Biological mechanisms and individual variation in fibrinolysis after major trauma.

OBJECTIVE: To understand more about the individual variation in the time course of fibrinolysis following major injury and to assess the potential for stratification of trauma patients for tranexamic acid (TXA) therapy. METHODS: A historical dataset (from 2004) was used, consisting of samples from 52 injured patients attended by a medical prehospital system. Blood samples were taken at the incident scene, on arrival in the emergency department, 2.5 hours after hospital arrival and 5 hours after hospital arrival. From the study database, we extracted values for tissue-type plasminogen activator (tPA; an activator of fibrinolysis), one of the plasminogen activator inhibitors (PAI-1; as a natural inhibitor of fibrinolysis) and D-dimer (as a marker of the extent of fibrinolysis). RESULTS: The changes over time in median tPA and PAI-1 were mirror images, with initial high tPA levels which then rapidly decreased and low initial PAI-1 levels which slowly increased. There were high levels of fibrinolytic activity (D-dimer) throughout. This pattern was present in patients across a broad range of injury severities. CONCLUSIONS: After major trauma, there seems to be an early 'antifibrinolytic gap' with the natural antifibrinolytic system lagging several hours behind the natural profibrinolytics. An early dose of exogenous antifibrinolytic (TXA) might have its effect by filling this gap. The finding that tPA and subsequent clot breakdown (illustrated by D-dimer formation) are raised in a broad range of patients, with little correlation between the initial fibrinolytic response and markers of injury severity, may be the reason that TXA is effective across a broad range of injured patients.

Effects of addition of tissue-type plasminogen activator in in vitro fertilization medium on bovine embryo development and quality.

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue-type plasminogen activator (t-PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus-oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t-PA and/or its inhibitor epsilon-aminocaproic acid (control, t-PA, t-PA+ε-ACA, ε-ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t-PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε-ACA. PAA in the treated group was significantly reduced by ε-ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t-PA-modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t-PA. In conclusion, it appears that excessive t-PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.

[The correlation study of the expression of VEGF with t-PA and PAI expression in plasma and intraocular tissue in proliferative diabetic retinopathy].

OBJECTIVE: To evaluate the correlation between the expression of VEGF with t-PA and PAI expression in plasma aqueous humor and vitreous of proliferative diabetic retinopathy (PDR). METHODS: It was an experimental study. The concentrations of VEGF t-PA and PAI in plasma, aqueous humor and vitreous body in PDR and normal group were detected by ELISA. RESULTS: The levels of VEGF (53.37 ng/L) and PAI (8.00 µg/L) in the plasma of PDR patients were higher than control group, and there was significant statistically difference between them (Z = -3.437, -5.816; P < 0.01). The levels of t-PA (24.32 µg/L) in plasma of PDR patients and control group was no statistically difference between them (Z = -1.715, P = 0.086). The levels of VEGF (335.00 ng/L) t-PA (5.70 µg/L) and PAI (0.63 µg/L) in the aqueous humor of PDR patients were higher than control group, and there was statistically difference between them (Z = -4.805, -1.967, -4.018; P < 0.05). The levels of VEGF (691.69 ng/L) t-PA (13.05 µg/L) and PAI (4.01 µg/L) in the vitreous of PDR patients were higher than control group , and there was statistically difference between them (Z = -2.807, -2.642, -2.230;P < 0.05). Compare the plasma aqueous humor and vitreous of PDR patients. The levels of VEGF is: vitreous>aqueous humor>plasma. The levels of t-PA is: plasma>vitreous>aqueous humor. The levels of PAI is: plasma and vitreous>aqueous humor. The expression of VEGF was highly correlated with t-PA and PAI in plasma aqueous humor and vitreous of PDR (P < 0.05). CONCLUSIONS: VEGF PAI in plasma, VEGF t-PA PAI in aqueous humor and vitreous were increases. The expression of VEGF was positive correlated with t-PA and PAI in plasma aqueous humor and vitreous of PDR.

The majority of type 1 plasminogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissue-type plasminogen activator.

