Prostaglandins & other Lipid Mediators 116–117 (2015) 131–135
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Prostaglandins and Other Lipid Mediators
Original Research Article
Prostacyclin receptor expression on platelets of humans with type 2 diabetes is inversely correlated with hemoglobin A1c levels Stephanie M. Knebel, Randy S. Sprague, Alan H. Stephenson ∗ Department of Pharmacological and Physiological Science, Saint Louis University, St. Louis, MO 63104, United States
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Article history: Available online 21 January 2015 Keywords: Prostacyclin receptor Platelet cAMP Type 2 diabetes Radioligand binding Western blot
a b s t r a c t Inappropriate platelet aggregation can result in thrombosis and tissue ischemia. When compared to healthy human platelets, those of humans with type 2 diabetes (DM2) exhibit increased aggregation when stimulated. Activation of the platelet prostacyclin receptor (IPR) results in cAMP accumulation and inhibition of platelet aggregation. We hypothesized that DM2 platelets express decreased IPR when compared to platelets of healthy humans, resulting in decreased IPR agonist-induced cAMP accumulation. We measured IPR expression with radioligand binding of [3 H]-iloprost, a stable prostacyclin analog, and with Western blotting of the IPR protein. Iloprost-stimulated platelet cAMP levels were used to identify the functional response to IPR activation. IPR binding, expression of the IPR protein and the levels of cAMP in platelets incubated with iloprost were significantly decreased in DM2 platelets when compared to platelets of healthy humans. IPR expression decreased in platelets as glycemic control of the subjects worsened, as indicated by increased hemoglobin A1c levels. Taken together, these findings suggest that reduced IPR expression in DM2 platelets may contribute to platelet hyperactivity in humans with type 2 diabetes. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Atherothrombotic disease affecting the coronary vessels, cerebral circulation and lower limb peripheral arteries is accelerated in subjects with type 2 diabetes [1–4]. It has been proposed that heightened platelet activation contributes to these events [1,2,5]. The evidence supporting this proposal is derived from studies demonstrating that platelets obtained from humans with type 2 diabetes aggregate more readily to stimulation with prothrombotic agonists such as collagen, ADP and platelet activating factor [1,6–8]. Moreover, platelets of humans with type 2 diabetes are less sensitive to the anti-aggregatory effects of prostacyclin [8–10] and nitric oxide [11,12]. This defect is exacerbated by the fact that the vascular endothelium of humans with type 2 diabetes releases decreased amounts of these important mediators [11,13,14]. Although many studies have focused on mechanisms that mediate the increased platelet activation associated with
∗ Corresponding author at: Saint Louis University, Department of Pharmacological & Physiological Science, School of Medicine, 1402 South Grand Blvd, St. Louis, MO 63104, United States. Tel.: +1 314 977 6400; fax: +1 314 977 6410. E-mail addresses: [email protected] (S.M. Knebel), [email protected] (R.S. Sprague), [email protected] (A.H. Stephenson). http://dx.doi.org/10.1016/j.prostaglandins.2014.12.002 1098-8823/© 2015 Elsevier Inc. All rights reserved.
diabetes [15], fewer studies have identified possible mechanisms that mediate the decreased sensitivity to prostacyclin or nitric oxide. In the present study, we hypothesized that one mechanism responsible for decreased sensitivity to prostacyclin in platelets of humans with type 2 diabetes may result from decreased expression of the prostacyclin receptor (IPR). We investigated this hypothesis by comparing IPR expression by both radioligand binding and IPR protein measurements from Western blots in platelets of healthy humans with that of humans with type 2 diabetes. As a functional consequence of altered IPR expression, we also examined the ability of these platelets to increase cyclic AMP levels in response to iloprost, a stable IPR agonist. 2. Methods 2.1. Isolation of platelets from whole blood Human blood was obtained by venipuncture and collected into a syringe containing heparin (500 U/30 ml of blood). Blood from humans with type 2 diabetes was obtained from patients at the Endocrinology Clinic at Saint Louis University Hospital. Hemoglobin A1c (HbA1c) levels were determined using 10 l of whole blood with an A1cNow® kit (Bayer Healthcare). Blood was centrifuged at 300 × g for 10 min at 4 ◦ C. The platelet rich plasma
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was removed and added to a tube containing an additional 500 U heparin in acid-citrate-dextrose (ACD) solution to prevent platelet aggregation [16] and centrifuged for 20 min at 1000 × g at 4 ◦ C. After centrifugation, the supernatant was aspirated and discarded. The platelet pellet was resuspended in ACD solution, and centrifuged for an additional 20 min at 1000 × g at 4 ◦ C. The supernatant was discarded and the pellet was resuspended in assay buffer (50 mM Tris–HCl, 5 mM MgCl2 , final pH 7.4 at 4 ◦ C) for radioligand binding assays and cAMP assays, or the pellet was resuspended in Western blot extraction buffer (25 mM HEPES, 300 mM NaCl, 10 mM EDTA, tetrasodium, 1.5 mM MgCl2 , 20 mM -glycerophosphate, 0.1 mM sodium vanadate, final pH of 7.4 at 4 ◦ C). One tablet of Complete® Protease Inhibitor Cocktail (Roche) was added per 10 ml of Western blot extraction buffer. Blood from all subjects was obtained with informed consent and the protocol for its removal was approved by the Institutional Review Board of Saint Louis University. 2.2. Platelet IPR binding experiments Platelets, lysed by sonication at 4 ◦ C were suspended in assay buffer (400 g of protein/100 l of lysate) and aliquotted into siliconized glass tubes. Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay (Pierce). [3 H]-iloprost (1–100 nM) was added to reaction tubes, followed by unlabelled iloprost or vehicle (saline). Platelets were incubated for 20 min at room temperature, followed by 15 min at 4 ◦ C. Following incubation, the mixture was rapidly vacuum-filtered (Millipore) through a glass microfiber filter (GF/C, Whatman), pre-soaked with buffer. The filters were rapidly washed twice with 4 ◦ C buffer (1 ml). Washed filters were placed inside scintillation vials with 10 ml scintillation fluid (MP Biochemicals). Radioactivity was counted after 24 h to allow for complete elution of [3 H]-iloprost into the scintillation fluid and to reduce chemiluminescence.
