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At least 487 records · Page 27Linked to original sources
PubMed2001

Close relationship of tissue plasminogen activator-plasminogen activator inhibitor-1 complex with multiple organ dysfunction syndrome investigated by means of the artificial pancreas.

BACKGROUND: Glucose tolerance (GT) has not been taken into consideration in investigations concerning relationships between coagulopathy and multiple organ dysfunction syndrome (MODS), and endothelial cell activation/endothelial cell injury (ECA/ECI) in septic patients, although coagulopathy is known to be influenced by blood glucose level. We investigated those relationships under strict blood glucose control and evaluation of GT with the glucose clamp method by means of the artificial pancreas in nine septic patients with glucose intolerance. The relationships between GT and blood stress related hormone levels (SRH) were also investigated. METHODS: The amount of metabolized glucose (M value), as the parameter of GT, was measured by the euglycemic hyperinsulinemic glucose clamp method, in which the blood glucose level was clamped at 80 mg/dl under a continuous insulin infusion rate of 1.12 mU/kg per min, using the artificial pancreas, STG-22. Multiple organ failure (MOF) score was calculated using the MOF criteria of Japanese Association for Critical Care Medicine. Regarding coagulopathy, the following parameters were used: disseminated intravascular coagulation (DIC) score (calculated from the DIC criteria of the Ministry of Health and Welfare of Japan) and the parameters used for calculating DIC score, protein-C, protein-S, plasminogen, antithrombin III (AT-III), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator-PAI-1 (tPA-PAI-1) complex. Thrombomodulin (TM) was measured as the indicator of ECI. RESULTS: There were no significant correlations between M value and SRH, parameters indicating coagulopathy and the MOF score. The MOF score and blood TM levels were positively correlated with DIC score, thrombin-AT-III complex and tPA-PAI-1 complex, and negatively correlated with blood platelet count. CONCLUSIONS: GT was not significantly related to SRH, coagulopathy and MODS under strict blood glucose control. Hypercoagulability was closely related to MODS and ECI. Among the parameters indicating coagulopathy, tPA-PAI-1 complex, which is considered to originate from ECA, seemed to be a sensitive parameter of MODS and ECI, and might be a predictive marker of MODS. The treatment for reducing hypercoagulability and ECA/ECI were thought to be justified as one of the therapies for acutely ill septic patients.

Blood Coagulation Disorders↗
PubMed1996

Coordinated expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 during corpus luteum formation and luteolysis in the adult pseudopregnant rat.

Proteolytic activity generated by the plasminogen activator (PA) system is associated with many biological processes. Using an adult pseudopregnant rat model, we have studied how two components of the PA system, tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1), are expressed temporally and spatially during different developmental stages of the corpus luteum (CL). Northern blot analysis, in situ hybridization, in situ zymography, and fibrin overlay were used to analyze the expression and distribution of tPA and PAI-1 messenger RNA (mRNA) as well as PA activity in CL of different ages. We demonstrated that during the luteinization period (approximately days 1-2), tPA mRNA was highly and evenly expressed in newly formed CL, whereas PAI-1 mRNA was mainly detected in the central part of the same CL. In accordance with these findings, proteolytic activity generated by tPA was detected in the outer region of newly formed CL by in situ zymography. During the luteotropic period (approximately days 3-10), tPA mRNA expression was very low. PAI-1 mRNA expression was also low, but increased on day 10. As expected, proteolytic activity was very low during this period. During functional luteolysis (days 13-14) and subsequent structural luteolysis, tPA mRNA was elevated. PAI-1 mRNA was also expressed during this period. Moreover, the net PA activity, as determined by fibrin overlay, was relatively high during this period. Our studies indicate that tPA and PAI-1 are coordinately expressed in the CL, resulting in increased proteolytic activities during the luteinization and luteolytic periods. PA-mediated proteolysis may, therefore, play a role in both CL formation and luteolysis in rats.

Animals↗
PubMed2005

Plasminogen activators and plasminogen activator inhibitors in endometriosis.