The interactions between exogenously added tissue-type plasminogen activator (t-PA) and the active form of type 1 plasminogen activator inhibitor (PAI-1) produced by and present in cultured human umbilical vein endothelial cells (HUVECs) were investigated. Immunoblotting analysis of the conditioned media obtained from monolayers of HUVECs treated with increasing concentrations of t-PA (less than or equal to 10 micrograms/ml) revealed a dose-dependent formation of both t-PA/PAI-1 complexes, and of a 42,000-Mr cleaved or modified form of the inhibitor. Immunoradiometric assays indicated that t-PA treatment resulted in a fourfold increase in PAI-1 antigen present in the conditioned media. This increase did not result from the release of PAI-1 from intracellular stores, but rather reflected a t-PA-dependent decrease in the PAI-1 content of the Triton X-100 insoluble extracellular matrix (ECM). Although the rate of t-PA-mediated release of PAI-1 was increased by the removal of the monolayer, similar quantities of PAI-1 were removed in the presence or absence of the cells. These results suggest that the cells only represent a semipermeable barrier between ECM-associated PAI-1 and exogenous t-PA. Treatment of HUVECs with t-PA (1 microgram/ml, 2 h) to deplete the ECM of PAI-1 did not affect the subsequent rate of PAI-1 production and deposition into the ECM. Immunogold electron microscopy of HUVECs not only confirmed the location of PAI-1 primarily in the region between the culture substratum and ventral cell surface but failed to demonstrate significant (less than 1%) PAI-1 on the cell surface. Thus, the majority of PAI-1 associated with cultured HUVEC monolayers is present under the cells in the ECM and is accessible to solution-phase t-PA.

Melatonin administration increased plasminogen activator activity in ram spermatozoa.

The aim of the present study was to investigate the effect of melatonin on plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in ram spermatozoa and seminal plasma, in correlation with changes in blood testosterone. Melatonin implants (18 mg) were placed subcutaneously in sixteen Chios rams in autumn and spring. Semen samples for spectrophotometrical assays were collected 36 h before the implantation of melatonin and thereafter once a week, for 17 weeks. Blood samples for testosterone assay (RIA) were collected 8h before implantation (one sample/30 min x 7.5 h) and thereafter every 15 days for 105 days after implantation. For each ram, six parameters of testosterone were estimated: mean value, basal level, number of peaks, peak amplitude, peak duration and mean testosterone concentration during peaks. Melatonin implantation during autumn induced an increase in PAA and t-PAI in spermatozoa; melatonin implantation in spring induced an additional increase in u-PAI and PI; no change in PAA, PAI or PI was found in seminal plasma, during autumn or spring. The melatonin-induced increase of PAA, PAI and PI in spermatozoa was in positive correlation with the increase of testosterone mean value, basal level and number of peaks; the increase of testosterone parameters was greater in autumn compared to spring. Changes of PAA, PAI and PI of spermatozoa, under the influence of melatonin, might indicate changes in the fertilizing ability of spermatozoa, since plasminogen activators and their inhibitors are present on the plasma and the outer acrosomal membrane of spermatozoa and are released during the acrosome reaction.

Differential protease expression by cutaneous squamous and basal cell carcinomas.

To assess the postulated role of plasminogen activation in tumor invasion, we have investigated the cellular sites of synthesis for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators and their inhibitors (PAI-1 and PAI-2) in two human cutaneous neoplasia that differ in their metastatic potential. The combined use of zymography on tissue sections and in situ hybridization demonstrates that uPA is produced by malignant cells of squamous cell carcinomas (SCC) but not by basal cell carcinomas (BCC), whereas tPA is detected exclusively in nonmalignant dermal tissue. In addition, we show that SCC neoplastic cells simultaneously produce variable amounts of PAI-1, and that PAI-1 production correlates inversely with uPA enzymatic activity. These observations establish that invasive human malignant cells in vivo can activate plasminogen through uPA production during the early phases of tumor growth; they also demonstrate that the proteolytic activity of tumor cells can be modulated by the concomitant production of PAI-1. Because SCC have a higher invasive and metastatic potential than BCC, our findings lend further support to the involvement of plasminogen activation in malignant behavior.

Plasminogen activator system in oral squamous cell carcinoma.

