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Heparin modulates the conformation and signaling of platelet integrin ?IIb?3.

ABSTRACT: The glycosaminoglycan heparin has been shown to bind to platelet integrin ?IIb?3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and ?IIb?3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets.We assessed conformational changes in ?IIb?3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express ?IIb?3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the ?3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified ?IIb?3, and of recombinant fragments of ?IIb and ?3, by surface plasmon resonance.Heparin binding results in conformational changes in ?IIb?3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin ?3 cytoplasmic domain, but the presence of heparin increased both ?3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from ?IIb bound heparin, while recombinant ?3 did not bind. This pattern of heparin binding, compared to the crystal structure of ?IIb?3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of ?IIb. Binding of heparin to these clusters may stabilize the transition of ?IIb?3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.

SUBMITTER: Yagi M

PROVIDER: S-EPMC3340540 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Heparin modulates the conformation and signaling of platelet integrin αIIbβ3.

Yagi Mayumi M Murray Jacqueline J Strand Kurt K Blystone Scott S Interlandi Gianluca G Suda Yasuo Y Sobel Michael M

Thrombosis research 20111223 6

<h4>Introduction</h4>The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbβ3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbβ3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets.<h4>Methods</h4>We assessed conformational changes in αIIbβ3 by flow cytometry of platelets exposed to unfractionated heparin. In human ...[more]

PMID: 22197178

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Project description:BackgroundThe prevalence of cardiovascular diseases (CVDs) is increasing at a high rate, and the available treatment options, sometimes, have complications which necessitates the need to develop safer and efficacious approaches. Ethnomedicinal applications reportedly reduce CVD risk. Ulmus parvifolia Jacq. (Ulmaceae) commonly known as Chinese Elm or Lacebark Elm, is native to China, Japan, and Korea. It exhibits anti-inflammatory, antiviral, and anticancer properties, but its anti-platelet properties have not yet been elucidated.PurposeTo investigate the pharmacological anti-platelet and anti-thrombotic effects of U. parvifolia bark extract.Study design and methodsHuman and rat washed platelets were prepared; light transmission aggregometry and scanning electron microscopy was performed to assess platelet aggregation and the change in platelet shape, respectively. Intracellular calcium mobilization, ATP release, and thromboxane-B2 production were also measured. Integrin αIIbβ3 activation was analyzed in terms of fibrinogen binding, fibronectin adhesion, and clot retraction. The expression of MAPK, Src, and PI3K/Akt pathway proteins was examined. Cyclic nucleotide signaling pathway was evaluated via cAMP elevation and VASP phosphorylation. Anti-thrombotic activity of the extract was evaluated in vivo using an arteriovenous shunt rat model, whereas its effect on hemostasis in mice was assessed via bleeding time assay.ResultsU. parvifolia extract significantly inhibited human and rat platelet aggregation in a dose-dependent manner along with inhibition of calcium mobilization, dense granule secretion, and TxB2 production. Integrin αIIbβ3 mediated inside-out and outside-in signaling events, as evidenced by the inhibition of fibrinogen binding, fibronectin adhesion, and clot retraction. The extract significantly reduced phosphorylation of Src, MAPK (ERK, JNK, and p38MAPK), and PI3K/Akt pathway proteins. Cyclic-AMP levels were elevated in U. parvifolia-treated platelets, while PKAαβγ and VASPser157 phosphorylation was enhanced. U. parvifolia reduced thrombus weight in rats and moderately increased bleeding time in mice.ConclusionU. parvifolia modulates platelet responses and inhibit thrombus formation by regulating integrin αIIbβ3 mediated inside-out and outside-in signaling events and cAMP signaling pathway.

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Heparin modulates the conformation and signaling of platelet integrin ?IIb?3.

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Heparin modulates the conformation and signaling of platelet integrin αIIbβ3.

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