Endometriosis is one of the most frequent benign gynecological diseases that affect women. Little is known about the pathogenesis and etiology of endometriosis, despite the numerous studies performed in this field. Although endometriosis is a benign disease, the endometrial tissue, after attachment to the peritoneum, has the ability to grow and invade the surrounding tissues. Similar to neoplastic growth, local extracellular proteolysis might take place, and therefore, the fibrinolytic system may be involved. An altered expression of several components of the fibrinolytic system in the endometrium and peritoneal fluid of women with the disease has been suggested as a key factor in the establishment of the endometriotic lesions. There is evidence of increased fibrinolytic activity in the eutopic endometrium of these women, resulting in endometrial fragments with a high potential to degrade the extracellular matrix and facilitate implantation. The peritoneum possesses an inherent fibrinolytic activity that is responsible for the degradation of the fibrin deposits originated after an injury. This physiological function allows a correct repair of the mesothelium, and therefore, prevents the formation of adhesions. Peritoneal fluid of women with endometriosis and pelvic adhesions has shown to have an increased fibrinolytic activity that may be implicated in reducing the formation of new adhesions. Endometriotic tissue has abnormal proteolytic capacity, which is determined by modifications of the fibrinolytic parameters in this tissue. Proteolytic status is determined by the imbalance between plasminogen activators and plasminogen activator inhibitors, which are expressed differently depending on the type of lesion considered and the stage of the disease. The aim of the present study is to review the role of the plasminogen activator system in endometriosis, consider the clinical implications and focus on possible further research efforts and therapeutic applications in this disease.

Animals↗
PubMedDate not supplied

Urokinase-type plasminogen activator and plasminogen activator inhibitor-1 as a prognostic factor in human colorectal carcinomas.

BACKGROUND/AIMS: The urokinase pathway of plasminogen activation is known to be involved in proteolytic degradation of the extracellular matrix during carcinoma invasion. METHODOLOGY: We immunohistochemically examined 97 colorectal carcinomas for the expression of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) to investigate whether uPA and PAI-1 expressions could serve as prognostic parameters; the gene expression of uPA and PAI-1 in human colon cancer tissues was also analyzed using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The relative expression levels of uPA and PAI-1 mRNAs were well correlated with immunoreactivities of uPA and PAI-1, respectively (p < 0.05). In immunohistochemical staining, diffuse specific staining was observed in the cytoplasm of carcinoma cells. uPA expression was detected in 57 carcinoma specimens (58.8%) and PAI-1 expression was detected in 36 specimens (37.1%). With regard to 5-year overall survival rate, patients whose tumors had positive uPA and negative PAI-1 immunoreactivities had a significantly poorer prognosis (p < 0.05). In multivariate analysis, the combined variable of uPA and PAI-1 was shown to be an independent prognostic indicator. CONCLUSIONS: Our results suggest that immunohistochemical combined analysis of uPA and PAI-1 may be a useful prognostic factor in colorectal carcinoma patients.

Adult↗
PubMed2002

Plasminogen activator inhibitor-1 and tissue-type plasminogen activator are up-regulated during unilateral ureteral obstruction in adult rats.

PURPOSE: The plasminogen activator (PA)-plasmin system has been shown to influence turnover of the extracellular matrix in various tissues. We examined the alteration of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (t-PA) in kidneys with unilateral ureteral obstruction in rats. MATERIALS AND METHODS: Female rats that underwent ligation of the left ureter were sacrificed 12 hours, 1, 5 or 10 days later. The expressions of PAI-1 and t-PA was determined by reverse transcription-polymerase chain reaction and immunohistochemical studies in the obstructed and contralateral kidneys in each group. RESULTS: Control kidneys showed no PAI-1 messenger (m)RNA expression. After days 1 through 10 of unilateral ureteral obstruction the amount of PAI-1 mRNA significantly increased in obstructed compared with contralateral kidneys (p <0.01). Meanwhile, slight polymerase chain reaction products of t-PA were observed in control kidneys. After 12 hours through 10 days of unilateral ureteral obstruction, t-PA mRNA in obstructed and contralateral kidneys was significantly elevated compared with in control kidneys (p <0.05). No significant difference in t-PA was observed in the obstructed and contralateral kidneys in each group. Immunoreactivity to PAI-1 and t-PA was identified in obstructed kidneys. CONCLUSIONS: PAI-1 and t-PA are up-regulated in obstructed rat kidneys. Our results indicate that the PA-plasmin system has a role in the process of matrix accumulation and degradation during rat obstructive nephropathy.

Animals↗
PubMed2002

Quantitative real-time reverse transcription-PCR assay for urokinase plasminogen activator, plasminogen activator inhibitor type 1, and tissue metalloproteinase inhibitor type 1 gene expressions in primary breast cancer.