BACKGROUND: The plasminogen activator system consists of two plasminogen activators, urokinase (uPA) and tissue (tPA); PA inhibitors (PAI-1, and -2), and a cell surface receptor for uPA (uPAR). The plasminogen activator system is involved at many stages of the metastatic cascade, including matrix remodelling, cell proliferation, and migration. AIMS: To compare tissue concentrations of the components of the plasminogen activator system in paired tumour tissue and normal tissue in patients with oral squamous cell carcinoma, and to correlate these with the histopathological grading of the tumour. METHODS: Thirty-eight paired tissue samples were analysed by enzyme-linked immunosorbent assays (ELISA; ng/mg protein) for uPA, tPA, uPAR, PAI-1, and PAI-2. RESULTS: Concentrations of uPA, uPAR, PAI-1, and PAI-2 were significantly higher in tumour than in normal oral tissue (median in uPAR tumour 1.6 (range; 0.1-7.5) ng/mg protein; normal=0.2 (0-2.3), p<0.05). There were strong correlations between the concentrations of certain components of the plasminogen activator system in particular between uPA, uPAR, and PAI-1 (p<0.05). Tissue concentrations of some components of the plasminogen activator system correlated with clinical and pathological indexes of aggression of tumours, including differentiation and T-stage. CONCLUSION: The relation between components of the plasminogen activator system, in particular uPA, uPAR, and PAI-1 in invasion, metastasis, prognosis, and survival, requires further investigation in oral squamous cell carcinomas.

Mulberry anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside, exhibited an inhibitory effect on the migration and invasion of a human lung cancer cell line.

Anthocyanins, present in various fruits and vegetables as natural colorant, have been well characterized to be involved in various bioactive properties and are wildly used for their antioxidant properties. Furthermore, recent studies have revealed pleiotropic anticancer and antiproliferative capabilities of anthocyanin. Berry extract contains high amounts of anthocyanins and is commonly used in diet or in some therapeutic applications. In this study, we first observed that cyanidin 3-rutinoside and cyanidin 3-glucoside (extracted from Morus alba L.) exerted a dose-dependent inhibitory effect on the migration and invasion, of highly metastatic A549 human lung carcinoma cells in absence of cytotoxicity. The results showed that cyanidin 3-glucoside and cyanidin 3-rutinoside treatments could decrease the expressions of matrix matalloprotinase-2 (MMP-2) and urokinase-plasminogen activator (u-PA) in a dose-dependent manner and enhance the expression of tissue inhibitor of matrix matalloprotinase-2 (TIMP-2) and plasminogen activator inhibitor (PAI). Further analysis with semi-quantitative RT-PCR showed that these alterations were all on the transcriptional level. Further, a treatment of cyanidin 3-rutinoside and cyanidin 3-glucoside also resulted in an inhibition on the activation of c-Jun and NF-kappaB. Together, these result suggested that anthocyanins could decrease the in vitro invasiveness of cancer cells and therefore, may be of great value in developing a potential cancer therapy.

Plasminogen activators and their inhibitor gene expression in cutaneous NF1-related neurofibromas.

Cutaneous neurofibromatosis 1 (NF1)-related neurofibromas are benign tumors and composed of Schwann cells, perineurial cells and/or fibroblasts, endothelial cells, mast cells and macrophages. Extracellular proteolysis namely plasminogen activation (PA) operates in many tissue destructive processes. We wanted to study plasminogen activators, urokinase (uPA) and tissue type (tPA) and their inhibitor PAI-1, which have not earlier been studied comprehensively in cutaneous NF1-related tumors. We analyzed the distribution of uPA, tPA and PAI-1 antigen level by immunohistochemistry and mRNA level by in situ hybridization, to identify which cells are primarily involved in proteolytic activity and plasminogen activation. Twelve NF1 skin tumor samples from six patients were obtained during the operations. Mast cells, macrophages and endothelial cells were distributed only locally. Their expression levels of PA components were not so notable. Large extent of tumor cells of Schwann cell origin and prominent expression levels of uPA, tPA and PAI-1 indicated that these cells are responsible for the main source of PA components in cutaneous NF1-related neurofibromas.

Protein and messenger RNA levels of plasminogen activators and inhibitors analyzed in 22 human tumor cell lines.

