Figure 1.
In silico analysis of Simplagrin.
Transcriptome analysis of female salivary glands from the black fly S. nigrimanum revealed the presence of an abundant transcript distantly related to Aegyptin, a salivary collagen binding protein from Aedes aegypti. (A) Amino acid alignment of mature Simplagrin with Aegyptin reveals low overall identity (25%) between both proteins. No putative conserved domain was found. (B) Three-dimensional structure prediction of Simplagrin showing a ribbon diagram of the model generated using PyMOL. Coordinates were generated automatically by I-TASSER software. (C) Surface map of Simplagrin generated by PyMOL APBS tools. Electrostatic potential surfaces of the model showing the positively (blue) and negatively (red) charged surfaces.
Figure 2.
Expression of recombinant Simplagrin and biophysical analysis.
(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).
Figure 3.
Simplagrin specifically binds to collagen.
(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.
Figure 4.
Kinetic and thermodynamic analysis of Simplagrin collagen interaction.
(A) and (B) Simplagrin at different concentrations was flowed over immobilized collagen for 180 seconds at 30 µL/mL and the collagen Simplagrin dissociation monitored for 600 seconds. The response data were fitted to a 1∶1 interaction model using global analysis. Simplagrin displays high affinity for collagen type I and III. (C) and (D) Thermodynamic parameters of Simplagrin collagen (I and III) were obtained from independent kinetic experiments using surface plasmon resonance (SPR). ΔH and ΔS were obtained using the van't Hoff equation. All SPR experiments were carried out in triplicate. Results are representative of typical sensograms. Molar concentrations of collagen were calculated assuming an MW of 250 kDa.
Figure 5.
Affinity values for Simplagrin with different collagen types.
(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.
Table 1.
Surface plasmon resonance analysis of Simplagrin-collagen interaction.
Table 2.
Binding constant of Simplagrin with different types of collagen.
Figure 6.
Simplagrin does not induce any significant conformational change upon binding to collagen type I.
To verify whether any major conformational change occurs upon collagen Simplagrin interaction, a circular dichroism (CD) spectroscopy of the complex was analyzed. (A) Collagen with or without equal molar concentration of Simplagrin showing no significant conformational change in the 1∶1 complex. (B) CD spectrum of collagen type I in equimolar concentration of Aegyptin showing the structural changes of the collagen molecules, resembling the CD spectrum of heat denatured soluble collagen type I. The CD analysis suggests that a conformational change of the collagen molecule, most likely due to reduction in poly proline II structure, results in a significant decrease of ellipticity of the collagen molecule. This unwinding of collagen may result in loss of collagen interaction with its physiological ligands. All experiments were carried out at 25°C in TBS with 6 µM concentration of collagen and Simplagrin or Aegyptin. Molar concentration of collagen was calculated assuming an average MW of 250 kDa. Sum represents the mathematic sum of measured CD spectra of individual molecules.
Table 3.
Thermodynamic analysis of Simplagrin-collagen interaction measured by surface plasmon resonance.
Figure 7.
Effect of Simplagrin on platelet aggregation.
(A) Human platelet rich plasma from healthy donors was incubated with Simplagrin at different concentrations for two minutes followed by addition of platelet agonists as described under Methods. Platelet aggregation was estimated by turbidimetry under stirring conditions at 37°C. No effect on collagen induced platelet aggregation was observed at 10 µg/mL of collagen or 5 µg/mL of CRP. (B) Simplagrin (1 µM) shows no inhibitory effect in platelet aggregation induced by other agonists. PMA, phorbol 12-myristate 13-acetate (0.5 µM), ADP (10 µM), convulxin (100 pM), thrombin receptor activating peptide (TRAP) (5 µM), U46619 thromboxane A2 analog (0.7 µM), arachidonic acid (Ara acid) (1.5 mM), epinephrine (50 µM), ristocetin (1 mg/mL), and thrombin (0.1 U/mL).
Figure 8.
Simplagrin binds to RGQOGVMGF, the von Willebrand Factor (vWF) binding site on collagen.
(A) Surface plasmon resonance binding analysis of immobilized Simplagrin with CRP (cross-linked GPO10), vWFpep (cross linked RGQOGVMGF), Iα2β1pep (cross-linked GFOGER) and Col-I (collagen type I). (B) Solid phase binding assay shows that Simplagrin binds in a dose response manner to wells coated with RGQOGVMGF peptides. Binding was detected using rabbit anti-Simplagrin antibodies. (C) Kinetic analysis showing that Simplagrin displays high affinity (KD 11.1±0.59 nM) for RGQOGVMGF peptide. Analyte concentration ranging from 1–500 nM of RGQOGVMGF were flowed over immobilized Simplagrin at 30 µL/minute for 180 seconds, and the complex dissociation was monitored for 600 seconds before regeneration of the sensor surface. A global 1∶1 reaction model was used to calculate kinetic parameters.
Table 4.
Surface plasmon resonance analysis of Simplagrin-RGQOGVMGF peptide.
Figure 9.
Simplagrin blocks von Willebrand Factor (vWF) interaction to collagen but not GPVI to collagen.
(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.
Figure 10.
Effect of Simplagrin on platelet adhesion to fibrillar collagen under high shear stress.
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).
Figure 11.
Effects of Simplagrin on thrombosis and bleeding.
(A) Simplagrin prevents thrombus formation in vivo. Photochemical induced carotid artery injury: female mice were treated with PBS (control) and two different doses (20 and 100 µg/kg) of Simplagrin 15 minutes before thrombosis was induced, and blood flow was measured as described in Methods. Inset: Hematoxylin and eosin stained cross-sections of injured carotid artery from mice treated with PBS and Simplagrin (100 µg/kg). Arrows show the collagen in the arteries. (B) Simplagrin does not increase bleeding in tail transection assay. Mice (n = 5) were intravenously treated with PBS (control) or Simplagrin (20 and 100 µg/kg). After 15 minutes, the distal 2 mm of the tail was surgically removed and blood loss monitored for 30 minutes. Mean and SEM are shown. P<0.05 was considered statistically significant.
Table 5.
Summarized comparison between Aegyptin and Simplagrin main features.