by electrophoresis, then transferred to polyvinyldiene difluoride (PVDF) membranes. PVDF membranes were blocked for 1 h at room temperature in Starting Block® (Thermo Scientific) containing 0.01% Tween-20 and incubated overnight (16 h) at 4 ◦ C with a monoclonal antibody (1:200, Abnova) directed against the N-terminal portion of the IPR followed by washing and incubation for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, GE Healthcare). IPR protein expression was visualized using Super Signal West Pico chemiluminescent substrate (Pierce) on photographic film. PVDF membranes were then stripped of antibody using Restore® stripping buffer (Thermo Scientific), blocked for 30 min at room temperature in Starting Block® (Thermo Scientific) containing 0.01% Tween-20 and incubated for 1 h at room temperature with primary antibody for -actin (1:5000, Sigma) followed by washing and incubation with a HRP-conjugated secondary antibody (1:5000, GE Healthcare). -Actin protein expression was visualized by enhanced chemiluminescence (ECL, Pierce) on photographic film. The intensity of the chemiluminescent signals captured on photographic film was converted to pixel density using QuantiScan software. The pixel density of IPR protein expression was normalized to the pixel density of -actin protein expression. 2.6. Statistical analysis Statistical significance between groups was determined using an analysis of variance (ANOVA). In the event that the F ratio indicated that a change had occurred, a Fisher’s Least Significant Difference (LSD) test was performed to identify individual differences between groups. Results are reported as the means ± the standard error (SE). Linear regression analysis was performed using GB STAT software, and the Line of Best Fit was plotted.
2.3. Determination of Bmax and Kd 3. Results Values obtained from saturation binding experiments were entered into GraphPad Prism software and values for the number of binding sites (Bmax ) and the dissociation constant (Kd ) were calculated with non-linear regression by fitting a hyperbola directly to the saturation isotherm. Scatchard Plots were drawn to visualize the saturation binding data. 2.4. cAMP assays Vehicle (saline) or 1 M iloprost was added to the platelets prior to incubation for 5 min at room temperature. After incubation, chilled acidified ethanol (4 ml, 4 ◦ C) was added to the platelet suspension. The suspension was centrifuged at 21,000 × g for 10 min at 4 ◦ C. The supernatant was removed and dried under vacuum centrifugation. Samples were reconstituted in assay buffer and cAMP was measured using a cAMP Enzyme Immunoassay (GE Healthcare) in which cAMP values were normalized to 5 × 108 platelets. Platelet counts were performed in a hemacytometer. 2.5. Western blot analysis Platelets in Western blot extraction buffer were sonicated briefly while on ice. After 20 min incubation on ice, the platelet lysate was centrifuged at 4 ◦ C for 30 min 500 × g. Platelet lysates (10 l) were aliquotted and stored at −80 ◦ C until the day of use. Platelet lysates (50 g protein) were solubilized in 2× Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, 0.125 M Tris–HCl, pH 6.8, Sigma) and boiled for 5 min. Samples were chilled on ice for 5 min and loaded onto pre-cast 4–20% SDS-polyacrylamide gels (Pierce), resolved
3.1. Characteristics of the subjects Individuals with type 2 diabetes were identified by physicians at Saint Louis University in the Endocrinology Clinic. A patient history was collected for each individual including a detailed listing of current medications and the patient’s age and gender. The subjects studied were healthy human volunteers (n = 17, 9 female, 8 male) and humans with type 2 diabetes (n = 15, 8 female, 7 male) with a mean age of 47 years (range 23–76 years) and 50 years (range 31–74 years), respectively. The average HbA1c of humans with type 2 diabetes in this study was 7.9 ± 0.4%. Patients with type 2 diabetes were treated with multiple drugs in various combinations including aspirin (n = 10), angiotensin converting enzyme inhibitors or angiotensin receptor blockers (n = 11), -adrenergic receptor blockers (n = 5), oral hypoglycemic agents (n = 12), insulin (n = 13), lipid lowering drugs (n = 9), calcium channel blockers (n = 3) and diuretics (n = 3). The nature of the patients’ illnesses precluded discontinuation of medications. Record keeping was in compliance with HIPAA (Health Insurance Portability and Accountability Act) regulations. 3.2. Platelets of humans with type 2 diabetes exhibit decreased saturation binding of 3 H-iloprost when compared to platelets of healthy humans Saturation binding studies using increasing concentrations (10–100 nM) of [3 H]-iloprost demonstrated that specific binding with 100 nM [3 H]-iloprost was significantly decreased (P < 0.05) in
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Fig. 1. Saturation binding of [3 H]-iloprost (ILO) to platelets of healthy humans (solid line, HH, n = 4) and humans with type 2 diabetes (broken line, DM2, n = 4). Platelets (400 g) were incubated with increasing concentrations of [3 H]-ILO (1–100 nM) for 20 min at room temperature, followed by 15 min incubation on ice. Non-specific binding (NSB) was measured with 1000-fold unlabelled iloprost. Specific binding was plotted as total binding minus NSB using GraphPad Prism software. Values are means ± SE. *Different from HH, P < 0.05. Mean HbA1c of humans with DM2 = 8.8 ± 1.1.
platelets from humans with type 2 diabetes when compared to platelets obtained from healthy humans (Fig. 1). Scatchard analysis of the IPR saturation binding experiments was linear and revealed the presence of a single class of high affinity, low capacity receptors. The number of binding sites (Bmax ) for the IPR in platelets of humans with type 2 diabetes (668.0 ± 189.6 fmol/mg protein) was significantly lower (P < 0.05) than the Bmax from platelets of healthy humans (1501.5 ± 131.86 fmol/mg protein). The Kd of the IPR did not differ significantly between platelets of humans with type 2 diabetes and healthy humans.
