PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes

Abstract

Background: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages.

Methods: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages.

Results: We found that MPs CD62P⁺ (PMP) and CD142⁺ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca²⁺-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway.

Conclusions: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Keywords: Extracellular vesicles; Glycated hemoglobin; NF-kB; Platelet activation; THP-1 transformed macrophages.

Fig. 1

Circulating microparticles characterization in plasma from T2DM. Plasma-derived microparticles were isolated from 23 subjects with normal glucose tolerance (NGT), 28 diabetic patients with HbA1c ≤ 7.0% (GGC) and 31 diabetic patients with HbA1c > 7.0% (PGC). a–d Microparticles were labelled with specific antibodies: a for platelet-derived MPs (CD62P⁺) (ANOVA, p < 0.0001), b for tissue factor bearing (CD142⁺) (ANOVA, p = 0.009), c for endothelial-derived MPs (CD62E⁺) (ANOVA, p < 0.0001), and d for leukocyte-derived MPs (CD45⁺) (ANOVA, p = 0.64). Values are mean ± SEM. The p-values were evaluated by ANOVA followed by a post-hoc Bonferroni test. e, f Correlations between CD62P⁺ MPs and glycated hemoglobin, and between CD62P + MPs and IL-6 plasma levels, respectively. Statistical significance was determined with linear regression. Dotted lines indicated the 95% of interval confidence

Fig. 2

PAR-4 receptor expression increased the release of PMP from diabetic patients with PGC. a Platelets from T2DM with GGC (HbA1c ≤ 7.0%, n = 28) and with PGC (HbA1c > 7.0%, n = 30) were incubated for 30 min with thrombin (1 U/mL) and collagen (10 µg/mL) co-stimulus, or with Ca²⁺ ionophore A23187 (10 µM). PMP were isolated and were analyzed by flow cytometer. b, c Representative Western blots and densitometric analysis of PAR-1 and PAR-4 protein expression in platelets from T2DM with GGC and with PGC. The results were expressed relative to the control on the same blot, defined as 100%, and by the protein of interest/β actin densitometric ratio (n = 21). d Representative scatter plot of MPs derived from platelets of (left) GGC and (right) PGC T2DM treated with (up) TRAP-6 (PAR-1 agonist, 20 μM) and (down) AY-NH₂ (PAR-4 agonist, 200 μM), labelled with CD62P (PMP), and Calcein-AM; double positive MPs are shown (PMP CD62P⁺/Calcein⁺); CD62P⁺ MPs (PMP) are shown in green, CD62P⁻ MPs are shown in light violet. e Numbers of PMP released by platelets from T2DM with GGC and with PGC (n = 25), treated with TRAP-6 and with AY-NH₂, determined by flow cytometry. Values are mean ± SEM; The p-values were evaluated by t-test

Fig. 3

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. 4

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. 5

Effect of PAR-4 treated-platelet MPs on IL-6 and NF-kB in THP-1, blunted by calpain inhibition. THP-1 cells were incubated for 24 h with PMP from PGC T2DM, unstimulated and treated with AY-NH₂ a PAR-4 agonist, in presence and in absence of ALLN, a calpain inhibitor. a IL-6 gene expression was determined by qPCR, and b IL-6 release was measured by ELISA assay (n = 8). c 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). d Representative Western blots of THP-1 cells incubated with PMP from platelets of GGC and PGC, treated with AY-NH2 a PAR-4 agonist; PGC platelets were also incubated in presence and in absence of ALLN. TNFα (10 ng/mL) was used as positive control. Values are mean ± SEM. The p-values were evaluated by ANOVA (p < 0.0001) followed by a post-hoc Bonferroni test

Fig. 6

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