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Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells.

Smadja DM, Basire A, Amelot A, Conte A, Bièche I, Le Bonniec BF, Aiach M, Gaussem P - J. Cell. Mol. Med. (2008)

Bottom Line: The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration.These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1.Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix.

View Article: PubMed Central - PubMed

Affiliation: AP-HP, Hôpital Européen Georges Pompidou, Service d'Hématologie Biologique A, Paris, France.

ABSTRACT
Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34(+) cells. Fibrin networks generated in microplates by adding CaCl(2) to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs.

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Characterization of EPCs derived from human cord blood (A). Representative flow cytometric histograms of detached EPCs after immunolabelling with a control antibody (black line) and specific antibodies (blue line) to endothelial markers (CD146, CD31), haematopoietic markers (CD34 and CD133), leucocyte markers (CD14 and CD45), thrombin receptors (PAR-1 and CD141 = thrombomodulin) and endothelial protein C receptor (EPCR). (B). Immunofluorescence was performed on unpermeabilized EPCs, with anti-EPCR antibodies. Bar = 50 μm (magnification × 40)
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fig01: Characterization of EPCs derived from human cord blood (A). Representative flow cytometric histograms of detached EPCs after immunolabelling with a control antibody (black line) and specific antibodies (blue line) to endothelial markers (CD146, CD31), haematopoietic markers (CD34 and CD133), leucocyte markers (CD14 and CD45), thrombin receptors (PAR-1 and CD141 = thrombomodulin) and endothelial protein C receptor (EPCR). (B). Immunofluorescence was performed on unpermeabilized EPCs, with anti-EPCR antibodies. Bar = 50 μm (magnification × 40)

Mentions: Sorted human cord blood CD34+ cells were cultured with specific endothelial growth factors (EGM-2 medium), yielding small colonies of so-called late EPCs after 7 to 14 days [19]. At confluence, EPCs exhibited a cobblestone morphology (a growth pattern typical of the endothelial lineage) and expressed CD146 (S-Endo1) and CD31 (Fig. 1A). EPCs expressed CD34 antigen and, as expected, were negative for the haematopoietic stem cell antigen CD133 and for the leucocyte markers CD14 and CD45. Flow cytometry also showed that, respectively, 94% and 89% of EPCs expressed the thrombin receptors PAR-1 and thrombomodulin (CD141). EPCR was expressed by 91% of EPCs. This expression was homogeneously distributed over the cell surface, as shown by confocal microscopy (Fig. 1B).


Thrombin bound to a fibrin clot confers angiogenic and haemostatic properties on endothelial progenitor cells.

Smadja DM, Basire A, Amelot A, Conte A, Bièche I, Le Bonniec BF, Aiach M, Gaussem P - J. Cell. Mol. Med. (2008)

Characterization of EPCs derived from human cord blood (A). Representative flow cytometric histograms of detached EPCs after immunolabelling with a control antibody (black line) and specific antibodies (blue line) to endothelial markers (CD146, CD31), haematopoietic markers (CD34 and CD133), leucocyte markers (CD14 and CD45), thrombin receptors (PAR-1 and CD141 = thrombomodulin) and endothelial protein C receptor (EPCR). (B). Immunofluorescence was performed on unpermeabilized EPCs, with anti-EPCR antibodies. Bar = 50 μm (magnification × 40)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401136&req=5

fig01: Characterization of EPCs derived from human cord blood (A). Representative flow cytometric histograms of detached EPCs after immunolabelling with a control antibody (black line) and specific antibodies (blue line) to endothelial markers (CD146, CD31), haematopoietic markers (CD34 and CD133), leucocyte markers (CD14 and CD45), thrombin receptors (PAR-1 and CD141 = thrombomodulin) and endothelial protein C receptor (EPCR). (B). Immunofluorescence was performed on unpermeabilized EPCs, with anti-EPCR antibodies. Bar = 50 μm (magnification × 40)
Mentions: Sorted human cord blood CD34+ cells were cultured with specific endothelial growth factors (EGM-2 medium), yielding small colonies of so-called late EPCs after 7 to 14 days [19]. At confluence, EPCs exhibited a cobblestone morphology (a growth pattern typical of the endothelial lineage) and expressed CD146 (S-Endo1) and CD31 (Fig. 1A). EPCs expressed CD34 antigen and, as expected, were negative for the haematopoietic stem cell antigen CD133 and for the leucocyte markers CD14 and CD45. Flow cytometry also showed that, respectively, 94% and 89% of EPCs expressed the thrombin receptors PAR-1 and thrombomodulin (CD141). EPCR was expressed by 91% of EPCs. This expression was homogeneously distributed over the cell surface, as shown by confocal microscopy (Fig. 1B).

Bottom Line: The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration.These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1.Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix.

View Article: PubMed Central - PubMed

Affiliation: AP-HP, Hôpital Européen Georges Pompidou, Service d'Hématologie Biologique A, Paris, France.

ABSTRACT
Recent data suggest that endothelial progenitor cells (EPCs) are involved in recanalizing venous thrombi. We examined the impact of a fibrin network, and particularly of adsorbed thrombin, on EPCs derived from cord blood CD34(+) cells. Fibrin networks generated in microplates by adding CaCl(2) to platelet-depleted plasma retained adsorbed thrombin at the average concentration of 4.2 nM per well. EPCs expressed high levels of endothelial cell protein C receptor and thrombomodulin, allowing the generation of activated protein C on the fibrin matrix in the presence of exogenous human protein C. The fibrin matrix induced significant EPC proliferation and, when placed in the lower chamber of a Boyden device, strongly enhanced EPC migration. These effects were partly inhibited by hirudin by 41% and 66%, respectively), which suggests that fibrin-adsorbed thrombin interacts with EPCs via the thrombin receptor PAR-1. Finally, spontaneous lysis of the fibrin network, studied by measuring D-dimer release into the supernatant, was inhibited by EPCs but not by control mononuclear cells. Such an effect was associated with a 10-fold increase in plasminogen activator inhibitor-1 (PAI-1) secretion by EPCs cultivated in fibrin matrix. Overall, our data show that EPCs, in addition to their angiogenic potential, have both anticoagulant and antifibrinolytic properties. Thrombin may modulate these properties and contribute to thrombus recanalization by EPCs.

Show MeSH
Related in: MedlinePlus