A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis

Abstract

A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbβ3 and endothelial cell αVβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 μM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.

Figure 1. Purification of Tablysin-15 from the horsefly salivary gland extract (SGE)

(A) SGE aliquot of 0.41 g was dissolved in 10 mL 0.1 M PBS, pH 6.0, and then was applied to a Sephadex G-75 (Superfine; Amersham Biosciences, Arlington Heights, IL) 2.6 × 100-cm gel filtration column equilibrated with 0.1 M PBS. Elution was performed with the same buffer, collecting fractions of 3.0 mL. The absorbance of the eluate was monitored at 280 nm. (B) Fraction 3 from Figure 1A was subjected to AKTA Mono S cationic exchange equilibrated with 0.02 M PBS, pH 6.0. The elution was performed at a flow rate of 1 ml/min with the indicated NaCl gradient. Inset in Figure 1B: SDS-PAGE analysis of fraction containing platelet-inhibitory properties. R, reduced; UR, non-reduced; M, molecular weight marker.

Figure 2. cDNA encoding Tablysin-15 and deduced amino acid (aa) sequence

(A) The aa sequences of peptide fragments determined by Edman degradation are underlined. The predicted signal peptide is italic. *, stop codon. (B) Comparison of the N-terminal partial aa sequence of Tablysin-15 with other disintegrins from arthropods saliva and snakes venoms. RGD or KGD sequences are bold. (C) SDS-PAGE analysis of recombinant Tablysin-15. Lane1: The lysate of inclusion bodies of recombinant Tablysin-15 were solubilized in 20 mM Tris–HCl, pH 7.9 with 6 M urea. Lane 2: The elute of recombinant Tablysin-15 inclusion bodies lysate from the Ni-NTA resin. Lane 3: Purified recombinant Tablysin-15 after AKTA Mono S chromatography. Lane 4: molecular weight marker. All these samples were subjected to reduced SDS-PAGE.

Figure 3. Interactions of Tablysin-15 and platelets

(A) Tablysin-15 inhibits human platelet aggregation induced by collagen, ADP and convulxin. Human platelet-rich plasma (2 × 10⁵/µl) was incubated with Tablysin-15 (tracings a, 0 nM; b, 85 nM; c, 670 nM) for 1 min followed by addition of platelet agonists as indicated. Platelet aggregation was estimated by turbidimetry under test tube stirring conditions. The tracings represent a typical experiment. (B) Tablysin-15 inhibits thrombus formation under flow conditions. Anticoagulated (PPACK) whole blood was perfused over immobilized fibrillar collagen for 240 s at a shear rate of 1500 s⁻¹ in the presence of 0 (panel b), 0.3 (panel c), and 3.0 µM (panel d) of Tablysin-15; panel a is a collagen-coated slides without perfused blood. (C) Dose-response curve for Tablysin-15 inhibition of thrombus formation under flow conditions. (D) Tablysin-15 (0–10 µM) inhibit platelet adhesion to fibronectin, human fibrinogen, without significant effects on adhesion to soluble or fibrillar collagen. EDTA (20 mM) and abciximab (20 µg/mL) were used as controls. Experiments were repeated 3–4 times.

Figure 4. Interactions of Tablysin-15 and platelet integrin αIIbβ3

(A) Platelets adhere to Tablysin-15 coated in a 96-well plate with concentration range from 0.01 µg/well to 3 µg/well (n = 5). (B) Different concentrations of abciximab (0–20 µg/mL) markedly inhibit platelet adhesion to 2 µg of Tablysin-15-coated wells. Controls included EDTA and mouse isotype IgG. (C) Solid-phase assays shows integrin αIIbβ3 binding to immobilized Tablysin-15 and fibrinogen but not to SVEP-E2 (E. coli-expressed salivary protein). Purified human platelet αIIbβ3 was added to each well followed by detection with mouse anti-human integrin β3 antibody. BSA was used as negative control. All experiments were performed in triplicates.

Figure 5. Platelet α_IIbβ₃ receptors are occupied by Tablysin-15 analyzed by flow cytometry

(A) Native Tablysin-15 occupies platelet α_IIbβ₃ receptor; (B) Recombinant Tablysin-15 occupies platelet α_IIbβ₃ receptor in a dose–dependent manner (n = 5). The values for numbers of free platelet α_IIbβ₃ treated by Tablysin-15 are significant different from the value for the control (*P < 0.05 and **p < 0.01).

Figure 6. Anti-angiogenesis properties of Tablysin-15

(A) Tablysin-15 was incubated with MVEC and the mixture added to vitronectin-, fibronectin-, or soluble collagen-coated wells. (B) Tablysin-15 was added to MVEC in the presence of FBS and FGF and proliferation was estimated 72 hrs after with MTT. (C) Tube formation was estimated as described in Methods using growth-factor reduced matrigel. Experiments were performed in quadruplicates or quintuplicates.

Figure 7. Antithrombotic activities of Tablysin-15 in vivo

(A) Antithrombotic effects of native and recombinant Tablysin-15, or urokinase in the rat arteriovenous shunt thrombosis model. Bleeding time induced by intravenous injection of Tablysin-15 was evaluated in dose- (B) and time-dependent manners (C). Experiments were performed as described in NT, native Tablysin-15; RT, recombinant Tablysin-15; UK, urokinase. Methods (*, P < 0.05; **, P < 0.01).