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PubMed1993

Plasminogen and plasminogen activator assembly on the human endothelial cell.

Through assembly of plasminogen and its activators, the endothelial cell surface may provide a favorable environment for constitutive generation of plasmin. This system may be regulated at multiple levels. Abundant expression of a 40-kDa protein with dual ligand-binding capacity may promote cell surface plasmin formation by colocalizing t-PA and plasminogen in a catalytically favorable configuration. Conversion of Glu-PLG to the preactivated form Lys-PLG, in the vicinity of the cell surface, may also precede plasmin formation. Physiologic concentrations of Lp(a), furthermore, may serve to modulate plasminogen activation at the cell surface by competing for binding sites, whereas elevated levels of Lp(a) might suppress this mechanism and lead to a subclinical prothrombotic state. Finally, cell surface binding sites for both plasmin and t-PA appear to protect these molecules from their physiologic antagonists, alpha 2-plasmin inhibitor and plasminogen activator inhibitor, type-1, respectively. Plasmin formation may contribute to the nonthrombogenicity of the blood vessel wall.

Animals↗

PubMed2004

[Expression of urokinase plasminogen activator, its receptor and plasminogen activator inhibitor type 1 in different types of atherosclerotic lesion in human aorta].

The role of plasminogen activators in the regulation of key processes of atherosclerosis progression stays unclear. The aim of this study was to evaluate the expression of urokinase plasminogen activator (uPA), its receptor (uPAR) and the plasminogen activator inhibitor type 1 (PAI-1) in human aorta, and to balance them with the stage of atherosclerotic lesion. We have shown that uPA and uPAR in normal aorta are mostly expressed by intimal smooth muscle cells. The expression of these proteins was up-regulated in diseased aorta compared to normal artery. The most part of cells in both fatty streak and fibro-fatty lesion were monocytes/macrophages, and about 60% of these cells expressed uPA and its receptor. PAI-1 was mostly localized on the lumonal part of the aorta and in the extracellular matrix of the intima. We observed a moderate increase of PAI-1 expression in atherosclerotic lesion. Thus, our data indicate participation of plasminogen system in atherogenesis.

Adult↗

PubMed1980

Increased neurite development and plasminogen activator expression by exposure of human neuroblastoma cells to plasminogen-deficient growth medium.

Growth of human neuroblastoma strain SK-N-SH in a plasminogen-deficient medium results in about a 40% increase in the number of differentiated cells (cells with a neurite-like process at least 50 micrometers in length) and about a five-fold increase in the amount of plasminogen activator liberated per cell. Plasminogen deficiency has no effect on the growth rate of SK-N-SH cells. These results are consistent with the hypothesis that plasminogen activator is involved in neuroblast development.

Cell Differentiation↗

PubMed1993

The construction and expression of chimeric urokinase-type plasminogen activator genes containing kringle domains of human plasminogen.

A series of chimeric urokinase-type plasminogen activator (uPA) genes, which contain combinations of kringle domains of human plasminogen (HPg) in place of the uPA kringle (KuPA), has been constructed and expressed. Some of the resulting recombinant (r) variant uPA chimeras contain modules that potentially mediate the macroscopic binding of HPg to its activation effectors, fibrin(ogen) and 6-aminohexanoic acid (EACA). Such binding sites are not possessed by KuPA, but are present in certain of the HPg kringles, viz., kringle 1 (K1HPg), kringle 4 (K4HPg), and kringle 5 (K5HPg). The recombinant (r) chimeras constructed included molecules with replacements of KuPA with K1HPg (r-[KuPA-->K1HPg]uPA), and with KuPA replaced by double kringle combinations of K1HPgK4HPg (r-[KuPA-->K1HPgK4HPg]uPA), K2HPgK3HPg (r-[KuPA-->K2HPgK3HPg]uPA), and K4HPgK5HPg (r-[KuPA-->K4HPgK5HPg]uPA). All of these variant genes, along with their wild-type (wt) r-uPA counterparts, were expressed in human kidney 293 cells. In cases wherein EACA-binding kringles from HPg have been placed in uPA, this property has been retained in the chimeric molecule and employed as an essential part of the purification procedures for the variants. The steady state amidolytic activity of two-chain (tc) wtr-uPA toward the chromogenic substrate, H-D-pyroglutamyl-Gly-L-Arg-p-nitroanilide (S2444), is characterized by a kcat/KM (pH 7.4, 37 degrees C) of 120 s-1 mM-1. This value ranges from 92 s-1 mM-1 (tcr-[KuPA-->K1HPg]uPA) to 166 s-1 mM-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) for each of the variants, demonstrating that the catalytic efficiency of the active site is altered only in a small way by changes in the noncatalytic domain of uPA. Small differences are also observed in the abilities of these tcr variants to interact with the fast-acting plasma inhibitor of uPA, viz., plasminogen activator inhibitor-1 (PAI-1). The second-order rate constant for the interaction of PAI-1 with tcr-uPA, 0.46 x 10(7) M-1s-1 (pH 7.4, 10 degrees C), ranges from 0.29 x 10(7) M-1s-1 (tcr-[KuPA-->K1HPgK4HPg]uPA) to 1.08 x 10(7) M-1s-1 (tcr-[KuPA-->K4HPgK5HPg]uPA), for the tcr-chimeric variants. Neither wtr-uPA nor any of its chimeric r-variants interacted macroscopically with a fibrin clot under conditions that allowed binding of 74% of single-chain r-tissue-type plasminogen activator. However, the tcr-chimeric uPA variants provided HPg-enriched clot lysis times between 0.2 (r-[KuPA-->K1HPgK4HPg]uPA) and 2.4 (r-[KuPA-->K2HPgK3HPg]uPA) relative to that of wtr-uPA.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminocaproic Acid↗

