Fig 2: Effect of PAR agonists on Ca2+ mobilization and calpain activity in platelets from T2DM. a–d Effect of PAR agonists on intracellular Ca2+ in platelets of GGC and PGC T2DM. Cells were loaded with Fura-2 and placed in a thermostated cuvette in a physiological buffer. a, b Representative tracing of intracellular Ca2+ (Ca2+i) from platelets, by fluorimetric measurements. TRAP-6 (PAR-1 agonist, 20 µM) and AY-NH2 (PAR-4 agonist, 200 µM) were added when baseline fluorescence was stable. Red lines indicate Ca2+i of platelets of GGC T2DM, and blue lines indicate Ca2+i of platelets of PGC T2DM. c, d Ca2+ peak values, and time of Ca2+ recovery in platelets from T2DM with GGC and with PGC, respectively. t 50%, half-time of recovery; t 100% total time of recovery. e Calpain activity in platelets from T2DM with PGC, stimulated with AY-NH2 (200 µM) and in the presence of calpain inhibitor (ALLN, 100 µM). Calpain activity was determined as the value of luminescence recorded as relative light units (RLU) per µg of protein lysate. f Counts of platelets-derived microparticles (PMP) released by platelets from T2DM with PGC treated with AY-NH2, (PAR-4 agonist), in the presence and in absence of calpain inhibitor, ALLN (n = 21). Values are mean ± SEM. The p-values were evaluated by t-test (c, d, f) or ANOVA (p < 0.0001) followed by a post-hoc Bonferroni test (e)

Fig 3: Effect of PMP from PAR-4 treated platelets on IL-6 release and NF-kB activation in THP-1. Uptake of microparticles (1000 MPs/µL) into THP-1 transformed macrophages obtained from PAR-4 treated platelets of T2DM with GGC and with PGC for 24 h. a Representative confocal fluorescence images obtained from Zeiss microscopy implemented with the ApoTome attachment (×630) of PMP into THP-1 transformed macrophages, labelled with Calcein-AM (1 µM) for 40 min and co-cultured with macrophages for 4 h. Images of THP-1 (left) in absence and (right) in presence of PMP treatment. Calcein-AM+ MPs are shown in green, while the nucleus is stained in blue with DAPI. b–d THP-1 cells were incubated for 24 h with PMP from platelets of GGC and PGC T2DM, treated with AY-NH2 a PAR-4 agonist. b IL-6 gene expression was measured by qPCR, and c IL-6 release was measured by ELISA assay (n = 8). d NF-kB activation was determined as the acetylation level of p65 subunit (Ac p65 NF-kB) normalized for p65 NF-kB protein (total p65 NF-kB) (n = 6). Values are mean ± SEM. The p-values were evaluated by t-test

Fig 4: Study design and summary. In vivo, circulating Microparticles (MPs) characterization was performed in T2DM with good (HbA1c=7.0%; GGC) or poor (HbA1c>7.0%; PGC) glycemic control. In vitro, platelets from GGC and PGC were treated with thrombin and PAR agonists (PAR1/4-AP) to generate MPs (PMP), and to investigate their molecular pathways. PMP from PAR-4 stimulated platelets were incubated into THP-1 transformed macrophages to test their pro-inflammatory effects

Fig 5: Subcellular localization of proteins following decidualization.Decidualization was induced with cAMP (0.5 mM) and MPA (10µM) for three days (A) Cells were then harvested and cytoplasmic/nuclear extraction was performed following manufacturer protocol. Western blot was performed to qualify the change in Par-4, p65 and FoxO1. PARP was used as nuclear fraction control while GAPDH was used as cytoplasmic fraction control. The presented blot is of one representative experiment. (B) HIESC were seeded in 6-wells containing coverslips and grown in presence of cAMP (0.5 mM) and MPA (10µM) for three days. p65 and Par-4 localization were then observed by confocal microscopy. Presented images are of one representative experiment.