Human plasma fibronectin as a substrate for human urokinase

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK.

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Effect of fibronectin on permeability of normal and TNF-treated lung endothelial cell monolayers

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1993.264.1.r90 ◽

1993 ◽

Vol 264 (1) ◽

pp. R90-R96 ◽

Cited By ~ 7

Author(s):

E. M. Wheatley ◽

P. A. Vincent ◽

P. J. McKeown-Longo ◽

T. M. Saba

Keyword(s):

Extracellular Matrix ◽

Tumor Necrosis Factor ◽

Vascular Permeability ◽

Human Plasma ◽

Tumor Necrosis ◽

Plasma Fibronectin ◽

Endothelial Monolayers ◽

Lung Vascular Permeability ◽

Protein Permeability ◽

Necrosis Factor

Fibronectin is found in a soluble form in plasma and lymph and in an insoluble form in the extracellular matrix. Plasma fibronectin can incorporate into the tissue pool of fibronectin where its adhesive properties may influence cell-cell interaction, cell adhesion to a collagenous matrix, and vascular integrity. Elevation of plasma fibronectin can attenuate the increase in lung vascular permeability in sheep during postoperative gram-negative bacteremia, and plasma fibronectin deficiency can magnify the increase in lung vascular permeability with postoperative sepsis. Using pulmonary endothelial monolayers, we determined if exogenous human plasma fibronectin (pFn) would influence the protein permeability of pulmonary endothelial monolayers as determined by transendothelial clearance (microliters/min) of 125I-albumin after they were exposed to human recombinant tumor necrosis factor-alpha. Treatment of endothelial monolayers with tumor necrosis factor (TNF) (200 U/ml) for 18 h resulted in a significant (P < 0.05) increase in protein permeability. Addition of intact purified human plasma fibronectin to normal confluent endothelial monolayers to yield a medium concentration of 300, 600, and 900 micrograms/ml for 18 h had no effect on baseline protein permeability. In contrast, whereas addition of lower amounts of human plasma fibronectin (300 micrograms/ml) did not attenuate the TNF-induced increase in monolayer permeability, the higher concentrations of 600 or 900 micrograms pFn/ml significantly decreased (P < 0.05) protein permeability. The ability of soluble plasma fibronectin to attenuate the TNF-induced increase in endothelial protein permeability required an incubation time of at least 2-3 h, perhaps due to a lag time required for its incorporation into the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

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Incorporation of fibronectin into matrix decreases TNF-induced increase in endothelial monolayer permeability

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.265.2.l148 ◽

1993 ◽

Vol 265 (2) ◽

pp. L148-L157 ◽

Cited By ~ 4

Author(s):

E. M. Wheatley ◽

P. J. McKeown-Longo ◽

P. A. Vincent ◽

T. M. Saba

Keyword(s):

Extracellular Matrix ◽

Human Plasma ◽

Tnf Alpha ◽

Plasma Fibronectin ◽

Endothelial Monolayer ◽

Necrosis Factor Alpha ◽

The Matrix ◽

Monolayer Permeability ◽

Adhesive Proteins ◽

Protein Permeability

Plasma fibronectin, a dimeric adhesive protein in blood, incorporates into the subendothelial and interstitial matrix in the lung especially during vascular injury. Fibronectin in the matrix is believed to influence cell-cell interaction and endothelial cell adhesion to the collagen-rich extracellular matrix. We previously observed that addition of purified soluble human plasma fibronectin (hFn) to cultured pulmonary endothelial monolayers attenuates the increase in protein permeability of such monolayers exposed to tumor necrosis factor-alpha (TNF-alpha). In the current study, we determined the specificity of this permeability response to fibronectin by comparing hFn to two other purified adhesive proteins in human plasma, i.e., vitronectin (Vn) and fibrinogen (Fg). We also determined whether matrix incorporation was essential for this hFn-mediated protective response by comparing normal intact hFn to either hFn alkylated with N-ethylmaleimide (NEM) or to purified 160/180-kDa hFn fragments, since these alternate forms of fibronectin are believed to exhibit limited ability to incorporate into matrix. Calf pulmonary artery endothelial (CPAE) monolayers (3-4 days postseeding) were exposed to human recombinant TNF-alpha for 18 h at a medium concentration of 200 U/ml followed by assessment of protein permeability using transendothelial 125I-labeled albumin clearance. Dimeric hFn (600 micrograms/ml) significantly (P < 0.05) reduced the TNF-induced increase in endothelial monolayer permeability. Vn or Fg, added at equal molar concentrations to the hFn, were unable to attenuate endothelial permeability. Immunofluorescent analysis utilizing antibodies specific to either hFn, human Vn, or human Fg revealed incorporation of the exogenous hFn into the extracellular matrix, but no matrix incorporation of Vn or Fg. Both NEM-treated dimeric hFn as well as purified 160/180-kDa fragments of hFn, which cannot incorporate into the matrix, were also unable to prevent the TNF-induced increase in protein permeability. Thus the ability for soluble hFn to reduce the TNF-induced increase in lung endothelial monolayer permeability was specific and dependent on its incorporation into the extracellular matrix.

