scholarly journals Binding of tissue plasminogen activator to human endothelial cells. Importance of the B-chain as a ligand

1992 ◽  
Vol 287 (2) ◽  
pp. 407-413 ◽  
Author(s):  
X F Cheng ◽  
O Bäck ◽  
T K Nilsson ◽  
E Nylander Lundqvist ◽  
G Pohl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Binding Sites ◽  
Human Umbilical Cord ◽  
Cultured Endothelial Cells ◽  
Tissue Plasminogen ◽  
A Chain ◽  
Pai 1

The aim of the present study was to investigate the binding of tissue plasminogen activator (tPA) to cultured endothelial cells and to characterize binding structures present in the cultures. Studies on the binding of 125I-tPA to cultured endothelial cells from human umbilical-cord veins (HUVEC) indicated that the number of sites for specific binding of tPA is 8 x 10(5) per cell. Treatment with an excess of antibodies against plasminogen-activator inhibitor type 1 (PAI-1) caused an 80% decrease in the binding, leaving about 1.6 x 10(5) unoccupied binding sites per cell, which appeared to be different from PAI-1. About 1.9 x 10(5) binding sites/cell for tPA were found on the surface of HUVEC that had been detached from the matrix. This indicates that only minor amounts of PAI-1 occur on the surface of the cells. In addition, immunocytochemical analysis showed that PAI-1 antigen is present almost exclusively in the cytoplasm but was not observed on the surface of the cells, whereas tPA antigen is abundant on the plasma membrane of tPA-treated cells as well as intracellularly. Competition studies using unlabelled compounds showed that native tPA and tPA B-chain (the proteinase domain), as well as the inactive derivatives, B-chain inactivated with D-Phe-Pro-Arg-chloromethane and tPA-PAI-1 complex, caused a considerable quenching of the binding of 125I-tPA to HUVEC, whereas the isolated A-chain had no demonstrable effect. Two components (apparent molecular masses 38 kDa and 56 kDa) reacting with tPA but lacking PAI-1 antigen determinants were identified. Thus the data suggest that tPA binds to HUVEC by two principally different mechanisms. One is mediated by PAI-1, which binds and inactivates tPA with a functional active site. The other binding is achieved by components which react with sites on the activator molecule other than structures of the A-chain or the active site.

Tissue Plasminogen Activator Expression in Endothelial Cells Exposed to Cyclic Strain in Vitro

1992 ◽  
Vol 1 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Toshiaki Iba ◽  
Bauer E. Sumpio
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Cyclic Strain ◽  
Neutral Position ◽  
Messenger Ribonucleic Acid ◽  
Tissue Plasminogen ◽  
Pai 1

The effects of cyclic strain on the production of tissue plasminogen activator (tPA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured endothelial cells (EC) were examined. Human saphenous vein EC were seeded in selective areas of culture plates with flexible membrane bottoms (corresponding to specific strain regions) and grown to confluence. Membranes were deformed by vacuum (-20 kPa) at 60 cycles/min (0.5 s strain alternating with 0.5 s relaxation in the neutral position) for 5 days. EC grown in the periphery were subjected to 7-24% strain, while cells grown in the center experienced less than 7% strain. The results show a significant increase in immunoreactive tPA production on days 1, 3 and 5 compared to day 0 in EC subjected to more than 7% cyclic strain. There was no significant elevation of tPA in the medium of EC subjected to less than 7% strain. tPA activity could only be detected in the medium of EC subjected to more than 7% cyclic strain. PAI-1 levels in the medium were not significantly different in either group. In addition, immunocytochemical detection of intracellular tPA and messenger ribonucleic acid (mRNA) expression of tPA (assessed by the reverse transcriptase polymerase chain reaction utilizing tPA specific sense and antisense primers) was significantly increased in EC subjected to more than 7% cyclic strain. We conclude that a 60 cycles/min regimen of strain that is greater than 7% can selectively stimulate tPA production by EC in vitro and may contribute to the relative nonthrombogenicity of the endothelium in vivo.

