Sappanchalcone Attenuates Platelet Activation and Clot Retraction Through Coordinated Modulation of Cyclic Nucleotides and PI3K/MAPK Signaling Pathways.

BACKGROUND: Aberrant platelet activation is a major contributor to thrombotic disorders, prompting interest in naturally derived candidates that can safely regulate platelet function. Sappanchalcone, a chalcone-type flavonoid derived from Caesalpinia sappan L., has diverse biological activities; however, its effects on platelet activation and clot retraction remain unclear. This study evaluated the antiplatelet potential of sappanchalcone and its underlying mechanisms in human platelets. METHODS: Washed human platelets and platelet-rich plasma were used to assess platelet activation and clot retraction, respectively. Samples were pretreated with sappanchalcone (16-128 μM, depending on the assay) and stimulated with collagen or thrombin. Platelet aggregation and cytotoxicity were assessed by light transmission aggregometry and lactate dehydrogenase (LDH) leakage assay. cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and thromboxane B2 (TXB2) levels were quantified using enzyme-linked immunosorbent assay (ELISA) kits, and intracellular Ca2+ mobilization was measured by Fura-2/AM-based fluorescence analysis. Protein phosphorylation was examined by Western blotting, whereas integrin αIIbβ3-fibrinogen binding was analyzed by flow cytometry. Dense granule secretion was evaluated using adenosine triphosphate (ATP) luminescence and serotonin ELISA assays. Thrombin-induced clot retraction was quantified by ImageJ (v1.54k, U.S. National Institutes of Health, Bethesda, MD, USA)-based residual clot area analysis. Data are presented as mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc test (n = 4; p < 0.05). RESULTS: Sappanchalcone concentration-dependently inhibited collagen-induced platelet aggregation, reducing aggregation from 83.0% to 10.7% at 128 μM, with a half-maximal inhibitory concentration (IC50) of 57.16 μM, without cytotoxicity. At 128 μM, sappanchalcone increased cAMP from 7.02 ± 0.85 to 11.96 ± 0.57 pmol/108 platelets and reduced Ca2+ mobilization from 711.97 ± 47.29 to 66.11 ± 6.83 nM. Vasodilator-stimulated phosphoprotein (VASP) Ser157 and Ser239 phosphorylation increased by 2.97- and 2.07-fold, respectively, whereas fibrinogen binding decreased from 74.43 ± 5.67% to 21.90 ± 1.25%. Phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 phosphorylation was reduced to 0.69-, 0.21-, 1.81-, 0.27-, and 0.49-fold, respectively. ATP secretion, serotonin release, TXB2 levels reflecting thromboxane A2 (TXA2) generation, cytosolic phospholipase A2 (cPLA2) phosphorylation, and thrombin-induced clot retraction were also suppressed. CONCLUSIONS: Sappanchalcone limits platelet activation, aggregation, and clot retraction by enhancing cyclic nucleotide-mediated inhibitory signaling and suppressing PI3K/Akt and mitogen-activated protein kinase (MAPK) phosphorylation. These findings provide mechanistic evidence supporting sappanchalcone as a potential naturally derived antiplatelet candidate.