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The Marine-Derived Kinase Inhibitor Fascaplysin Exerts Anti-Thrombotic Activity.

Ampofo E, Später T, Müller I, Eichler H, Menger MD, Laschke MW - Mar Drugs (2015)

Bottom Line: This was associated with a decreased platelet aggregation.Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation.Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical and Experimental Surgery, Saarland University, 66421 Homburg/Saar, Germany. emmanuel.ampofo@uks.eu.

ABSTRACT

Background: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis.

Methods: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time.

Results: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time.

Conclusion: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound.

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Related in: MedlinePlus

Effect of fascaplysin on platelet viability and PI3K signaling. (A) Washed platelets (WP) were incubated with different concentrations of fascaplysin (black bars, n = 4) or vehicle dimethyl sulfoxide (DMSO), white bars, n = 4) for 0.5 h. Untreated platelets served as negative control (grey bars, n = 4). Platelet viability was assessed by flow cytometry. Data are given in % of vehicle. Mean ± SEM. * p < 0.05 vs. vehicle; (B,C) Western blot analysis of CDK4, p85 and α-tubulin expression in untreated human dermal microvascular endothelial cells (HDMEC) and WP. The shown Western blots are representative of experiments conducted in triplicate; (D) Western blot analysis of ERK, p-ERK and α-tubulin expression in WP, which were incubated with 10 μM fascaplysin for 0.5 h followed by stimulation with protease-activated receptor-1-activating peptide (PAR-1-AP). The shown Western blots are representative of experiments conducted in triplicate.
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marinedrugs-13-06774-f001: Effect of fascaplysin on platelet viability and PI3K signaling. (A) Washed platelets (WP) were incubated with different concentrations of fascaplysin (black bars, n = 4) or vehicle dimethyl sulfoxide (DMSO), white bars, n = 4) for 0.5 h. Untreated platelets served as negative control (grey bars, n = 4). Platelet viability was assessed by flow cytometry. Data are given in % of vehicle. Mean ± SEM. * p < 0.05 vs. vehicle; (B,C) Western blot analysis of CDK4, p85 and α-tubulin expression in untreated human dermal microvascular endothelial cells (HDMEC) and WP. The shown Western blots are representative of experiments conducted in triplicate; (D) Western blot analysis of ERK, p-ERK and α-tubulin expression in WP, which were incubated with 10 μM fascaplysin for 0.5 h followed by stimulation with protease-activated receptor-1-activating peptide (PAR-1-AP). The shown Western blots are representative of experiments conducted in triplicate.

Mentions: In a first set of experiments, we analyzed the effect of fascaplysin on platelet viability by flow cytometry. We found that fascaplysin doses up to 10 μM did not exert any toxic effects, whereas higher concentrations of 25 and 50 μM significantly reduced the viability of platelets (Figure 1A). Based on these results, we decided to use only fascaplysin doses up to 10 μM for all following in vitro assays to investigate the effects of the kinase inhibitor in a non-toxic dose range.


The Marine-Derived Kinase Inhibitor Fascaplysin Exerts Anti-Thrombotic Activity.

Ampofo E, Später T, Müller I, Eichler H, Menger MD, Laschke MW - Mar Drugs (2015)

Effect of fascaplysin on platelet viability and PI3K signaling. (A) Washed platelets (WP) were incubated with different concentrations of fascaplysin (black bars, n = 4) or vehicle dimethyl sulfoxide (DMSO), white bars, n = 4) for 0.5 h. Untreated platelets served as negative control (grey bars, n = 4). Platelet viability was assessed by flow cytometry. Data are given in % of vehicle. Mean ± SEM. * p < 0.05 vs. vehicle; (B,C) Western blot analysis of CDK4, p85 and α-tubulin expression in untreated human dermal microvascular endothelial cells (HDMEC) and WP. The shown Western blots are representative of experiments conducted in triplicate; (D) Western blot analysis of ERK, p-ERK and α-tubulin expression in WP, which were incubated with 10 μM fascaplysin for 0.5 h followed by stimulation with protease-activated receptor-1-activating peptide (PAR-1-AP). The shown Western blots are representative of experiments conducted in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663553&req=5

marinedrugs-13-06774-f001: Effect of fascaplysin on platelet viability and PI3K signaling. (A) Washed platelets (WP) were incubated with different concentrations of fascaplysin (black bars, n = 4) or vehicle dimethyl sulfoxide (DMSO), white bars, n = 4) for 0.5 h. Untreated platelets served as negative control (grey bars, n = 4). Platelet viability was assessed by flow cytometry. Data are given in % of vehicle. Mean ± SEM. * p < 0.05 vs. vehicle; (B,C) Western blot analysis of CDK4, p85 and α-tubulin expression in untreated human dermal microvascular endothelial cells (HDMEC) and WP. The shown Western blots are representative of experiments conducted in triplicate; (D) Western blot analysis of ERK, p-ERK and α-tubulin expression in WP, which were incubated with 10 μM fascaplysin for 0.5 h followed by stimulation with protease-activated receptor-1-activating peptide (PAR-1-AP). The shown Western blots are representative of experiments conducted in triplicate.
Mentions: In a first set of experiments, we analyzed the effect of fascaplysin on platelet viability by flow cytometry. We found that fascaplysin doses up to 10 μM did not exert any toxic effects, whereas higher concentrations of 25 and 50 μM significantly reduced the viability of platelets (Figure 1A). Based on these results, we decided to use only fascaplysin doses up to 10 μM for all following in vitro assays to investigate the effects of the kinase inhibitor in a non-toxic dose range.

Bottom Line: This was associated with a decreased platelet aggregation.Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation.Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical and Experimental Surgery, Saarland University, 66421 Homburg/Saar, Germany. emmanuel.ampofo@uks.eu.

ABSTRACT

Background: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis.

Methods: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time.

Results: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time.

Conclusion: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound.

Show MeSH
Related in: MedlinePlus