Effect of Peptides on the Inactivation of Tissue Plasminogen Activator by Plasminogen Activator Inhibitor-1 and on the Binding of Tissue Plasminogen Activator to Endothelial Cells

1990 ◽  
Vol 64 (02) ◽  
pp. 270-275 ◽  
Author(s):  
A J Wittwer ◽  
M A Sanzo
Keyword(s):  
Endothelial Cells ◽  
Thrombolytic Therapy ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Amino Acid Sequences ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tissue Plasminogen ◽  
Two Factors ◽  
Pai 1

SummaryThe effectiveness of tissue plasminogen activator (tPA) in thrombolytic therapy is dependent upon the rate at which therapeutically administered tPA reaches the clot site and the proportion of that tPA which is enzymatically active. Interactions between tPA and its main plasma inhibitor (PAI-1) and between tPA and the endothelial cells lining blood vessels are two factors which may limit efficacy. In an attempt to identify the regions of the tPA molecule involved in these interactions, we have examined a series of synthetic peptides with amino acid sequences corresponding to different regions of the tPA molecule for their ability to protect tPA from inactivation by PAI-1 and for their ability to reduce the binding of tPA to endothelial cells. Three peptides were identified which were especially effective at maintaining tPA activity in the presence of PAI-1 and three others were found which had a lesser effect. These same peptides were also found to inhibit the binding of tPA to endothelial cells. This suggests that the same regions of the tPA molecule are involved in both processes. None of the peptides inhibited the binding of tPA to fibrin. These peptides may serve as models for the development of agents for enhancing the activity of both endogenous tPA and of tPA administered in thrombolytic therapy.

Interaction of Activators, Inhibitors and Plasminogen with Each other and with Fibrin

1979 ◽  
Author(s):  
P Wallén ◽  
M Rånby
Keyword(s):  
Plasminogen Activator ◽  
Active Site ◽  
Binding Sites ◽  
Plasminogen Activators ◽  
Weak Effect ◽  
Terminal Part ◽  
Functional Importance ◽  
Tissue Plasminogen ◽  
A Chain ◽  
Rate Of Activation

Fibrin seems to have an important regulating function for both activating and inhibiting processes. Both plasminogen and plasminogen activators are specifically adsorbed to fibrin. This is in particular the case for the tissue plasminogen activator (TA). which is very strongly adsorbed to fibrin. Fibrin in complex with plasminogen and TA greatly enhances the activation. Thus the rate of activation is increased about 40-fold in the presence of a soluble fibrin polymer whereas fibrinogen only has a weak effect. Fibrin prepared from fragment X lacking the C-terminal parts of both α-chains has significantly lower stimulating power than native fibrin. This is in accordance with the finding that a fragment with affinity for TA has been isolated from the C-terminal part of the A-chain. If native plasminogen is replaced by low Mw-plasminogen obtained by elastase cleavage the stimulating activity of fibrin disappears indicating that the lysine binding sites in the N-terminal part of plasminogen are of functional importance for the TA-induced fibrinolysis. Recent studies indicate that fibrin competes with α2 -antiplasmin for lysine binding sites and the active site in plasmin (Wiman and Collen, Nature 272, 549, 1978). Thus plasmin occupied with fibrin degradation would largely be unaccessible for α2-antiplasmin whereas free plasmin is very rapidly inactivated.

scholarly journals Astrocyte Regulation of Endothelial Tissue Plasminogen Activator in a Blood-Brain Barrier Model

1998 ◽  
Vol 18 (12) ◽  
pp. 1316-1324 ◽  
Author(s):  
Nam D. Tran ◽  
Steven S. Schreiber ◽  
Mark Fisher
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Mrna Levels ◽  
Brain Capillary ◽  
Blood Brain ◽  
Tissue Plasminogen ◽  
Pai 1

