and the associated pathways of conformational transitions. Whereas in the absence of Triton X-100, the observed PAI-1 stability was independent on the source of PAI-1, the addition of Triton X-100 revealed major glycosylation-dependent differences in the inactivation process of PAI-1. Incubation at 0°C in the presence of Triton X-100 resulted in a conversion of the active conformation to a substrate-like conformation for all PAI-1 molecules examined. The rate (k) of this conversion was 0.00016 s–1and 0.00121 s–1for non-glycosylated PAI-1 and CHO-PAI-1, respectively. When incubating non-glycosylated PAI-1 with 0.2% Triton X-100 at 37°C, two consecutive conformational transitions occur ultimately resulting in a complete conversion to the latent conformation. However, incubation of recombinant CHO-PAI-1 under these conditions yielded significantly different pathways of conformational transitions, i.e. a rapid conversion (k1>0.035 s–1) of part (39%) of the active conformation into a stable substrate conformation and a slower conversion (k1=0.0004 s–1) of the remaining part (61%) of the active conformation into the latent conformation, revealing the existence of two distinct active conformations. In conclusion, this is the first report describing significant differences between glycosylated and non-glycosylated PAI-1. Both the rate and the pathways of induced conformational transitions and concomitant inactivation of PAI-1 depend strongly on the glycosylation.
Fibrin plays a key role in fibrinolysis, acting not only as a substrate but also as a regulator of activity. As well as stimulating plasminogen activation, it controls interactions between proteases and inhibitors, both protecting proteases from inhibition and, conversely, localizing inhibitors. The principal inhibitor of plasmin, α2-antiplasmin, is cross-linked to fibrinogen and fibrin, inhibiting fibrinolysis. Our studies have shown that a second inhibitor, PAI-2, is also cross-linked to fibrinogen and fibrin, by either factor XIIIa or tissue transglutaminase. These inhibitors are both members of the serpin family but the cross-linking sites are quite unrelated. Cross-links are formed between glutamine residues in the inhibitors and lysine residues in fibrin(ogen). The Gln residues involved are at position 2 in the N-terminus of α2-AP and at position 83 and 86 in PAI-2, located in a loop between helices C and D. All cross-linking observed was to the Aα chain of fibrin(ogen). The two inhibitors did not compete for cross-linking sites. α2-AP binds only to Lys 303 of the Aα chain and a 30-residue peptide based on the sequence around this Lys competed with fibrinogen for cross-linking to α2-AP but not for cross-linking to PAI-2. PAI-2 was cross-linked to several Lys residues (but not Lys 303) in the Aα chain, as shown by tryptic digestion and mass spectrometry. PAI-2 was cross-linked to Lys 148, 176, 183 and 467 by tissue transglutaminase and to Lys 148, 176, 230 and 413 by factor XIIIa. The activity of PAI-2 was not affected by cross-linking, so that this is a mechanism whereby it can be covalently bound to fibrinogen and retained in a fibrin clot, without loss of activity towards u-PA and two-chain t-PA. PAI-1, the other major inhibitor of fibrinolysis, also binds to fibrin but we find no evidence for its being cross-linked. All three inhibitors achieve high local concentrations on fibrin, which they protect from lysis by t-PA, u-PA and plasmin. The inhibitors differ in their major sources in blood, with α2-AP present at high concentrations in plasma, PAI-1 primarily in platelets, and PAI-2 a product of stimulated monocytes, giving them distinct and complementary roles in stabilizing fibrin in different physiological and pathological locations.
We have used atomic force microscopy (AFM) in order to study the ultrastructure of fibrin fibre dissolution in real time. Thin purified fibrin gels and plasma clots were prepared on glass surfaces and overlaid with isotonic saline or heparinized plasma in an AFM fluid-cell. Fibrinolysis was initiated by introducing plasmin or recombinant tissue-type plasminogen activator (rt-PA) into the solution bathing the clots. Microscopy was performed serially in real time on the Nanoscope III Atomic Force Microscope operating in the tapping or contact mode. The acquisition time for a single image was 2–8 min and the clots were imaged for up to 1 h with fields of view ranging from 128 × 128 μm to 0.7 × 0.7 μm with a resolution of 512 × 512 pixels. In the smallest fields of view fibrin fibres were seen to be composed of globules 40–70 nm in diameter. The diameter of composite fibrin fibres in purified gels depended on the concentration of NaCl in the fibrinogen solution: 250 ± 155 nm in 150 mmol/l NaCl vs. 1.42 ± 0.19 μm in 50 mmol/l NaCl. Plasma clots were composed of thick fibres with interspersed thinner fibres. In clots from platelet-rich plasma both the thick and the thin fibres had significantly smaller diameters than the corresponding fibre types in clots from platelet-depleted plasma (620 ± 195 nm vs. 965 ± 200 nm, and 195 ± 30 nm vs. 260 ± 60 nm, P< 0.001 for both comparisons). Fibrinolysis of both thick and thin fibres proceeded predominantly by lateral section of the whole fibre thickness at a given site, regardless of whether it was initiated by plasmin or by rt-PA. The time to complete fibre section by 2.5 U/ml of plasmin did not differ between thin and thick fibrin fibres (7.6 ± 3.7 min vs. 6.4 ± 4.2 min). With a low concentration of plasmin (0.17 U/ml) some fibrin fibres became thinner along their entire observed length before they were cleaved. The rate of fibre thinning was 3-times faster in the thicker fibres than in the thinner ones. We conclude that the 'cut-through' pattern is the predominant way of fibrinolysis in purified gels and in plasma clots, and that proteolysis leading towards fibre cleavage proceeds more efficiently in thick than in thin composite fibrin fibres.
