Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B
- University of Birmingham, United Kingdom
- Wellcome Trust Sanger Institute, United Kingdom
- University Hospital Würzburg, Germany
- Université de Strasbourg, Institut National de la Santé et de la Recherche Médicale, Etablissement Français du Sang Grand Est, Unité Mixte de Recherche-S 1255, Fédération de Médecine Translationnelle de Strasbourg, France
- Sygnature Discovery Limited, United Kingdom
- University of Helsinki, Helsinki University Hospital, Finland
- Aplagon Oy, Finland
- Helsinki University Hospital Research Institute, Finland
- University of Westminster, United Kingdom
- Alderley Park, United Kingdom
- Dudley Group NHS Foundation Trust, United Kingdom
- Aston University, United Kingdom
Figures
Figure 1 with 3 supplements
Prominent binding of mG6b-B-Fc to the vessel wall.
Immunohistochemistry staining of frozen mouse tissue sections with mG6b-B-Fc or human IgG-Fc fragments (control). Bound protein was visualized using a secondary anti-human-Fc-HRP antibody and DAB …
Figure 1—figure supplement 1
Overview sections of tissues stained with mG6b-B-Fc or negative control.
Overview sections of the tissues depicted in Figure 1. Boxed areas mark the sections shown in Figure 1.
Figure 1—figure supplement 2
Overview sections of tissues stained with mG6b-B-Fc or negative control.
Overview sections of the tissues depicted in Figure 1. Boxed areas mark the sections shown in Figure 1.
Figure 1—figure supplement 3
Pull-down of G6b-B binding partners from vena cava lysates.
Vena cava homogenates were incubated with G6b-B-Fc or Fc control protein as well as protein G sepharose beads to precipitate G6b-B binding partners. Following multiple washing steps, samples were …
Figure 2 with 1 supplement
G6b-B-Fc binds to heparan sulfate side-chains of perlecan.
(A) 96-well plates were coated with the indicated substrates (5 µg/ml) and incubated with mouse G6b-B-Fc (10 µg/ml), human G6b-B-Fc (30 µg/ml) or Fc-control (10 µg/ml). Bound protein was detected …
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Figure 2—source data 1
Source data for graphs shown in Figure 2A–C.
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Figure 2—figure supplement 1
Loss of heparin sulfation impairs interaction with G6b-B.
96-well plates were coated with streptavidin followed by incubation of biotinylated heparin. Binding of the huG6b-B monomer–anti-G6b-B antibody complex (1 µg/ml each) was measured in the presence of …
Figure 3
The heparan sulfate biosynthesis pathway is required for G6b-B binding to HEK293 cells.
(A) Recombinant G6b-B, produced as a monomeric biotinylated protein and conjugated to streptavidin-PE to generate an avid probe, binds to HEL, HEK293 and COLO-320-HSR cells. (B) A genome-wide …
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Figure 3—source data 1
Raw read counts from the screen carried out in HEK293 cells.
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Figure 3—source data 2
MAGeCK output for gene-wise ranking from the screen carried out in HEK293 cells.
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Figure 3—source data 3
Raw read counts from the screen carried out in HEL cells.
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Figure 3—source data 4
MAGeCK output for gene-wise ranking from the screen carried out in HEL cells.
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Figure 4 with 3 supplements
Ribbon representation of the ternary complex of the extracellular domain (ECD) of human G6b-B bound to heparin and the Fab fragment of a G6b-B-specific antibody.
(A) Overview of the structure, with G6b-B colored in magenta and dark green, heparin shown as spheres, and the Fab fragment chains in light green/light blue, respectively. The assembly represents …
Figure 4—figure supplement 1
Mutations in G6b-B abolish heparin binding.
(A) G6b-B model. (i) Ribbon representation showing predicted locations of basic residues K54, K58, R60 and R61 in green. (ii) Superposition of the G6b-B model (dark blue) with Siglec-7 (cyan) in …
Figure 4—figure supplement 2
Representation of the crystal lattice.
(A) Crystal lattice of the ternary complex of G6b-B bound to heparin and the G6b-B-specific Fab fragment. The top and bottom halves of the view are related by a 90° rotation about the horizontal …
Figure 4—figure supplement 3
Unbiased σA-weighted difference density map demonstrating the presence of the O-linked glycosyl groups at Thr73.
https://doi.org/10.7554/eLife.46840.021
Figure 5
Heparin induces G6b-B dimer formation.