Expression of tissue plasminogen activator (tPA) substantially determines endothelial-dependent fibrinolysis. We used a blood-brain barrier (BBB) model to analyze regulation of brain capillary endothelial tPA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). This model consists of coculture of murine astrocytes with bovine brain capillary endothelial cells grown as capillary-like structures (CS); after 1 week, astrocytes become extensively associated with CS, and the BBB-associated enzyme γ-glutamyl transpeptidase is present. We measured tPA and PAI-1 mRNA and tPA activity in this model. Reverse transcription-polymerase chain reaction (RT-PCR) studies showed similar tPA and PAI-1 mRNA levels after 1 day mono-culture (endothelial cells only) versus astrocyte-endothelial coculture preparations. After 7 days (i.e., when elements of the BBB are present), astrocyte-endothelial cocultures (compared with endothelial mono-cultures) showed a 50.7% ± 27.1% (mean ± SD) reduction in tPA mRNA ( P < 0.03) and a 183.3% ± 86.9% increase in PAI-1 mRNA expression ( P < 0.02). Moreover, 7-day cocultures demonstrated reduced tPA activity compared with mono-cultures (14.6 ± 2.9 IU/mL versus 30.2 ± 7.7 IU/mL, P < 0.01); 1-day cocultures and mono-cultures had similar tPA activity. These findings demonstrate that astrocytes regulate brain capillary endothelial expression of tPA when elements of the BBB phenotype are present in this model. These data suggest an important role for astrocytes in the regulation of brain capillary endothelial fibrinolysis.

INTERACTION OF HUMAN ENDOTHELIAL CELLS WITH HEPARIN-LIKE POLYMERS : INSOLUBLE SULPHONATED POLYSTYRENE RESINS

1987 ◽  
Author(s):  
M Najab ◽  
Ch Jeanneau ◽  
H Serne ◽  
M Jozefowicz ◽  
Y Sultan
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Cellular Growth ◽  
Coagulation System ◽  
Human Umbilical Cord ◽  
Cell Extracts ◽  
Tissue Plasminogen ◽  
Polystyrene Resins ◽  
Cells Cultured

It was previously demonstrated that insoluble sulphonated polystyrene resins possessed an anticoagulant heparin-like activity in the presence of plasma. The antithrombic activity is dependent on the surface density of the sulphonated groups and involves plasmatic anti T-III. This anticoagulant activity makes this material interesting for blood compatibility concerning its effect on the coagulation system. As these anticoagulant biomaterials may be used “in vivo”, their compatibility with endothelial cells (EC) is of great interest. In the present study, EC from human umbilical cord vein were cultured in 96 well plates in presence and absence of cryoprecipitate considered as the reference culture surfaces, and in wells covered with sulphonated polystyrene beads (SPB). Cell growth in this various conditions was observed and the following parameters were compared : rate of growth of EC, presence of Von Willebrand factor (VWF) by immunofluorescence, release and synthesis of EC specific antigens : VWF, tissue plasminogen activator (T-PA) and tissue plasminogen activator inhibitor (PAI). On SPB, cellular growth was found to be in a normal range but cell morphology was somewhat different. VWF antigen was identified in cells grown either on SPB or on reference wells. Non stimulated cells, incubated in serum free medium released a basic level of VWF in the supernatant. Thrombin enhanced the release of VWF from cells cultured on coated or uncoated dishes and from cells cultured on wells covered with SPB as well. In paralell, VWF in cell extracts decreased after thrombin stimulation and no difference was observed with cells cultured in presence or in absence of SPB. Without stimulation a small amount of t-PA was only observed in the supernatant 24 H samples. Thrombin stimulation induced a comparable release of t-PA from cells cultured either on SPB or reference surfaces. t-PA synthesis, measured in cell extracts did not show significant differences. In contrast, SPB inhibited the release of PAI from EC stimulated or not stimulated by thrombin. Studies are in progress to determine whether PAI is released and absorbed by SPB or absent from the cells.

scholarly journals Binding of tissue plasminogen activator to human aortic endothelial cells

1990 ◽  
Vol 269 (2) ◽  
pp. 475-482 ◽  
Author(s):  
M A Sanzo ◽  
S C Howard ◽  
A J Wittwer ◽  
H M Cochrane
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Binding Sites ◽  
Specific Binding ◽  
Chinese Hamster Ovary Cells ◽  
Type I ◽  
Homogeneous Population ◽  
Tissue Plasminogen ◽  
Pai 1 ◽  
Aortic Endothelial

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

1989 ◽  
Vol 61 (03) ◽  
pp. 409-414 ◽  
Author(s):  
M Rånby ◽  
G Nguyen ◽  
P Y Scarabin ◽  
M Samama
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Degradation Products ◽  
Gel Filtration ◽  
Free Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Pai 1

SummaryAn enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2, α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasffi, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Effect of Lipoprotein(a) and LDL on Plasminogen Binding to Extracellular Matrix and on Matrix-dependent Plasminogen Activation by Tissue Plasminogen Activator

