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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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Impact of thrombin bound to a fibrin clot on EPC proliferation and migration (A) EPCs plated at a density of 5 × 104/well were cultured for 24 hrs on a fibrin network (pre-treated or not with hirudin for 30 min. in unsupple-mented EBM-2 medium. The cells were then washed twice in ice-cold PBS and lysed overnight at 4°C in 1 N NaOH. DNA synthesis was determined by measuring the incorporation of 5′-[3H]-thymidine for 4 hrs with a Betamatic counter. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05.(B) EPC migration was measured in modified Boyden chambers with 8-μm pore-size filters. EPCs were seeded at a density of 5 × 104 per well in 200 μl of EBM-2 medium/1% SVF and were allowed to migrate for 5 hrs at 37°C toward a fibrin matrix placed in the lower chamber. Fibrin networks were pre-treated 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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fig03: Impact of thrombin bound to a fibrin clot on EPC proliferation and migration (A) EPCs plated at a density of 5 × 104/well were cultured for 24 hrs on a fibrin network (pre-treated or not with hirudin for 30 min. in unsupple-mented EBM-2 medium. The cells were then washed twice in ice-cold PBS and lysed overnight at 4°C in 1 N NaOH. DNA synthesis was determined by measuring the incorporation of 5′-[3H]-thymidine for 4 hrs with a Betamatic counter. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05.(B) EPC migration was measured in modified Boyden chambers with 8-μm pore-size filters. EPCs were seeded at a density of 5 × 104 per well in 200 μl of EBM-2 medium/1% SVF and were allowed to migrate for 5 hrs at 37°C toward a fibrin matrix placed in the lower chamber. Fibrin networks were pre-treated with 100 nM hirudin for 30 min. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05

Mentions: When placed on the fibrin matrix, EPCs strongly adhered and proliferated, without the need for exogenous angiogenic growth factors (EBM2 medium). During the first hours of culture on the fibrin matrix, EPCs formed pseudo-tubes, but these rapidly disorganized owing to the rapid cell proliferation (data not shown). EPC proliferation was significantly reduced by hirudin, as measured by 3H thymidine incorporation (77,634 ± 15,644 versus 45,352 ± 12,822 counts per minute (cpm) for EPC proliferation on fibrin network in the absence or in the presence of hirudin, respectively, Fig. 3A), suggesting that thrombin bound to a fibrin clot is able to activate PAR-1 and to induce cell prolif-eration. We also examined whether EPCs were able to migrate towards a fibrin network in a modified Boyden chamber. Fibrin attracted EPCs through the chamber membrane, and this effect was inhibited by fibrin pretreatment with hirudin (100%versus 58 ± 5% for EPC migration towards fibrin in the absence or in the presence of hirudin, respectively, Fig. 3B). The partial inhibition of EPC proliferation and migration by hirudin suggests the involvement of mechanisms other than PAR-1 activation by thrombin bound to fibrin.


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)

Impact of thrombin bound to a fibrin clot on EPC proliferation and migration (A) EPCs plated at a density of 5 × 104/well were cultured for 24 hrs on a fibrin network (pre-treated or not with hirudin for 30 min. in unsupple-mented EBM-2 medium. The cells were then washed twice in ice-cold PBS and lysed overnight at 4°C in 1 N NaOH. DNA synthesis was determined by measuring the incorporation of 5′-[3H]-thymidine for 4 hrs with a Betamatic counter. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05.(B) EPC migration was measured in modified Boyden chambers with 8-μm pore-size filters. EPCs were seeded at a density of 5 × 104 per well in 200 μl of EBM-2 medium/1% SVF and were allowed to migrate for 5 hrs at 37°C toward a fibrin matrix placed in the lower chamber. Fibrin networks were pre-treated with 100 nM hirudin for 30 min. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05
© Copyright Policy
Related In: Results  -  Collection

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

fig03: Impact of thrombin bound to a fibrin clot on EPC proliferation and migration (A) EPCs plated at a density of 5 × 104/well were cultured for 24 hrs on a fibrin network (pre-treated or not with hirudin for 30 min. in unsupple-mented EBM-2 medium. The cells were then washed twice in ice-cold PBS and lysed overnight at 4°C in 1 N NaOH. DNA synthesis was determined by measuring the incorporation of 5′-[3H]-thymidine for 4 hrs with a Betamatic counter. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05.(B) EPC migration was measured in modified Boyden chambers with 8-μm pore-size filters. EPCs were seeded at a density of 5 × 104 per well in 200 μl of EBM-2 medium/1% SVF and were allowed to migrate for 5 hrs at 37°C toward a fibrin matrix placed in the lower chamber. Fibrin networks were pre-treated with 100 nM hirudin for 30 min. Results are expressed as mean ± SEM of at least three separate experiments.*P < 0.05
Mentions: When placed on the fibrin matrix, EPCs strongly adhered and proliferated, without the need for exogenous angiogenic growth factors (EBM2 medium). During the first hours of culture on the fibrin matrix, EPCs formed pseudo-tubes, but these rapidly disorganized owing to the rapid cell proliferation (data not shown). EPC proliferation was significantly reduced by hirudin, as measured by 3H thymidine incorporation (77,634 ± 15,644 versus 45,352 ± 12,822 counts per minute (cpm) for EPC proliferation on fibrin network in the absence or in the presence of hirudin, respectively, Fig. 3A), suggesting that thrombin bound to a fibrin clot is able to activate PAR-1 and to induce cell prolif-eration. We also examined whether EPCs were able to migrate towards a fibrin network in a modified Boyden chamber. Fibrin attracted EPCs through the chamber membrane, and this effect was inhibited by fibrin pretreatment with hirudin (100%versus 58 ± 5% for EPC migration towards fibrin in the absence or in the presence of hirudin, respectively, Fig. 3B). The partial inhibition of EPC proliferation and migration by hirudin suggests the involvement of mechanisms other than PAR-1 activation by thrombin bound to fibrin.

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