Size exclusion chromatography of G6b-B ECD. Protein was either analyzed immediately or incubated at 4°C for 1.5 hr in the presence of dp12 before analysis on a Superdex 75 10/300 GL column. …
Figure 6 with 1 supplement
Electrostatic surface potential of the G6b-B ECD and representation of non-covalent contacts between heparin and G6b.
(A) The G6b-B dimer is shown with a translucent surface colored according to electrostatic surface potential (calculated using CCP4mg). The heparin ligand is shown as a stick model and polar …
Figure 6—figure supplement 1
Unbiased σA-weighted difference density map demonstrating the presence of the heparin ligand.
The electron map has been calculated with phases from a structure model lacking the heparin atoms and with amplitudes (Fo – Fc), contouring the map at 2.8 σ above the mean.
Figure 7
High-affinity interaction between G6b-B and its ligands.
Representative traces of the surface plasmon resonance experiments, results of which are presented in Table 3. (A) Binding of the indicated compound to immobilized dimeric G6b-B in the standard …
Figure 8 with 1 supplement
Heparan sulfate removal of perlecan facilitates platelet adhesion.
The indicated substrates were coated alone or in combination onto wells in 96-well plates (2.5 µg/ml collagen and 10 µg/ml for all other substrates) overnight. Where indicated, wells were treated …
Figure 8—figure supplement 1
Mean surface area of individual adherent platelets.
Mean surface area of the individual platelets analyzed in Figure 8C,D. Each dot represents an individual platelet from a total of (A) five human donors or (B) 5–7 mice/condition/genotype from 2 to 3 …
Figure 9 with 2 supplements
Megakaryocytes come into contact with perlecan in the bone marrow.
(A) Analysis of immunofluorescent images of murine femur sections from WT and Mpig6b–/–mice. Sinusoids were marked using anti-endoglin (CD105) and MKs by anti-GPIX antibodies. Perlecan is abundantly …
Figure 9—figure supplement 1
Overview sections of the bone marrow from WT and Mpig6b–/–mice.
Overview sections of the bone marrow in femora of WT and Mpig6b–/–depicted in Figure 9A are shown.
Figure 9—figure supplement 2
Mpig6b–/–megakaryocytes form clusters.
Megakaryocytes are indicated with a transparent orange overlay and endothelial cells with a transparent blue overlay. *indicates the sinusoidal lumen; MK, megakaryocyte. Bars: 10 µm.
Figure 10
G6b knockout megakaryocytes show enhanced spreading on perlecan.
Adhesion of WT and Mpig6b–/–MKs on perlecan. (i) Mean surface area of MKs was quantified with ImageJ. n = 4–6 mice/condition/genotype from three independent experiments; total cell numbers analyzed …
Figure 11
Effects of G6b-B ligands on platelet aggregation.
Human platelet rich plasma (PRP) was incubated with the indicated compound for 90 s prior to agonist addition. Aggregation traces were recorded on a Chronolog four channel aggregometer. Averaged …
Figure 12
APAC inhibits CLEC-2-mediated degranulation in WT but not Mpig6b KO platelets.
Mouse blood, diluted 1:10 in Tyrode’s buffer was incubated with the indicated compounds (0.05 µM) in the (A) absence or (B) presence of a stimulating CLEC-2 (3 µg/ml) for the indicated time. Samples …
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Figure 12—source data 1
Source data for graphs shown in Figure 12A–C.
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Figure 13 with 1 supplement
APAC induces G6b-B phosphorylation and downstream signaling.
(A) Washed human platelets (5 × 108/ml) were incubated for 90 s with 0.05 µM APAC, 0.7 µM heparin or buffer in the presence of 10 µM integrilin. Where indicated, platelets were additionally …
Figure 13—figure supplement 1
Effects of G6b-B ligands on G6b-B phosphorylation.
(A,B) Washed human platelets (5 × 108/ml) were incubated for 90 s with the indicated compounds in the presence of 10 µM integrillin. Whole cell lysates (WCL) subjected to immunoprecipitation (IP) …
Figure 14 with 1 supplement
Simplified model of glycan-mediated regulation of G6b-B function.