1996 ◽  
Vol 75 (03) ◽  
pp. 497-502 ◽  
Author(s):  
Hadewijch L M Pekelharing ◽  
Henne A Kleinveld ◽  
Pieter F C.C.M Duif ◽  
Bonno N Bouma ◽  
Herman J M van Rijn
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Single Step ◽  
Plasminogen Activation ◽  
Structural Homology ◽  
Tissue Plasminogen ◽  
Umbilical Vein Endothelial Cells

SummaryLp(a) is an LDL-like lipoprotein plus an additional apolipoprotein apo(a). Based on the structural homology of apo(a) with plasminogen, it is hypothesized that Lp(a) interferes with fibrinolysis. Extracellular matrix (ECM) produced by human umbilical vein endothelial cells was used to study the effect of Lp(a) and LDL on plasminogen binding and activation. Both lipoproteins were isolated from the same plasma in a single step. Plasminogen bound to ECM via its lysine binding sites. Lp(a) as well as LDL were capable of competing with plasminogen binding. The degree of inhibition was dependent on the lipoprotein donor as well as the ECM donor. When Lp(a) and LDL obtained from one donor were compared, Lp(a) was always a much more potent competitor. The effect of both lipoproteins on plasminogen binding was reflected in their effect on plasminogen activation. It is speculated that Lp(a) interacts with ECM via its LDL-like lipoprotein moiety as well as via its apo(a) moiety.

Herpes Simplex Virus Decreases Endothelial Cell Plasminogen Activator Inhibitor

1993 ◽  
Vol 69 (03) ◽  
pp. 253-258 ◽  
Author(s):  
Robert A Bok ◽  
Harry S Jacob ◽  
Jozsef Balla ◽  
Margaret Juckett ◽  
Theresa Stelle ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Umbilical Vein ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Plasminogen ◽  
Simplex Virus ◽  
Activator Inhibitor ◽  
Pai 1

SummaryHerpes simplex virus (HSV) infection is histopathologically associated with vascular injury, fibrinoid necrosis and inflammatory cell infiltrates. We have previously shown in vitro that HSV infection of human umbilical vein endothelial cells (HUVEC) promotes a procoagulant phenotype manifest by the induction of tissue factor, the loss of thrombomodulin, and an increase in platelet adhesion. In these studies we examined the effects of HSV infection on HUVEC plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (t-PA). HSV infection caused the loss of PAI-1 in the extracellular matrix (ECM) and that released into the supernatant of HUVEC. Both activity and antigen levels of the Serpin inhibitor are diminished as a result of HSV infection. The loss of inhibitor is not secondary to diminished vitronectin (Vn), the primary binding protein of PAI-1 in the ECM, but appears to be secondary to decreased synthesis at the RNA level. Tissue plasminogen activator (t-PA). synthesis is also decreased in endothelial HSV infection. PAI-1 loss may further promote a procoagulant phenotype in HSV infection in vivo.

Localization of Fibrinolytic Activators and Inhibitors in Normal and Atherosclerotic Vessels

1996 ◽  
Vol 75 (06) ◽  
pp. 933-938 ◽  
Author(s):  
Marten Fålkenberg ◽  
Johan Tjärnstrom ◽  
Per Örtenwall ◽  
Michael Olausson ◽  
Bo Risberg
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Vasa Vasorum ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Tissue Plasminogen ◽  
The Media ◽  
Activator Inhibitor ◽  
Pai 1

SummaryLocal fibrinolytic changes in atherosclerotic arteries have been suggested to influence plaque growth and promote mural thrombosis on ruptured or ulcerated plaques. Increased levels of plasminogen activator inhibitor (PAI-1) have been found in atherosclerotic arteries. In this study tissue plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA) and PAI-1 were localized in arterial biopsies of healthy and atherosclerotic vessels by immunohistochemis-try. The expression of fibrinolytic regulators was related to the distribution of endothelial cells (EC) and macrophages. Results: t-PA was expressed in vasa vasorum. PAI-1 was positive in endothelial cells, in the media and in the adventitia. Increased expression of t-PA, u-PA and PAI-1 was found in atherosclerotic vessels. t-PA, u-PA, PAI-1 and macrophages were co-localized in plaques. These results support the concept that macrophages can be important in the local regulation of fibrinolysis in atherosclerotic vessels.

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