Anti-Thrombotic Activity of 3-Deoxysappanchalcone via Inhibiting Platelet Aggregation and Thrombin (FIIa)/Activated Factor X (FXa) Activity

Abstract

Naturally occurring plant-based compounds are increasingly being explored for their therapeutic potential in treating a wide range of conditions, particularly those related to vascular health. The compound 3-deoxysappanchalcone (3-DSC), derived from Caesalpinia sappan L., has been proven to exhibit anti-inflammatory, anti-influenza, and anti-allergic properties, though its role in thrombosis and haemostasis remains unexplored. This study aimed to evaluate the anti-thrombotic potential of 3-DSC in both in vitro and in vivo models. The anticoagulant activities of 3-DSC were assessed using activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin (FIIa) and activated factor X (FXa) activity assays, as well as fibrin polymerization and platelet aggregation tests. Its effects on plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) expression were evaluated in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). The results demonstrated that 3-DSC extended aPTT and PT, suppressed thrombin and FXa activities, reduced their production in HUVECs, inhibited thrombin-induced fibrin polymerization and platelet aggregation, and exerted anticoagulant effects in mice. Furthermore, 3-DSC significantly decreased the PAI-1 to t-PA ratio. These findings suggest that 3-DSC possesses potent anti-thrombotic properties by modulating coagulation pathways and fibrinolysis. Its therapeutic potential warrants further investigation for the development of novel anticoagulant agents.

Keywords: 3-deoxysappanchalcone; Caesalpinia sappan L.; anti-thrombotic activity; fibrinolysis.

Figure 1

Evaluation of effect of 3-DSC on plasma fibrin formation and cellular toxicity. (A) Fibrin polymerization triggered by thrombin was assessed in the presence of various concentrations of 3-DSC, using a catalytic assay detailed in Section 4. Results are reported as V_max values, represented as percentages relative to the untreated control. (B) The impact of 3-DSC on platelet aggregation stimulated by thrombin (3 U/mL) was analyzed in mouse platelets. D = 0.2% DMSO served as the vehicle control. (C) Cell viability in response to 3-DSC treatment was determined via the MTT assay. Data represent the mean±SD of three independent experiments performed in triplicate. * p < 0.01 vs. Th alone.

Figure 2

Effects of 3-DSC on the inhibition and generation of thrombin and factor Xa. (A) Thrombin (Th) activity was assessed in the presence of 3-DSC using a chromogenic substrate-based assay, following procedures outlined in Section 4. (B) Similarly, factor Xa (FXa) inhibition by 3-DSC was evaluated via chromogenic analysis. Argatroban (for (A)) and rivaroxaban (for (B)) served as reference inhibitors. (C) HUVEC monolayers were pre-treated with factor Va (FVa, 100 pM) and FXa (1 nM) for 10 min along with increasing concentrations of 3-DSC. Prothrombin (1 μM) was added, and thrombin formation was measured after 30 min as per established protocols. (D) To assess FXa generation, HUVECs were pre-treated with varying doses of 3-DSC for 10 min, then stimulated with TNF-α (10 ng/mL for 6 h), followed by incubation with FVIIa (10 nM) and FX (175 nM) in the presence or absence of anti-tissue factor (TF) IgG (25 μg/mL), with FXa activity analyzed as described in Section 4. * p < 0.01 vs. 0 (A–C) or TNF-α alone (D).

Figure 3

Effects of 3-DSC on the release of PAI-1 and t-PA. (A) HUVECs were incubated with or without TNF-α (10 ng/mL) in the presence or absence of 3-DSC for 18 h, after which the levels of PAI-1 in the culture supernatants were quantified according to the protocol outlined in Section 4. (B) Under the same experimental conditions, t-PA concentrations in the conditioned media were also measured. (C) The ratio of PAI-1 to t-PA was calculated from the data in panels (A,B) to assess the impact of 3-DSC on fibrinolytic balance in TNF-α-treated HUVECs. * p < 0.01 vs. TNF-α alone or D; n.s., not significant.