(A) In the absence of any ligand, G6b-B is mainly present in a monomeric state and phosphorylated to a low degree. (B) Small soluble ligands, for example heparin, induce the dimerization of the …
Figure 14—figure supplement 1
Selected structures of proteins with a heparin ligand.
Side-by-side views of Cα traces (ribbon representation) and electrostatic surfaces of proteins bound to heparin or a heparin analog. The subset includes structures in which the heparin ligand …
Author response image 1
Relative numbers of MKs located at the vessel.
https://doi.org/10.7554/eLife.46840.043
Author response image 2
A pentameric version of the G6b-B protein is used.
CBFH refers to the COMP-β-lactamase-FLAG-3XHis tag. Binding assay was performed nine days post transduction with a sgRNA targeting EXTL3 as described before (Sharma et al., Genome Res., 2018). Two …
Author response image 3
Comparison of genome-wide screens performed for G6b-B (top panel) vs a CS-binding protein (bottom panel).
G6B-BLH screened on HEL cells. Note the presence of HS-specific genes (EXTL3, EXT1, EXT2) only for G6B and CHST11 only for the CS-binding protein.
Author response image 4
Binding behaviour of a CS binding protein.
Targeting EXTL3 has no effect on binding whereas targeting SLC35B2 clearly abrogates binding.
Tables
Table 1
List of proteins immunoprecipitated with mG6b-B-Fc from vena cava lysates
https://doi.org/10.7554/eLife.46840.006| Accession number | Name | Peptides | Protein score | Protein score negative control | FE |
|---|---|---|---|---|---|
| E9PZ16 | Basement membrane-specific heparan sulfate proteoglycan core protein (perlecan) | 131 | 607.22 | n.d. | |
| E9QPE7 | Myosin-11 | 103 | 468.02 | 719.71 | 0.7 |
| F8VQJ3 | Laminin subunit gamma-1 | 75 | 434.43 | 9.66 | 45.0 |
| Q5SX39 | Myosin-4 | 80 | 328.62 | 587.14 | 0.6 |
| Q8VDD5 | Myosin-9 | 81 | 318.18 | 513.68 | 0.6 |
| P97927 | Laminin subunit alpha-4 | 56 | 285.20 | n.d. | |
| Q61292 | Laminin subunit beta-2 | 63 | 262.37 | n.d. | |
| B2RWX0 | Myosin, heavy polypeptide 1, skeletal muscle, adult | 61 | 244.66 | 446.14 | 0.5 |
| P02469 | Laminin subunit beta-1 | 57 | 236.87 | n.d. | |
| J3QQ16 | Protein Col6a3 | 61 | 232.99 | 14.76 | 15.8 |
| G3UW82 | MCG140437, isoform CRA_d | 54 | 214.75 | 378.87 | 0.6 |
| B7FAU9 | Filamin, alpha | 58 | 202.67 | 139.51 | 1.5 |
| Q3UHL6 | Putative uncharacterized protein — fibronectin | 48 | 192.76 | n.d. | |
| Q9JKF1 | Ras GTPase-activating-like protein IQGAP1 | 31 | 107.79 | 68.57 | 1.6 |
| M0QWP1 | Agrin | 21 | 84.47 | n.d. | |
| P19096 | Fatty acid synthase | 27 | 74.68 | 23.81 | 3.1 |
| Q61001 | Laminin subunit alpha-5 | 23 | 73.24 | n.d. | |
| E9QPX1 | Collagen alpha-1(XVIII) chain | 16 | 59.16 | n.d. | |
| A2AJY2 | Collagen alpha-1(XV) chain | 14 | 53.53 | n.d. | |
| B7ZNH7 | Collagen alpha-1(XIV) chain | 15 | 43.27 | 3.09 | 14.0 |
| P26039 | Talin-1 | 11 | 42.29 | 29.93 | 1.4 |
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Fold enrichment (FE)=score G6b-B-FC precipitation/score negative control; n.d. = not detectable. Proteins that are prominently present in the negative control (FE < 2) are shown in italic. The protein score was calculated using the SEQUEST HT search algorithm and is the sum of all peptide Xcorr values above the specified score threshold (0.8 + peptide_charge × peptide_relevance_factor where peptide_relevance_factor is a parameter with a default value of 0.4). The full data set, including the mass spectrometry result for the respective band of a G6b-B-FC only sample, is found in Table 1—source data 1–3. A picture of a gel and the bands excised for mass-spectrometric analysis are shown in Figure 1—figure supplement 3.
