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Extract of Seaweed Codium fragile Inhibits Integrin αIIbβ3-Induced Outside-in Signaling and Arterial Thrombosis.

ABSTRACT: Seaweeds are thought to be promising candidates for functional foods and to help prevent thrombotic and related cardiovascular diseases. Codium fragile (Suringer) Hariot has been traditionally used as a culinary ingredient, and it possesses a range of biological activities, including the inhibition of platelet function. However, the mechanism of this inhibition is unclear. The aim of this study was to examine the inhibitory effect of C. fragile in platelet function. The antiplatelet activity of C. fragile on agonist-activated platelet aggregation, granule secretion, calcium mobilization, platelet spreading, and clot retraction was assessed. The phosphorylation of c-Src, Syk, PLCγ2, and several proteins involving in the αIIbβ3 integrin outside-in signaling pathway were also studied in thrombin and CRP-stimulated platelets. The antithrombotic effect was investigated in mice using ferric chloride-induced arterial thrombus formation in vivo. Transection tail bleeding time was used to evaluate whether C. fragile inhibited primary hemostasis. The main components and contents of C. fragile ethanol extract were confirmed by GC-MS analysis. C. fragile significantly impaired agonist-induced platelet aggregation granule secretion, calcium mobilization, platelet spreading, and clot retraction. Biochemical analysis revealed that C. fragile inhibited the agonist-induced activation of c-Src, Syk, and PLCγ2, as well as the phosphorylation of PI3K, AKT, and mitogen-activated protein kinases (MAPKs). The inhibitory effect of C. fragile resulted from an inhibition of platelet αIIbβ3 integrin outside-in signal transduction during cell activation. Oral administration of C. fragile efficiently blocked FeCl₃-induced arterial thrombus formation in vivo without prolonging bleeding time. GC-MS analysis revealed that phytol was the main constituent and the total content of isomers was 160.8 mg/kg. Our results demonstrated that C. fragile suppresses not only the inside-out signaling of αIIbβ3 integrin but also outside-in signal transmission. Therefore, C. fragile could be an effective antiplatelet therapeutic candidate.

SUBMITTER: Kim TI

PROVIDER: S-EPMC8283197 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundBidirectional integrin αIIbβ3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function.ResultsAn antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin αIIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin αIIbβ3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling.ConclusionsThese findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin αIIbβ3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.

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Extract of Seaweed Codium fragile Inhibits Integrin αIIbβ3-Induced Outside-in Signaling and Arterial Thrombosis.

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