Fibrin γ/γ' influences the secretion of fibrinolytic components and clot structure

Abstract Background In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. Methods The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3%, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). Results The kinetic of fibrin formation on HMEC-1 with 3% and 10% fibrinogen γ/γ' was similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3% to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. Conclusion A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.

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Fibrin γ/γ' influences the secretion of fibrinolytic components and clot structure

10.21203/rs.2.11393/v3 ◽

2019 ◽

Author(s):

Miriam Cantero ◽

Héctor Rojas ◽

Eduardo Anglés-Cano ◽

Rita Marchi

Keyword(s):

Plasminogen Activator ◽

Laser Scanning ◽

Plasminogen Activator Inhibitor ◽

Laser Scanning Confocal Microscopy ◽

Fibrinogen Concentration ◽

Scanning Confocal Microscopy ◽

Activator Inhibitor Type ◽

Culture Supernatants ◽

Pai 1 ◽

Fibrin Structure

Abstract Background In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. Methods The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3%, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). Results The kinetic of fibrin formation on HMEC-1 with 3% and 10% fibrinogen γ/γ' was similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3% to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. Conclusion A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.

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Fibrin γ/γ' influences the secretion of fibrinolytic components and clot structure

10.21203/rs.2.11393/v1 ◽

2019 ◽

Author(s):

Rita Marchi ◽

Miriam Cantero ◽

Héctor Rojas ◽

Eduardo Anglés-Cano

Keyword(s):

Plasminogen Activator ◽

Laser Scanning ◽

Plasminogen Activator Inhibitor ◽

Laser Scanning Confocal Microscopy ◽

Fibrinogen Concentration ◽

Scanning Confocal Microscopy ◽

Activator Inhibitor Type ◽

Culture Supernatants ◽

Pai 1 ◽

Fibrin Structure

Abstract Background In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. Methods The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3%, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). Results The kinetic of fibrin formation on HMEC-1 with 3% and 10% fibrinogen γ/γ' was similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3% to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. Conclusion A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.

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225 The influence of fibrin formation and degradation on the kinetics of inhibition of tissue-type plasminogen activator (T-PA) by plasminogen activator inhibitor 1 (PAI-1)

Fibrinolysis and Proteolysis ◽

10.1016/0268-9499(88)90572-3 ◽

1988 ◽

Vol 2 ◽

pp. 96 ◽

Cited By ~ 2

Author(s):

M. Røder ◽

M. Philips ◽

E. Suenson ◽

S. Thorsen

Keyword(s):

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Tissue Type ◽

Plasminogen Activator Inhibitor 1 ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Kinetics Of ◽

Kinetics Of Inhibition ◽

Activator Inhibitor ◽

Pai 1

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Haemostatic Properties of Human Pulmonary and Cerebral Microvascular Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656009 ◽

1997 ◽

Vol 77 (03) ◽

pp. 585-590 ◽

Cited By ~ 29

Author(s):

Georges E Grau ◽

Philippe de Moerloose ◽

Oana Bulla ◽

Jinning Lou ◽

Zheng Lei ◽

...

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Microvascular Endothelial Cells ◽

Plasminogen Activator Inhibitor 1 ◽

E Coli ◽

Microvascular Endothelial ◽

Factor Α ◽

Kinetics Of ◽

Pai 1

SummaryLittle is known on the haemostatic profiles of human microvascular endothelial cells (MVEC) from different tissues. In addition it is not known whether MVEC from patients display the same haemostatic pattern as MVEC coming from healthy controls. To address these questions MVEC from human lung and brain were isolated and stimulated with tumour necrosis factor α (TNF) and E. coli lipopolysaccharide (LPS) for 24 h. The level and the kinetics of procoagulant activity (PCA) and thrombomodulin (TM) expression were found to be different depending on the tissue of origin and on the agonist used. In particular, the inducible PCA was higher in lung than in brain MVEC, an observation that may be related to the frequency of lung involvement in septic shock. Differences were also observed for tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) with MVEC supernatants or cell lysates. These variables were then measured in lung MVEC purified from patients with acute respiratory distress syndrome (ARDS) and compared to controls. Cells from ARDS patients constitutively expressed more PCA and PAI-1 than controls. The fibrinolytic potential, expressed as t-PA/PAI-1 ratio, was lower in ARDS than in lung MVEC. It is concluded that MVEC display different haemostatic features depending on the tissue they come from and that lung MVEC from ARDS patients present a procoagulant profile when compared with those from controls.

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ON THE KINETICS OF THE INHIBITION OF PLASMINOGEN ACTIVATORS BY THE PLASMINOGEN ACTIVATOR INHIBITOR PAI-1

10.1055/s-0038-1642808 ◽

1987 ◽

Author(s):

J Chmielewska ◽

B Wiman

Keyword(s):

