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An ?IIb?3- and phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis.

ABSTRACT: Blood platelets are required for normal wound healing, but they are also involved in thrombotic diseases, which are usually managed with anticoagulant drugs. Here, using genetic engineering, we coupled the disintegrin protein echistatin, which specifically binds to the platelet integrin ?IIb?3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin-annexin V fusion protein (r-EchAV). Lipid binding and plasma coagulation studies revealed that r-EchAV dose-dependently binds PS and delays plasma clotting time. Moreover, r-EchAV inhibited ADP-induced platelet aggregation in a dose-dependent manner and exhibited potent antiplatelet aggregation effects. r-EchAV significantly prolonged activated partial thromboplastin time, suggesting that it primarily affects the in vivo coagulation pathway. Flow cytometry results indicated that r-EchAV could effectively bind to the platelet ?IIb?3 receptor, indicating that r-EchAV retains echistatin's receptor-recognition region. In vivo experiments in mice disclosed that r-EchAV significantly prolongs bleeding time, indicating a significant anticoagulant effect in vivo resulting from the joint binding of r-EchAV to both PS and the ?IIb?3 receptor. We also report optimization of the r-EchAV production steps and its purification for high purity and yield. Our findings indicate that r-EchAV retains the active structural regions of echistatin and annexin V and that the whole molecule exhibits multitarget-binding ability arising from the dual functions of echistatin and annexin V. Therefore, r-EchAV represents a new class of anticoagulant that specifically targets the anionic membrane-associated coagulation enzyme complexes at thrombogenesis sites and may be a potentially useful antithrombotic agent.

SUBMITTER: Jing J

PROVIDER: S-EPMC6497940 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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An αIIbβ3- and phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis.

Jing Jian J Sun Yanna Y

The Journal of biological chemistry 20190225 17

Blood platelets are required for normal wound healing, but they are also involved in thrombotic diseases, which are usually managed with anticoagulant drugs. Here, using genetic engineering, we coupled the disintegrin protein echistatin, which specifically binds to the platelet integrin αIIbβ3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin-annexin V fusion pr ...[more]

PMID: 30803987

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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.

Project description:Integrin alpha(IIb)beta(3) plays a pivotal role in hemostasis and thrombosis by mediating adhesive interactions of platelets. Binding of alpha(IIb)beta(3) to its physiological ligands, immobilized fibrinogen and fibrin, induces outside-in signaling in platelets, leading to their adhesion and spreading even without prior stimulation by agonists. Implicit in these phenomena is a requirement for the linkage between integrins' cytoplasmic tails and intracellular proteins. However, the nature of the initiating signal has not been established. In this study, we examined whether binding of alpha(IIb)beta(3) to immobilized fibrin(ogen), per se, triggers interaction of the integrin with cytoplasmic proteins. Using the integrin-binding skelemin fragment as a marker of exposure of residues involved in the clasp between alpha(IIb) and beta(3) cytoplasmic tails, we showed that its binding site in the membrane-proximal beta(3) 715-730 segment is cryptic and becomes exposed as a result of binding of isolated alpha(IIb)beta(3) to immobilized ligands. Furthermore, the skelemin-like protein present in platelets and CHO cells does not associate with alpha(IIb)beta(3) in resting platelets or suspended alpha(IIb)beta(3)-expressing CHO cells but is recruited to integrin during cell adhesion. In addition, not only beta(3) but also the membrane-proximal 989-1000 segment of the alpha(IIb) cytoplasmic tail binds the skelemin fragment. Finally, the same residues, alpha(IIb) Val(990), alpha(IIb) Arg(995), and beta(3) His(722), involved in the formation of the clasp between the tails are also required for skelemin binding. These studies suggest that ligation of alpha(IIb)beta(3) by immobilized ligands during platelet adhesion induces a transmembrane conformation change in the integrin, resulting in unclasping of the complex between the membrane-proximal parts of cytoplasmic tails, thereby unmasking residues involved in binding the skelemin-like protein. Thus, the junction between alpha(IIb) and beta(3) cytoplasmic tails may contain the critical structural information for the initiation of outside-in signaling.

Dataset Information

An ?IIb?3- and phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis.

Publications

An α<sub>IIb</sub>β<sub>3</sub>- and phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis.

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