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Dok-1 negatively regulates platelet integrin ?IIb?3 outside-in signalling and inhibits thrombosis in mice.

ABSTRACT: Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin ?3, but the underlying effects of Dok-1 on ?IIb?3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in ?IIb?3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of ?IIb?3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect "inside-out" platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p

SUBMITTER: Niki M

PROVIDER: S-EPMC4854776 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Dok-1 negatively regulates platelet integrin αIIbβ3 outside-in signalling and inhibits thrombosis in mice.

Niki Masaru M Nayak Manasa K MK Jin Hong H Bhasin Neha N Plow Edward F EF Pandolfi Pier Paolo PP Rothman Paul B PB Chauhan Anil K AK Lentz Steven R SR

Thrombosis and haemostasis 20160121 5

Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin β3, but the underlying effects of Dok-1 on αIIbβ3 signalling, platelet activation and thrombosis remain to be elucidated. In the present stud ...[more]

PMID: 26790499

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Project description: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.

Project description:Essentials peroxisome proliferator-activated receptor γ (PPARγ) agonists inhibit platelet function. PPARγ agonists negatively regulate outside-in signaling via integrin αIIbβ3. PPARγ agonists disrupt the interaction of Gα13 with integrin β3. This is attributed to an upregulation of protein kinase A activity.SummaryBackground Agonists for the peroxisome proliferator-activated receptor (PPARγ) have been shown to have inhibitory effects on platelet activity following stimulation by GPVI and GPCR agonists. Objectives Profound effects on thrombus formation led us to suspect a role for PPARγ agonists in the regulation of integrin αIIbβ3 mediated signaling. Both GPVI and GPCR signaling pathways lead to αIIbβ3 activation, and signaling through αIIbβ3 plays a critical role in platelet function and normal hemostasis. Methods The effects of PPARγ agonists on the regulation of αIIbβ3 outside-in signaling was determined by monitoring the ability of platelets to adhere and spread on fibrinogen and undergo clot retraction. Effects on signaling components downstream of αIIbβ3 activation were also determined following adhesion to fibrinogen by Western blotting. Results Treatment of platelets with PPARγ agonists inhibited platelet adhesion and spreading on fibrinogen and diminished clot retraction. A reduction in phosphorylation of several components of αIIbβ3 signaling, including the integrin β3 subunit, Syk, PLCγ2, focal adhesion kinase (FAK) and Akt, was also observed as a result of reduced interaction of the integrin β3 subunit with Gα13. Studies of VASP phosphorylation revealed that this was because of an increase in PKA activity following treatment with PPARγ receptor agonists. Conclusions This study provides further evidence for antiplatelet actions of PPARγ agonists, identifies a negative regulatory role for PPARγ agonists in the control of integrin αIIbβ3 outside-in signaling, and provides a molecular basis by which the PPARγ agonists negatively regulate platelet activation and thrombus formation.

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Dok-1 negatively regulates platelet integrin ?IIb?3 outside-in signalling and inhibits thrombosis in mice.

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Dok-1 negatively regulates platelet integrin αIIbβ3 outside-in signalling and inhibits thrombosis in mice.

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