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Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

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ABSTRACT

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

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Effect of annonacinone and tiplaxtinin in thromboelastography on a plasma pool at 50 μM.Addition of tPA in plasma (closed square) resulted in quick lysis of the clot, further addition of PAI-1 resulted in complete inhibition of tPA fibrinolytic effect (closed dot). Addition of annonacinone at 50 μM (closed triangle) and tiplaxtinin at 50 μM (closed diamond) inhibited PAI-1 antifibrinolytic effect and reduced clot lysis time.
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f1: Effect of annonacinone and tiplaxtinin in thromboelastography on a plasma pool at 50 μM.Addition of tPA in plasma (closed square) resulted in quick lysis of the clot, further addition of PAI-1 resulted in complete inhibition of tPA fibrinolytic effect (closed dot). Addition of annonacinone at 50 μM (closed triangle) and tiplaxtinin at 50 μM (closed diamond) inhibited PAI-1 antifibrinolytic effect and reduced clot lysis time.

Mentions: After demonstrating annonacinone activity in vitro, its effect in a plasma global coagulation assay, thromboelastography (TEG), was investigated. The most relevant parameters to study the effect of PAI-1 inhibition in TEG were the LY30 and the LY60, which measure the percentage of clot lysis 30 minutes and 60 minutes after the maximal amplitude (MA). Experiments conditions were set as addition of tPA in plasma resulted in quick lysis of the clot (LY60 = 96.8%), whereas further addition of PAI-1 completely inhibited the fibrinolytic effect of tPA (LY60 = 0%). Pre-incubation of PAI-1 with tiplaxtinin and annonacinone resulted in shortened lysis time. At 50 μM, LY60 was shortened by 50% in presence of tiplaxtinin while complete inhibition (LY60 = 94%) of the PAI-1 effect was observed with annonacinone (Fig. 1). No significant effects were observed on the other parameters (clotting time R, clot formation time K, angle, maximal amplitude MA) (see Supplementary Table S1). The LY60 values of three independent experiments were plotted against annonacinone concentration to calculate the IC50, 2.3 ± 1 μM.


Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1
Effect of annonacinone and tiplaxtinin in thromboelastography on a plasma pool at 50 μM.Addition of tPA in plasma (closed square) resulted in quick lysis of the clot, further addition of PAI-1 resulted in complete inhibition of tPA fibrinolytic effect (closed dot). Addition of annonacinone at 50 μM (closed triangle) and tiplaxtinin at 50 μM (closed diamond) inhibited PAI-1 antifibrinolytic effect and reduced clot lysis time.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120274&req=5

f1: Effect of annonacinone and tiplaxtinin in thromboelastography on a plasma pool at 50 μM.Addition of tPA in plasma (closed square) resulted in quick lysis of the clot, further addition of PAI-1 resulted in complete inhibition of tPA fibrinolytic effect (closed dot). Addition of annonacinone at 50 μM (closed triangle) and tiplaxtinin at 50 μM (closed diamond) inhibited PAI-1 antifibrinolytic effect and reduced clot lysis time.
Mentions: After demonstrating annonacinone activity in vitro, its effect in a plasma global coagulation assay, thromboelastography (TEG), was investigated. The most relevant parameters to study the effect of PAI-1 inhibition in TEG were the LY30 and the LY60, which measure the percentage of clot lysis 30 minutes and 60 minutes after the maximal amplitude (MA). Experiments conditions were set as addition of tPA in plasma resulted in quick lysis of the clot (LY60 = 96.8%), whereas further addition of PAI-1 completely inhibited the fibrinolytic effect of tPA (LY60 = 0%). Pre-incubation of PAI-1 with tiplaxtinin and annonacinone resulted in shortened lysis time. At 50 μM, LY60 was shortened by 50% in presence of tiplaxtinin while complete inhibition (LY60 = 94%) of the PAI-1 effect was observed with annonacinone (Fig. 1). No significant effects were observed on the other parameters (clotting time R, clot formation time K, angle, maximal amplitude MA) (see Supplementary Table S1). The LY60 values of three independent experiments were plotted against annonacinone concentration to calculate the IC50, 2.3 ± 1 μM.

View Article: PubMed Central - PubMed

ABSTRACT

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

No MeSH data available.


Related in: MedlinePlus