In 22 human tumor cell lines the regulation of production of plasminogen activators urokinase (u-PA) and tissue-type (t-PA) and their inhibitors PAI-1 and PAI-2 was studied. These four components may determine the net plasminogen activator activity, which is often associated with tumor development and metastatic processes. The amount of specific mRNA and protein produced by the cells was measured for all four components. The frequent finding of t-PA (alone or in combination with u-PA) suggests that t-PA can also be a tumor-associated plasminogen activator. In 11 of the 22 cells PAI-1 mRNA and in 6 of the 22 cells PAI-2 mRNA was found, pointing to a possible role of plasminogen activator inhibitors in the tumor-related plasminogen activator activity. This study demonstrates that there are at least two important regulatory steps in the regulation of production of plasminogen activators and their inhibitors: (a) the regulation at the mRNA level, since a high protein amount is always correlated with a high mRNA amount found in the tumor cells; (b) there must be a significant regulatory step at the (post)translational level as can be concluded from differences in mRNA usage.

Modulation of the plasminogen activation system in murine macrophages.

We have dissected the state of fibrinolytic balance in the C57/BL mouse macrophages, by means of immunotrap assays and zymography. We have monitored the individual changes of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activities of cellular lysates and secretions of these macrophages, after they were stimulated by various exogenous agents. The resident peritoneal macrophages were found to have very little PA but high level of PAI, and are therefore highly anti-fibrinolytic in nature. Upon stimulation by thioglycollate, PA activity increased and PAI activity decreased, thus raising the fibrinolytic balance in these macrophages. Upon incubation of resident or thioglycollate-activated macrophages by lipopolysaccharide (LPS), the PA level was depressed while the PAI level was increased, resulting in a large drop in the total fibrinolytic balance of the activated cells. When resident or thioglycollate-activated macrophages were incubated with the anti-inflammatory agent dexamethasone, the drug depressed both the expressions of PA and PAI, in the lysate and conditioned medium of both cell types. Thus cell-bound or secreted forms of macrophage PA and PAI activities were either increased or decreased in response to thioglycollate, LPS or dexamethasone challenge. The changes in PA and PAI resulted in different state of fibrinolytic balance in macrophages, and could be related to the different functions of these macrophages at different stages of their development.

Early coronary reperfusion blunts the procoagulant response of plasminogen activator inhibitor-1 and von Willebrand factor in acute myocardial infarction.

The effects of early coronary recanalization on the plasma levels of two procoagulant acute phase proteins, the fastacting plasminogen activator inhibitor and von Willebrand factor, were investigated in 24 patients with myocardial infarction receiving intravenous recombinant tissue-type plasminogen activator (rt-PA) within 6 h of the onset of symptoms. Coronary angiography was performed before and 90 min after the start of rt-PA infusion. Continuous electrocardiographic recordings and 4 h plasma creatine kinase MB isoenzyme (CK MB) were performed over the first 24 h. Plasma plasminogen activator inhibitor activity, von Willebrand factor and C-reactive protein were measured before rt-PA infusion, daily for the first 3 days and after 90 days. In the entire group, plasminogen activator inhibitor activity peaked at 24 h (day 1), representing a significant increase over values at all other times (p = 0.03). von Willebrand factor was higher in the first 2 days of infarction compared with after 90 days (p = 0.001). C-reactive protein peaked on day 2, with an eightfold increase over values on admission (p = 0.001). In the 16 patients with a patent infarct-related artery at 90 min, infarct size estimated by integrated 24 h CK MB, time for ST segment elevation to decrease to half-maximum and peak C-reactive protein were reduced significantly by more than twofold compared with values in the 8 patients with an occluded artery at 90 min. The patients with early recanalization also had lower plasminogen activator inhibitor activity on day 2 (p = 0.05) and day 3 (p = 0.02) and lower 0 to 72 h averaged von Willebrand factor (p = 0.01). Thus, early coronary recanalization curtails the response of plasminogen activator inhibitor activity and von Willebrand factor to myocardial infarction, most likely by reducing the extent of ischemia and necrosis and the consequent acute phase reaction. By blunting the early postinfarction procoagulant state, prompt recanalization may reduce the risk of thromboembolic complications in the first days after myocardial infarction.