PubMed1996

Competition between plasminogen and procathepsin B as a probe to demonstrate the in vitro activation of procathepsin B by the tissue plasminogen activator.

The tissue plasminogen activator (tPA) was found to activate in vitro the procathepsin B purified from malignant ascitic fluids. This activation was time and dose dependent, and was associated with the processing of procathepsin B. The present study shows that tPA is a fast activator of procathepsin B in a neutral pH range, such that generation of cathepsin B activity and processing of procathepsin B are achieved after a 5-min incubation time at 37 degrees C, pH 7.4. In contrast, competition between plasminogen and procathepsin B was observed for the activation and processing by tPA. From these findings, a plasminogen activator pathway for procathepsin B activation related to the plasminogen concentration may exist. In vivo this pathway may be involved in a proteolytic cascade linked to invasion and metastasis.

Binding, Competitive↗

PubMed1995

Skin abnormalities in mice transgenic for plasminogen activator inhibitor 1: implications for the regulation of desquamation and follicular neogenesis by plasminogen activator enzymes.

Plasminogen activator enzymes have been implicated in the regulation of growth, migration, and differentiation which occur continually in normal epidermis and cyclically in the hair follicle. To elucidate further the importance of plasminogen activation in epidermal physiology, studies were conducted using mice transgenic for human plasminogen activator inhibitor 1 (PAI-1). The epidermis of the newborn (4-7 days) transgenic mice was flaky and showed delayed hair growth compared to that of their control littermates. Histologic analyses revealed a greatly thickened stratum corneum in the transgenics. By 2 weeks after birth, no differences in epidermal morphology were apparent between transgenic and control littermates. Using in situ hybridization, immunocytochemistry, and in situ reverse zymography techniques, epidermal PAI-1 expression was correlated temporally with the aberrant epidermal morphology. These data implicate plasminogen activator activity in the regulation of epidermal shedding and follicular neogenesis.

Animals↗

PubMed1996

Urokinase-type plasminogen activator-induced monocyte adhesion is modulated by kininogen, kallikrein, factor XII, and plasminogen.

Urokinase-type plasminogen activator (u-PA) was found to induce monocyte adhesion through a u-PA receptor (u-PAR)-mediated cAMP-dependent signal transduction pathway (J. Biol. Chem. 270, 30282-30285, 1995). In the present study, the effects of kininogen, kallikrein, factor XII, and plasminogen on u-PA-induced monocyte adhesion were examined since these proteins are abundant in plasma and closely related to u-PA in fibrinolysis and inflammation. Monocyte adhesion to a standard plastic surface by u-PA was shown to be inhibited by the activated, two-chain forms of kininogen (HKa) and kallikrein. The latter occurred only at higher, though physiological, concentrations and was dependent on its catalytic activity. Monocyte adhesion was promoted by factor XII and plasminogen via a noncatalytic mechanism. The findings indicated that u-PA-induced monocyte adhesion was downregulated by HKa and kallikrein and upregulated by factor XII and plasminogen at physiological concentrations. Therefore, these contact system proteins may be important modulators of u-PA-induced monocyte adhesion, a process which is involved in many pathophysiological events.