BACKGROUND: The plasminogen activation system and matrix metalloproteinases (MMPs) play a key role in the degradation of basement membrane and extracellular matrix in tissue remodeling, cancer cell invasion, and metastasis. METHODS: Quantitative real-time reverse-transcription-PCR (RT-PCR) assays were developed to quantify urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue metalloproteinase inhibitor type 1 (TIMP-1) mRNA in 54 breast cancer tissues. Gene fragments were amplified in a LightCycler real-time PCR system using gene-specific primers and SYBR Green I. The results were normalized to beta-actin mRNA. We also quantified antigen and functional concentrations of these components. RESULTS: The intra- and interassay variabilities for mRNA quantification showed mean SDs for the crossing point of 0.12 and 0.15 cycles, respectively. PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and PAI-1 and uPA functional concentrations increased with tumor severity; the increase was statistically significant for PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and for uPA functional concentrations. Node-positive patients showed significantly higher PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations than those who were node negative. CONCLUSIONS: Quantitative real-time RT-PCR is a highly sensitive, reproducible, and fast method for measuring gene expression of PAI-1, uPA, and TIMP-1 in breast cancer. These components may be involved in breast cancer development, and increased mRNA expression may be associated with a worse prognosis.

Adult↗
PubMed2005

Gene expression of fibrinolytic factors urokinase plasminogen activator and plasminogen activator inhibitor-1 in rabbit temporo-mandibular joint cartilage with disc displacement.

BACKGROUND: The urokinase plasminogen activator system is believed to play an important role in degradation of the extracellular matrix associated with cartilage and bone destruction; however its precise roles in temporomandibular disorders have not yet been clarified. The aims of this study were to investigate the gene expression of fibrinolytic factors urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the articular cartilage of rabbit temporomandibular joint (TMJ) with disc displacement (DD) and to probe the relationship between fibrinolytic activity and cartilage remodeling. METHODS: Disc displacement of right joints was performed in 36 of 78 rabbits under investigation. The animals were sacrificed at 4 days and 1, 2, 4, 8 and 12 weeks after surgery, respectively. The right joints of these animals were harvested and processed for the examination of mRNA expression of uPA and PAI-1 in articular cartilage using in situ hybridization techniques. RESULTS: The expression of uPA and PAI-1 was co-expressed weakly in the chondrocytes from transitive zone to hypertrophic zone and mineralized zone, while no hybridizing signals were shown in proliferative zone and superficial zone in control rabbits. The most striking was the up-regulation of uPA and PAI-1 mRNA in 4-day rabbits postoperatively at the onset of cartilage degeneration. The strongest hybridizing signals for uPA and PAI-1 were seen in 2-week rabbits postoperatively. After 2 weeks, the expression of uPA and PAI-1 began to decrease and reached nearly normal level at 12 weeks. CONCLUSIONS: The expression of the uPA/PAI-1 system coincides with the pathological changes in condylar cartilage after DD. The uPA/PAI-1 system may be one of the essential mediators in articular cartilage remodeling.

Animals↗
PubMed1992

Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a noninhibitory substrate for tissue-type plasminogen activator.

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, is a serine proteinase inhibitor (serpin) that forms a 1:1 stoichiometric complex with its target proteinase leading to the formation of a stable inactive complex. The active, inhibitory form of PAI-1 spontaneously converts to a latent form that can be reactivated by protein denaturants. In the present study we have isolated another molecular form of intact PAI-1 that, in contrast with active PAI-1, does not form stable complexes with t-PA but is cleaved at the P1-P1' bond (Arg346-Met347). Other serine proteinases, e.g. urokinase-type plasminogen activator and thrombin, also cleaved this "substrate" form of PAI-1. Fluorescence spectroscopy revealed conformational differences between the latent, active, and substrate forms of PAI-1. This observation confirms our hypothesis that the three functionally different forms of PAI-1 are the consequence of conformational transitions. Thus PAI-1 may occur in three interconvertible conformations: latent, inhibitor, and substrate PAI-1. The identification of two distinct conformations of PAI-1 which interact with their target protease either as an inhibitor or as a substrate is a previously unrecognized phenomenon among the serpins. Conversion of substrate PAI-1 to its inactive degradation product may constitute a pathway for the physiological regulation of PAI-1 activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

[Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in children with ulcerative colitis].