Thrombosis-related markers in unstable angina pectoris.

While thrombus formation has been implicated in the pathogenesis of unstable angina, the value of thrombus-related markers for distinguishing unstable from stable angina is not well defined. Fibrin D-dimer and plasminogen activator inhibitor were prospectively analyzed in the peripheral blood of 46 patients (26 with unstable angina and 20 with stable angina or normal coronary arteries). Baseline blood samples were drawn within 24 h after rest pain in patients with unstable angina and in 19 of these 26 patients in less than 6 h. In patients with unstable angina, mean +/- SD (median) values for fibrin D-dimer and plasminogen activator inhibitor values measured 0.09 +/- 0.06 (0.07) microgram/ml and 9.1 +/- 9.6 (5.9) IU, respectively, compared to 0.11 +/- 0.10 (0.05) microgram/ml and 5.5 +/- 1.9 (5.0) IU/ml, in patients in the control group (p = NS for all comparisons between the two groups). Recurrent in-hospital pain, coronary anatomy and need for intervention showed no relation to the levels of these markers. In 19 additional patients (9 with unstable angina and 10 control patients) samples from the coronary sinus and the peripheral blood were also analyzed. Again, in patients with unstable angina all samples were drawn less than 24 h after rest pain; in six of nine patients samples were drawn in less than 6 h. A coronary sinus to peripheral blood gradient for either of these markers could not be demonstrated. The differences between peripheral and coronary sinus D-dimer and plasminogen activator inhibitor concentrations were also similar in patients with unstable angina and control patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinolytic system during long-distance running in IDDM patients and in healthy subjects.

OBJECTIVE: Endurance exercise has been advocated in diabetes mellitus to improve both metabolic control and prevent atherosclerotic complications. The response of the fibrinolytic system during prolonged exercise has not been studied in diabetes. RESEARCH DESIGN AND METHODS: In seven male marathon runners with IDDM and eight healthy nondiabetic male control subjects, matched for age and degree of training, we studied fibrinolytic and coagulation parameters during a 3-h 32-km outdoor running session. Measurements included t-PA, u-PA, PAI-1, PAP (as a measure of in vivo activation of fibrinolysis), FbDPs, FGN, vWF, and VIII:C. RESULTS: In both IDDM and control subjects, levels of t-PA, u-PA, PAP, vWF, and VIII:C continued to rise throughout the exercise, whereas PAI-1 showed a similar decline in both groups. FbDP rose slightly in both groups, and FGN remained unchanged. t-PA levels during exercise correlated closely with exercise intensity. These findings indicate that continued stimulation by exercise does not deplete endothelial PA stores. Differences between IDDM and control subjects were seen only for t-PA, vWF, and u-PA. The AUC during exercise (AUC0.5-3.0) of t-PA in IDDM was insignificantly lower than in control subjects (53 +/- 19 vs. 67 +/- 31 ng.ml-1.h), but the ratio of t-PA to exercise intensity was lower in IDDM (0.24 +/- 0.11 vs. 0.31 +/- 0.13, P less than 0.05). The AUC0.5-3.0 of vWF was lower in IDDM than in control subjects (569 +/- 268 vs. 880 +/- 265%.h, P less than 0.05). The AUC0.5-3.0 of u-PA was higher in IDDM than in control subjects (15.1 +/- 3.5 vs. 11.2 +/- 1.8 ng.ml-1.h, P less than 0.05). CONCLUSIONS: Despite a defect in the exercise-induced endothelial release of vWF and t-PA, the overall potential to activate fibrinolysis is intact in IDDM, possibly by enhancement of u-PA after exercise. Our data suggest that in IDDM, as in nondiabetic subjects, long-distance running may slow the progression of atherosclerosis by stimulating fibrinolysis.

Differential effects of platelet-derived growth factor isoforms on plasminogen activator activity in fetal rat osteoblasts due to isoform-specific receptor functions.