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Table 1—source data 1
Mass spectrometry results for proteins precipitated from vena cava lysates with mG6b-B-Fc.
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Table 1—source data 2
Mass spectrometry results for proteins precipitated from vena cava lysates with Fc control protein.
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Table 1—source data 3
Mass spectrometry results for the proteins detected at the respective height after loading mG6b-B-Fc only (no vena cava lysate).
- https://doi.org/10.7554/eLife.46840.009
Table 2
Crystallographic data collection and refinement statistics for the G6b-B ECD–dp12–Fab complex.
https://doi.org/10.7554/eLife.46840.022| X-ray diffraction data | |
|---|---|
| Beamline | I03, Diamond Light Source |
| Wavelength (Å) | 0.97624 |
| Space group | C2 |
| Cell parameters (Å) | 183.8, 72.34, 131.0, β = 124.5° |
| Complexes per asymmetric unit | 1 |
| Resolution range (Å) | 65.27–3.13 |
| High resolution shell (Å) | 3.18–3.13 |
| Rmerge (%)* | 17.0 (146.6) |
| Total observations, unique reflections | 74,255/24,543 |
| I/σ(I)* | 4.0 (0.7) |
| Completeness (%)* | 97.2 (98.2) |
| Multiplicity* | 3.0 (3.1) |
| CC1/2*, † | 0.991 (0.348) |
| Refinement | |
| Resolution range | 63.1–3.13 |
| Unique reflections | 24,543 |
| Rcryst, Rfree (%) | 22.6, 26.0 |
| Number of non-H atoms | 7852 |
| RMSD bonds (Å) | 0.01 |
| RMSD angles (°) | 1.18 |
| B-factors | |
| Wilson (Å2) | 77.5 |
| Average overall (Å2) | 84.7 |
| RMSD B-factors (Å2) | 5.737 |
| Ramachandran statistics‡ | |
| Favored regions (%) | 91.2 |
| Allowed regions (%) | 8.3 |
| Disallowed (%) | 0.5 |
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* parentheses refer to the high resolution shell.
† as defined in Karplus and Diederichs (2012).
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‡ calculated using molprobity (Williams et al., 2018).
Table 3
Surface plasmon resonance affinities.
https://doi.org/10.7554/eLife.46840.027| Immobilized G6b-B receptor (standard configuration) | ||||
|---|---|---|---|---|
| Ligand | G6b-B | Kon | Koff | KD (M) |
| Heparin | Monomer | 1.12 ± 0.39×106 | 2.01 ± 0.54×10−3 | 2.00 ± 1.17×10−9 |
| Dimer | 0.60 ± 0.56×106 | 3.16 ± 1.17×10−3 | 7.76 ± 5.30×10−9 | |
| Fractionated HS | Monomer | 1.33 ± 0.01×105 | 9.99 ± 0.16×10−4 | 7.47 ± 0.17×10−9 |
| Dimer | 1.20 ± 0.08×105 | 1.71 ± 1.11×10−3 | 14.0 ± 8.26×10−9 | |
| Perlecan | Monomer | 1.94 ± 1.72×102 | 1.01 ± 0.37×10−4 | 7.32 ± 4.64×10−7 |
| Dimer | 5.79 ± 6.94×103 | 2.28 ± 2.51×10−3 | 4.74 ± 1.34×10−7 | |
| dp12 | Monomer | 0.31 ± 0.27×106 | 2.39 ± 1.79×10−3 | 8.12 ± 1.22×10−9 |
| Dimer | 2.50 ± 2.72×106 | 4.60 ± 5.01×10−3 | 1.84 ± 0.01×10−9 | |
| Immobilized ligand (reversed configuration) | ||||
| Ligand | G6b-B | Kon | Koff | KD (M) |
| Heparin | Monomer | 1.30 ± 0.29×105 | 8.85 ± 0.40×10−2 | 6.99 ± 1.25×10−7 |
| Dimer | 3.28 ± 0.53×105 | 1.73 ± 0.04×10−3 | 5.33 ± 0.75×10−9 | |
| Fractionated HS | Monomer | 9.22 ± 2.67×103 | 6.40 ± 0.33×10−3 | 7.31 ± 2.47×10−7 |