Objective: Elevation of several molecular markers of the haemostatic/fibrinolytic system and of soluble adhesion molecules has been associated with acute or chronic phases of atherosclerosis. Speculating that markers associated with atherosclerosis would increase with age, we determined the age-dependency of some of these markers in clinically healthy individuals. Methods: 129 healthy persons were enrolled [division: younger (≤34 years)/older group (>34 years)]. Tissue-type plasminogen activator (t-PA) concentration, plasmin/α2-antiplasmin complex (PAP), D-dimer (DD), prothrombin fragment F1+2 (F1+2), thrombin-antithrombin III complex (TAT), soluble (sICAM-1) intercellular adhesion molecule-1, soluble (sVCAM-1) vascular cell adhesion molecule-1 and sP-selectin were measured with enzyme-linked immuno assays, plasminogen activator inhibitor-1 (PAI-1), plasma kallikrein-like activity (KK), and factor XII (FXII) with chromogenic substrate tests, fibrinogen with the Clauss method. Results: Fibrinogen (P < 0.01), F1+2 (P<0.01), KK (P<0.05) were significantly higher in the older vs. the younger group and correlated significantly with age (P<0.05, P<0.01). DD showed significantly higher values in the older vs. the younger group, whereas T-PA, PAP, FXII, TAT did not. PAI-1 tended towards higher values in the older vs. the younger persons. T-PA, PAI-1, DD correlated significantly with age P-selectin, sICAM-1, sVCAM-1 did not differ between both groups, nor could a significant age-dependency be found. Conclusion: This study indicates that plasma levels of several molecular markers of the haemostatic and fibrinolytic system increase with age. The age-dependency of these markers has to be taken into account in respect to their clinical use in order to characterize patients with suspected risk of atherosclerotic events.
Fibrin clot lysis is a dynamic process in which transport of fibrinolytic proteins plays an important role. Various recently established transport processes for plasminogen as well as for tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively) are reviewed.During internal lysis of a plasma clot, plasminogen is translocated from the fluid phase to the surface of the lysing fibrin fibres. During external lysis, plasminogen strongly accumulates on the moving surface of the clot. In both types of lysis, binding takes place on C-terminal lysine residues in partially degraded fibrin that are generated by plasmin. The recently discovered thrombin activatable fibrinolysis inhibitor (TAFI) inhibits plasminogen binding by removing the C-terminal lysine residues. TAFI is a plasma carboxypeptidase B that is activated by thrombin and that links the coagulation system and the fibrinolytic system.Transport of plasminogen activators is, in particular, essential for external clot lysis as it occurs during thrombolytic therapy. Because transport of proteins is not only mediated by diffusion, but also by fluid permeation, flow strongly promotes clot lysis. Penetration of plasminogen activators into clots is hampered by fibrin binding. While t-PA sticks to the surface of a clot, u-PA is able to enter a clot unhindered.Recent studies show that ultrasound promotes plasminogen activator-induced clot lysis. A variety of mechanisms have been proposed to explain this promotion. Results indicate that an increased transport of fibrinolytic proteins significantly contributes to the acceleration of fibrinolysis by ultrasound. (C) 2000 Harcourt Publishers Ltd.
