Absence of specific cell-surface binding of tissue plasminogen activator in uterine cells

1990 ◽  
Vol 5 (1) ◽  
pp. 7-14 ◽  
Author(s):  
M. Ayub ◽  
N. Jenkins ◽  
J. O. White
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Carcinoma Cells ◽  
Scatchard Analysis ◽  
Specific Cell ◽  
Dependent Manner ◽  
Surface Binding ◽  
Tissue Plasminogen ◽  
Liver Membranes ◽  
Fold Excess

ABSTRACT Tissue plasminogen activator (tPA), an arginine-specific serine protease, is an oestrogen-regulated protein in uterine and breast cancer tissue. It contains a domain which shares homology with epidermal growth factor (EGF). The aim of the present study was to determine whether specific tPA receptors or EGF receptors mediate the binding of tPA to cells and whether tPA possesses intrinsic mitogenic activity. The binding of 125I-labelled tPA to rat uterine and liver membranes was shown to be non-specific and could not be displaced by unlabelled tPA or EGF. Furthermore, acid washing of cell membranes did not unmask specific tPA-binding sites. In contrast, 125I-labelled EGF binding to both rat uterine and liver membranes was displaced in a dose-dependent manner by unlabelled EGF, and Scatchard analysis of the binding data revealed dissociation constant (Kd) values of 2·4 and 0·71 nm respectively. Unlabelled tPA (up to 20 000-fold excess) did not displace 125I-labelled EGF binding to these membranes. A study of the binding of 125I-labelled tPA and 125I-labelled EGF to endometrial carcinoma cells (Ishikawa), cervical carcinoma cells (HOG-1) and vulval carcinoma cells (A431) showed that up to a 100-fold excess of EGF or a 1000-fold excess of tPA did not displace 125I-labelled tPA binding to these cells. In contrast, 125I-labelled EGF binding was displaced by unlabelled EGF (Kd values for Ishikawa and HOG-1 cells were 2·72 and 1·92 nm respectively) but not by unlabelled tPA (1000-fold excess). Treatment of Ishikawa and HOG-1 cells with EGF (20 ng/ml) for up to 6 days stimulated [3H]thymidine incorporation 1·3- to 2-fold and 3·5- to 4·7-fold respectively. Human recombinant tPA (10 IU/ml) was without effect on both cell lines over 6 days of treatment. It was concluded that although tPA contains an EGF-like domain it does not bind specifically to membrane preparations from rat uterine or liver cells or to cultured uterine cancer cells, and does not displace bound 125I-labelled EGF. Furthermore, tPA does not have mitogenic properties similar to those of EGF.

scholarly journals Tissue plasminogen activator is a ligand of cation-independent mannose 6-phosphate receptor and consists of glycoforms that contain mannose 6-phosphate

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James J. Miller ◽  
Richard N. Bohnsack ◽  
Linda J. Olson ◽  
Mayumi Ishihara ◽  
Kazuhiro Aoki ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Dependent Manner ◽  
Plasminogen Activation ◽  
Urokinase Plasminogen Activator Receptor ◽  
Plasminogen Activation System ◽  
Extracellular Region ◽  
Glycosylation Sites ◽  
Tissue Plasminogen ◽  
Mannose 6 Phosphate

AbstractPlasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

scholarly journals Activation of tissue plasminogen activator by metastasis-inducing S100P protein

2017 ◽  
Vol 474 (19) ◽  
pp. 3227-3240 ◽  
Author(s):  
Christopher J. Clarke ◽  
Stephane R. Gross ◽  
Thamir M. Ismail ◽  
Philip S. Rudland ◽  
Morteta Al-Medhtiy ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Cell Invasion ◽  
Negative Control ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Tissue Plasminogen ◽  
First Time ◽  
Rate Of Invasion

S100P protein in human breast cancer cells is associated with reduced patient survival and, in a model system of metastasis, it confers a metastatic phenotype upon benign mammary tumour cells. S100P protein possesses a C-terminal lysine residue. Using a multiwell in vitro assay, S100P is now shown for the first time to exhibit a strong, C-terminal lysine-dependent activation of tissue plasminogen activator (tPA), but not of urokinase-catalysed plasminogen activation. The presence of 10 μM calcium ions stimulates tPA activation of plasminogen 2-fold in an S100P-dependent manner. S100P physically interacts with both plasminogen and tPA in vitro, but not with urokinase. Cells constitutively expressing S100P exhibit detectable S100P protein on the cell surface, and S100P-containing cells show enhanced activation of plasminogen compared with S100P-negative control cells. S100P shows C-terminal lysine-dependent enhancement of cell invasion. An S100P antibody, when added to the culture medium, reduced the rate of invasion of wild-type S100P-expressing cells, but not of cells expressing mutant S100P proteins lacking the C-terminal lysine, suggesting that S100P functions outside the cell. The protease inhibitors, aprotinin or α-2-antiplasmin, reduced the invasion of S100P-expressing cells, but not of S100P-negative control cells, nor cells expressing S100P protein lacking the C-terminal lysine. It is proposed that activation of tPA via the C-terminal lysine of S100P contributes to the enhancement of cell invasion by S100P and thus potentially to its metastasis-promoting activity.