Blood Coagulation Factors↗

PubMed1992

Tissue plasminogen activator, plasminogen activator inhibitor, and other parameters of fibrinolysis in the early stages of taurocholate acute pancreatitis in rats.

It is well known that fibrinolytic activity in the early stages of acute experimental pancreatitis (AEP) as assessed by euglobulin lysis time (ELT) is depressed. The aim of this study was to evaluate changes in the fibrinolytic system in the early stages of taurocholate AEP in rats. Tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor 1 (PAI-1) activity, plasminogen, alpha 1 proteinase inhibitor (alpha 1 PI), alpha 2 antiplasmin (alpha 2 AP), antithrombin III (AT III), fibrinogen, and ELT were measured 0.5, 1, 3, and 6 h after the induction of taurocholate AEP in rats, as well as in sham-operated animals and the control group, which was not submitted to any operation. T-PA activity decreased significantly after 3 and 6 h of AEP; PAI activity had a time course reverse to t-PA and was parallel to alpha 1 PI activity. ELT was slightly prolonged after 0.5, 1, and 3 h, whereas alpha 2 AP activity and plasminogen levels increased significantly; AT III activity was increased after 1 h in comparison to control group. Sham operation caused nonsignificant changes in fibrinolysis. Increase of PAI activity and decrease of t-PA could be a reasonable explanation for inhibited plasma euglobulin fibrinolytic activity noted in the early period of AEP.

Acute Disease↗

PubMedDate not supplied

Mechanism of enhancement by fucoidan and CNBr-fibrinogen digest of the activation of glu-plasminogen by tissue plasminogen activator.

The interactions of fucoidan with human glutamic type plasminogen (Glu-Plg), porcine pancreatic elastase digested plasminogen fractions and two chain tissue plasminogen activator t-PA) were investigated using fucoidan-Sepharose affinity chromatography. The results showed a high degree of affinity between fucoidan-Sepharose and Glu-Plg or PlgK(1-3) but not with PlgK4 or mini-Plg. Fucoidan-Sepharose also showed a high affinity for t-PA, which was largely reversed by 0.002 M 6-aminohexanoic acid (6-AH). The addition of fucoidan and CNBr-fibrinogen digest (CNBr-Fbg) gave the highest enhancement of the in vitro activation of Glu-Plg by t-PA in the presence of 0.002 M 6-AH. The results of affinity chromatography and enhancement studies suggested a template mechanism, since increasing the concentrations of any one of the two cofactors reversed the enhancement. Enzyme kinetic studies, using double reciprocal plots, showed that the addition of fucoidan-6-AH increased Kcat by 7-fold without affecting Km and addition of CNBr-Fbg lowered Km by 5-fold without significantly affecting Kcat while addition of the two cofactors lowered Km by 16-fold without significantly affecting Kcat. The enhancement by fucoidan-6-AH or by CNBr-Fbg of the in vitro activation of Glu-Plg by t-PA was reversed by plasminogen activator inhibitor 1 (PAI-1). Fucoidan-Sepharose affinity chromatography revealed that the binding of PAI-1 with fucoidan may be responsible for the reversal of the enhancement by fucoidan-6-AH.

Chromatography, Affinity↗

PubMed1992

Modulation of plasminogen activator and plasminogen activator inhibitor expression in the human U373 glioblastoma/astrocytoma cell line by inflammatory mediators.

The human U373 glioblastoma/astrocytoma cell line was found to constitutively produce and secrete a plasminogen activator and a plasminogen activator inhibitor. The plasminogen activator was identified as urokinase based on apparent molecular weight, immunoblotting with anti-urokinase antibodies, and Northern blotting with a human urokinase cDNA probe. The inhibitor secreted by U373 cells was found to be related to the PAI-1 molecule based on reactivity with anti-human PAI-1 antibodies, apparent molecular weight, and Northern blot analysis with a human PAI-1 cDNA probe. The expression of both urokinase and the PAI-1-like molecule by U373 cells could be modulated by phorbol myristate acetate or by inflammatory mediators such as interferon-gamma and interleukin-1. In the case of interleukin-1, the alpha form exhibited no detectable effect while the beta form not only elevated inhibitor levels, it also appeared to induce the production of tissue plasminogen activator. Thus, in these cells interleukin-1 beta induces alterations in PA and PAI expression and interleukin-1 alpha does not, even though the two forms are reported to utilize the same cellular receptor.

