Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin alpha2beta1, and von Willebrand factor

Abstract

Blood-sucking arthropods have evolved a number of inhibitors of platelet aggregation and blood coagulation. In this study we have molecularly and functionally characterized aegyptin, a member of the family of 30-kDa salivary allergens from Aedes aegypti, whose function remained elusive thus far. Aegyptin displays a unique sequence characterized by glycine, glutamic acid, and aspartic acid repeats and was shown to specifically block collagen-induced human platelet aggregation and granule secretion. Plasmon resonance experiments demonstrate that aegyptin binds to collagen types I-V (K(d) approximately 1 nm) but does not interact with vitronectin, fibronectin, laminin, fibrinogen, and von Willebrand factor (vWf). In addition, aegyptin attenuates platelet adhesion to soluble or fibrillar collagen. Furthermore, aegyptin inhibits vWf interaction with collagen type III under static conditions and completely blocks platelet adhesion to collagen under flow conditions at high shear rates. Notably, aegyptin prevents collagen but not convulxin binding to recombinant glycoprotein VI. These findings suggest that aegyptin recognizes specific binding sites for glycoprotein VI, integrin alpha2beta1, and vWf, thereby preventing collagen interaction with its three major ligands. Aegyptin is a novel tool to study collagen-platelet interaction and a prototype for development of molecules with antithrombotic properties.

Fig. 1

Molecular characterization of aegyptin and identification in the Ae. aegypti salivary gland. (A) Schematic representation of aegyptin with typical acidic N-terminus, (GEEDA) repeats, and basic carboxyterminal depicted. (B) Purification of recombinant aegyptin after Ni²⁺ agarose and gel-filtration columns. Inset: NU-PAGE of purified aegyptin under denaturating conditions. (C) Plasmon resonance experiments: aegyptin interacts with collagen (I-III) but not with vitronectin, laminin, fibronectin, vWf, and fibrinogen. (D) Gel filtration of salivary gland homogenate (—) and identification of the active fraction that binds to collagen (–•–). The elution pattern of recombinant aegyptin is superimposed (–•–). (E) Anion-exchange chromatography of the active fraction obtained in (D) and identification of collagen-binding activity (–•–). The elution pattern of recombinant aegyptin is superimposed (–•–). Inset: Western blot for detection of aegyptin in the salivary gland using polyclonal antiaegyptin antibodies.

Fig. 2

Aegyptin specifically inhibits human platelet aggregation and granule secretion induced by collagen. (A) Human platelet-rich plasma (2×10⁵/ml) was incubated with aegyptin (in nM; a, 0; b, 30; c, 60; d, 120) for 1 minute followed by addition of platelet agonists as indicated. Platelet aggregation was estimated by turbidimetry under test-tube stirring conditions. Washed human platelets were used when thrombin was used as an agonist. (B) Dose-response inhibition of collagen-induced platelet aggregation and ATP release by aegyptin. The tracings represent a typical experiment (n = 6).

Fig. 3

Aegyptin binds to collagen I and III. Typical sensorgrams of aegyptin interaction with collagen type I (A) or type III (B). Different concentrations of recombinant aegyptin (in nM: a, 5; b, 2.5; c, 1.25; d, 0.625; and e, 0.31) were injected over immobilized collagen for 180 s. Dissociation of aegyptin-collagen complex was monitored for 1800 s, and a global two-state binding model was used to calculate kinetic parameters. (C) Ae. aegypti saliva (0.07 μg/ml) was injected over immobilized collage type I (a) and III (b) for 120 s at a flow rate of 20 μl/minute. Sensorgrams are representative of triplicate experiments.

Fig. 4

Aegyptin inhibits collagen, but not convulxin, interaction with GPVI. (A) Collagen type I was injected at different concentrations (in μg/ml: a, 50; b, 25, c, 12.5; d, 6.25; and e, 3.175) over immobilized GPVI. (B) Collagen type I was incubated with buffer (sensorgram a) or 500 nM aegyptin (sensorgrams c–g) at the following concentrations (in μg/ml: a and c, 50; d, 25, e, 12.5; f, 6.25; and g, 3.175). The mixtures were injected over immobilized GPVI. Sensorgram b shows that aegyptin at 500 nM does not bind to immobilized GPVI. (C) Convulxin was injected at different concentrations (a, 10 nM; b, 5 nM and c, 2.5 nM) over immobilized GPVI. (D) Convulxin (a, 10 nM; b, 5 nM and c, 2.5 nM) was saturated with 500 nM of aegyptin and the mixture injected over immobilized GPVI (n = 3).

Fig. 5

Aegyptin prevents platelet adhesion to fibrillar and soluble collagen under static conditions. Washed human platelets (2 × 10⁵/ml) incubated with (A) fibrillar or (C) soluble collagen for 45 minutes in presence of aegyptin at various concentrations: a, 0 μM; b, 0.1 μM; c, 0.3 μM; d, 1 μM; and e, 3 μM. In f, coverslips were coated with denatured BSA in absence of collagen. (B) and (D) are dose-responses curves for aegyptin-mediated inhibition of platelet adhesion to fibrillar or soluble collagen, respectively. Typical experiments are shown (n = 3).

Fig. 6

Aegyptin inhibits interaction of vWf with collagen under static and flow conditions. (A) Inhibition of vWf binding to soluble collagen III was estimated by ELISA in the presence of indicated concentrations of aegyptin. (B) Anticoagulated whole blood was perfused over immobilized fibrillar collagen for 240 s at a shear rate of 1500 s^-1 in the presence of a, 0; b, 0.1; c, 0.3; and d, 1 μM of aegyptin. (C) Dose-response curve for aegyptin-mediated inhibition of platelet adhesion to collagen under flow conditions. Typical experiments are shown (n = 3).