We examined receptor binding and metabolic effects of the platelet-derived growth factor (PDGF) isoforms AA, AB, and BB in cultures of osteoblastic cells from fetal rat calvaria. Saturation binding experiments demonstrated the presence of 6,000 binding sites for PDGF-AA, 42,000 for PDGF-AB, and 60,000 for PDGF-BB. Binding competition experiments were compatible with the recently postulated model of three PDGF receptor subtypes with differential affinity for the PDGF isoforms. The effects of the PDGF isoforms on DNA synthesis, collagen synthesis, and alkaline phosphatase activity in osteoblasts strictly correlated with the number of available binding sites. Accordingly, PDGF-BB was the most potent isoform, PDGF-AB was slightly less potent, and PDGF-AA was the least potent. In contrast, we found that PDGF-BB was less potent than PDGF-AB in increasing plasminogen activator activity in the osteoblast-conditioned medium. Our data strongly suggest that the PDGF receptor subtypes in fetal rat osteoblasts not only selectively bind one or more PDGF isoforms, but are also capable of responding differently to these isoforms.

Angiotensin II stimulation of plasminogen activator inhibitor-1 gene expression in astroglial cells from the brain.

In this study, we investigated the mechanism of angiotensin II (Ang II) induced secretion of plasminogen activator inhibitor-1 (PAI-1) from astroglial cells prepared from 21-day-old rat brain. Competition-inhibition experiments with the use of selective antagonists for Ang II receptor subtypes indicated that astroglial cells contain chiefly Ang II type 1 (AT1) receptors. The interaction of Ang II with AT1 receptors resulted in a time- and concentration-dependent stimulation of PAI-1 gene expression. A maximal, 20-fold induction of PAI-1 messenger RNA (mRNA) steady-state levels was observed with 10 nM Ang II. This effect of Ang II was blocked by DuP753, an AT1 receptor antagonist, but not by PD123177, an AT2 receptor antagonist. Raise in PAI-1 mRNA levels was followed by an elevation in PAI-1 concentration in culture media reaching its maximum after 24 h. Interaction of Ang II with AT1 receptors also resulted in a time- and concentration-dependent stimulation of inositol phospholipid (IP) hydrolysis. A maximal, 3- to 5-fold stimulation of IP hydrolysis was observed with 10 nM Ang II. The time course experiments indicated that Ang II-induced stimulation of IP hydrolysis precedes the stimulation of PAI-1 mRNA. This suggested that activation of phospholipase C, IP hydrolysis system and possibly protein kinase C (PKC) may mediate Ang II's effect on PAI-1 mRNA. Direct stimulation of PKC by phorbol ester, phorbol 12,13-dibutyrate (PDB), resulted in a time- and concentration-dependent elevation of PAI-1 mRNA levels, similar to that caused by Ang II (maximal stimulation of 20-fold with 100 nM PDB for 4 h). This effect was totally blocked by the protein kinase C inhibitor, H7. In addition, Ang II stimulation of PAI-1 mRNA was also blocked by H7. In contrast, Ang II did not elevate PAI-1 mRNA levels in astroglial cultures from neonatal rat brains. However, treatment of neonatal cultures with PDB increased levels of this mRNA species. These observations indicate that the coupling of AT1 receptors with IP hydrolysis and PKC activation may be important for Ang II stimulation of PAI-1 gene expression. The lack of Ang II's effect on PAI-1 mRNA in neonatal astroglia may be explained either by a low coupling efficiency between AT1 receptors and the second messenger system, or by a low AT1 to AT2 receptor level ratio.

Transcriptional antagonism of phorbol ester-mediated induction of plasminogen activator inhibitor types 1 and 2 by cyclic adenosine 3',5'-monophosphate.

Gene expression of plasminogen activator inhibitor (PAI) types 1 and 2 is modulated by the protein kinase-C (PKC) and cAMP-dependent protein kinase-A (PKA) signal transduction pathways. To determine whether the PKC and PKA pathways functionally interact during modulation of PAI gene expression, we assessed changes in gene transcription rates, mRNA, and antigen levels of PAI-1 and PAI-2 in HT-1080 fibrosarcoma cells treated with the PKC activator phorbol 12-myristate 13-acetate (PMA), alone or in combination with cAMP agonists and analogs. PMA produced a transient increase in PAI-1 and a sustained increase in PAI-2, which was evident at the level of gene transcription and mRNA. Treatment with the cAMP agonist forskolin or the cAMP analog 8-bromo-cAMP decreased constitutive and PMA-mediated expression of PAI-1 mRNA. PAI-2 mRNA was below detection limits in nontreated and cAMP-treated cells. However, elevated levels of cAMP reduced the stimulatory effect of PMA on PAI-2 mRNA. The antagonism of the PMA effect by cAMP was evident at the level of gene transcription, suggesting that the end point of the functional interplay between the PKC and PKA pathways requires modulation of a nuclear transcription factor(s). Our results suggest that the PKC- and PKA-dependent signaling pathways have counteractive effects on transcriptional expression of the PAI-1 and PAI-2 genes in HT-1080 cells.