Astrocytes↗

PubMed1990

Production of plasminogen activator and plasminogen activator inhibitor by bovine lymphatic endothelial cells: modulation by TNF-alpha.

We have investigated whether lymphatic endothelial cells in culture produce plasminogen activators (PAs) and their inhibitors (PAIs) and if these activities can be modulated by the inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). Examination by reverse fibrin autography of the conditioned medium from these cells revealed a PAI of Mr 50 kDa. Also evident by fibrin autography were two species of PAs, of Mr 110 kDa and Mr 60 kDa. The 110 kDa protein co-migrated with the PA-PAI complexes and the 60 kDa protein co-migrated with tissue Plasminogen Activator (tPA). Functional and immunological assays indicated the human TNF-alpha increased the type 1 plasminogen activator inhibitor (PAI-1) in a time dependent manner. Treatment of the cells with recombinant human TNF-alpha for 24 hours resulted in a 3 to 7 fold increase in the amount of PAI released into the conditioned media. Immunoblot analysis identified the PAI in the TNF-alpha treated cell conditioned media, as PAI-1. Deposition of PAI-1 in the extracellular matrix then became apparent. TNF-alpha increased 4 fold the amount of tPA-PAI-1 complexes (Mr 110 kDa) detected in the conditioned media. Free tPA (Mr 60 kDa) decreased to 1/5 of control. Net fibrinolytic activity, as determined by a chromogenic substrate assay, decreased after TNF-alpha treatment. No urokinase type Plasminogen Activator (uPA) activity was detected in control or treated cells. This fibrinolytic activity may be important in maintaining free fluid movement in the interstitium and lymphatic vessels and in inflammatory states this potential may be decreased by the increase in PAI-1.

Animals↗

PubMed1999

Local insulin infusion stimulates expression of plasminogen activator inhibitor-1 and tissue-type plasminogen activator in normal subjects.

PURPOSE: Plasma levels of plasminogen activator inhibitor-1 are increased in obesity, hypertension, and diabetes. Their correlation with insulin levels supports the hypothesis that hypofibrinolysis may affect the development of atherosclerotic complications in patients with insulin resistance. To investigate the effect of insulin on fibrinolysis, we evaluated levels of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) antigens during insulin infusion in the forearm vascular beds of 8 healthy subjects. MATERIALS AND METHODS: Insulin was infused in the brachial artery of each subject to raise local venous concentrations to approximately 100 microU/mL. Blood samples were obtained from the brachial artery and vein at baseline, after 30, 60, 90, and 120 minutes of infusion, and 30 minutes after the end of the infusion. RESULTS: Following intra-arterial infusion of insulin, forearm blood flow (mean +/- SD) increased progressively from 2.7 +/- 0.6 to 4.0 +/- 0.6 mL/dL/min (P <0.01) and did not return to baseline after the end of the infusion. Plasminogen activator inhibitor-1 balance increased (345 +/- 160 versus 8 +/- 152 fmol/dL/min, P <0.02) at 60 minutes, reaching baseline levels after the end of the infusion. After 90 minutes, tPA balance increased (40 +/- 26 versus 7 +/- 29 fmol/dL/min, P <0.01) with a profile similar to forearm blood flow. CONCLUSIONS: Local hyperinsulinemia induces regional vasodilation and expression of PAI-1 and tPA antigens. An alteration of this physiological process could be involved in the development of hypofibrinolysis and atherosclerosis in states of insulin resistance.

Adult↗

PubMed2002

Plasminogen mRNA induction in the mouse brain after kainate excitation: codistribution with plasminogen activator inhibitor-2 (PAI-2) mRNA.

