Actin pools and actin microfilament organization in cultured human endothelial cells after exposure to thrombin

Galdal, Kjell Sverre; Evensen, Stein A.; Höglund, Anders; Nilsen, Erling

doi:10.1111/j.1365-2141.1984.tb06108.x

Cited by 17 publications

(9 citation statements)

References 13 publications

(6 reference statements)

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“…That MARCKS may be involved in this process is consistent with several observations of agonist-induced endothelial cell responses. First, treatment with a-thrombin in the 0.01-0.1 U/ml activity range alters the integrity of endothelial cell monolayers and increases solute permeability (Galdal et al, 1984;Laposata et al, 1983;Killackey et al, 1986;De Michele et al, 1990). These concentrations are similar to those which we report here for MARCKS phosphorylation and are approximately 10-fold less than those required for maximal elevation of [Ca2'], or vWF secretion (Brock and Capasso, 1988;Zavoico et al, 1989).…”

Section: Discussionmentioning

confidence: 99%

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine‐rich C‐kinase substrate in human umbilical vein endothelial cells: Evidence for distinct patterns of protein kinase activation

Jacobson¹,

Pober

Fenton

et al. 1992

Journal Cellular Physiology

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Human alpha-thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine-rich C-kinase substrate (MARCKS). Stimulation by 0.1-10 U/ml of alpha-thrombin produces a time-dependent, sustained (plateau 3-5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42-55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like alpha-thrombin, produces sustained phosphorylation of MARCKS (plateau 3-5 min). In contrast, 100 microM histamine results in rapid but transient MARCKS phosphorylation (peak 1-3 min). HUVEC treated with 100 microM histamine for 5 min can be restimulated by alpha-thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both alpha-thrombin- and histamine-induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine-induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist-specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC.

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Section: Discussionmentioning

confidence: 99%

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine‐rich C‐kinase substrate in human umbilical vein endothelial cells: Evidence for distinct patterns of protein kinase activation

Jacobson¹,

Pober

Fenton

et al. 1992

Journal Cellular Physiology

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“…Exogenous thrombin addition to unstimulated and stimulated HUVECs caused cells to lose their normal polygonal shape due to actin polymerization. 26 To prevent actin polymerization and cell contraction, confluent HUVECs were treated for 30 minutes with the Rhoassociated kinase inhibitor Y-27632 (10M, final) in culture media; this inhibitor does not prevent fibrin from interacting with ␤3 integrins. 27 Fibrinolysis assays were performed by adding tissue plasminogen activator (tPA; 250 ng/mL, final) to PFP at the reaction start.…”

Section: Clot Formation and Lysis Assaysmentioning

confidence: 99%

Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability

Campbell

Overmyer

Selzman

et al. 2009

Blood

132

159

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Fibrin is essential for hemostasis; however, abnormal fibrin formation is hypothesized to increase thrombotic risk. We previously showed that in situ thrombin generation on a cell's surface modulates the 3-dimensional structure and stability of the fibrin network. Currently, we compared the abilities of extravascular and intravascular cells to support fibrin formation, structure, and stability. Extravascular cells (fibroblasts, smooth muscle) supported formation of dense fibrin networks that resisted fibrinolysis, whereas unstimulated intravascular (endothelial) cells produced coarse networks that were susceptible to fibrinolysis. All 3 cell types produced a fibrin structural gradient, with a denser network near, versus distal to, the cell surface. Although fibrin structure depended on cellular procoagulant activity, it did not reflect interactions between integrins and fibrin. These findings contrasted with those on platelets, which influenced fibrin structure via interactions between ␤3 integrins and fibrin. Inflammatory cytokines that induced prothrombotic activity on endothelial cells caused the production of abnormally dense fibrin networks that resisted fibrinolysis. Blocking tissue factor activity significantly reduced the density and stability of fibrin networks produced by cytokine-stimulated endothelial cells. Together, these findings indicate fibrin structure and stability reflect the procoagulant phenotype of the endogenous cells, and suggest abnormal fibrin structure is a novel link between inflammation and thrombosis. (Blood. 2009;114:4886-4896)

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“…Exposure of EC to enzymatically active thrombin at a concentration of0.1 U/ml (8 X 10-10 mol/liter) or greater has been shown to cause cell contraction (12,33), and a decrease in unpolymerized actin associated with increased formation of stress fibers was found after exposure to 2 U/ml (33). In our cell spreading experiments, we prepared fibrin using a thrombin concentration of 0.5 U/ml, and < 1% bound to the surface, resulting in the incorporation of < 1 X l0-' mol (0.001 U) per well or slide.…”

Section: Discussionmentioning

confidence: 99%

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Bunce

Sporn²,

Francis³

1992

J. Clin. Invest.

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Adhesion and spreading ofcultured human umbilical vein endothelial cells on fibrin surfaces of varying structure were characterized to understand better the interactions occurring between endothelium and fibrin at sites of vascular injury. Fibrin prepared with reptilase, which cleaves only fibrinopeptide A from fibrinogen, and fibrin prepared with thrombin, which cleaves both fibrinopeptide A and fibrinopeptide B, equally supported endothelial cell adhesion. In contrast, only fibrin made with thrombin mediated endothelial cell spreading, as assessed by fluorescence microscopy of cells stained with rhodamine phalloidin to identify actin stress fibers or by scanning electron microscopy. Fibrin prepared with reptilase failed to support cell spreading. To further investigate the role of the amino terminus of the fibrin 6 chain after fibrinopeptide B cleavage in promoting cell spreading, protease III from Crotalus atrox venom was used to specifically cleave the amino-terminal 42 residues of the fibrinogen B,) chain. After clotting with thrombin, this fibrin derivative lacking B#1-42 failed to support significant cell spreading. Spreading on fibrin was unaffected by depletion of Weibel-Palade bodies from endothelial cells, indicating that the spreading was independent of stimulated von Willebrand factor release. We conclude that endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and involves residues 1542 of the fibrin 8 chain. (J. Clin. Invest.

show abstract

Actin pools and actin microfilament organization in cultured human endothelial cells after exposure to thrombin

Cited by 17 publications

References 13 publications

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine‐rich C‐kinase substrate in human umbilical vein endothelial cells: Evidence for distinct patterns of protein kinase activation

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine‐rich C‐kinase substrate in human umbilical vein endothelial cells: Evidence for distinct patterns of protein kinase activation

Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

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