| Dimer | 3.76 ± 4.69×104 | 4.58 ± 6.32×10−4 | 7.70 ± 7.21×10-9 | |
| Perlecan | Monomer | 6.73 ± 3.38×103 | 1.28 ± 0.24×10−3 | 2.28 ± 1.51×10−7 |
| Dimer | 4.90 ± 2.16×104 | 6.78 ± 2.57×10−4 | 1.41 ± 0.09×10−8 | |
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Values are means ± SD from two independent experiments.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Mpig6b–/– | PMID: 23112346 | Dr. Yotis Senis (University of Birmingham and EFS Grand Est, Inserm UMR-S1255) | |
| Genetic reagent (M. musculus) | Mpig6bdiYF/diYF | PMID: 29891536 | Dr. Yotis Senis (University of Birmingham and EFS Grand Est, Inserm UMR-S1255) | |
| Cell line (Cricetulus griseus) | A5 CHO | other | provided by Dr. Ana Kasirer-Friede and Dr. Sanford Shattil (University of California, San Diego) | |
| Antibody | anti-perlecan (rat monoclonal) | Santa Cruz Biotechnologies | clone A7L6; sc-33707; RRID:AB_627714 | (1:100); used for IF staining of bone marrow (BM) |
| Antibody | anti-mouse CD105 (Endoglin) (rat monoclonal) | eBioscience/Thermo Fisher Scientific | #MA5-17943; clone MJ7/18; RRID:AB_2539327 | (1:100); used for IF staining of BM |
| Antibody | anti-GPIX-Alexa488 (rat monoclonal) | other | clone 56F8 | 1.4 µg/ml; used for IF staining of BM, custom made lab reagent |
| Antibody | anti rat IgG Alexa 647 (goat polyclonal) | Invitrogen | #A-21247; RRID:AB_141778 | (1:300); used for IF staining of BM |
| Antibody | anti rat IgG Alexa 546 (goat polyclonal) | Invitrogen | #A-11081; RRID:AB_141738 | (1:300); used for IF staining of BM |
| Antibody | anti-actin (mouse monoclonal) | Sigma-Aldrich | #A4700, clone AC-40; RRID:AB_476730 | (1:1000) |
| Antibody | Anti-α-tubulin (mouse monoclonal) | Sigma-Aldrich | #T6199, clone DM1A; RRID:AB_477583 | (1:1000) |
| Antibody | anti-GAPDH (rabbit monoclonal) | Cell Signaling Technology | #2118, clone: 14C10; RRID:AB_561053 | (1:10) dilution, on 0.05 mg/ml lysates for Wes |
| Antibody | anti-Src p-Tyr418 (rabbit polyclonal) | Sigma-Aldrich | #44660G; RRID:AB_1500523 | (1:10) dilution, on 0.05 mg/ml lysates for Wes |
| Antibody | anti-Shp1 p-Tyr564 (rabbit monoclonal) | Cell Signaling Technology | #8849, clone: D11G5; RRID:AB_11141050 | (1:10) dilution, on 0.2 mg/ml lysates for Wes |
| Antibody | anti-Shp2 p-Tyr542 (rabbit polyclonal) | Cell Signaling Technology | #3751; RRID:AB_330825 | (1:10) dilution, on 0.2 mg/ml lysates for Wes |
| Antibody | anti-Shp2 p-Tyr580 (rabbit polyclonal) | Cell Signaling Technology | #3703; RRID:AB_2174962 | (1:10) dilution, on 0.2 mg/ml lysates for Wes |
| Antibody | anti-Syk p-Tyr525/6 (rabbit polyclonal) | Cell Signaling Technology | #2711; RRID:AB_2197215 | (1:50) dilution, on 0.2 mg/ml lysates for Wes |
| Antibody | anti-SH-PTP1/Shp-1 (rabbit polyclonal) | Santa Cruz | sc-287 (C19); RRID:AB_2173829 | (1:1000) |
| Antibody | anti-SH-PTP2/Shp-2 (rabbit polyclonal) | Santa Cruz | sc-280 (C18); RRID:AB_632401 | (1:1000) |
| Antibody | anti-phosphotyrosine (mouse monoclonal) | Merck-Millipore | 05–321, clone 4G10; RRID:AB_309678 | (1:1000) |