Plasminogen Activator ◽

Rate Constant ◽

Rate Constants ◽

Plasminogen Activator Inhibitor ◽

Plasminogen Activators ◽

Order Rate Constant ◽

Association Reaction ◽

The One ◽

Kinetics Of ◽

Pai 1

The kinetics of the inhibition of the following plasminogen activators: one- and two-chain tissue plasminogen activator (t-PA) and low and high molecular weight urokinase (UK) by PAI-1 was studied. For this purpose direct systems were employed and the reactions were studied in the presence of different concentrations of plasminogen activator chromogenic substrates. The second-order rate constant of the association reaction was estimated from the initial decline in plasminogen activator activity. Determination of the rate constants in the absence of substrates was performed by plotting the rate constants versus the substrate concentrations and extrapolation to zero concentration. The rate constants with all plasminogen activators were very similar and estimated as 2 - 4 x 107 M-1 x s-1. The reactions were also studied in the presence of 6-aminohexanoic acid, lysine, arginine, guanidinium chloride (final concentrations for all substances about 1 mmol/L) and heparin (10 mg/L), without any significant effect on the rate constants. The effect of soluble fibrin (bathroxobin-digested fibrinogen in urea) at 10 - 300 nmol/L was also studied. With one-chain t-PA the rate constant was decreased about 10-fold with the highest fibrin concentration and about 2-fold at 30 nmol/L. In contrast, the reactions with urokinase or two-chain t-PA were not influenced by fibrin at these concentrations. These findings may have a physiological significance: the one-chain t-PA adsorbed to the fibrin surface and actively involved in fibrinolysis would be protected against inactivation by PAI. This phenomenon adds further to the physiological fibrin specificity of one-chain t-PA.

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Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

1990 ◽

Vol 48 (3) ◽

pp. 166-167

Author(s):

J. Holy ◽

G. Schatten

Keyword(s):

Confocal Microscopy ◽

Mitotic Spindle ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Two Dimensions ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Cleavage Division ◽

Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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Automated 3-D cell population analysis in thick tissue sections using laser-scanning confocal-microscopy data

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100148642 ◽

1993 ◽

Vol 51 ◽

pp. 562-563

Author(s):

Hakan Ancin

Keyword(s):

Laser Scanning ◽

Population Analysis ◽

Three Dimensional ◽

Large Data ◽

Laser Scanning Confocal Microscopy ◽

Tissue Slices ◽

Scanning Confocal Microscopy ◽

Tissue Sections ◽

Spatially Adaptive ◽

Microscopy Data

This paper presents methods for performing detailed quantitative automated three dimensional (3-D) analysis of cell populations in thick tissue sections while preserving the relative 3-D locations of cells. Specifically, the method disambiguates overlapping clusters of cells, and accurately measures the volume, 3-D location, and shape parameters for each cell. Finally, the entire population of cells is analyzed to detect patterns and groupings with respect to various combinations of cell properties. All of the above is accomplished with zero subjective bias.In this method, a laser-scanning confocal light microscope (LSCM) is used to collect optical sections through the entire thickness (100 - 500μm) of fluorescently-labelled tissue slices. The acquired stack of optical slices is first subjected to axial deblurring using the expectation maximization (EM) algorithm. The resulting isotropic 3-D image is segmented using a spatially-adaptive Poisson based image segmentation algorithm with region-dependent smoothing parameters. Extracting the voxels that were labelled as "foreground" into an active voxel data structure results in a large data reduction.

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943: Plasminogen Activator Inhibitor 1 (PAI-1) is Increased in Human Peyronie's Disease

The Journal of Urology ◽

10.1016/s0022-5347(18)35099-7 ◽

2005 ◽

Vol 173 (4S) ◽

pp. 255-255 ◽

Cited By ~ 1

Author(s):

Hugo H. Davila ◽

Thomas R. Magee ◽

Freddy Zuniga ◽

Jacob Rajfer ◽

Nestor F. GonzalezCadavid

Keyword(s):

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Peyronie’S Disease ◽

Plasminogen Activator Inhibitor 1 ◽

Peyronie's Disease ◽

Activator Inhibitor ◽

Pai 1

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Relationship of Plasminogen Activator Inhibitor-1 Levels following Thrombolytic Therapy with rt-PA as Compared to Streptokinase and Patency of Infarct Related Coronary Artery

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614637 ◽

1999 ◽

Vol 82 (07) ◽

pp. 104-108 ◽

Cited By ~ 13

Author(s):

Franck Paganelli ◽

Marie Christine Alessi ◽

Pierre Morange ◽

Jean Michel Maixent ◽

Samuel Lévy ◽

...

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Thrombolytic Therapy ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Control Group ◽

Plasminogen Activator Inhibitor 1 ◽

Vessel Patency ◽

Activator Inhibitor ◽

Pai 1

Summary Background: Type 1 plasminogen activator inhibitor (PAI-1) is considered to be risk factor for acute myocardial infarction (AMI). A rebound of circulating PAI-1 has been reported after rt-PA administration. We investigated the relationships between PAI-1 levels before and after thrombolytic therapy with streptokinase (SK) as compared to rt-PA and the patency of infarct-related arteries. Methods and Results: Fifty five consecutive patients with acute MI were randomized to strep-tokinase or rt-PA. The plasma PAI-1 levels were studied before and serially within 24 h after thrombolytic administration. Vessel patency was assessed by an angiogram at 5 ± 1days. The PAI-1 levels increased significantly with both rt-PA and SK as shown by the levels obtained from a control group of 10 patients treated with coronary angioplasty alone. However, the area under the PAI-1 curve was significantly higher with SK than with rt-PA (p <0.01) and the plasma PAI-1 levels peaked later with SK than with rt-PA (18 h versus 3 h respectively). Conversely to PAI-1 levels on admission, the PAI-1 levels after thrombolysis were related to vessel patency. Plasma PAI-1 levels 6 and 18 h after SK therapy and the area under the PAI-1 curve were significantly higher in patients with occluded arteries (p <0.002, p <0.04 and p <0.05 respectively).The same tendency was observed in the t-PA group without reaching significance. Conclusions: This study showed that the PAI-1 level increase is more pronounced after SK treatment than after t-PA treatment. There is a relationship between increased PAI-1 levels after thrombolytic therapy and poor patency. Therapeutic approaches aimed at quenching PAI-1 activity after thrombolysis might be of interest to improve the efficacy of thrombolytic therapy for acute myocardial infarction.

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