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Fig. 2. Platelets of humans with type 2 diabetes (DM2, n = 8) exhibit decreased expression of monomeric isoforms and all isoforms combined of the prostacyclin receptor (IPR) when compared to platelets of healthy humans (HH, n = 8). Panel A: IPR MW isoforms of platelets from representative HH and DM2 resolved by Western blot analysis. MW bands at 40–65 kDa represent monomeric isoforms of the IPR, with differing amounts of N-glycosylation. MW bands at 65 kDa likely represent the highly N-glycosylated monomeric IPR isoforms. MW bands at 100 kDa represent dimeric isoforms of the IPR. Panel B: densitometric analysis of the IPR Western blots was used to compare levels of IPR protein expression between platelet lysates of HH and humans with DM2. Values were normalized to levels of -actin expression. G, glycosylated IPR; NG, non-glycosylated IPR; Mono, monomeric IPR isoforms; Di, dimeric IPR isoforms. *Different from HH, P < 0.05. Mean HbA1c values of humans with DM2 = 8.3 ± 1.2.
were analyzed alone and when combined glycosylated and nonglycosylated isoforms were included in the regression. However, when non-glycosylated isoforms alone were analyzed by regression analysis, there was no significant relationship between IPR protein expression and HbA1c.
3.3. Platelets of humans with type 2 diabetes demonstrate reduced IPR protein expression Western immunoblots of platelet lysates from humans exhibited multiple bands representing monomeric, dimeric and glycosylated isoforms of the IPR (Fig. 2A) as described previously [17,18]. Densitometric comparisons of IPR protein expression were performed on the non-glycosylated monomeric isoforms (40 kDa), the combined non-glycosylated and glycosylated monomeric isoforms (40–65 kDa) and all IPR isoforms combined (40–100 kDa). Higher molecular weight non-glycosylated dimeric isoforms could not be adequately resolved from the glycosylated dimeric isoforms in our Western immunoblots. Platelets of humans with type 2 diabetes exhibited significantly less of the monomeric isoforms when the glycosylated and non-glycosylated isoforms were combined. However, the expression of the non-glycosylated IPR was not different between platelets of healthy humans and those with type 2-diabetes (Fig. 2B). The combined monomeric and dimeric IPR isoforms also differed between healthy humans and humans with type 2 diabetes (Fig. 2B). Linear regression analysis identified an inverse correlation between HbA1c and protein expression of both the monomeric IPR variants and all IPR isoforms combined (Fig. 3), indicating that as glycemic control worsens, as evidenced by increases in HbA1c, IPR protein expression decreases in platelets from humans with type 2 diabetes. This significant relationship between IPR expression and HbA1c was present when the glycosylated isoforms
Fig. 3. Decreased platelet prostacyclin receptor (IPR) protein expression correlates with increased HbA1c levels. Linear regression analysis demonstrates an inverse correlation between IPR protein expression of both the monomeric IPR isoforms (y = −0.867x + 8.685, R2 = 0.852, P < 0.01) and all IPR isoforms combined (y = −0.599x + 5.817, R2 = 0.962, P < 0.01) and HbA1c levels. n = 7, mean HbA1c = 7.3 ± 0.4.
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Fig. 4. Platelets from humans with type 2 diabetes (DM2, n = 3) exhibit decreased iloprost (ILO)-induced increases in cAMP levels when compared to platelets from healthy humans (HH, n = 5). cAMP was measured in platelets incubated for 5 min with vehicle (saline) or 1 M ILO. Values are means ± SE. *Different from respective vehicle controls, P < 0.05; † different from HH, P < 0.05. Mean HbA1c of humans with DM2 = 6.4 ± 0.6.
3.4. Platelets of humans with type 2 diabetes exhibit a decreased ability to increase cAMP levels upon incubation with iloprost The lower number of platelet IPR binding sites in humans with type 2 diabetes suggests that these platelets would also exhibit attenuated IPR-mediated increases in cAMP levels. Indeed, in response to 1 M iloprost, platelets from humans with type 2 diabetes exhibited significantly smaller (P < 0.05) iloprost-induced increases in cAMP when compared to those of healthy humans (Fig. 4). 4. Discussion Prostacyclin-mediated inhibition of platelet aggregation within the vasculature is an important mechanism for preventing inappropriate thrombus formation [19,20]. Prostacyclin is derived predominantly from the vascular endothelium [20]. In humans with type 2 diabetes, vascular prostacyclin synthase expression is decreased and prostacyclin release from the vascular endothelium is known to be compromised [11,13,14], increasing the likelihood for thrombosis. In the present study, we identified a decreased level of IPR expression in platelets of humans with type 2 diabetes using two different methods, i.e., [3 H]-iloprost radioligand binding and IPR protein expression by Western blot. As reported previously using [3 H]-Iloprost as the radioligand, we found the platelet IPR of healthy humans to be expressed as a single class of high affinity receptors exhibiting a Bmax midway between values described in two previous reports [21,22]. Platelets of humans with type 2 diabetes exhibited a significantly lower Bmax than those of healthy humans, indicating fewer membrane-associated IP receptors. Decreased platelet IPR expression in humans with type 2 diabetes has not been reported previously. The findings of the binding studies presented here are supported by measurements of IPR protein expression using Western immunoblotting. Interestingly, two previous reports suggested that IPR expression in platelets of humans with type 2 diabetes is not different from IPR expression in healthy humans [23,24]. However, both of those reports relied solely on binding studies to measure IPR expression and both studies used the endogenous, but chemically unstable [3 H]-prostacyclin as their radioligand. The half-life of authentic
