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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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Fibrin network lysis by EPCs.(A) D-dimer release by EPCs and mononuclear cells (MNCs) cultured on fibrin matrices for 72 hrs. The control corresponds to spontaneous clot lysis in the absence of cells (EBM-2 medium). Closed histograms show the results of fibrin network pre-treatment with 100 nM hirudin for 30 min. before adding EPCs. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05.(B) PAI-1 levels in supernatants of EPCs and MNCs placed on fibrin clots for 72 hrs. The control corresponds to basal levels of PAI-1 in the supernatant of the clot in the absence of cells (EBM-2 medium). When indicated, thrombin was inhibited by pre-treating the fibrin network with 100 nM hirudin for 30 min. Results are expressed as mean ±SEM of at least three separate experiments.*P < 0.05.
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fig04: Fibrin network lysis by EPCs.(A) D-dimer release by EPCs and mononuclear cells (MNCs) cultured on fibrin matrices for 72 hrs. The control corresponds to spontaneous clot lysis in the absence of cells (EBM-2 medium). Closed histograms show the results of fibrin network pre-treatment with 100 nM hirudin for 30 min. before adding EPCs. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05.(B) PAI-1 levels in supernatants of EPCs and MNCs placed on fibrin clots for 72 hrs. The control corresponds to basal levels of PAI-1 in the supernatant of the clot in the absence of cells (EBM-2 medium). When indicated, thrombin was inhibited by pre-treating the fibrin network with 100 nM hirudin for 30 min. Results are expressed as mean ±SEM of at least three separate experiments.*P < 0.05.

Mentions: In an attempt to explain these data, we first evaluated the expression level of genes encoding the main fibrinolytic system actors before and after PAR-1 activation. We used real-time quantitative RT-PCR to measure the expression of u-PA, u-PAR, t-PA, PAI-1 and PAI-2. All these genes were expressed at high levels by EPCs and were over-expressed 4 hrs after PAR-1 activation by thrombin or by the SFLLRN peptide (Table 2). The corresponding protein levels were determined in EPC lysates and supernatants by using ELISA. After thrombin and SFLLRN peptide treatment, u-PAR antigen levels in cell lysates increased by 283% and 331%, respectively (Fig. 4A). The increase in u-PAR membrane expression was confirmed by flow cytometry (data not shown). The level of secreted u-PA increased by 537% and 258%, respectively (Table 2). The level of PAI-1, a potent t-PA and u-PA inhibitor, also significantly increased after PAR-1 activation (Table 2). These results suggest that, in EPCs, PAR-1 activation is associated with modification of fibrinolytic balance.


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)

Fibrin network lysis by EPCs.(A) D-dimer release by EPCs and mononuclear cells (MNCs) cultured on fibrin matrices for 72 hrs. The control corresponds to spontaneous clot lysis in the absence of cells (EBM-2 medium). Closed histograms show the results of fibrin network pre-treatment with 100 nM hirudin for 30 min. before adding EPCs. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05.(B) PAI-1 levels in supernatants of EPCs and MNCs placed on fibrin clots for 72 hrs. The control corresponds to basal levels of PAI-1 in the supernatant of the clot in the absence of cells (EBM-2 medium). When indicated, thrombin was inhibited by pre-treating the fibrin network with 100 nM hirudin for 30 min. Results are expressed as mean ±SEM of at least three separate experiments.*P < 0.05.
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Related In: Results  -  Collection

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

fig04: Fibrin network lysis by EPCs.(A) D-dimer release by EPCs and mononuclear cells (MNCs) cultured on fibrin matrices for 72 hrs. The control corresponds to spontaneous clot lysis in the absence of cells (EBM-2 medium). Closed histograms show the results of fibrin network pre-treatment with 100 nM hirudin for 30 min. before adding EPCs. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05.(B) PAI-1 levels in supernatants of EPCs and MNCs placed on fibrin clots for 72 hrs. The control corresponds to basal levels of PAI-1 in the supernatant of the clot in the absence of cells (EBM-2 medium). When indicated, thrombin was inhibited by pre-treating the fibrin network with 100 nM hirudin for 30 min. Results are expressed as mean ±SEM of at least three separate experiments.*P < 0.05.
Mentions: In an attempt to explain these data, we first evaluated the expression level of genes encoding the main fibrinolytic system actors before and after PAR-1 activation. We used real-time quantitative RT-PCR to measure the expression of u-PA, u-PAR, t-PA, PAI-1 and PAI-2. All these genes were expressed at high levels by EPCs and were over-expressed 4 hrs after PAR-1 activation by thrombin or by the SFLLRN peptide (Table 2). The corresponding protein levels were determined in EPC lysates and supernatants by using ELISA. After thrombin and SFLLRN peptide treatment, u-PAR antigen levels in cell lysates increased by 283% and 331%, respectively (Fig. 4A). The increase in u-PAR membrane expression was confirmed by flow cytometry (data not shown). The level of secreted u-PA increased by 537% and 258%, respectively (Table 2). The level of PAI-1, a potent t-PA and u-PA inhibitor, also significantly increased after PAR-1 activation (Table 2). These results suggest that, in EPCs, PAR-1 activation is associated with modification of fibrinolytic balance.

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