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Synthesis of fibronectin by cultured human endothelial cells.

Journal of Experimental Medicine ◽

10.1084/jem.147.6.1779 ◽

1978 ◽

Vol 147 (6) ◽

pp. 1779-1791 ◽

Cited By ~ 311

Author(s):

E A Jaffe ◽

D F Mosher

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Human Plasma ◽

Culture Medium ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Basement Membranes ◽

Plasma Fibronectin ◽

Human Endothelial Cells ◽

Cultured Endothelial Cells

Plasma fibronectin is probably the major nonimmune particulate opsonin in blood and is cross-linked to fibrin during the final stage of blood coagulation. Fibronectin also occurs in an insoluble form in basement membranes especially those underlying endothelial cells and in loose connective tissue. Fibronectin was demonstrated in cultured human endothelial cells and in the surrounding extracellular matrix by immunofluorescence microscopy by using antibody to human plasma fibronectin. Cultured human endothelial cells released fibronectin into the culture medium which was immunologically identical to the fibronectin in human plasma. Cultured human endothelial cells were labeled with [3H] leucine. The radioactive fibronectin present in the endothelial postculture medium and in urea extracts of cellular monolayers was isolated with either anti-fibronectin coupled to Protein A-Sepharose or double antibody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When reduced, the [3H] fibronectin synthesized by cultured endothelial cells had the same mol wt (approximately 200,000) as plasma fibronectin. Unreduced, the [3H] fibronectin synthesized by endothelial cells migrated as a dimer, as did plasma fibronectin. Fibronectin accounted for approximately 15% of the protein synthesized and released by endothelial cells into the culture medium. Thus, cultured endothelial cells synthesize fibronectin, secrete it into the culture medium, and incorporate it into extracellular matrix. The results suggest that the endothelial cell is potentially a major site of synthesis of circulating plasma fibronectin. In addition, fibronectin derived from endothelial cells may be an important structural component of the subendothelium.

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Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling

Analytical Biochemistry ◽

10.1016/j.ab.2007.07.027 ◽

2008 ◽

Vol 372 (1) ◽

pp. 62-71 ◽

Cited By ~ 32

Author(s):

Celine Hoffmann ◽

Johanne Leroy-Dudal ◽

Salima Patel ◽

Olivier Gallet ◽

Emmanuel Pauthe

Keyword(s):

Extracellular Matrix ◽

Human Plasma ◽

Fluorescein Isothiocyanate ◽

Extracellular Matrix Remodeling ◽

Matrix Remodeling ◽

Plasma Fibronectin

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Degradation of human plasma and extracellular matrix fibronectin by tissue type plasminogen activator and urokinase

The International Journal of Biochemistry & Cell Biology ◽

10.1016/1357-2725(96)00055-6 ◽

1996 ◽

Vol 28 (10) ◽

pp. 1141-1150 ◽

Cited By ~ 23

Author(s):

Eleonora Marchina ◽

Sergio Barlati

Keyword(s):

Extracellular Matrix ◽

Human Plasma ◽

Plasminogen Activator ◽

Tissue Type ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650304 ◽

1996 ◽

Vol 75 (03) ◽

pp. 497-502 ◽

Cited By ~ 10

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.

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Studies on Activator Formation in Human Plasma with Streptokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649088 ◽

1973 ◽

Vol 30 (02) ◽

pp. 381-392

Author(s):

M Martin ◽

Keyword(s):

Human Plasma ◽

Plasminogen Activator ◽

Mass Action ◽

Activator Concentration ◽

Kinetic Effect ◽

Mass Action Law

SummaryThe plasminogen-streptokinase complex called “activator” was present in diluted plasma in the form of a largely dissociated mixture. More than ⅞ of the streptokinase and plasminogen molecules were available for further activator formation.The activator is probably a dissociated complex of the formulaStreptokinase + Plasminogen ⇄ Activator.The fact that an increase in activator concentration by x times is obtained by multiplying either the streptokinase content by the factor y or the plasminogen concentration by the same factor y would point to a kinetic effect along the lines of the mass action law.

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Absence of Synergism Between Tissue-Type Plasminogen Activator (t-PA), Single-Chain UrokinaseType Plasminogen Activator (scu-PA) and Urokinase on Clot Lysis in a Plasma Milieu In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661598 ◽

1986 ◽

Vol 56 (01) ◽

pp. 035-039 ◽

Cited By ~ 42

Author(s):

D Collen ◽

F De Cock ◽

E Demarsin ◽

H R Lijnen ◽

D C Stump

Keyword(s):

Human Plasma ◽

Plasminogen Activator ◽

Dose Response ◽

Fibrinolytic System ◽

Tissue Type ◽

Clot Lysis ◽

Single Chain ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator

SummaryA potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scuPA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in titrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and α2-antiplasmin.t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant α2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal.

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