prostacyclin is approximately 3.5 min at pH 7.5 and 25 ◦ C [25], the conditions used for the 10 min binding incubations reported by Modesti et al. [23]. Under those conditions, more than 80% of the added [3 H]-prostacyclin would have been hydrolysed to 6-ketoPGF1␣ , which is not an IPR ligand, before the fraction of ligand bound to the IPR receptor was isolated. In the binding studies of Shepherd et al. [24], the half-life of [3 H]-prostacyclin was extended to greater than 2 h by increasing the incubation pH to 8.5, but by measuring platelet IPR binding under this non-physiological condition, it is unclear whether essential agonist-receptor interactions were preserved. In the present study, we also observed a significant inverse correlation between IPR protein expression and HbA1c, suggesting that as glycemic control worsens, platelets of humans with type 2 diabetes would become even less sensitive to the anti-aggregatory effects of prostacyclin. The inverse correlation of decreased prostacyclin sensitivity with HbA1c in platelets of humans with type 2 diabetes has been described previously by Davi et al. [8] without examining a mechanism for the decreased sensitivity. Published studies also reported that increased platelet aggregation [8] and augmented platelet TXA2 synthesis [26,27] correlated with the level of glycemic control in humans with type 2 diabetes, contributing to the greater risk for thrombosis in these individuals. In the previously mentioned studies of Modesti et al. [23] and Shepherd et al. [24] in which IPR expression was reported to be unaltered in humans with type 2 diabetes, the glycemic control of the subjects was not taken into consideration. Prostacyclin is the main physiological stimulator of cAMP production in platelets [19]. Agonist occupation of the IPR stimulates adenylyl cyclase activity, increasing the synthesis of platelet cAMP which results in reduced platelet activation [28–30]. As would be expected, decreased IPR expression in the platelets of humans with type 2 diabetes was associated with decreased levels of cAMP in response to incubation with iloprost. The reduced platelet IPR expression could be a factor contributing to the reduced prostacyclin-mediated increases in cAMP levels, which are required for prostacyclin-mediated inhibition of platelet aggregation. The IPR in platelets of humans has been reported to be Nglycosylated at two asparagine sites (N7 and N78 ) [17]. When these sites were mutated to prevent their N-linked glycosylation, surface expression of the IPR was reduced while expression of the IPR within the cytoplasm was increased; co-localized within the Golgi [17], and iloprost-induced activation of adenylyl cyclase was reduced relative to the extent of receptor glycosylation. Other studies have also correlated surface expression of the platelet IPR with increased agonist-induced cAMP accumulation [31,32]. In our study, inclusion of the glycosylated IPR isoforms was necessary to observe the reduced IPR protein expression in platelets of humans with type 2 diabetes when compared to healthy human controls. Because glycosylation at one or both asparagine sites is required for optimal platelet IPR surface expression, agonist binding and signal transduction, decreased expression of the glycosylated forms of the IPR in humans with type 2 diabetes could be responsible for the reduced prostacyclin-mediated increases in platelet cAMP, resulting in enhanced platelet aggregation [17,33]. Other interpretations for reduced prostacyclin-mediated stimulation of cAMP synthesis in platelets of humans with type 2 diabetes have been linked to “reduced sensitivity” of the platelets to prostacyclin [8,9]. Mechanisms of reduced sensitivity have been attributed to changes in platelet membrane lipid composition [34] or insulin resistance [35]. Platelets of humans with type 2 diabetes may contain elevated membrane cholesterol levels due to the association of hyperlipidemia with type 2 diabetes. Although increased platelet cholesterol levels have been reported to decrease human platelet IPR expression, human subjects in the present study who exhibited elevated blood cholesterol levels were treated with lipid lowering
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drugs that would tend to normalize the effects of hypercholesterolemia on platelet IPR [34]. Incubation of platelets with insulin at physiologically relevant concentrations has been reported to increase the number of IPR on the surface of platelets obtained from healthy humans and to restore normal anti-aggregatory effects of IPR agonists to platelets from humans with type 2 diabetes [31,36,37]. Although insulin itself had no effect on platelet cAMP levels in a study in which insulin was incubated with isolated healthy human platelets [31], agonist stimulation of the IPR in the insulin-treated platelets resulted in an increase in cAMP accumulation comparable to the increase in IPR receptor expression. Therefore, insulin resistance associated with type 2 diabetes may result in decreased expression of the IPR in platelets of humans with type 2 diabetes. The reduced sensitivity to insulin, at physiological concentrations, may be responsible for the decreased iloprost-mediated increases in platelet cAMP described in the present study. A mechanism proposed for insulin-mediated increases in platelet IPR expression requires insulin-mediated ADP-ribosylation of platelet Gi␣ [36]. Interestingly, platelets of humans with type 2 diabetes express normal levels of Gs, the G-protein coupled to the IPR, but their levels of Gi␣2 and Gi␣3 were reported to be only 49% and 75%, respectively, of those levels identified in platelets obtained from healthy human control subjects, suggesting that a defect in Gi expression in platelets of subjects with type 2 diabetes may be a component of insulin resistance that reduces IPR expression in their platelets. Taken together, these findings suggest that reduced IPR expression in platelets may contribute to platelet hyperactivity in humans with type 2 diabetes. Acknowledgements The authors also wish to thank E.A. Bowles and J. Schreiweis of Saint Louis University for technical support and assistance in reviewing this manuscript. This study was supported by a grant from the American Diabetes Association (BS-150). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prostaglandins. 2014.12.002. References [1] Colwell JA, Winocour PD, Lopes-Virella M, Halushka PV. New concepts about the pathogenesis of atherosclerosis in diabetes mellitus. Am J Med 1983;75:67–80. [2] Pyorala K, Laakso M, Uusitupa M. Diabetes and atherosclerosis: an epidemiologic view. Diabetes Metab Rev 1987;3:463–524. [3] Winer N, Sowers JR. Epidemiology of diabetes. J Clin Pharmacol 2004;44:397–405. [4] Bethel MA, Sloan FA, Belsky D, Feinglos MN. Longitudinal incidence and prevalence of adverse outcomes of diabetes mellitus in elderly patients. Arch Intern Med 2007;167:921–7. [5] Ferroni P, Basili S, Falco A, Davi G. Platelet activation in type 2 diabetes mellitus. J Thromb Haemost 2004;2:1282–91. [6] Li Y, Woo V, Bose R. Platelet hyperactivity and abnormal Ca(2+) homeostasis in diabetes mellitus. Am J Physiol Heart Circ Physiol 2001;280:H1480–9. [7] Bern MM. Platelet functions in diabetes mellitus. Diabetes 1978;27: 342–50. [8] Davi G, Rini GB, Averna M, et al. Thromboxane B2 formation and platelet sensitivity to prostacyclin in insulin-dependent and insulin-independent diabetics. Thromb Res 1982;26:359–70.