| Antibody | anti-human G6b-B (mouse monoclonal) | other | clone 17–4 | 10 µg/ml, custom-made lab reagent |
| Peptide, recombinant protein | purified human IgG-Fc fragment | Bethyl Laboratories | P80-104 | |
| Peptide, recombinant protein | recombinant Mouse Syndecan-2/CD362 protein, CF | R&D Systems | 6585-SD-050 | |
| Peptide, recombinant protein | recombinanthuman Agrin protein, N-terminal, CF | R&D Systems | 8909-AG-050 | |
| Peptide, recombinant protein | rec. human laminin 111 | Biolamina | LN111-02 | |
| Peptide, recombinant protein | rec. human laminin 411 | Biolamina | LN411-02 | |
| Peptide, recombinant protein | rec. human laminin 421 | Biolamina | LN421-02 | |
| Peptide, recombinant protein | rec. human laminin 511 | Biolamina | LN511-02 | |
| Peptide, recombinant protein | rec. human laminin 521 | Biolamina | LN521-02 | |
| Chemical compound, drug | heparan sulfate proteoglycan | Sigma-Aldrich | H4777 | alternative name: perlecan |
| Chemical compound, drug | heparin | Iduron | HEP001 | https://iduron.co.uk/product/Heparin-1 |
| Chemical compound, drug | heparin oligosaccharide dp4 | Iduron | HO04 | https://iduron.co.uk/product/Heparin-1 |
| Chemical compound, drug | heparin oligosaccharide dp8 | Iduron | HO08 | https://iduron.co.uk/product/Heparin-1 |
| Chemical compound, drug | heparin oligosaccharide dp12 | Iduron | HO12 | https://iduron.co.uk/product/Heparin-1 |
| Chemical compound, drug | heparin oligosaccharide dp20 | Iduron | HO20 | https://iduron.co.uk/product/Heparin-1 |
| Chemical compound, drug | 2-O-desulphated heparin | Iduron | DSH001/2 | |
| Chemical compound, drug | 6-O-desulphated heparin | Iduron | DSH002/6 | |
| Chemical compound, drug | N desulphated heparin | Iduron | DSH003/N | |
| Chemical compound, drug | N-desulphated re N-acetylated heparin | Iduron | DSH004/Nac | |
| Chemical compound, drug | heparan sulphate | Iduron | GAG-HS01 | |
| Chemical compound, drug | HS fraction III approx. mol. wt. 9 kDa | Iduron | GAG-HS III | |
| Chemical compound, drug | APAC | Aplagon Oy | ||
| Chemical compound, drug | heparinase III (heparitinase I) Flavobacterium heparinum (EC 4.2.2.8) | AMSBiotechnology | AMS.HEP-ENZ III | |
| Chemical compound, drug | Heparin−biotin sodium salt | Sigma-Aldrich | B9806-10MG | |
| Chemical compound, drug | fibronectin | Cabiochem | Cat #341631 | |
| Chemical compound, drug | fibrinogen | Enzyme Research Laboratories | Fib 3 3496L | |
| Chemical compound, drug | collagen I | Takeda | 1130630 | collagen reagens horms |
| Chemical compound, drug | Cultrex Mouse Collagen IV | Trevigen | 3410-010-01 | purchased via R & D Systems |
| Chemical compound, drug | Laminin from EHS murine sarcoma basement membrane | Sigma-Aldrich | L2020 | refers to mouse laminin-111 in this study |
| Chemical compound, drug | streptavidin | Sigma-Aldrich | S4762 | |
| Software | Cell Profiler (2.2.0) | Broad Institute | http://cellprofiler.org/ RRID:SCR_007358 | |
| Software | Fiji | PMID: 22743772 | https://imagej.net/Fiji; RRID:SCR_002285 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.46840.041