UNLABELLED: The analysis of bibliography referring to the role of haemostasis disturbances in pathogenesis of ulcerative colitis in children revealed considerable discrepancies in the obtained findings of the study and some controversies in their interpretation. The aim of the study was the assessment of tissue plasminogen activator (t-PA) and its inhibitor (PAI-1) in children with ulcerative colitis. MATERIAL AND METHODS: The 22 children with ulcerative colitis were included in our investigation, there were 7 girls and 15 boys whose mean age was 15, 55 (+/- 3.0) years. The reference group was made up of 22 children suffering from celiac disease covering the period of full compensation in the range of similar age and sex. Blood was drawn from the cubital vein in the morning with minimal stasis into a tube containing 3.2% sodium citrate at the ratio of 1:10. The levels of t-PA and PAI-1 were measured with ELISA. RESULTS: In children with ulcerative colitis and in the control group we found out similar concentration of antigen tissue plasminogen activator (t-PA:Ag). In the group of children with ulcerative colitis under investigation a mean concentration of antigen inhibitor plasminogen activator (PAI-1:Ag) was statistically higher. CONCLUSIONS: Elevation of PAI-1:Ag concentration in plasma of the children suffering from ulcerative colitis may confirm the participation of PAI-1 in inflammatory reaction of disease- induced changes in colon mucosa.

Adolescent↗
PubMed2006

[Urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor in the seminal plasma and sperm of fertile and oligoasthenozoospermia males].

OBJECTIVE: To find the difference between the levels of urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor(uPAR) in the seminal plasma and sperm of fertile and oligoasthenozoospermia men, and to understand their correlation with male fertility. METHODS: The levels of uPA in the seminal plasma and sperm of 22 normospermic males and 44 oligoasthenozoospermia patients were measured by ELISA. RESULTS: (1) The average level of uPA in the seminal plasma and sperm of the normospermic group, ([4803.69 +/- 602.78] mU/L) and ([30.29 +/- 3.16] mU/10(6) sperm) were higher than those of the oligoasthenozoospermia group, ([4061.35 +/- 736.23] mU/L), and ([20.51 +/- 4.2] mU/10(6) sperm) (P < 0.01). (2) The average level of uPAR in the sperm of the normospermic group ([12.97 +/- 3.11] mU/10(6) sperm) was significantly higher than that of the oligoasthenozoospermia group, ([6.09 +/- 1.45] mU/10(6) sperm) (P < 0.01). (3) The levels of uPA and uPAR in the sperm and the content of uPA in the seminal plasma were positively correlated with sperm motility and viability. CONCLUSION: Urokinase-type plasminogen activator is related with male fertility and the levels of uPA and uPAR vary in the seminal plasma and sperm of fertile and oligoasthenozoospermia males.

Adult↗
PubMed1992

Kinetics of plasmin activation of single chain urinary-type plasminogen activator (scu-PA) and demonstration of a high affinity interaction between scu-PA and plasminogen.

The activation kinetics of single chain urinary-type plasminogen activator (scu-PA) by plasmin have been studied in detail. Nonstandard Michaelis-Menten kinetics were observed. To explain our results, we propose a model in which plasmin can exist in two conformations of lower activity (kcat/Km = 1.4 x 10(6) M-1 s-1) or higher activity (kcat/Km = 16.7 x 10(6) M-1 s-1) depending on whether a lysine binding site is occupied or free, respectively. These kinetic studies demonstrate that scu-PA interacts at this binding site (KD approximately 30 nM) and so is able to act as both a substrate and effector in this reaction. Binding was also demonstrated between scu-PA and Glu- or Lys-plasminogen at a high affinity site (KD approximately 65 nM), sensitive to the presence of lysine analogs. This suggests that scu-PA may be almost completely bound to plasminogen in plasma under normal physiological conditions and provides a possible explanation for the fibrin specificity of this activator, as discussed.

Aminocaproic Acid↗
PubMedDate not supplied

Levels of three hemostatic factors in relation to serum lipids. Monocyte procoagulant activity, tissue plasminogen activator, and type-1 plasminogen activator inhibitor.

To explore the relationship between blood lipid levels and a predisposition to thrombosis, levels of three hemostatic factors were measured in 41 human subjects and correlated with serum lipids. Procoagulant activity associated with peripheral blood monocytes isolated and purified after a 2-hour incubation in whole blood was not significantly related to lipid levels. However, activity in monocytes incubated with 100 ng/ml of bacterial endotoxin was significantly correlated with high density lipoprotein (HDL) cholesterol (r = 0.55, p less than 0.005), while net procoagulant activity (endotoxin-challenged minus basal) was significantly correlated with both HDL cholesterol (r = 0.61, p less than 0.005) and total cholesterol (r = 0.50, p less than 0.01). Plasma levels of the fibrinolytic factor, tissue plasminogen activator, were significantly correlated with total cholesterol (r = 0.41, p less than 0.01), while those of the type-1 plasminogen activator inhibitor were significantly correlated with both total cholesterol (r = 0.46, p less than 0.01) and total triglycerides (r = 0.31, p less than 0.05). The balance between the fibrinolytic factors was not significantly related to serum lipids. These results suggest that the expression of procoagulant activity by peripheral blood monocytes exposed to endotoxin may be enhanced in cases where HDL cholesterol levels are high. In addition, these results suggest that hypertriglyceridemia may be associated with a decreased fibrinolytic capacity due to elevated secretion of plasminogen activator inhibitor.

