c5ar1 antagonist pmx205  (Tocris)

Journal: bioRxiv

Article Title: Platelet C5aR1 Aggravates Myocardial Infarction through Platelet–Neutrophil Interactions and CXCL4-Dependent NET Release

doi: 10.64898/2026.01.12.699090

Figure Lengend Snippet: (A) Experimental design of permanent left anterior descending (LAD) coronary artery ligation in Pf4^cre+^ C5ar1^fl/fl^ mice and Cre-negative littermate controls, followed by serial echocardiography and terminal analyses up to day 14. (B) Representative transverse left ventricular sections stained with TTC on day 14 after myocardial infarction, with infarcted myocardium appearing white and viable myocardium appearing red. Infarcted areas are indicated by a solid white outline (scale bar = 2 mm). (C) Quantification of infarct size expressed as percentage of left ventricle (LV). (D) Representative M-mode echocardiographic images. (E) Echocardiographic assessment of left ventricular ejection fraction (EF; left) and fractional shortening (FS; right) on day 1 and day 13 after myocardial infarction. (F) Representative immunofluorescence images of collagen I (red) and DNA (DAPI, blue) on day 14. (G) Quantification of collagen I–positive area (collagen I, % of LV section). (H) Representative CD31 (green) immunostaining with DNA staining (DAPI, blue) in the peri-infarct region on day 14. (I) Quantification of capillary density (CD31-positive area, %). Data are shown as mean ± SD; each dot represents one mouse. n = 4 mice per group for TTC and immunofluorescence analyses; n = 9–10 mice per group for echocardiography. Statistical analysis was performed using one-way ANOVA for EF and FS and two-tailed unpaired t-tests for all other comparisons. P < 0.05, P < 0.01, P < 0.001.

Article Snippet: For pharmacological inhibition experiments, wild-type mice only received the selective C5aR1 antagonist PMX205 (Tocris) or PBS vehicle.

Techniques: Ligation, Staining, Immunofluorescence, Immunostaining, Two Tailed Test

Journal: bioRxiv

Article Title: Platelet C5aR1 Aggravates Myocardial Infarction through Platelet–Neutrophil Interactions and CXCL4-Dependent NET Release

doi: 10.64898/2026.01.12.699090

Figure Lengend Snippet: (A) Representative immunofluorescence images of peri-infarct myocardium stained for Ly6G (red) and CD42b (green) with DNA staining (DAPI, blue), showing myocardial platelet–neutrophil complexes (PNCs). (B) Quantification of myocardial PNC density (mm⁻²) in the peri-infarct region (see also Supplementary Figure 10 for platelet and neutrophil infiltration). (C) Representative immunofluorescence images of peri-infarct myocardium stained for myeloperoxidase (MPO, red) and citrullinated histone H3 (H3Cit, green) with DNA (DAPI, blue), showing neutrophil extracellular traps (NETs). (D) Quantification of NET burden expressed as percentage of H3Cit⁺ area of the left ventricle (LV). (E) Flow cytometric gating strategy for platelet–neutrophil complexes (PNCs) in whole blood. Neutrophils were identified by Ly6G, and PNCs were defined as Ly6G⁺CD42b⁺ events. (F) Circulating PNCs expressed as percentage of CD42b⁺ events among Ly6G⁺ neutrophils on day 1 and day 14 after myocardial infarction in Pf4^cre+ C5ar1^fl/fl mice and Cre-negative littermate controls. (G) Spearman correlation analysis demonstrating an inverse relationship between circulating PNCs on day 1 after myocardial infarction and myocardial PNC density on day 14 across individual mice (r = −0.83, P = 0.02). Data are shown as mean ± SD; each dot represents one mouse. n = 4 mice per group for immunofluorescence analyses (A–D) and n = 9 mice per group for flow cytometric analyses (E–F). Statistical analysis was performed using two-tailed unpaired t-tests for immunofluorescence data, one-way ANOVA for flow cytometric analyses, and Spearman’s rank correlation for association analysis (G). P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Scale bars, 20 µm (A) and 10 µm (C).