Substantial data have been collected for numerous types of solid cancer, including cancer of the breast, the gastrointestinal and urological tract, the lung, and the brain, demonstrating a strong clinical value of the plasminogen activation system in predicting disease recurrence and survival in cancer patients. Elevated levels of certain members of the plasminogen activation system, the serine protease uPA (urokinase-type plasminogen activator), its receptor (uPA-R; CD87), and inhibitor (PAI-1), in tumour tissue or blood emphasize their fundamental role in tumour invasion and metastasis and provide the rationale for novel therapeutic strategies. uPA, besides its proteolytic action toward the extracellular matrix, in concert with uPA-R, PAI-1, and integrins contributes to tumour cell proliferation, adhesion, and migration. Several technical methods of affecting tumour growth and metastasis by targeting the uPA-system in cancer patients at the gene and protein level have been explored: (1) antisense oligodeoxynucleotides to uPA, uPA-R, or PAI-1; (2) antisense oligonucleotides to signal transduction pathway components such as Rel (NF-κ B), affecting uPA but not PAI-1 synthesis; (3) viral vectors delivering genes for components of the plasminogen activation system; (4) soluble, recombinant uPA-R as a scavenger for uPA; (5) monoclonal antibodies directed to uPA or uPA-R blocking uPA/uPA-R interaction; (6) enzymatically inactive uPA to compete for active uPA binding to uPA-R; (7) linear and cyclic uPA-derived peptides to block uPA/uPA-R interaction; (8) toxins, coupled to uPA or fractions thereof to kill tumour cells; (9) naturally occurring inhibitors to uPA and its derivatives for inhibition of uPA proteolytic activity; and (10) synthetic inhibitors to uPA to inhibit uPA proteolytic activity. There is substantial hope that substances designed to affect or turn off the plasminogen activation system will eventually be administered to cancer patients thereby opening a new vista for tumour biology-based, individualized cancer therapy.
Plasminogen activator inhibitor type 1 (PAI-1) is associated with tumour invasion, angiogenesis and metastatic spread. It is also a strong prognostic factor for relapse in a number of human cancers. This study was designed to investigate the effects of overexpression of PAI-1 on lung colonization by human HT-1080 fibrosarcoma cells. Full length PAI-1 cDNA was transfected in a non-aggressive HT-1080 clonal cell line (1–3C). Stable transfected clones were isolated of which one (3F52) secreted a 29-fold increase in PAI-1 protein levels (29.1±6.5 μg/106cells/24 h) as compared with control mock transfected cells (1.0±0.2 μg/106cells/24 h). 3F52 cells were significantly better able to form lung colonies after i.v. tail vein injection of athymic mice (mean number of colonies±SE 238±105) as compared with control cells (8±7). In addition, PAI-1 overexpressing cells were between 1.5 and 2.5 fold more adhesive to extracellular matrix membrane (ECM) proteins than mock transfected cells in an in vitro cell adhesion assay. These findings provide experimental support that PAI-1 facilitates tumour cell lodgement in vivo and adhesion of HT-1080 cells in vitro.
In a substudy on patients undergoing thrombolytic therapy for deep venous thrombosis with different doses of recombinant tissue-type plasminogen activator (Alteplase; Actilyse(R), Boehringer Ingelheim, Germany) within a multicentre trial, several haemostatic parameters were determined serially in an attempt to correlate changes of these parameters with clinical events, such as therapeutic outcome and bleeding complications.The main finding of our study was that the consumption of the inhibitors of fibrinolytic activity, PAI-1 and plasmin-inhibitor (formerly alpha (2)-antiplasmin) during continuous thrombolysis for deep venous thrombosis was associated with a significant increase of bleeding complications. In addition we found a trend towards lower recanalization rates and more frequent bleeding complications in patients with enhanced activation of the plasmatic coagulation system, reflected by higher concentrations of the activation peptides thrombin-antithrombin-complex, fibrin(ogen)-degradation-product and d-dimer.As bleeding represents the major limitation to a wider application of thrombolytic therapy in deep vein thrombosis it might be worthwhile to evaluate a concept of individualized thrombolytic therapy, adjusted for parameters associated with enhanced bleeding risk and low recanalization rates. (C) 2000 Harcourt Publishers Ltd.
The mammalian organism is composed of an inter-dependent series of tissue compartments separated from each other by an extracellular matrix (ECM). This ECM functions as both a determinant of tissue architecture and a mechanical barrier to cellular invasion. ECM proteolysis facilitates tissue penetration, and a distinctive property of many malignant tumor cells is the capacity to invade host tissues and establish metastatic foci. Malignant cells produce a spectrum of matrix-degrading proteinases with activities directed against the major ECM proteins. These enzymes are identical to those normally involved in physiologic processes; however, proteinase regulation is altered such that enzyme expression and/or activity are inappropriately controlled. The purpose of this review is to highlight biochemical mechanisms commonly utilized by tumor cells to regulate proteinase activity and to discuss the potential functional consequences with respect to tumor cell behavior. Specific examples will be provided to illustrate the concepts of regulation via limited proteolysis, enzyme-inhibitor binding, compartmentalization, and alteration of proteinase expression.
