The Platelet Anaphylatoxin Receptor C5aR1 (CD88) Is a Promising Target for Modulating Vessel Growth in Response to Ischemia a

stimulation. 28 Recently, we demonstrated that C5aR1-induced CXCL4 release modulates revascularization. 14 Here, we present further data on the importance of C5aR1 on platelets for the modulation of tissue revascularization.


Mouse hindlimb ischemia model
We used a previously described protocol to induce hindlimb ischemia (2,4).Briefly, the femoral artery of mice aged 10-12 weeks was ligated twice distal to the branch point of the caudal femoral artery and the epigastric artery.Afterwards, the femoral artery was intersected between both ligations.Tissue perfusion was assessed preoperatively, immediately post-ligation, and at 2, 4, 6, 8, 10 and 14 days after the surgical intervention, as previously described (5).Blood flow in the hindlimb was analyzed using an infrared laser Doppler imager (LDI2-IR, Moor Instruments, Axminster, UK) at 37°C under medetomidine/midazolam (0.5/5 mg/kg) anesthesia.Mice were left for a standard amount of time on the measuring heat mat to ensure consistency of results.
Data were analyzed with Moor LDI image processing software (Moor Instruments) and reported as the ratio of flow in the ischemic versus nonischemic hindlimb.For the display of the LDI images, the colour scale was adapted to ensure maximum informativity of displayed results.
The above-mentioned flow cytometry experiments were performed using a Beckman Coulter Cytoflex S 4-laser cytometer (Beckman Coulter, Krefeld, Germany) immediately after sample preparation and staining.Unless otherwise stated, specific monoclonal antibody binding was expressed as the geometric mean fluorescence intensity (geo.MFI) of 25,000 events in the target gate, and data were analyzed using CytExpert software (v.2.4,Beckman Coulter).
Human platelets from peripheral artery disease (PAD) patients were stained in whole blood within 20 minutes of blood withdrawal using CPDA tubes.Blood was drawn from patients suffering for asymptomatic PAD.All subjects consented freely to donate blood and all experiments were conducted in accordance with the specific ethical codes of the University of Lübeck, Germany.Asymptomatic PAD is defined as peripheral artery disease indicated by pathological ankle-brachial index below 0.9 as well as atherosclerotic arterial stenosis assessed by sonography, exclusion of diabetes mellitus and no walking pain in the patient history or during a six-minute walking test, respectively.Asymptomatic patients were matched with respect to gender and age with symptomatic patients Fontained stage IIb, verified by performing 6-minute walking test, during which all patients developed walking pain.Here, diabetes mellitus was excluded as well.Data were analyzed using Kaluza Analysis Software (ver.2.1, Beckman Coulter).We measured C5aR1 expression in 24 patients.As C5aR1 should be quantified in resting platelets, we remove 4 patients with preactivated platelets from the analysis.

Immunofluorescence microscopy studies
From ischemic murine hindlimb muscle tissue, 8-μm-thick sections of the gastrocnemius muscle were processed for immunofluorescence staining.Matrigel sections or sections of human coronary thrombi were processed accordingly.Snapfrozen tissue was rehydrated in PBS, fixed for 10 min in ice-cold acetone and blocked with 10% goat or donkey serum and 1% BSA for 30 min at room temperature.
The stained sections were mounted on glass slides, and image acquisition of muscle sections was performed as described earlier.Higher magnification images were taken using a Zeiss LSM 800 confocal laser scanning microscope with Zeiss ZEN 2.3 (blue edition) software.Subsequent image analysis was performed with Image Pro Plus (Ver.

In vivo Matrigel plug assay
The in vivo Matrigel plug assay was performed as previously described ( 9) with some modifications.Two aliquots of Matrigel (0.5 ml, Corning, Tewksbury, MA, USA) containing recombinant hirudin (22.4 U/ml, Merck, Darmstadt, Germany) supplemented with basic fibroblast growth factor (bFGF, 150 ng/ml, PeproTech, Rocky Hill, NJ, USA) were subcutaneously injected into the mid-abdominal region of mice, one aliquot on each side.Hirudin was used as an anticoagulant because the classical anticoagulant heparin, that is usually used in Matrigel plug assays, has been shown to inhibit complement activation (10).In some experiments, Matrigel was supplemented with freshly isolated murine platelets (10 8 /ml Matrigel) from various knockout mice, in addition to bFGF, as indicated in the figure legends.After 7 days, mice were sacrificed, and the Matrigel plugs were fixed with 4% PFA, processed for histology (frozen sections) and stained with H&E using standard staining protocols and reagents.Brightfield images were obtained with a Nikon Optishot-2 microscope equipped with a 2x plan-apochromat (N.A. 0.08) objective lens and a digital sight DS-5M camera using the Nikon NIS elements BR software (v.3.2,Nikon Instruments, Tokyo, Japan) for image acquisition and analysis.For readout, the ratio of the vessel area to the Matrigel plug area was calculated and expressed as neovascularization area fraction.