Article Snippet: For pharmacological inhibition experiments, wild-type mice only received the selective C5aR1 antagonist PMX205 (Tocris) or PBS vehicle.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Platelet C5aR1 Aggravates Myocardial Infarction through Platelet–Neutrophil Interactions and CXCL4-Dependent NET Release

doi: 10.64898/2026.01.12.699090

Figure Lengend Snippet: (A) Representative single-plane confocal immunofluorescence images of fixed and permeabilized resting wild-type (WT) and C5aR1-deficient platelets stained for P-selectin (CD62P, green) and CXCL4 (red), illustrating altered α-granule organization. (B) Quantification of intracellular α-granule content per platelet, expressed as total area of P-selectin–positive granules and colocalized P-selectin/CXCL4 granules, measured by confocal microscopy. Data are shown as individual platelet values pooled from n = 3 independent experiments. Outliers were identified and removed using the ROUT method (Q = 1%) prior to analysis. (C) Flow cytometric analysis of platelet surface P-selectin expression 24 h after myocardial infarction following ex vivo stimulation of whole blood with 100 nM phorbol 12-myristate 13-acetate (PMA), expressed as geometric mean fluorescence intensity (GMFI) of CD42b⁺ platelets. (D) Flow cytometric analysis of platelet integrin GPIIb/IIIa activation under the same conditions, expressed as percentage of activated GPIIb/IIIa among CD42b⁺ platelets. (E) Plasma CXCL4 concentrations after MI in Pf4^cre+^ C5aR1^fl/fl^ mice and Cre-negative littermate controls. (F) Schematic of the in vitro platelet–neutrophil co-incubation assay. WT or C5aR1-deficient platelets were stimulated with C5a and co-incubated with neutrophils, followed by confocal immunofluorescence staining for myeloperoxidase (MPO), citrullinated histone H3 (H3Cit), and DNA (DAPI) to assess NET formation, in the presence or absence of low-dose heparin or recombinant CXCL4 (rCXCL4). (G) Representative immunofluorescence images of neutrophils after co-incubation, stained for MPO (red), H3Cit (green), and DNA (DAPI, blue), illustrating NET formation under the indicated conditions (see Supplementary Figure 13 for neutrophil-intrinsic and platelet-mediated control conditions). (H) Quantification of NET formation expressed as percentage of H3Cit⁺ neutrophils. Data are shown as mean ± SD unless otherwise indicated; each dot represents one biological replicate or mouse, as indicated. For α-granule analyses (A–B), data are shown as individual platelet values pooled from n = 3 independent experiments and analyzed using two-tailed unpaired t-tests following ROUT-based outlier exclusion (Q = 1%). Flow cytometry data (C–D) were analyzed using one-way ANOVA across time points and genotypes (see also Supplementary Figures for day 14 analyses). Plasma CXCL4 measurements (E) were analyzed using two-tailed unpaired t-tests. NET formation assays (H) were analyzed using one-way ANOVA with appropriate post hoc correction. P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Scale bars, 2 µm (A) and 20 µm (G).

Article Snippet: For pharmacological inhibition experiments, wild-type mice only received the selective C5aR1 antagonist PMX205 (Tocris) or PBS vehicle.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Expressing, Ex Vivo, Fluorescence, Activation Assay, Clinical Proteomics, In Vitro, Incubation, Recombinant, Control, Two Tailed Test, Flow Cytometry

Journal: bioRxiv

Article Title: Platelet C5aR1 Aggravates Myocardial Infarction through Platelet–Neutrophil Interactions and CXCL4-Dependent NET Release