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Prostaglandins and Other Lipid Mediators
Original Research Article
Prostacyclin receptor expression on platelets of humans with type 2 diabetes is inversely correlated with hemoglobin A1c levels Stephanie M. Knebel, Randy S. Sprague, Alan H. Stephenson ∗ Department of Pharmacological and Physiological Science, Saint Louis University, St. Louis, MO 63104, United States
a r t i c l e
i n f o
Article history: Available online 21 January 2015 Keywords: Prostacyclin receptor Platelet cAMP Type 2 diabetes Radioligand binding Western blot
a b s t r a c t Inappropriate platelet aggregation can result in thrombosis and tissue ischemia. When compared to healthy human platelets, those of humans with type 2 diabetes (DM2) exhibit increased aggregation when stimulated. Activation of the platelet prostacyclin receptor (IPR) results in cAMP accumulation and inhibition of platelet aggregation. We hypothesized that DM2 platelets express decreased IPR when compared to platelets of healthy humans, resulting in decreased IPR agonist-induced cAMP accumulation. We measured IPR expression with radioligand binding of [3 H]-iloprost, a stable prostacyclin analog, and with Western blotting of the IPR protein. Iloprost-stimulated platelet cAMP levels were used to identify the functional response to IPR activation. IPR binding, expression of the IPR protein and the levels of cAMP in platelets incubated with iloprost were significantly decreased in DM2 platelets when compared to platelets of healthy humans. IPR expression decreased in platelets as glycemic control of the subjects worsened, as indicated by increased hemoglobin A1c levels. Taken together, these findings suggest that reduced IPR expression in DM2 platelets may contribute to platelet hyperactivity in humans with type 2 diabetes. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Atherothrombotic disease affecting the coronary vessels, cerebral circulation and lower limb peripheral arteries is accelerated in subjects with type 2 diabetes [1–4]. It has been proposed that heightened platelet activation contributes to these events [1,2,5]. The evidence supporting this proposal is derived from studies demonstrating that platelets obtained from humans with type 2 diabetes aggregate more readily to stimulation with prothrombotic agonists such as collagen, ADP and platelet activating factor [1,6–8]. Moreover, platelets of humans with type 2 diabetes are less sensitive to the anti-aggregatory effects of prostacyclin [8–10] and nitric oxide [11,12]. This defect is exacerbated by the fact that the vascular endothelium of humans with type 2 diabetes releases decreased amounts of these important mediators [11,13,14]. Although many studies have focused on mechanisms that mediate the increased platelet activation associated with
∗ Corresponding author at: Saint Louis University, Department of Pharmacological & Physiological Science, School of Medicine, 1402 South Grand Blvd, St. Louis, MO 63104, United States. Tel.: +1 314 977 6400; fax: +1 314 977 6410. E-mail addresses: [email protected] (S.M. Knebel), [email protected] (R.S. Sprague), [email protected] (A.H. Stephenson). http://dx.doi.org/10.1016/j.prostaglandins.2014.12.002 1098-8823/© 2015 Elsevier Inc. All rights reserved.
diabetes [15], fewer studies have identified possible mechanisms that mediate the decreased sensitivity to prostacyclin or nitric oxide. In the present study, we hypothesized that one mechanism responsible for decreased sensitivity to prostacyclin in platelets of humans with type 2 diabetes may result from decreased expression of the prostacyclin receptor (IPR). We investigated this hypothesis by comparing IPR expression by both radioligand binding and IPR protein measurements from Western blots in platelets of healthy humans with that of humans with type 2 diabetes. As a functional consequence of altered IPR expression, we also examined the ability of these platelets to increase cyclic AMP levels in response to iloprost, a stable IPR agonist. 2. Methods 2.1. Isolation of platelets from whole blood Human blood was obtained by venipuncture and collected into a syringe containing heparin (500 U/30 ml of blood). Blood from humans with type 2 diabetes was obtained from patients at the Endocrinology Clinic at Saint Louis University Hospital. Hemoglobin A1c (HbA1c) levels were determined using 10 l of whole blood with an A1cNow® kit (Bayer Healthcare). Blood was centrifuged at 300 × g for 10 min at 4 ◦ C. The platelet rich plasma
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was removed and added to a tube containing an additional 500 U heparin in acid-citrate-dextrose (ACD) solution to prevent platelet aggregation [16] and centrifuged for 20 min at 1000 × g at 4 ◦ C. After centrifugation, the supernatant was aspirated and discarded. The platelet pellet was resuspended in ACD solution, and centrifuged for an additional 20 min at 1000 × g at 4 ◦ C. The supernatant was discarded and the pellet was resuspended in assay buffer (50 mM Tris–HCl, 5 mM MgCl2 , final pH 7.4 at 4 ◦ C) for radioligand binding assays and cAMP assays, or the pellet was resuspended in Western blot extraction buffer (25 mM HEPES, 300 mM NaCl, 10 mM EDTA, tetrasodium, 1.5 mM MgCl2 , 20 mM -glycerophosphate, 0.1 mM sodium vanadate, final pH of 7.4 at 4 ◦ C). One tablet of Complete® Protease Inhibitor Cocktail (Roche) was added per 10 ml of Western blot extraction buffer. Blood from all subjects was obtained with informed consent and the protocol for its removal was approved by the Institutional Review Board of Saint Louis University. 2.2. Platelet IPR binding experiments Platelets, lysed by sonication at 4 ◦ C were suspended in assay buffer (400 g of protein/100 l of lysate) and aliquotted into siliconized glass tubes. Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay (Pierce). [3 H]-iloprost (1–100 nM) was added to reaction tubes, followed by unlabelled iloprost or vehicle (saline). Platelets were incubated for 20 min at room temperature, followed by 15 min at 4 ◦ C. Following incubation, the mixture was rapidly vacuum-filtered (Millipore) through a glass microfiber filter (GF/C, Whatman), pre-soaked with buffer. The filters were rapidly washed twice with 4 ◦ C buffer (1 ml). Washed filters were placed inside scintillation vials with 10 ml scintillation fluid (MP Biochemicals). Radioactivity was counted after 24 h to allow for complete elution of [3 H]-iloprost into the scintillation fluid and to reduce chemiluminescence.
by electrophoresis, then transferred to polyvinyldiene difluoride (PVDF) membranes. PVDF membranes were blocked for 1 h at room temperature in Starting Block® (Thermo Scientific) containing 0.01% Tween-20 and incubated overnight (16 h) at 4 ◦ C with a monoclonal antibody (1:200, Abnova) directed against the N-terminal portion of the IPR followed by washing and incubation for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000, GE Healthcare). IPR protein expression was visualized using Super Signal West Pico chemiluminescent substrate (Pierce) on photographic film. PVDF membranes were then stripped of antibody using Restore® stripping buffer (Thermo Scientific), blocked for 30 min at room temperature in Starting Block® (Thermo Scientific) containing 0.01% Tween-20 and incubated for 1 h at room temperature with primary antibody for -actin (1:5000, Sigma) followed by washing and incubation with a HRP-conjugated secondary antibody (1:5000, GE Healthcare). -Actin protein expression was visualized by enhanced chemiluminescence (ECL, Pierce) on photographic film. The intensity of the chemiluminescent signals captured on photographic film was converted to pixel density using QuantiScan software. The pixel density of IPR protein expression was normalized to the pixel density of -actin protein expression. 2.6. Statistical analysis Statistical significance between groups was determined using an analysis of variance (ANOVA). In the event that the F ratio indicated that a change had occurred, a Fisher’s Least Significant Difference (LSD) test was performed to identify individual differences between groups. Results are reported as the means ± the standard error (SE). Linear regression analysis was performed using GB STAT software, and the Line of Best Fit was plotted.
2.3. Determination of Bmax and Kd 3. Results Values obtained from saturation binding experiments were entered into GraphPad Prism software and values for the number of binding sites (Bmax ) and the dissociation constant (Kd ) were calculated with non-linear regression by fitting a hyperbola directly to the saturation isotherm. Scatchard Plots were drawn to visualize the saturation binding data. 2.4. cAMP assays Vehicle (saline) or 1 M iloprost was added to the platelets prior to incubation for 5 min at room temperature. After incubation, chilled acidified ethanol (4 ml, 4 ◦ C) was added to the platelet suspension. The suspension was centrifuged at 21,000 × g for 10 min at 4 ◦ C. The supernatant was removed and dried under vacuum centrifugation. Samples were reconstituted in assay buffer and cAMP was measured using a cAMP Enzyme Immunoassay (GE Healthcare) in which cAMP values were normalized to 5 × 108 platelets. Platelet counts were performed in a hemacytometer. 2.5. Western blot analysis Platelets in Western blot extraction buffer were sonicated briefly while on ice. After 20 min incubation on ice, the platelet lysate was centrifuged at 4 ◦ C for 30 min 500 × g. Platelet lysates (10 l) were aliquotted and stored at −80 ◦ C until the day of use. Platelet lysates (50 g protein) were solubilized in 2× Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, 0.125 M Tris–HCl, pH 6.8, Sigma) and boiled for 5 min. Samples were chilled on ice for 5 min and loaded onto pre-cast 4–20% SDS-polyacrylamide gels (Pierce), resolved
3.1. Characteristics of the subjects Individuals with type 2 diabetes were identified by physicians at Saint Louis University in the Endocrinology Clinic. A patient history was collected for each individual including a detailed listing of current medications and the patient’s age and gender. The subjects studied were healthy human volunteers (n = 17, 9 female, 8 male) and humans with type 2 diabetes (n = 15, 8 female, 7 male) with a mean age of 47 years (range 23–76 years) and 50 years (range 31–74 years), respectively. The average HbA1c of humans with type 2 diabetes in this study was 7.9 ± 0.4%. Patients with type 2 diabetes were treated with multiple drugs in various combinations including aspirin (n = 10), angiotensin converting enzyme inhibitors or angiotensin receptor blockers (n = 11), -adrenergic receptor blockers (n = 5), oral hypoglycemic agents (n = 12), insulin (n = 13), lipid lowering drugs (n = 9), calcium channel blockers (n = 3) and diuretics (n = 3). The nature of the patients’ illnesses precluded discontinuation of medications. Record keeping was in compliance with HIPAA (Health Insurance Portability and Accountability Act) regulations. 3.2. Platelets of humans with type 2 diabetes exhibit decreased saturation binding of 3 H-iloprost when compared to platelets of healthy humans Saturation binding studies using increasing concentrations (10–100 nM) of [3 H]-iloprost demonstrated that specific binding with 100 nM [3 H]-iloprost was significantly decreased (P < 0.05) in
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Fig. 1. Saturation binding of [3 H]-iloprost (ILO) to platelets of healthy humans (solid line, HH, n = 4) and humans with type 2 diabetes (broken line, DM2, n = 4). Platelets (400 g) were incubated with increasing concentrations of [3 H]-ILO (1–100 nM) for 20 min at room temperature, followed by 15 min incubation on ice. Non-specific binding (NSB) was measured with 1000-fold unlabelled iloprost. Specific binding was plotted as total binding minus NSB using GraphPad Prism software. Values are means ± SE. *Different from HH, P < 0.05. Mean HbA1c of humans with DM2 = 8.8 ± 1.1.
platelets from humans with type 2 diabetes when compared to platelets obtained from healthy humans (Fig. 1). Scatchard analysis of the IPR saturation binding experiments was linear and revealed the presence of a single class of high affinity, low capacity receptors. The number of binding sites (Bmax ) for the IPR in platelets of humans with type 2 diabetes (668.0 ± 189.6 fmol/mg protein) was significantly lower (P < 0.05) than the Bmax from platelets of healthy humans (1501.5 ± 131.86 fmol/mg protein). The Kd of the IPR did not differ significantly between platelets of humans with type 2 diabetes and healthy humans.
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Fig. 2. Platelets of humans with type 2 diabetes (DM2, n = 8) exhibit decreased expression of monomeric isoforms and all isoforms combined of the prostacyclin receptor (IPR) when compared to platelets of healthy humans (HH, n = 8). Panel A: IPR MW isoforms of platelets from representative HH and DM2 resolved by Western blot analysis. MW bands at 40–65 kDa represent monomeric isoforms of the IPR, with differing amounts of N-glycosylation. MW bands at 65 kDa likely represent the highly N-glycosylated monomeric IPR isoforms. MW bands at 100 kDa represent dimeric isoforms of the IPR. Panel B: densitometric analysis of the IPR Western blots was used to compare levels of IPR protein expression between platelet lysates of HH and humans with DM2. Values were normalized to levels of -actin expression. G, glycosylated IPR; NG, non-glycosylated IPR; Mono, monomeric IPR isoforms; Di, dimeric IPR isoforms. *Different from HH, P < 0.05. Mean HbA1c values of humans with DM2 = 8.3 ± 1.2.
were analyzed alone and when combined glycosylated and nonglycosylated isoforms were included in the regression. However, when non-glycosylated isoforms alone were analyzed by regression analysis, there was no significant relationship between IPR protein expression and HbA1c.
3.3. Platelets of humans with type 2 diabetes demonstrate reduced IPR protein expression Western immunoblots of platelet lysates from humans exhibited multiple bands representing monomeric, dimeric and glycosylated isoforms of the IPR (Fig. 2A) as described previously [17,18]. Densitometric comparisons of IPR protein expression were performed on the non-glycosylated monomeric isoforms (40 kDa), the combined non-glycosylated and glycosylated monomeric isoforms (40–65 kDa) and all IPR isoforms combined (40–100 kDa). Higher molecular weight non-glycosylated dimeric isoforms could not be adequately resolved from the glycosylated dimeric isoforms in our Western immunoblots. Platelets of humans with type 2 diabetes exhibited significantly less of the monomeric isoforms when the glycosylated and non-glycosylated isoforms were combined. However, the expression of the non-glycosylated IPR was not different between platelets of healthy humans and those with type 2-diabetes (Fig. 2B). The combined monomeric and dimeric IPR isoforms also differed between healthy humans and humans with type 2 diabetes (Fig. 2B). Linear regression analysis identified an inverse correlation between HbA1c and protein expression of both the monomeric IPR variants and all IPR isoforms combined (Fig. 3), indicating that as glycemic control worsens, as evidenced by increases in HbA1c, IPR protein expression decreases in platelets from humans with type 2 diabetes. This significant relationship between IPR expression and HbA1c was present when the glycosylated isoforms
Fig. 3. Decreased platelet prostacyclin receptor (IPR) protein expression correlates with increased HbA1c levels. Linear regression analysis demonstrates an inverse correlation between IPR protein expression of both the monomeric IPR isoforms (y = −0.867x + 8.685, R2 = 0.852, P < 0.01) and all IPR isoforms combined (y = −0.599x + 5.817, R2 = 0.962, P < 0.01) and HbA1c levels. n = 7, mean HbA1c = 7.3 ± 0.4.
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Fig. 4. Platelets from humans with type 2 diabetes (DM2, n = 3) exhibit decreased iloprost (ILO)-induced increases in cAMP levels when compared to platelets from healthy humans (HH, n = 5). cAMP was measured in platelets incubated for 5 min with vehicle (saline) or 1 M ILO. Values are means ± SE. *Different from respective vehicle controls, P < 0.05; † different from HH, P < 0.05. Mean HbA1c of humans with DM2 = 6.4 ± 0.6.
3.4. Platelets of humans with type 2 diabetes exhibit a decreased ability to increase cAMP levels upon incubation with iloprost The lower number of platelet IPR binding sites in humans with type 2 diabetes suggests that these platelets would also exhibit attenuated IPR-mediated increases in cAMP levels. Indeed, in response to 1 M iloprost, platelets from humans with type 2 diabetes exhibited significantly smaller (P < 0.05) iloprost-induced increases in cAMP when compared to those of healthy humans (Fig. 4). 4. Discussion Prostacyclin-mediated inhibition of platelet aggregation within the vasculature is an important mechanism for preventing inappropriate thrombus formation [19,20]. Prostacyclin is derived predominantly from the vascular endothelium [20]. In humans with type 2 diabetes, vascular prostacyclin synthase expression is decreased and prostacyclin release from the vascular endothelium is known to be compromised [11,13,14], increasing the likelihood for thrombosis. In the present study, we identified a decreased level of IPR expression in platelets of humans with type 2 diabetes using two different methods, i.e., [3 H]-iloprost radioligand binding and IPR protein expression by Western blot. As reported previously using [3 H]-Iloprost as the radioligand, we found the platelet IPR of healthy humans to be expressed as a single class of high affinity receptors exhibiting a Bmax midway between values described in two previous reports [21,22]. Platelets of humans with type 2 diabetes exhibited a significantly lower Bmax than those of healthy humans, indicating fewer membrane-associated IP receptors. Decreased platelet IPR expression in humans with type 2 diabetes has not been reported previously. The findings of the binding studies presented here are supported by measurements of IPR protein expression using Western immunoblotting. Interestingly, two previous reports suggested that IPR expression in platelets of humans with type 2 diabetes is not different from IPR expression in healthy humans [23,24]. However, both of those reports relied solely on binding studies to measure IPR expression and both studies used the endogenous, but chemically unstable [3 H]-prostacyclin as their radioligand. The half-life of authentic
prostacyclin is approximately 3.5 min at pH 7.5 and 25 ◦ C [25], the conditions used for the 10 min binding incubations reported by Modesti et al. [23]. Under those conditions, more than 80% of the added [3 H]-prostacyclin would have been hydrolysed to 6-ketoPGF1␣ , which is not an IPR ligand, before the fraction of ligand bound to the IPR receptor was isolated. In the binding studies of Shepherd et al. [24], the half-life of [3 H]-prostacyclin was extended to greater than 2 h by increasing the incubation pH to 8.5, but by measuring platelet IPR binding under this non-physiological condition, it is unclear whether essential agonist-receptor interactions were preserved. In the present study, we also observed a significant inverse correlation between IPR protein expression and HbA1c, suggesting that as glycemic control worsens, platelets of humans with type 2 diabetes would become even less sensitive to the anti-aggregatory effects of prostacyclin. The inverse correlation of decreased prostacyclin sensitivity with HbA1c in platelets of humans with type 2 diabetes has been described previously by Davi et al. [8] without examining a mechanism for the decreased sensitivity. Published studies also reported that increased platelet aggregation [8] and augmented platelet TXA2 synthesis [26,27] correlated with the level of glycemic control in humans with type 2 diabetes, contributing to the greater risk for thrombosis in these individuals. In the previously mentioned studies of Modesti et al. [23] and Shepherd et al. [24] in which IPR expression was reported to be unaltered in humans with type 2 diabetes, the glycemic control of the subjects was not taken into consideration. Prostacyclin is the main physiological stimulator of cAMP production in platelets [19]. Agonist occupation of the IPR stimulates adenylyl cyclase activity, increasing the synthesis of platelet cAMP which results in reduced platelet activation [28–30]. As would be expected, decreased IPR expression in the platelets of humans with type 2 diabetes was associated with decreased levels of cAMP in response to incubation with iloprost. The reduced platelet IPR expression could be a factor contributing to the reduced prostacyclin-mediated increases in cAMP levels, which are required for prostacyclin-mediated inhibition of platelet aggregation. The IPR in platelets of humans has been reported to be Nglycosylated at two asparagine sites (N7 and N78 ) [17]. When these sites were mutated to prevent their N-linked glycosylation, surface expression of the IPR was reduced while expression of the IPR within the cytoplasm was increased; co-localized within the Golgi [17], and iloprost-induced activation of adenylyl cyclase was reduced relative to the extent of receptor glycosylation. Other studies have also correlated surface expression of the platelet IPR with increased agonist-induced cAMP accumulation [31,32]. In our study, inclusion of the glycosylated IPR isoforms was necessary to observe the reduced IPR protein expression in platelets of humans with type 2 diabetes when compared to healthy human controls. Because glycosylation at one or both asparagine sites is required for optimal platelet IPR surface expression, agonist binding and signal transduction, decreased expression of the glycosylated forms of the IPR in humans with type 2 diabetes could be responsible for the reduced prostacyclin-mediated increases in platelet cAMP, resulting in enhanced platelet aggregation [17,33]. Other interpretations for reduced prostacyclin-mediated stimulation of cAMP synthesis in platelets of humans with type 2 diabetes have been linked to “reduced sensitivity” of the platelets to prostacyclin [8,9]. Mechanisms of reduced sensitivity have been attributed to changes in platelet membrane lipid composition [34] or insulin resistance [35]. Platelets of humans with type 2 diabetes may contain elevated membrane cholesterol levels due to the association of hyperlipidemia with type 2 diabetes. Although increased platelet cholesterol levels have been reported to decrease human platelet IPR expression, human subjects in the present study who exhibited elevated blood cholesterol levels were treated with lipid lowering
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drugs that would tend to normalize the effects of hypercholesterolemia on platelet IPR [34]. Incubation of platelets with insulin at physiologically relevant concentrations has been reported to increase the number of IPR on the surface of platelets obtained from healthy humans and to restore normal anti-aggregatory effects of IPR agonists to platelets from humans with type 2 diabetes [31,36,37]. Although insulin itself had no effect on platelet cAMP levels in a study in which insulin was incubated with isolated healthy human platelets [31], agonist stimulation of the IPR in the insulin-treated platelets resulted in an increase in cAMP accumulation comparable to the increase in IPR receptor expression. Therefore, insulin resistance associated with type 2 diabetes may result in decreased expression of the IPR in platelets of humans with type 2 diabetes. The reduced sensitivity to insulin, at physiological concentrations, may be responsible for the decreased iloprost-mediated increases in platelet cAMP described in the present study. A mechanism proposed for insulin-mediated increases in platelet IPR expression requires insulin-mediated ADP-ribosylation of platelet Gi␣ [36]. Interestingly, platelets of humans with type 2 diabetes express normal levels of Gs, the G-protein coupled to the IPR, but their levels of Gi␣2 and Gi␣3 were reported to be only 49% and 75%, respectively, of those levels identified in platelets obtained from healthy human control subjects, suggesting that a defect in Gi expression in platelets of subjects with type 2 diabetes may be a component of insulin resistance that reduces IPR expression in their platelets. Taken together, these findings suggest that reduced IPR expression in platelets may contribute to platelet hyperactivity in humans with type 2 diabetes. Acknowledgements The authors also wish to thank E.A. Bowles and J. Schreiweis of Saint Louis University for technical support and assistance in reviewing this manuscript. This study was supported by a grant from the American Diabetes Association (BS-150). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prostaglandins. 2014.12.002. References [1] Colwell JA, Winocour PD, Lopes-Virella M, Halushka PV. New concepts about the pathogenesis of atherosclerosis in diabetes mellitus. Am J Med 1983;75:67–80. [2] Pyorala K, Laakso M, Uusitupa M. Diabetes and atherosclerosis: an epidemiologic view. Diabetes Metab Rev 1987;3:463–524. [3] Winer N, Sowers JR. Epidemiology of diabetes. J Clin Pharmacol 2004;44:397–405. [4] Bethel MA, Sloan FA, Belsky D, Feinglos MN. Longitudinal incidence and prevalence of adverse outcomes of diabetes mellitus in elderly patients. Arch Intern Med 2007;167:921–7. [5] Ferroni P, Basili S, Falco A, Davi G. Platelet activation in type 2 diabetes mellitus. J Thromb Haemost 2004;2:1282–91. [6] Li Y, Woo V, Bose R. Platelet hyperactivity and abnormal Ca(2+) homeostasis in diabetes mellitus. Am J Physiol Heart Circ Physiol 2001;280:H1480–9. [7] Bern MM. Platelet functions in diabetes mellitus. Diabetes 1978;27: 342–50. [8] Davi G, Rini GB, Averna M, et al. Thromboxane B2 formation and platelet sensitivity to prostacyclin in insulin-dependent and insulin-independent diabetics. Thromb Res 1982;26:359–70.
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