Simplagrin, a Platelet Aggregation Inhibitor from <i>Simulium nigrimanum</i> Salivary Glands Specifically Binds to the Von Willebrand Factor Receptor in Collagen and Inhibits Carotid Thrombus Formation <i>In Vivo</i>

<div><p>Background</p><p>Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (<i>Simulium nigrimanum</i>), named Simplagrin.</p><p>Methods and Findings</p><p>Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2–15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα₂β₁ interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (K_D 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation <i>in vivo</i> without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease.</p><p>Conclusion</p><p>Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods.</p></div

Simplagrin blocks von Willebrand Factor (vWF) interaction to collagen but not GPVI to collagen.

<p>(A) In solution competition using surface plasmon resonance (SPR) shows that Simplagrin inhibits interaction of collagen and RGQOGVMGF peptide on immobilized vWF. Collagen type I (0.1 µM) or RGQOGVMGF peptide (1 µM) in the presence or absence of Simplagrin (0.2 µM) were flowed over immobilized vWF. No detectable Simplagrin-vWF binding was observed. (B) Preincubation Simplagrin with saturating concentrations of RGQOGVMGF peptide abrogates Simplagrin-collagen interaction in SPR experiments. (C) Solid-phase assay showing that Simplagrin blocks, in a dose-response manner, collagen vWF interaction. (D) Simplagrin partially blocks GPVI-collagen interaction. SPR in solution competition shows that preincubation of collagen or CRP with Simplagrin at 1∶20 or 1∶40 molar ratios only reduces the response binding of collagen to GPVI approximately 60%; however, Simplagrin fails to block CRP-GPVI interaction. This can be explained by steric hindrance of Simplagrin binding to RGQOGVMGF sequence in collagen. (E) Control experiment showing that Simplagrin does not affect convulxin-GPVI interaction. All SPR experiments were carried out in triplicate.</p

Surface plasmon resonance analysis of Simplagrin-RGQOGVMGF peptide.

<p>Kinetic and binding constants were calculated using a 1:1 interaction binding model. Experiments were carried out in triplicate.</p

Binding constant of Simplagrin with different types of collagen.

<p>The steady-state affinity constant was calculated using an equilibrium binding model.</p

Affinity values for Simplagrin with different collagen types.

<p>(A-F) Soluble human collagen type I, III, IV, V, VI and rat tail I were flowed over immobilized Simplagrin. Fitting of steady-state responses from Simplagrin collagen interaction measured by surface plasmon resonance. The steady-state signal reached at the end of the analyte injection (240 seconds at 30 µL/minute) was plotted against the analyte concentration and the resulting curve fitted with a Langmuir 1∶1 binding model.</p

Surface plasmon resonance analysis of Simplagrin-collagen interaction.

<p>Kinetic and binding constants were calculated using a 1:1 interaction binding model. Experiments were carried out in triplicate.</p

Simplagrin specifically binds to collagen.

<p>(A) Surface plasmon resonance: initial screening over immobilized Simplagrin, showing it exclusively binds to collagen (human type I, III, IV, V, and type I from rat tail). No detectable binding was observed to (1–8) coagulation factors IIa, Va and Xa, vitronectin, laminin, fibronectin, vWf, and fibrinogen. (B) Solid-phase assay shows that Simplagrin binds to immobilized collagens on 96 well plates. Binding was detected by ELISA using rabbit anti-Simplagrin antibodies. (C) Visualization of Simplagrin collagen interaction. Fluorescent microscopy showing direct binding of FITC labeled Simplagrin on coverslip coated with fibrillar collagen. Collagen coated coverslips were incubated with 1 µM of FITC labeled Simplagrin for 15 minutes and then washed 5 times with PBS before mounting. Collagen fibrils revealed by bright-field and fluorescence shows that it lacks of autofluorescence.</p

Effect of Simplagrin on platelet adhesion to fibrillar collagen under high shear stress.

<p>Simplagrin inhibits platelet collagen interaction under high shear stress in a dose dependent fashion. Anticoagulated whole blood from healthy patients was perfused over immobilized fibrillar collagen for 240 seconds at a shear rate of 1500⁻¹ in the presence of different doses of Simplagrin and immediately perfused with Tyrode's buffer at the same shear rate to remove loosely bound platelets. Coverslips were mounted and analyzed under bright-field microscopy. Representative results of a typical experiment (n = 6).</p

Expression of recombinant Simplagrin and biophysical analysis.

<p>(A) Recombinant Simplagrin was expressed as a secreted protein in HEK293 cells. Supernatant containing Simplagrin was concentrated and loaded onto a Ni⁺²-Hitrap column for affinity purification and further purified to homogeneity by size exclusion chromatography. Inset: Coomassie stained NuPAGE and western blot using rabbit anti-Simplagrin antibodies. (B) Circular dichroism (CD) spectroscopy analysis of Simplagrin shows that it mainly comprises α-helix (59%) followed by unordered/disorganized (29%) secondary structures. Inset shows the calculated percentages of secondary structures determined by CD analysis. (C) Analytical size exclusion chromatography shows that Simplagrin runs at a higher than expected molecular weight. (D) Hydrodynamic property of Simplagrin demonstrates its monomeric, elongated form with a hydrodynamic radius of 5.6 nm. The calculated molecular weight of Simplagrin by dynamic scattering plot was 32 kDa (blue line in the chromatogram). (E) Recombinant Simplagrin is not glycosylated in HEK293 cells. Evaluation of putative glycosylation of recombinant Simplagrin was evaluated using DeGlycoMx kit. Fifteen µg of Simplagrin or Lundep (positive control) were heat denatured and treated with an enzymatic deglycosylase mix. After three hours at 37°C, samples were electrophoresed in a NuPAGE-MES and stained with Coomassie blue. Lane 1: Lundep, 2: Lundep+DeGlycoMx, 3: Simplagrin, 4: Simplagrin+DeGlycoMx, 5: mW standard (SeeBlue Plus2 in kDa).</p

Summarized comparison between Aegyptin and Simplagrin main features.

<p>Summarized comparison between Aegyptin and Simplagrin main features.</p