Isolation of human and murine platelets and generation of platelet releasate
Human and mouse washed platelets were isolated from human or mouse blood as previously described (16,17).Briefly, human venous blood was donated by healthy volunteers.All subjects consented freely to donate blood and all experiments were conducted in accordance with the specific ethical codes of the University of Tübingen and Lübeck, Germany.Blood was drawn from the antecubital vein into acid-citrate-dextrose (ACD) buffer and centrifuged at 430x g for 20 min.PRP was removed and added to HEPES-buffered Tyrode's solution (2.5 mM HEPES, 150 mM NaCl, 1 mM KCl, 2.5 mM NaHCO3, 0.36 mM NaH2PO4, 5.5 mM glucose and 1 mg/ml BSA, pH 6.5) and subsequently centrifuged at 900x g for 10 min.The resulting platelet pellet was resuspended in HEPES-buffered Tyrode's solution (pH 7.4, supplemented with 1 mM CaCl2 and 1 mM MgCl2).Subsequently, platelets were used for further experiments.
To isolate murine platelets, blood was drawn from the retroorbital plexus, collected in ACD buffer and centrifuged at 1800 rpm for 5min (Hettich EBA-3S, Germany).Supernatant was transferred, filled up with HEPES-buffered Tyrode's solution (pH7.4) and centrifuged at 800 rpm for 5min.Platelet rich plasma (PRP) was removed, filled up with Tyrode's solution then centrifuged at 2800 rpm for 5 min.The platelet pellet was carefully resuspended in HEPES-buffered Tyrode's solution (pH 7.4).The platelet content was quantified using a Sysmex cytometer (Sysmex KX-21N, Görlitz, Germany) and was adjusted to the required concentration.Subsequently, the platelets were either stimulated, and the supernatant was collected or lysed; the platelets were used for single-cell staining; or the platelets were used at specific concentrations for coincubation with endothelial cells, resuspended in Matrigel for injection into mice or used in flow cytometric analyses.For some experiments, platelet isolation was performed using activation inhibitors prostacyclin (0.5 µM, Sigma Aldrich) and apyrase (0.02U/mL, Sigma Aldrich).
As agonists for stimulation of murine as well as human platelets, we used C5a (R&D).
For some experiments, platelets were preincubated with the C5aR1 antagonist PMX205 (used at 15 µM for 30 minutes at 37°C, Tocris) or control peptide.Subsequently, platelets were stimulated with C5a.
If not otherwise stated, stimulation with C5a (R&D) was performed at a concentration of 20 nM for 10 min at 37°C as previously described for other cell types (9).Following C5a stimulation, the supernatant was collected by centrifugation at 14,000x g at 4°C and analyzed or used to stimulate endothelial cells.

Flow chamber assay
Heparinized whole blood was perfused through a transparent flow chamber over a coated surface (fibrinogen (50 μg/ml; #341576, Merck Milipore) with moderate (1.000 s −1 ) shear rates for 10 min (18).3 reference images at 3 different positions were taken using a a VWR IT414 microscope and the area of adherent platelets was quantified using ImageJ.

Scratched wound assay and tube formation assay
For the in vitro tube formation assay, MHEC-5T (6x10 4 -cells/well) were plated onto Matrigel-coated (Corning, NY) Angiogenesis µ-slides (ibidi, Planegg, Germany) in endothelial culture medium containing 2% FBS.Cells were with platelet supernatant, recombinant mouse CXCL4 (Biolegend, San Diego, CA) or anti-mouse CD183 antibody (eBioscience, San Diego, CA, 10µg/ml).After 6h tube formation was imaged by phase-contrast microscopy.Tube Formation was analyzed using Angiogenesis analyzer for ImageJ as described before and total branching length as well as number of nodes per area were assessed (19).An example analysis is shown in Supplementary Fig. 11.