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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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Effect of fascaplysin and heparin on thrombus formation and tail-vein bleeding time. (A) Intravital fluorescent microscopic images of a postcapillary venule in the dorsal skinfold chamber of a vehicle-treated BALB/c mouse before (baseline) and after photochemically induced thrombus formation (asterisk). Blue-light epi-illumination with contrast enhancement by 5% fluorescein isothiocyanate (FITC)-labeled dextran 150,000. Scale bar: 50 μm. (B) Complete occlusion time of postcapillary and collecting venules upon photochemically induced thrombus formation in dorsal skinfold chambers of heparin-treated (upper black bar, n = 3), fascaplysin-treated (lower black bar, n = 6), and vehicle-treated BALB/c mice (upper white bar, saline, n = 3 and lower white bar, DMSO, n = 6), as assessed by intravital fluorescence microscopy. Mean ± SEM. * p < 0.05 vs. vehicle. (C) Tail-vein bleeding time of heparin-treated (upper black bar, n = 3), fascaplysin-treated (lower black bar, n = 6), and vehicle-treated BALB/c mice (upper white bar, saline, n = 3 and lower white bar, DMSO, n = 6). Mean ± SEM. * p < 0.05 vs. vehicle.
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marinedrugs-13-06774-f006: Effect of fascaplysin and heparin on thrombus formation and tail-vein bleeding time. (A) Intravital fluorescent microscopic images of a postcapillary venule in the dorsal skinfold chamber of a vehicle-treated BALB/c mouse before (baseline) and after photochemically induced thrombus formation (asterisk). Blue-light epi-illumination with contrast enhancement by 5% fluorescein isothiocyanate (FITC)-labeled dextran 150,000. Scale bar: 50 μm. (B) Complete occlusion time of postcapillary and collecting venules upon photochemically induced thrombus formation in dorsal skinfold chambers of heparin-treated (upper black bar, n = 3), fascaplysin-treated (lower black bar, n = 6), and vehicle-treated BALB/c mice (upper white bar, saline, n = 3 and lower white bar, DMSO, n = 6), as assessed by intravital fluorescence microscopy. Mean ± SEM. * p < 0.05 vs. vehicle. (C) Tail-vein bleeding time of heparin-treated (upper black bar, n = 3), fascaplysin-treated (lower black bar, n = 6), and vehicle-treated BALB/c mice (upper white bar, saline, n = 3 and lower white bar, DMSO, n = 6). Mean ± SEM. * p < 0.05 vs. vehicle.

Mentions: Finally, we tested the anti-thrombotic activity of fascaplysin and heparin (positive control) in the dorsal skinfold chamber model of photochemically induced thrombus formation [31]. For this purpose, we analyzed postcapillary and collecting venules with a diameter of 15–25 μm, which exhibited comparable centerline red blood cell (RBC) velocities and wall shear rates (Table 1). As expected, heparin effectively increased complete vessel occlusion time and tail-vein bleeding time (Figure 6B,C), indicating that the present experimental setting is suitable to measure anti-thrombotic effects of test compounds. In line with our in vitro results, we detected a significantly prolonged complete vessel occlusion time within dorsal skinfold chambers of fascaplysin-treated mice when compared to vehicle-treated controls (Figure 6A,B). Moreover, treatment with fascaplysin also increased the tail-vein bleeding time (Figure 6C).


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 and heparin on thrombus formation and tail-vein bleeding time. (A) Intravital fluorescent microscopic images of a postcapillary venule in the dorsal skinfold chamber of a vehicle-treated BALB/c mouse before (baseline) and after photochemically induced thrombus formation (asterisk). Blue-light epi-illumination with contrast enhancement by 5% fluorescein isothiocyanate (FITC)-labeled dextran 150,000. Scale bar: 50 μm. (B) Complete occlusion time of postcapillary and collecting venules upon photochemically induced thrombus formation in dorsal skinfold chambers of heparin-treated (upper black bar, n = 3), fascaplysin-treated (lower black bar, n = 6), and vehicle-treated BALB/c mice (upper white bar, saline, n = 3 and lower white bar, DMSO, n = 6), as assessed by intravital fluorescence microscopy. Mean ± SEM. * p < 0.05 vs. vehicle. (C) Tail-vein bleeding time of heparin-treated (upper black bar, n = 3), fascaplysin-treated (lower black bar, n = 6), and vehicle-treated BALB/c mice (upper white bar, saline, n = 3 and lower white bar, DMSO, n = 6). Mean ± SEM. * p < 0.05 vs. vehicle.
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marinedrugs-13-06774-f006: Effect of fascaplysin and heparin on thrombus formation and tail-vein bleeding time. (A) Intravital fluorescent microscopic images of a postcapillary venule in the dorsal skinfold chamber of a vehicle-treated BALB/c mouse before (baseline) and after photochemically induced thrombus formation (asterisk). Blue-light epi-illumination with contrast enhancement by 5% fluorescein isothiocyanate (FITC)-labeled dextran 150,000. Scale bar: 50 μm. (B) Complete occlusion time of postcapillary and collecting venules upon photochemically induced thrombus formation in dorsal skinfold chambers of heparin-treated (upper black bar, n = 3), fascaplysin-treated (lower black bar, n = 6), and vehicle-treated BALB/c mice (upper white bar, saline, n = 3 and lower white bar, DMSO, n = 6), as assessed by intravital fluorescence microscopy. Mean ± SEM. * p < 0.05 vs. vehicle. (C) Tail-vein bleeding time of heparin-treated (upper black bar, n = 3), fascaplysin-treated (lower black bar, n = 6), and vehicle-treated BALB/c mice (upper white bar, saline, n = 3 and lower white bar, DMSO, n = 6). Mean ± SEM. * p < 0.05 vs. vehicle.
Mentions: Finally, we tested the anti-thrombotic activity of fascaplysin and heparin (positive control) in the dorsal skinfold chamber model of photochemically induced thrombus formation [31]. For this purpose, we analyzed postcapillary and collecting venules with a diameter of 15–25 μm, which exhibited comparable centerline red blood cell (RBC) velocities and wall shear rates (Table 1). As expected, heparin effectively increased complete vessel occlusion time and tail-vein bleeding time (Figure 6B,C), indicating that the present experimental setting is suitable to measure anti-thrombotic effects of test compounds. In line with our in vitro results, we detected a significantly prolonged complete vessel occlusion time within dorsal skinfold chambers of fascaplysin-treated mice when compared to vehicle-treated controls (Figure 6A,B). Moreover, treatment with fascaplysin also increased the tail-vein bleeding time (Figure 6C).

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