Adult↗
PubMed1987

Evaluation of fibrinolytic capacity by a combined assay system for tissue-type plasminogen activator antigen and function using monoclonal anti-tissue-type plasminogen activator antibodies.

An assay system has been developed that allows consecutive quantification of tissue-type plasminogen activator (t-PA) activity and t-PA antigen in the same plasma sample. In the first step t-PA is bound to an immobilized IgM monoclonal anti-t-PA antibody and functional activity of bound t-PA is quantified by its plasminogen-activating activity. In the second step the amount of bound t-PA antigen is determined by using a different peroxidase-labeled monoclonal anti-t-PA antibody. In this combined assay system t-PA functional activity was found to depend not only on the amount of t-PA antigen but also on the amount of plasminogen activator inhibitor (PAI), whereas in the t-PA antigen assay PAI did not affect the results. In plasma samples obtained from normal controls t-PA activity was detected only in post-venous occlusion plasma (3.7 +/- 2.5 IU/ml), whereas 2.7 +/- 0.5 ng/ml t-PA antigen was found before and 12.6 +/- 4.4 ng/ml after venous occlusion. Using this combined assay system to study plasma samples from patients who did not respond to venous occlusion with shortening of the euglobulin clot lysis time (ECLT), it was possible not only to confirm that in none of these patients could t-PA activity be detected in the postocclusion plasma samples but also to subdivide that group of patients into a group of about 39% not reacting with normal t-PA antigen release to venous occlusion and into a second group of about 61% that reacted with normal t-PA antigen release.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator.

Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.

Adenocarcinoma↗
PubMed1987

Plasminogen activator inhibitor-1 is the primary inhibitor of tissue-type plasminogen activator in pregnancy plasma.

The aim of the present work was to clarify to what extent plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) contribute to the increase in plasma inhibition of tissue-type plasminogen activator (t-PA) observed during pregnancy. It was demonstrated that a monoclonal antibody against PAI-1 almost completely quenched inhibition of single-chain t-PA and most of the inhibition of two-chain t-PA in plasma during the third trimester of pregnancy. The remaining inhibition of two-chain t-PA was to a great extent abolished by a PAI-2 antibody. The second order rate constant (k1) for inhibition of single-chain t-PA by the inhibitor neutralized by the PAI-1 antibody was about 4.8.10(6) M-1.s-1. The conversion of single-chain t-PA to the two-chain form increased the reaction rate with the inhibitor about 3-fold. These kinetic data are comparable with those obtained with PAI-1 in non-pregnancy plasma or with purified PAI-1. From the above results it is concluded that PAI-1 is the primary inhibitor of both single-chain and two-chain t-PA and that PAI-2 is the secondary inhibitor of two-chain t-PA in pregnancy plasma. The concentration of reactive PAI-1 versus gestation age was assayed in plasma from 6 women by binding of PAI-1 to 125I-labelled single-chain t-PA followed by quantitation of the labelled t-PA-PAI-1 complex after separation by SDS-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗
PubMed1994

The role of the finger domain of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) in initiation and progression of thrombolysis.

We further investigated the role of the finger (F) and the kringle-2 (K2) domains of tissue-type plasminogen activator (t-PA) in fibrin-stimulated plasminogen activation. To that end, the action of purified (wt) t-PA or of variants lacking F (del.F) or K2 (del.K2) was assessed either in a static, human whole blood clot-lysis system or in whole blood thrombi generated in the "Chandler loop". In both clot-lysis systems, significant differences were observed for the initiation of thrombolysis with equimolar concentrations of the t-PA variants. A relatively minor "lag phase" occurred in thrombolysis mediated by wt t-PA, whereas a 6.4-fold and 1.6-fold extension is found for del.F and del.K2, respectively. We observed identical lag-times, characteristic for each t-PA variant, in platelet-rich heads and in platelet-poor tails of thrombi. Since plasminogen activator inhibitor 1 (PAI-1) is preferentially retained in the platelet-rich heads, we conclude that the inhibitor does not interfere with the initial stage of thrombolysis but exerts its action in later stages, resulting in a reduction of the rate of clot lysis. A complementation clot-lysis assay was devised to study a potential interplay of del.F and del.K2. Accordingly, clot lysis was determined with combinations of del.F and del.K2 that were inversely varied in relation to equipotent dosage to distinguish between additive, antagonistic or synergistic effects of these variants. The isobole for combinations of del.F and del.K2 shows an independent, additive action of del.F and del.K2 in clot lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinolysis↗
PubMed1994

