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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 agonist-induced P-selectin expression and GPIIb/IIIa activation. (A–F) WP were incubated with different concentrations of fascaplysin (black bars, n = 6) or vehicle (DMSO, white bars, n = 6) for 0.5 h following stimulation with PAR-1-AP (A,B), adenosine diphosphate (ADP) (B,D), or phorbol-12-myristate-13-acetate (PMA) (E,F). Data are given in % of vehicle. Unstimulated platelets served as negative control (grey bars, n = 6). Surface levels of PAC-1 (recognizing the activated form of glycoprotein (GP)IIb/IIIa) and P-selectin were assessed by flow cytometry. Mean ± SEM. * p < 0.05 vs. vehicle.
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marinedrugs-13-06774-f002: Effect of fascaplysin on agonist-induced P-selectin expression and GPIIb/IIIa activation. (A–F) WP were incubated with different concentrations of fascaplysin (black bars, n = 6) or vehicle (DMSO, white bars, n = 6) for 0.5 h following stimulation with PAR-1-AP (A,B), adenosine diphosphate (ADP) (B,D), or phorbol-12-myristate-13-acetate (PMA) (E,F). Data are given in % of vehicle. Unstimulated platelets served as negative control (grey bars, n = 6). Surface levels of PAC-1 (recognizing the activated form of glycoprotein (GP)IIb/IIIa) and P-selectin were assessed by flow cytometry. Mean ± SEM. * p < 0.05 vs. vehicle.

Mentions: In a next step, we investigated the effect of fascaplysin on the expression of P-selectin and the activation of GPIIb/IIIa on platelets. For this purpose, washed platelets were incubated with concentrations of 2–10 μM fascaplysin, stimulated with the three agonists PAR-1-AP, adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) and analyzed by flow cytometry. We found that fascaplysin does not affect the expression of P-selectin after PAR-1-AP and ADP stimulation (Figure 2A,C). Moreover, only the higher dose of 10 μM fascaplysin significantly inhibited P-selectin expression on PMA-activated platelets (Figure 2E). In contrast, fascaplysin markedly suppressed the activation of GPIIb/IIIa, as indicated by reduced PAC-1 binding levels after PAR-1-AP, ADP or PMA stimulation (Figure 2B,D,F).


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 agonist-induced P-selectin expression and GPIIb/IIIa activation. (A–F) WP were incubated with different concentrations of fascaplysin (black bars, n = 6) or vehicle (DMSO, white bars, n = 6) for 0.5 h following stimulation with PAR-1-AP (A,B), adenosine diphosphate (ADP) (B,D), or phorbol-12-myristate-13-acetate (PMA) (E,F). Data are given in % of vehicle. Unstimulated platelets served as negative control (grey bars, n = 6). Surface levels of PAC-1 (recognizing the activated form of glycoprotein (GP)IIb/IIIa) and P-selectin were assessed by flow cytometry. Mean ± SEM. * p < 0.05 vs. vehicle.
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marinedrugs-13-06774-f002: Effect of fascaplysin on agonist-induced P-selectin expression and GPIIb/IIIa activation. (A–F) WP were incubated with different concentrations of fascaplysin (black bars, n = 6) or vehicle (DMSO, white bars, n = 6) for 0.5 h following stimulation with PAR-1-AP (A,B), adenosine diphosphate (ADP) (B,D), or phorbol-12-myristate-13-acetate (PMA) (E,F). Data are given in % of vehicle. Unstimulated platelets served as negative control (grey bars, n = 6). Surface levels of PAC-1 (recognizing the activated form of glycoprotein (GP)IIb/IIIa) and P-selectin were assessed by flow cytometry. Mean ± SEM. * p < 0.05 vs. vehicle.
Mentions: In a next step, we investigated the effect of fascaplysin on the expression of P-selectin and the activation of GPIIb/IIIa on platelets. For this purpose, washed platelets were incubated with concentrations of 2–10 μM fascaplysin, stimulated with the three agonists PAR-1-AP, adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) and analyzed by flow cytometry. We found that fascaplysin does not affect the expression of P-selectin after PAR-1-AP and ADP stimulation (Figure 2A,C). Moreover, only the higher dose of 10 μM fascaplysin significantly inhibited P-selectin expression on PMA-activated platelets (Figure 2E). In contrast, fascaplysin markedly suppressed the activation of GPIIb/IIIa, as indicated by reduced PAC-1 binding levels after PAR-1-AP, ADP or PMA stimulation (Figure 2B,D,F).

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