doi: 10.64898/2026.01.12.699090

Figure Lengend Snippet: (A) Experimental design for daily subcutaneous administration of the C5aR1 inhibitor PMX205 or PBS for 14 days following permanent left anterior descending (LAD) coronary artery ligation. (B) Representative TTC-stained transverse left ventricular sections on day 14 after myocardial infarction, with infarcted myocardium appearing white and viable myocardium appearing red. Infarcted areas are indicated by a solid white outline (left). Corresponding quantification of infarct size, expressed as percentage of left ventricular (LV) area (right). (C) Quantification of collagen I–positive area in immunofluorescence staining, expressed as percentage of LV area. (D) Representative M-mode echocardiographic images on day 13 after myocardial infarction. (E) Echocardiographic assessment of left ventricular ejection fraction (EF; left) and fractional shortening (FS; right) on day 1 and day 13 after myocardial infarction. (F) Representative immunofluorescence images of peri-infarct myocardium stained for Ly6G (red) and CD42b (green) with DNA (DAPI, blue), illustrating myocardial platelet–neutrophil complexes (PNCs). (G) Quantification of myocardial PNC density (mm⁻²). (H) Quantification of myocardial NET burden expressed as percentage of H3Cit⁺ neutrophils. (I) Representative immunofluorescence images of peri-infarct myocardium stained for myeloperoxidase (MPO), citrullinated histone H3 (H3Cit), and DNA (DAPI), illustrating NET deposition. (J) Representative flow cytometry plots of platelet surface P-selectin expression and activated GPIIb/IIIa (αIIbβ3) in PMA-stimulated whole blood 24 h after myocardial infarction. (K) Quantification of platelet surface P-selectin expression in PMA-stimulated whole blood 24 h after myocardial infarction. (L) Quantification of platelet GPIIb/IIIa activation in PMA-stimulated whole blood 24 h after myocardial infarction. Data are shown as mean ± SD; each dot represents one mouse. Statistical analysis was performed using two-tailed unpaired t-tests for infarct size and collagen I quantification (B, C) and for myocardial PNC and NET quantification (G, H). Echocardiographic parameters (E) and flow cytometric platelet activation analyses (K, L) were analyzed using one-way ANOVA across time points and treatment groups (see also Supplementary Information for day 14 analyses). P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Gating strategies are provided in the Supplementary Information. Scale bars, 2 mm (B) and 20 µm (F, I).

Article Snippet: For pharmacological inhibition experiments, wild-type mice only received the selective C5aR1 antagonist PMX205 (Tocris) or PBS vehicle.

Techniques: Ligation, Staining, Immunofluorescence, Flow Cytometry, Expressing, Activation Assay, Two Tailed Test

Journal: Journal of Neurochemistry

Article Title: Complement C3a and C5a Receptors Are Presented in Mouse Sciatic and Human Sural Nerves and Selectively Modulate the Neuronal Function of Large‐Caliber Fibers in Mice

doi: 10.1111/jnc.70129

Figure Lengend Snippet: C5aR1 is mainly localized in unmyelinated fibers. Longitudinal and cross‐sections of human sural nerves were labeled for C5aR1 (red, A; green, D), Caspr (green, B), and NCAM (E, red). Double‐stained images are merged (C, F). Teased mouse sciatic nerve images of labeled C5aR1 (red, G, J) and Caspr (green, H). Double‐stained mouse sciatic fibers were merged (I). Peripherin (green, K) merged (L). Caspr‐contactin‐associated protein; NCAM‐neural cell adhesion molecule; n = 10 mice; Scale bar 10, 20 μM.

Article Snippet: The C5aR1 agonist BM213 (1 μM, Cat. No. HY‐145237, MedChemExpress), the C5aR1 antagonist PMX205 (1 μM, Cat. No. 13607, Cayman Chemicals, Ann Arbor, Michigan, USA) and the combination were applied at the same final concentrations as described above.

Techniques: Labeling, Staining

Journal: Journal of Neurochemistry

Article Title: Complement C3a and C5a Receptors Are Presented in Mouse Sciatic and Human Sural Nerves and Selectively Modulate the Neuronal Function of Large‐Caliber Fibers in Mice

doi: 10.1111/jnc.70129

Figure Lengend Snippet: mRNA and protein expression in the mouse sciatic nerve. Quantitative PCR (qPCR) analysis and western blotting were performed on sciatic nerves from 13‐week‐old mice ( n = 6 nerves). Intrinsic expression of the complement receptors and factors is seen (A). The outcomes are presented relative to hypoxanthine guanine phosphoribosyltransferase expression using the 2 −ΔCT method calculation method. Proteins C3aR and C5aR1 are present in mouse sciatic nerves ( n = 6 nerves) (B, C). HPRT‐hypoxanthine guanine phosphoribosyltransferase; ** p < 0.01.

Article Snippet: The C5aR1 agonist BM213 (1 μM, Cat. No. HY‐145237, MedChemExpress), the C5aR1 antagonist PMX205 (1 μM, Cat. No. 13607, Cayman Chemicals, Ann Arbor, Michigan, USA) and the combination were applied at the same final concentrations as described above.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot