High Glucose Enhances Transient Receptor Potential Channel Canonical Type 6–Dependent Calcium Influx in Human Platelets via Phosphatidylinositol 3-Kinase–Dependent Pathway

Platelet Preparation and Fluorescence Measurements

Platelets were obtained from healthy control subjects or patients with type 2 diabetes mellitus free from intercurrent illness. All donors gave informed consent. The study was approved by the local ethics committee. Subjects did not take antiplatelet drugs including aspirin for at least 2 weeks. 20 mL blood was anticoagulated with 3.2% trisodium citrate according to previously described methods.^9,10 Blood was centrifuged at 240g for 10 minutes to obtain platelet rich plasma, which was centrifuged at 240g for 15 minutes. Platelets were resuspended in Hanks balanced salt solution containing (in mmol/L) NaCl 136, KCl 5.40, CaCl₂ 1, KH₂PO₄ 0.44, Na₂HPO₄ 0.34, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid 10, pH 7.4 and incubated with 1 μmol/L of the fluorescent dye 1-[2-(5-Carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N′N′-tetraacetic acid acetoxymethyester (fura2-AM; Merck Biosciences) for 60 minutes. After centrifugation at 240g for 15 minutes to remove extraneous dye, the platelet pellet was again resuspended in Hanks balanced salt solution. The platelet counts were adjusted to 1×10⁸ platelets/mL. To exclude that the isolation procedure (including centrifugation and washing procedures) affects platelet action we performed additional experiments where platelets were isolated in the presence of 25 mmol/L glucose. Then platelets were exposed to 5.5 mmol/L glucose or 25 mmol/L throughout all subsequent steps (including loading with fura 2-AM).

Fluorescence measurements were undertaken as previously described by our group using a 96-well fluorescent plate reader (Fluoroskan Ascent Fluorometer, Thermo LabSystems Oy) at 510 nm emission with excitation wavelengths of 340 nm and 380 nm, and the fluorescence ratio 340 nm/380 nm was calculated.^11,12

Fluorescence measurements were performed after platelets had been incubated for 60 minutes with buffer for control, or glucose (25 mmol/L final concentration) in the absence or presence of inhibitors of phosphatidylinositol 3-kinases, ie, wortmannin (final concentration 20 μmol/L) or 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002, final concentration 10 μmol/L). Increasing concentrations of the cell-permeable diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), were used to activate the platelets. The sustained calcium increase after administration of OAG was determined. Some experiments were conducted in the presence of an inhibitor of TRP channels, 1-[β-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365; Merck Biosciences; final concentration, 10 μmol/L)^11,13,14 or in the presence of the L-type calcium channel blocker, 2-[(2-Aminoethoxy)-methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedi-carboxylic-acid-3-ethyl-5-methyl-ester (amlodipine; Sigma-Aldrich; final concentration, 10 μmol/L).

Platelet expression of P-selectin was measured as previously described¹⁵ after platelets had been incubated for 60 minutes with buffer for control or 25 mmol/L glucose. After fixation in paraformaldehyde, platelets were washed and incubated with mouse IgG (nonspecific binding) and a monoclonal antibody against P-selectin (both from Coulter Immunotech), followed by a secondary fluorescein isothiocyanate (FITC)-labeled antibody (Sigma-Aldrich). Flow cytometry was thereafter performed (FACScan; Becton Dickinson). Data are given as the median immunofluorescence intensity (arbitrary units) after subtraction of the nonspecific mouse IgG binding.

Immunofluorescence of TRP Channels

For the identification of TRPC channels, quantitative immunofluorescence assays of human platelets were performed using the Odyssey infrared imaging system (Licor Biosciences). Human platelets in 96-well plates were incubated with rabbit anti-human TRPC1, TRPC3, TRPC4, TRPC5, or TRPC6-antibodies (1:1000, Alomone Labs, Jerusalem, Israel)^{11,12,16–18} for 2 hours, washed, incubated with IRDye800CW-infrared fluorescent dye-conjugated goat antirabbit antibodies (1:1000, Biomol) overnight at 4°C. Imaging was performed at 810 nm emission with an excitation wavelength of 780 nm. Monoclonal rat anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000; Serotec, Düsseldorf, Germany) antibodies were used as internal reference after permeabilization. After incubation with Alexa Fluor680-allophycocyanin-fluorescence-labeled goat anti-rat antibodies (1:1000, MoBiTec, Göttingen, Germany) imaging was performed at 700 nm emission with an excitation wavelength of 680 nm. TRPC6 expression was analyzed under control conditions and after administration of 25 mmol/L glucose. To exclude osmotic effects platelets were also exposed to 25 mmol/L urea or 25 mmol/L taurine.

Immunofluorescence staining of platelets from healthy subjects was performed with specific TRPC6 antibodies and fluorescent dye–labeled secondary antibodies and analyzed with a TE2000 Nikon microscopy. The fluorescence intensities obtained from measurements of single platelets were averaged.

All substances were obtained from Sigma-Aldrich or Merck Biosciences if not indicated otherwise.

Statistics

All data are presented as mean±SEM of at least 3 to 10 independent experiments, and were compared using a 2-tailed Student t test or ANOVA as appropriate. A 2-sided probability value less than 0.05 was used to indicate statistical significance.

Glucose Enhances the 1-oleoyl-2-acetyl-sn-glycerol (OAG)-Induced Calcium Influx

Cytosolic calcium was measured in fura2-loaded platelets that had been incubated in the absence and presence of 25 mmol/L glucose for 60 minutes (Figure 1A). Resting cytosolic calcium in platelets was not significantly different after incubation in the absence or presence of 25 mmol/L glucose (F340 nm/F380 nm fluorescence ratio, 1.8±0.1 U [n=37] versus 1.7±0.1 U [n=40]; P>0.05). Platelets were incubated under control conditions and with 25 mmol/L glucose for 60 minutes. The administration of 60 μmol/L OAG increased the F340 nm/F380 nm fluorescence ratio in glucose-treated platelets by 0.76±0.10 U and in control platelets by 0.40±0.06 U (each n=13). The OAG-induced calcium influx was significantly higher in glucose-treated platelets (P<0.05). Next, we used the calcium channel blocker amlodipine. In the presence of the L-type calcium channel blocker amlodipine the OAG-induced calcium influx was also higher in glucose-treated platelets compared to control conditions (0.61±0.03 U versus 0.51±0.02 U; n=10, P<0.05; Figure 1B). Furthermore, we used SKF-96365, an inhibitor of TRP channels.^11,13,14 In the presence of SKF-96365 the OAG-induced calcium influx in glucose-treated platelets was significantly reduced from 0.76±0.10 U (n=13) to 0.43±0.02 U (n=10; P<0.05). In the presence of SKF-96365 the OAG-induced calcium influx was not significantly different in glucose-treated platelets compared to control conditions (0.43±0.02 U versus 0.38±0.04 U; each n=10, P>0.05; Figure 1C). The glucose-enhanced OAG-induced calcium influx was concentration-dependent (Figure 1D). As shown in Figure 1E the effect of glucose on calcium influx could be observed with different OAG concentrations.

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Figure 1. 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced calcium influx in platelets treated with 25 mmol/L glucose (filled circles or bars) or control buffer (open circles or bars) under control conditions (A), in the presence of amlodipine (Amlo; B), or SKF-96365 (SKF; C). Dose-response curve for glucose (D) and for OAG (E). *P<0.05.

To exclude that the isolation procedure affects platelet action, the platelets were isolated in the presence of 25 mmol/L glucose. Then platelets were exposed to 5.5 mmol/L glucose or 25 mmol/L glucose throughout all subsequent steps. These experiments confirmed that exposure to 25 mmol/L glucose as compared to 5.5 mmol/L glucose increased OAG-induced calcium influx into platelets (0.86±0.05 U versus 0.48±0.03 U, each n=15, P<0.05).

Inhibition of Phosphatidylinositol 3-Kinase Attenuates the Glucose-Enhanced OAG-Induced Calcium Influx in Human Platelets

Because activation of phosphatidylinositol 3-kinase is thought to be necessary for the translocation of TRPC channels to the surface we examined the effects of specific inhibitors of the phosphatidylinositol 3-kinase, wortmannin or LY294002. In the presence of wortmannin or LY294002 the OAG-induced calcium increase after incubation with 25 mmol/L glucose was significantly attenuated (Figure 2A). Compared to control conditions, wortmannin significantly reduced the glucose-enhanced OAG-induced calcium increase to 42±7% (P<0.05; n=6) and LY294002 significantly reduced it to 36±1% (P<0.05; n=6; Figure 2B). As indicated in Figure 2C the inhibitory effect of wortmannin on glucose-enhanced OAG-induced calcium increase was dose-dependent. These data indicate that high glucose concentrations enhance OAG-induced calcium influx by a phosphatidylinositol 3-kinase-dependent mechanism.

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Figure 2. 1-oleoyl-2-acetyl-sn-glycerol (OAG)–induced calcium influx in platelets treated with 25 mmol/L glucose (filled circles or bars) plus wortmannin or plus LY294002 or control buffer (open circles). Original tracings (A), summary data (B), and dose-response curve for wortmannin. *P<0.05.

Glucose Increases TRPC6 Channel Expression in Platelets

It is well-known that OAG-induced calcium influx is at least in part mediated through TRPC channels. Therefore we evaluated whether glucose enhances the expression of TRPC channels on human platelets. Using quantitative immunofluorescence we observed an increased TRPC6 protein surface expression after incubation of platelets with 25 mmol/L glucose (Figure 3). The effects of glucose on TRPC6 expression were measured in 33 experiments. Glucose significantly increased the TRPC6 expression to 131±12% (n=33; P<0.05). On the other hand glucose did not significantly change the protein expression of TRPC1, TRPC3, TRPC4, or TRPC5 (Figure 3B). The administration of 25 mmol/L urea did not significantly affect TRPC6 protein surface expression (100±1%; n=4; P>0.05 compared to control conditions). The administration of 25 mmol/L taurine did not significantly affect TRPC6 expression (102±10%; n=5; P>0.05 compared to control), further excluding osmotic effects.

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Figure 3. Immunofluorescence assay of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 (green) normalized to GAPDH (red) and overlay (yellow) on the surface of platelets treated with glucose (filled bars) or control buffer (open bars). Representative assay (A), summary data (B). *P<0.05.

We also evaluated whether glucose increased the expression of P-selectin on human platelets using immunofluorescence and FACScan flow cytometry. Compared to control conditions, the administration of 25 mmol/L glucose did not significantly change the expression of P-selectin in platelets (4.2±0.2 arbitrary units versus 4.3±0.4 arbitrary units; each n=8; P>0.05).

To evaluate the effects of phosphatidylinositol 3-kinases on TRPC6 expression we examined TRPC6 expression after administration of glucose in the presence of inhibitors of the phosphatidylinositol 3-kinase, wortmannin or LY294002. As shown in Figure 4, glucose significantly increased the TRPC6 expression on the platelet surface to 151±9% (n=10; P<0.01). The increased TRPC6 expression after incubation with 25 mmol/L glucose was significantly attenuated in the presence of wortmannin to 99±6% (n=4), or in the presence of LY294002 it was significantly attenuated to 76±2% (n=4; each P<0.01 compared to incubation with 25 mmol/L glucose alone; Figure 4B). These results were also observed using immunofluorescence of single platelets which were analyzed by fluorescence microscopy (Figure 4C). Compared to control conditions the administration of 25 mmol/L glucose significantly increased the TRPC6 expression on the platelet surface (152±2%, n=102; versus 100±3%, n=70; P<0.001).

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Figure 4. Immunofluorescence assay of TRPC6 on the surface of platelets treated with buffer as control or with 25 mmol/L glucose, glucose plus wortmannin (Wort) or plus LY294002 (LY). Representative assay (A), summary data (B). **P<0.01 compared to control. ++P<0.01 compared to glucose. TRPC6 expression on single platelets treated with buffer (control) of glucose.

As shown in Figure 5 the glucose-induced increase of TRPC6 protein surface expression in platelets was time-dependent. TRPC6 protein expression was significantly increased after incubation with high glucose concentration for at least 40 minutes.

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Figure 5. Time-dependent effect of glucose on TRPC6 expression on platelet surface. Representative immunofluorescence assay (A), summary data (B). *P<0.05 compared to control; +P<0.05 comparing glucose vs glucose plus wortmannin. C, TRPC6 expression in nondiabetic control subjects (Control) and patients with diabetes mellitus (Diabetes). Values are mean±SEM from 5 subjects, *P<0.05.

Furthermore, we examined platelets from patients with type 2 diabetes mellitus (mean age, 61±3 years; fasting blood glucose 212±32 mg/dL) and age-matched nondiabetic individuals (mean age, 62±4 years; fasting blood glucose 98±9 mg/dL). Platelets from patients with type 2 diabetes mellitus showed increased TRPC6 expression compared to nondiabetic individuals (273±40% versus 100±36%, each n=5, P<0.05; Figure 5C).