Phorbol ester increases plasminogen activator inhibitor accumulation in cultures of human granulosa cells.

Proteolytic enzymes such as plasminogen activators (PAs) and collagenases are implicated in the process of ovarian follicle rupture. Data obtained in rats support the concept that PAs are both hormonally regulated and temporally related to the ovulatory process; however, such data are lacking in the human ovary. The recent identification of a family of PA inhibitors (PAIs) adds a new dimension to the control of PA activity, and in contrast to animal studies, the human preovulatory follicle is characterized by PA inhibitory activity. To initially examine the PA and PAI system in the human ovary, granulosa cells obtained from women undergoing gonadal hyperstimulation for in vitro fertilization were cultured in the presence or absence of the protein kinase-C activator, phorbol 12-myristate 13-acetate. Reverse fibrin autography of conditioned medium from control cells revealed the presence of a putative PAI with an apparent mol wt of 50,000. Phorbol ester stimulated the accumulation of this PAI in a specific and dose-dependent manner. Cumulus cells and noncumulus granulosa cells were similar in terms of presence and regulation of PAI. Immunoprecipitation with specific antisera revealed that this human granulosa cell PAI was immunochemically related to PAI-1. This identification was supported by quantitative analysis revealing a 3.7-fold increase in PAI-1 antigen, as assessed by a specific enzyme-linked immunoabsorbent assay. These findings are the first demonstration of the in vitro regulation of PAI activity in the human ovary.

Plasminogen activators and plasminogen activator inhibitors in human colorectal carcinoma tissues are not expressed by the tumour cells.

Plasminogen activators (PA) have been implicated with the degradation of extracellular matrix during the invasive growth of metastasising tumour cells. The significance of PA expression in tumour cells for the in vivo growth of malignant tumours is still a matter of debate. We, therefore, performed immunohistological studies on human colon tumours using monoclonal antibodies against urokinase- (u-PA) and tissue-type plasminogen activator (t-PA) as well as against plasminogen activator inhibitors 1 and 2 (PAI-1, PAI-2). Normal colorectal mucosa of seven samples was negative for all four constituents of the PA system. Tumour epithelium of 64 colorectal carcinomas and 10 liver metastases was consistently negative for both, PA and their inhibitors. However, two of four human colon carcinoma cell lines weakly expressed u-PA, PAI-1 and PAI-2. Intestinal dendritic or fibroblast-like cells within the tumour tissue strongly expressed u-PA and, at a lower level, also t-PA, PAI-1 and PAI-2. Vascular endothelial cells were weakly positive for all components of the PA system in colon carcinoma. Our findings indicate that colon carcinoma cells in their natural environment do not express constituents of the PA system. PA activity, previously found in colon carcinoma tissue, is most likely derived from interstitial cells.

Prognostic value of plasminogen activators and their inhibitors in colorectal cancer.

Colorectal tumorigenesis is associated with remarkable changes in the plasminogen activation system at the tissue level. The sequence of normal mucosa-adenomatous polyp-adenocarcinoma-metastasis is accompanied by an increase in the urokinase-type of plasminogen activator, the urokinase receptor and the inhibitors type-1 and type-2, with a concurrent decrease in the tissue-type plasminogen activator. Overall survival analysis of colorectal cancer patients, with a follow-up of more than 5 years, revealed that several of these components, in both the carcinomas and their corresponding normal mucosa, are of prognostic value independent of major clinicopathological parameters. Therefore, the plasminogen activation cascade not only contributes to the invasive and metastatic growth of colorectal tumours, but might also have a clinical impact with respect to adjuvant and intervention therapy.