Plasminogen (Plg), which can be converted to the active protease plasmin by plasminogen activators, has been previously implicated in brain plasticity and in toxicity inflicted in hippocampal pyramidal neurons by kainate. Here we have localized Plg. mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate injection (i.p.). The results indicated that Plg mRNA was undetectable in the normal brain, but after kainate injection it was induced in neuronal cells in multiple, but specific areas, including layers II-III of the neocortex; the olfactory bulb, anterior olfactory nucleus, and the piriform cortex; the caudate/putamen and accumbens nucleus shell; throughout the amygdaloid complex; and in the CAI/CA3 subfields of the hippocampus. Interestingly, this distribution pattern coincided with what we have recently described for the plasminogen activator inhibitor-2 (PAI-2) mRNA, however differing from that of the plasminogen activator inhibitor-1 (PAI-1) mRNA, as also shown here. These results suggest that enhanced Plg gene expression could be involved in events associated with olfactory, striatal, and limbic structures. Furthermore, because PAI-2 is thought to intracellularly counteract cytotoxic events, our results raise the possibility that PAI-2 can act in the brain as an intracellular neuroprotector against potential plasmin-mediated toxicity.

Animals↗

PubMed1979

Regulation of plasminogen activator secretion in mouse peritoneal macrophages. I. - Role of serum studied by a new spectrophotometric assay for plasminogen activators.

A chromogenic tripeptide - H-D-Val-Leu-Lys-p-nitroanilide-substrate of plasmin, can be used to follow plasminogen activation by an activator such as urokinase or the activator secreted by mouse peritoneal macrophages (thioglycolate-elicited). The acceleration of p-nitroaniline production is proportional to the initial rate of plasmin formation from plasminogen. Thus, at a given plasminogen concentration, this acceleration is proportional to the activator concentration. The acceleration can be evaluated from the spectrophotometer trace recording at 405 nm the appearance of p-nitroaniline, either by means of a computer program or by a plot of delta A405 vs.t2. The sensitivity of this assay allows detection of 0.003 CTA units of urokinase. Thioglycollate-elicited mouse peritoneal macrophages secrete plasminogen activator into the extracellular medium during in vitro cultivation only after a contact with serum.

Animals↗

PubMed2001

Enzymatic assays for native plasmin, plasminogen and plasminogen activators in bovine milk.

Rapid and sensitive assays for plasmin, plasminogen and plasminogen activators (PA) were developed and applied to bovine milk. The reaction medium was clarified by addition of a dissolving agent after hydrolysis of a fluorescent substrate specific for plasmin. This final step enabled the use of larger sample amount with higher substrate concentration than other methods, and avoided previous sample preparation. The use of 4 g gelatin/l in buffers preserved plasmin activity, thus avoiding risks of overestimation of the assays results. Sensitivity, detection level, repeatability and analysis run time of plasmin and plasminogen assay were improved over previous enzymatic methods with synthetic substrates. The PA assay was assessed by measuring conversion of exogenous plasminogen into plasmin. A new kinetic approach was used to enable the direct determination of global PA activities on raw milk samples without interference from indigenous plasmin.

Animals↗

PubMed1992

A region of tissue plasminogen activator that affects plasminogen activation differentially with various fibrin(ogen)-related stimulators.

The dissolution of blood clots by plasmin is normally initiated in vivo by the activation of plasminogen to plasmin through the activity of tissue plasminogen activator (t-PA). The rate of plasminogen activation can be stimulated several orders of magnitude by the presence of fibrin-related proteins. Here we describe the kinetic analysis of both recombinant human t-PA (wild-type) and a t-PA variant produced by site-directed mutagenesis in which the original sequence from amino acids 296 to 299, KHRR, has been altered to AAAA. This tetra-alanine variant form of t-PA, K296A/H297A/R298A/R299A t-PA, we refer to as "KHRR" t-PA here. The plasminogen activating kinetics of wild-type t-PA (Activase alteplase) showed a catalytic efficiency which changed over 100-fold dependent on the stimulator in the assay. The lowest rate was in the absence of a stimulator. The following stimulators showed increasing ability to accelerate the catalytic efficiency of the reaction: fibrinogen, fragments of fibrinogen obtained by digestion with plasmin, fibrin, and slightly degraded fibrin. This increase in efficiency was driven primarily by decreases in the Michaelis constant (KM) of the reaction, whereas the catalytic rate constant (kcat) of the reaction did not change significantly. The "KHRR" variant of t-PA displayed novel kinetics with all stimulators tested. In the absence of a stimulator or with the poorer stimulators (fibrinogen and fibrinogen fragments), the KM values of the reaction with Activase alteplase and "KHRR" t-PA were similar. The kcat however, was lower with "KHRR" t-PA than with wild-type t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1998

A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms.

Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.

Animals↗

PubMed2001

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts.

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.

Aggregatibacter actinomycetemcomitans↗