Fig 2: α-synuclein deficiency inhibits platelet granule secretion. Platelet-rich plasma (PRP) from WT (black bars) and α-synuclein−/− (white bars) mice were stimulated with thrombin (0.025–0.1 U/mL) for 20 min, and platelet alpha-granule (a), dense granule (b), and lysosome (c) secretion was assessed by using flow cytometry. Median fluorescence intensity (MFI) is presented as mean ± SEM. (d) t-Distributed Stochastic Neighbourhood Embedding (t-SNE) analysis was performed on flow cytometry data to visualise subpopulations of platelets based on granule secretion markers in WT (+/+) and α-synuclein−/− (−/−) platelets after stimulation with thrombin (0.1 U/mL). Heatmaps represent median fluorescence intensity (MFI) for CD62p (left column), CD63 (middle column), and LAMP-1 (right column). * p < 0.05, Mann–Whitney U test.

Fig 3: Mrgpra1 signaling dampens neutrophil anti-bacterial responses to Streptococcus pneumoniae.(A-E) BMNs from Mrgpra1+/+ and Mrgpra1−/− mice were cultured with Streptococcus pneumoniae (MOI 1) for 90 min. Frequency of DHR+ (A,B), and CD63+ (C,D) neutrophils, and (E) CFU of remaining bacteria. BMNs (pooled) from mice (3–4 per group) were plated in replicates and used for experiments. Data is mean+SEM and representative of at least 2–3 independent experiments.(F-L) Mrgpra1+/+ and Mrgpra1−/− mice given 2.5×105 Streptococcus pneumoniae i.t. and 24 h later, the numbers of (F) neutrophils, (G) DHR123+ neutrophils, (H) DHR123hi neutrophils, (I) CD63+ neutrophils, (J) DHR123+ AMs and (K) S. pneumoniae CFU in the lungs were quantified. (L) Survival of infected Mrgpra1+/+ (n=17) and Mrgpra1−/− (n=12) mice. Each dot represents an animal (n= 3–12) (F-K). Data is mean+SEM and representative of at least 2–3 independent experiments, or pooled from 2–3 experiments (K,L)For all experiments, both male and female mice were used. Data was analyzed by Student’s T test, one-way ANOVA followed by posthoc test or the Log-rank test for survival *p<0.05, **p<0.01. See also Figure S2.

Fig 4: A distinct population of neutrophils express NPFF, the Mrgpra1 ligand, and this populations correlates with an alternative program.(A,B) RNA-seq data (GSE164766) showing expression of (A) neuropeptides (NP) and (B) NP receptors in BMNs.(C) NP mRNAs from BMNs exposed in vitro to PBS or S. pneumoniae (S.p) (MOI 1) for 16 h determined by PCR.(D-G) Mice treated with saline or S. pneumoniae and 24 h later (D) number of various NPFF+ lung cells, (E) number of NPFF+ neutrophils in the BAL and (F) frequency of NPFF+ neutrophils. (G) Representative flow plots depicting NPFF staining in BAL neutrophils.(H-Q) Characteristics of NPFF− and NPFF+ BAL neutrophils 24h after saline or S. pneumoniae (S.p) exposure. (H) forward scatter, (I) side scatter, (J) Ly6G MFI and (K) CD11b MFI. Frequency and representative flow plots of (L) CD63+, (M) iNOS+, (N) CD24+, (O) FcεRI+, (P) CD204+, (Q) Arg1+.Data is mean+SEM, each dot (E-Q) represents individual animal (n=3–9) and is representative (H-Q) or pooled (D-F) from at least 2–4 independent experiments that used male and female mice. Data is analyzed by Student’s T test or two-way ANOVA followed by posthoc test. *p<0.05, **p<0.01, ***p<0.001, ***p<0.001. See also Figure S5.

Fig 5: Finding EVs and their immunophenotyping by IFC. ExoFlow-ONE™ labelling (A) revealed a significantly higher concentration of ‘gated’ EVs/mL in caput compared to cauda epididymosomes and seminal vesicle fluid EV samples. (B) Characterization of tetraspanin content of male reproductive tract EVs indicated a distinctive abundance of tetraspanins across the male reproductive tract EV subtypes. Seminal vesicle fluid EVs had significantly fewer percentages of CD9+ vesicles than caput and cauda epididymosomes. (C) Percentage of caput EVs (epididymosomes) expressing either two tetraspanins (CD9+/CD81+ or CD9+/CD63 + or CD63+/CD81+) or expressing all three tetraspanins (CD9+CD63+CD81+) in the whole subpopulation. (D) Percentage of male reproductive tract EVs that stained positively for CD9+/CD81+. Data are expressed as the mean ± SD. One-way ANOVA and unpaired two-tailed t-test, Tukey’s post-hoc analyses, *P < 0.05, **P < 0.01 and ***P < 0.001.