Enhancement of Tissue Plasminogen Activator-Catalyzed Plasminogen Activation by Escherichia coii S Fimbriae Associated with Neonatal Septicaemia and Meningitis

1991 ◽  
Vol 65 (05) ◽  
pp. 483-486 ◽  
Author(s):  
Jaakko Parkkinen ◽  
Jörg Hacker ◽  
Timo K Korhonen
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Surface Protein ◽  
Protein S ◽  
Aminocaproic Acid ◽  
Bacterial Invasion ◽  
Dependent Manner ◽  
Plasminogen Activation ◽  
Plasmin Activity ◽  
Tissue Plasminogen

SummaryThe effect of Escherichia coli strains isolated from blood and cerebrospinal fluid of septic infants on plasminogen activation was studied. These strains typically carry a filamentous surface protein, S fimbria, that has formerly been shown to bind to endothelial cells and interact with plasminogen. The bacteria effectively promoted plasminogen activation by tissue plasminogen activator (t-PA) which was inhibited by s-aminocaproic acid. A recombinant strain expressing S fimbriae accelerated t-PA-catalyzed plasminogen activation to a similar extent as did the wild-type strains whereas the nonfimbriate recipient strain had no effect. After incubation with t-PA and plasminogen, the S-fimbriate strain displayed bacterium-bound plasmin activity whereas the nonfimbriate strain did not. Bacterium-associated plasmin generation was also observed with a strain expressing mutagenized S fimbriae that lack the cell-binding subunit SfaS but not with a strain lacking the major subunit SfaA. Both t-PA and plasminogen bound to purified S fimbriae in a lysine-dependent manner and purified S fimbriae accelerated t-PA-catalyzed plasminogen activation. The results indicate that E. coli S fimbriae form a complex with t-PA and plasminogen which enhances the rate of plasminogen activation and generates bacterium-bound plasmin. This may promote bacterial invasion and persistence in tissues and contribute to the systemic activation of fibrinolysis in septicaemia.

TISSUE PLASMINOGEN ACTIVATOR INHIBITS THROMBIN-INDUCED AGGREGATION AND SHAPE CHANGE,BUT FACILITATES SECRETION,IN GEL-FILTERED PLATELETS ONLY

1987 ◽  
Author(s):  
D Blockmans ◽  
E Van Houtte ◽  
J Arnout ◽  
P Mombaerts ◽  
D Collen ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Light Transmission ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Calcium Ionophore ◽  
Dependent Manner ◽  
Subsequent Increase ◽  
Tissue Plasminogen ◽  
Induced Aggregation

Prolonged administration of tissue plasminogen activator (t-PA) has caused bleeding problems in some patients, that did not necessarily correlate with a significant drop of fibrinogen levels. We have therefore evaluated the effect of t-PA on platelet function in vitro.Incubation of gel-filtered platelets for one hour at 37°C with 180 μg/ml plasminogen and increasing concentrations of t-P(50-1600 ng/ml) significantly inhibited shapechange and aggregation induced by thrombinand the thromboxane mimetic U 46619 in a dose-dependent manner. In an EDTA milieu, whichbolishes aggregation, a dual effect of t-PA and plasminogen was observed in the aggregometer: the thrombin- or U 46619-elicited initial decrease in light transmission, reflectingthe disc-to-sphere transformation of platelets, was almost completely inhibited from 50 ng/ml t-PA upwards; the subsequent increase in light transmission, reflecting granule secretion, was however enhanced by small amounts of t-PA (up to 200 ng/ml). The latter finding was confirmed by direct measurement of secreted ATP:t-PA atconcentrations up to 200ng/ml enhanced thrombin- or U 46619-in-duced secretion. The amount of plasmin generated in the gel-filtered platelets-plasminogen-t-PA mixtures was quantified. The same amountsof plasmin, while also inhibiting the disc-to-sphere transformation of the platelets, did not enhance thrombin-or U 46619-induced ATP secretion. When whole blood or platelet-rich plasma or gel-filtered platelets resuspended in α-antiplas-min-depleted plasma waspreincubated with t-PA, aggregation and/or shape change induced by ristocetin, arachidonic acid, the calcium ionophore A 23187, adenosine diphosphate, U 46619, thrombin, serotonin or platelet activating factor were not modified.Our results suggest that in a purified system the effects of t-PA plus plasminogen onplatelets are distinct from those of plasmin;it appears that low pharmacological concentrations of t-PA enhance the secretory responses to stimuli.

Plasmin Induces Local Thrombolysis without Causing Hemorrhage: A Comparison with Tissue Plasminogen Activator in the Rabbit

2001 ◽  
Vol 86 (09) ◽  
pp. 739-745 ◽  
Author(s):  
Kyle Landskroner ◽  
Valery Novokhatny ◽  
Thomas Zimmerman ◽  
Mansze Kong ◽  
Joel Kanouse ◽  
...  
Keyword(s):  
Blood Flow ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Solid Phase ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Local Thrombolysis ◽  
Current Usage ◽  
Tissue Plasminogen ◽  
Flow Restoration

SummaryThe direct fibrinolytic enzyme, plasmin, was compared with tissue plasminogen activator (TPA) in rabbit models of local thrombolysis and fibrinolytic hemorrhage. Plasmin was produced by solid-phase urokinase activation of plasminogen and purified on benzamidine Sepharose. Applied as an intra-arterial infusion into the thrombosed abdominal aorta under conditions of unimpeded blood flow, plasmin (4 mg/kg) and TPA (2 mg/kg) achieved equivalent clot dissolution and flow restoration. Using the model of restricted blood flow into the thrombosed aorta, which limits local plasminogen supply, plasmin was superior to TPA in clot lysis and vascular reperfusion. Using similar dosages of plasmin (2 or 4 mg/kg) and TPA (1 or 2 mg/kg) in the earpuncture rebleed model. TPA induced rebleeding in a dose-dependent manner from prior puncture sites in 9 of 10 animals, while none of the 10 animals exposed to plasmin rebled from these sites.These results suggest that plasmin is an effective, unique thrombolytic agent, distinguished from the plasminogen activators in current usage by its striking safety profile.

Tissue plasminogen activator as a novel diagnostic aid in acute pulmonary embolism

VASA ◽  
2014 ◽  
Vol 43 (6) ◽  
pp. 450-458 ◽  
Author(s):  
Julio Flores ◽  
Ángel García-Avello ◽  
Esther Alonso ◽  
Antonio Ruíz ◽  
Olga Navarrete ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
Negative Predictive Value ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Predictive Value ◽  
Acute Pulmonary Embolism ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Utility ◽  
Tissue Plasminogen ◽  
D Dimer

Background: We evaluated the diagnostic efficacy of tissue plasminogen activator (tPA), using an enzyme-linked immunosorbent assay (ELISA) and compared it with an ELISA D-dimer (VIDAS D-dimer) in acute pulmonary embolism (PE). Patients and methods: We studied 127 consecutive outpatients with clinically suspected PE. The diagnosis of PE was based on a clinical probability pretest for PE and a strict protocol of imaging studies. A plasma sample to measure the levels of tPA and D-dimer was obtained at enrollment. Diagnostic accuracy for tPA and D-dimer was determined by the area under the receiver operating characteristic (ROC) curve. Sensitivity, specificity, predictive values, and the diagnostic utility of tPA with a cutoff of 8.5 ng/mL and D-dimer with a cutoff of 500 ng/mL, were calculated for PE diagnosis. Results: PE was confirmed in 41 patients (32 %). Areas under ROC curves were 0.86 for D-dimer and 0.71 for tPA. The sensitivity/negative predictive value for D-dimer using a cutoff of 500 ng/mL, and tPA using a cutoff of 8.5 ng/mL, were 95 % (95 % CI, 88–100 %)/95 % (95 % CI, 88–100 %) and 95 % (95 % CI, 88–100 %)/94 %), respectively. The diagnostic utility to exclude PE was 28.3 % (95 % CI, 21–37 %) for D-dimer and 24.4 % (95 % CI, 17–33 %) for tPA. Conclusions: The tPA with a cutoff of 8.5 ng/mL has a high sensitivity and negative predictive value for exclusion of PE, similar to those observed for the VIDAS D-dimer with a cutoff of 500 ng/mL, although the diagnostic utility was slightly higher for the D-dimer.

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