Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis.

We analyzed co-expression of the anaphylatoxin receptor C3aR with activated GPIIb/IIIa on platelets of 501 coronary artery disease patients using flow cytometry, detected C3aR expression in human or murine specimen by PCR, immunofluorescence, western blotting or flow cytometry and examined the importance of platelet C3aR by various in vitro platelet function tests, by in vivo bleeding time and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized using disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry.

Methods Please see the online-only Data

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Study approval

For experiments with human material, written informed consent was received from participants prior to inclusion in the study (approval number 270/2011B01). All animal experiments were approved by governmental authorities and performed in accordance with the German law guidelines of animal care (approval number M10/12).

Immunofluorescence staining

Human thrombi harvested from aspirates of the coronary arteries of patients with myocardial infarction undergoing percutaneous coronary intervention (PCI) were analyzed by immunofluorescence microscopy. Samples were stained with primary antibodies: anti-human C3aR and anti-human or corresponding control IgG followed by a secondary antibody conjugated with ALEXA-fluorochromes.

Flow cytometry analysis

Human platelets in freshly-collected blood were evaluated for the surface expression of C3aR and P-selectin (CD62P) after gating for the platelet specific marker CD42b. Isolated murine platelets for flow cytometric analysis were stained with the anti-mouse antibodies FITC-anti-P-selectin, FITC-anti-GPVI, FITC-anti-GPIbα, FITC-anti-integrin αIIbβIII, FITC-anti-integrin α5 and FITC-anti-integrin β3. Activated GPIIb/IIIa was detected using fibrinogen-Alexa488 or PE-anti-integrin GPIIb/IIIa. Samples were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany).

Mice

C57BL/6 mice and C3 −/− mice were obtained from The Jackson Laboratory. C3aR −/− mice were a kind gift from Dr. Rick Wetsel (University of Houston, Texas, USA) and C5aR −/− mice were kindly provided by Dr. Craig Gerard (Harvard Medical School, Boston, USA). Spa.1 −/− mice were provided by Dr. Yasutoshi Agata (Kyoto University, Kyoto, Japan) and bred with the C3aR −/− mice to obtain a Spa.1 −/− x C3aR −/− double knockout strain. GPIbalpha −/− mice were kindly provided by Dr. Jerry Ware (University of Arkansas for Medical Sciences, USA). All strains were on a C57BL/6 background and genotypes were confirmed by PCR. Littermates were used in all experiments as control animals.

Isolation of human and murine platelets

Human platelets were isolated according to standard protocols. Murine platelet preparations were obtained using a modified protocol. For activation, isolated platelets were stimulated with 20 µM ADP or 0.01 U/mL thrombin and/or C3a (as indicated in figure legends) at room temperature.

Western blotting

Whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis and subjected to western blot. Membranes were incubated with primary antibodies (anti-mouse C3aR, anti-human C3aR or anti-Spa-1, clone M-300 or anti-RAP1 or anti-GAPDH or anti-mouse phospho-PI3K or anti-mouse total PI3K or anti-Tubulin). Membranes were scanned with the Odyssey Infrared Imaging System (LI-COR, Bad Homburg, Germany) and analyzed.

Flow chamber assay

Isolated human platelets were stimulated with recombinant human C3a or scrambled C3a and perfused through a transparent flow chamber over a coated surface (collagen (20 µg/ml), vWF (20 µg/ml) or fibrinogen (50 µg/ml)) with high (1.700 s −1 ) shear rates for 10 min. 29 Experiments were recorded with a video recorder linked to a microscope (optical objectives ×20 and ×40; Carl Zeiss) and adherent platelets were quantified. Platelet Spreading Isolated murine platelets were incubated on fibrinogen-coated and BSA-blocked coverslides for 30 minutes at room temperature. Platelets were fixed and permeabilized with PHEM buffer containing PFA and NP-40, stained with rhodamin-phalloidin and analyzed with an Axio Observer Z.1 microscope (Zeiss). Scanning Ion Conductance Microscopy (SICM) Isolated WT and C3aR −/− platelets were stimulated with thrombin and allowed to adhere for 30 min to fibrinogen-coated culture plates. Platelets were then fixed with 4% PFA and mounted into the SICM setup. Platelet morphology was analysed using a custom-written software. Briefly, the cell contour was determined automatically using a height threshold of 50 nm and processed to calculate the morphology parameters (area A , height, and circularity C = 4π A / P 2 with perimeter P ).

Immunoprecipitation

Resting and stimulated platelets were lysed by adding an equal amount of lysis buffer. Samples were incubated with anti-C3aR antibody. Protein G-Sepharose was added and samples were incubated over night at 4 °C with rotation. Immunoblotting was performed as indicated. SDS PAGE and in-gel digestion Complete protein eluates of the pull-down experiments were submitted to a short run (1cm in length) on 1D SDS-PAGE. The proteins were visualized by staining and the corresponding gel sectors were excised and subjected to tryptic in-gel digestion. The resulting peptide mixtures were desalted before Liquid chromatography/Mass spectrometry (LC/MS) measurement.

Nanoscale liquid chromatography coupled to tandem mass spectrometry

(NanoLC-MS/MS) analysis LC-MS analysis was carried out on a nanoLC (Easy-nLC , Thermo Fisher Scientific, formerly Proxeon Biosystems) coupled to a LTQ-Orbitrap-XL (Thermo Fisher Scientific). MS data were processed using the MaxQuant software suite, peptide sequences were retrieved by using the Andromeda search engine, data were searched against the Uniprot mouse database.

Rap1 activation and pulldown assay

For detection and pulldown of activated Rap1 (GTP-Rap1), a commercially available kit (affinity precipitation assay, Merck Millipore) was used. Measurement of cytosolic Ca 2+ concentration Washed murine platelets were loaded with 5 μM fura-2 acetoxymethylester in the presence of 0.2 μg/ml pluronic F-127. Loaded platelets were activated with thrombin (0.01U) ± C3a (200 nM), ± C3aR Inhibitor SB290157 (10 µM). Calcium responses were measured under stirring with a spectrofluorimeter (LS 55; PerkinElmer) at alternate excitation wavelengths of 340 and 380 nm (37°C). The 340/380 nm ratio values were converted into concentrations of [Ca 2+ ]. Platelet aggregometry Aggregation of isolated human or murine platelets was estimated from light transmission determined with a luminoaggregometer (model 700; ChronoLog). Platelets were activated with 20 µM ADP, C3a in different doses or control protein (scrambled C3a) at the indicated concentrations. Analysis was performed with the Aggrolink8 software (ChronoLog). Bleeding time Mice were anesthetized and a 3-mm segment of the tail tip was removed with a scalpel. Tail bleeding was monitored by gentle absorption of the blood with filter paper at 20-s intervals without making contact with the wound site.

Intravital microscopy

Intravital microscopy and induction of platelet thrombus formation in vivo were carried out as described before. 29 Stroke model All stroke experiments were performed in accordance with the recently published ARRIVE guidelines (nc3rs) and performed as described before. 30 Myocardial infarction model All myocardial infarction experiments were performed as described before. 31 Statistics Data are provided as means±SD; n represents the number of experiments. All data were tested for significance using unpaired Student t-test and one-way or two-way ANOVA with Bonferroni’s post hoc test. Results with P < 0.05 were considered statistically significant.