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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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Protein C activation by EPCs on a fibrin clot, in the presence of exogenous protein C. Monolayer cultures of EPCs grown to confluence on a fibrin network in 96-well microtitre plates were overlaid with 50 μl of 50 mM Tris/150 mM NaCl2 (TBS) containing 5 mM calcium and 0.2% bovine serum albumin. Then, 64 nM human protein C (PC) was added with 30 mM benzamidine, 100 nM hirudin, or TBS, for 0 to 180 min. at 37°C. Twenty microlitres of supernatant was added to 80 μl of 100 nM hirudin solution to stop activated protein C (APC) generation, and APC was quantified by measuring the kinetics of hydrolysis of 500 μM S-2366 substrate at 405 nm.
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fig02: Protein C activation by EPCs on a fibrin clot, in the presence of exogenous protein C. Monolayer cultures of EPCs grown to confluence on a fibrin network in 96-well microtitre plates were overlaid with 50 μl of 50 mM Tris/150 mM NaCl2 (TBS) containing 5 mM calcium and 0.2% bovine serum albumin. Then, 64 nM human protein C (PC) was added with 30 mM benzamidine, 100 nM hirudin, or TBS, for 0 to 180 min. at 37°C. Twenty microlitres of supernatant was added to 80 μl of 100 nM hirudin solution to stop activated protein C (APC) generation, and APC was quantified by measuring the kinetics of hydrolysis of 500 μM S-2366 substrate at 405 nm.

Mentions: EPCs express the two cofactors of PC activation on their surface, implying that they might have anticoagulant functions. To determine whether EPCs express anticoagulant properties when adherent to fibrin, we studied APC generation on the surface of EPCs cultured on gelatin for 30 to 40 days then placed on a fibrin matrix prepared by recalcification of human plasma (see Methods). Part of the thrombin formed during the clotting process is adsorbed to the fibrin network. Fibrin-bound thrombin activity, measured by selective chromogenic substrate S2238 hydrolysis, averaged 4.2 nM per well. APC generation was then quantified in the presence of EPCs and exogenous immunopurified human PC, by determining the kinetics of hydrolysis of the chromogenic substrate S2366 (Fig. 2). In our experimental conditions, APC generation reached a plateau at 90 nM within 1 hr. APC formation was inhibited when the fibrin surface was pretreated with hirudin or benzamidine, inferring that thrombin adsorbed to the fibrin network plays a major role in APC generation on the EPC surface.


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)

Protein C activation by EPCs on a fibrin clot, in the presence of exogenous protein C. Monolayer cultures of EPCs grown to confluence on a fibrin network in 96-well microtitre plates were overlaid with 50 μl of 50 mM Tris/150 mM NaCl2 (TBS) containing 5 mM calcium and 0.2% bovine serum albumin. Then, 64 nM human protein C (PC) was added with 30 mM benzamidine, 100 nM hirudin, or TBS, for 0 to 180 min. at 37°C. Twenty microlitres of supernatant was added to 80 μl of 100 nM hirudin solution to stop activated protein C (APC) generation, and APC was quantified by measuring the kinetics of hydrolysis of 500 μM S-2366 substrate at 405 nm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401136&req=5

fig02: Protein C activation by EPCs on a fibrin clot, in the presence of exogenous protein C. Monolayer cultures of EPCs grown to confluence on a fibrin network in 96-well microtitre plates were overlaid with 50 μl of 50 mM Tris/150 mM NaCl2 (TBS) containing 5 mM calcium and 0.2% bovine serum albumin. Then, 64 nM human protein C (PC) was added with 30 mM benzamidine, 100 nM hirudin, or TBS, for 0 to 180 min. at 37°C. Twenty microlitres of supernatant was added to 80 μl of 100 nM hirudin solution to stop activated protein C (APC) generation, and APC was quantified by measuring the kinetics of hydrolysis of 500 μM S-2366 substrate at 405 nm.
Mentions: EPCs express the two cofactors of PC activation on their surface, implying that they might have anticoagulant functions. To determine whether EPCs express anticoagulant properties when adherent to fibrin, we studied APC generation on the surface of EPCs cultured on gelatin for 30 to 40 days then placed on a fibrin matrix prepared by recalcification of human plasma (see Methods). Part of the thrombin formed during the clotting process is adsorbed to the fibrin network. Fibrin-bound thrombin activity, measured by selective chromogenic substrate S2238 hydrolysis, averaged 4.2 nM per well. APC generation was then quantified in the presence of EPCs and exogenous immunopurified human PC, by determining the kinetics of hydrolysis of the chromogenic substrate S2366 (Fig. 2). In our experimental conditions, APC generation reached a plateau at 90 nM within 1 hr. APC formation was inhibited when the fibrin surface was pretreated with hirudin or benzamidine, inferring that thrombin adsorbed to the fibrin network plays a major role in APC generation on the EPC surface.

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