Heparin selectively inhibits synthesis of tissue type plasminogen activator and matrix deposition of plasminogen activator inhibitor 1 by human mesangial cells.

BACKGROUND: Mesangial changes in a variety of pathologic conditions involve mesangial cell proliferation and mesangial matrix remodelling. Heparin has been shown to prevent these processes in vivo. In vitro, heparin interferes with cell growth, proto-oncogene expression, synthesis of specific proteins, and extracellular matrix composition. In some cell types, it seems to interact with intracellular protein kinase C-dependent pathways. The effect of heparin on the mesangial plasminogen activating system (tissue type plasminogen activator, t-PA, and plasminogen activator inhibitor type 1, PAI-1), which is thought to be involved in matrix remodelling, has not been previously reported. EXPERIMENTAL DESIGN: Cultured human mesangial cells were stimulated by 10% fetal calf serum (FCS) or 16 nM phorbol myristate acetate (PMA) in the presence or absence of anticoagulant or nonanticoagulant heparins. Cell proliferation, synthesis of t-PA and PAI-1, cell morphology, and PAI-1 matrix deposition were studied using cell counting, [3H]thymidine incorporation, specific t-PA and PAI-1 enzyme-linked immunosorbent assay, Northern blot analysis, light microscopy, immunofluorescence and immunogold silver staining with combined bright-field and epipolarization microscopy. RESULTS: Heparin partially inhibited FCS-stimulated cell growth but not PMA-induced thymidine incorporation. FCS and PMA stimulated t-PA (p < 0.05 and p < 0.01, respectively) and PAI-1 synthesis (p < 0.05 and p < 0.01 respectively). Heparin selectively and partially inhibited FCS-stimulated t-PA, but not PAI-1 synthesis. It has no effect on PMA-stimulated t-PA or PAI-1 synthesis but prevented cell shape-changes induced by PMA, suggesting that heparin inhibits some but not all protein kinase C (PKC)-dependent effects and that heparin block in t-PA synthesis is distal to PKC activation. Heparin decreased PAI-1 matrix accumulation. Similar distal to PKC activation. Heparin decreased PAI-1 matrix accumulation. Similar results were observed with anticoagulant and nonanticoagulant heparin fragments. CONCLUSIONS: In human mesangial cells, anticoagulant and nonanticoagulant heparin exert an antiproliferative effect and may prevent mesangial matrix changes by decreasing FCS-stimulated t-PA synthesis and PAI-1 deposition in the matrix. Heparin is able to inhibit PKC-dependent cell shape changes but not PKC-dependent t-PA or PAI-1 synthesis. It also inhibits PKC-independent cell proliferation and t-PA synthesis. These results suggest multiple intracellular sites of action for heparin, unrelated or distal to PKC activation.

Blotting, Northern↗
PubMed1994

[Tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI)].

Tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) are the most important factors involved in the regulation of blood fibrinolysis. t-PA converts zymogen plasminogen to plasmin on the surface of endothelial cells to maintain blood fluidity and on the surface of the thrombus to efficiently lyse the thrombus. In circulating plasma, PAI-1 inactivates t-PA by forming an equimolar complex with t-PA to suppress the hyperfibrinolysis of the blood. Both proteins are synthesized by the vascular endothelial cells and secreted from the cells in resting state and in response to several stimuli such as thrombin, endotoxin, cytokines and growth factors. In various diseases such as DIC, thromboembolism and bacterial infection, the plasma concentrations of those two factors vary as a consequence of several stimuli, representing the altered fibrinolytic balance caused by the disease. Measurements of these factors and evaluation of the fibrinolytic balance will be important for determination of the most appropriate method of treatment. Moreover, the high plasma concentration of PAI-1 will be a prognostic marker of the disease and may be a risk factor of thrombotic events. In clinical treatment, t-PA is now widely used as a valuable fibrinolytic agent especially in myocardial infarction.

Endothelium, Vascular↗