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Inhibition of platelet GPIIb–IIIa and P-selectin expression by aspirin is impaired by stress hyperglycemia
Journal of Diabetes and Its Complications 23 (2009) 65 – 70 WWW.JDCJOURNAL.COM
Inhibition of platelet GPIIb–IIIa and P-selectin expression by aspirin is impaired by stress hyperglycemia Alexandre Le Guyadera,b, Garrett Pachecoa, Norma Seavera, Grace Davis-Gormana, Jack Copelanda, Paul F. McDonagha,4 a
Cardiovascular and Thoracic Surgery and the Sarver Heart Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA b Thoracic and Cardiovascular Surgery Department, Dupuytren University Hospital, University of Limoges, 87042 Limoges, France Received 14 March 2007; received in revised form 24 May 2007; accepted 1 June 2007
Abstract Increased aspirin resistance may contribute to the increase in thrombotic events observed in patients with type 2 diabetes. In this study, we examined if acute exposure to increased plasma glucose impaired the inhibitory effects of aspirin on platelet activation. Whole-blood samples were incubated with 100 (euglycemia), 200, 300, and 600 mg/dl glucose followed by incubation with aspirin [acetylsalicylic acid (ASA)]. Using flow cytometry, GPIIb–IIIa and P-selectin were analyzed in unstimulated and arachidonic acid (AA)-stimulated platelets. In euglycemic blood, AA caused a significant increase in platelet GPIIb–IIIa expression [unstimulated: 59.5F8.2 total fluorescence intensity (TFI), AA stimulated: 319.6F42.7 TFI, P=.002] and P-selectin (4.4F0.7 and 179.5F38.5 TFI, Pb.001). In vitro, ASA significantly inhibited both GPIIb–IIIa expression (36.5%) and P-selectin expression (81%; Pb.005). However, increased blood glucose (200 mg/dl) significantly impaired the inhibitory effect of ASA (84% for GPIIb–IIIa, Pb.005; 48% for P-selectin, P=NS). Increasing glucose to 600 mg/dl completely overwhelmed the inhibitory effect of ASA. A statistically significant interaction between glucose concentration and ASA dose was found ( Pb.001 for GPIIb–IIIa and P=.004 for P-selectin). In vitro, concentration-dependent stress hyperglycemia significantly impaired the inhibitory effects of aspirin on human platelet GPIIb–IIIa and P-selectin expression. Under acute hyperglycemic conditions, the effectiveness of ASA to inhibit platelets via the AA-activation pathway may be significantly reduced. D 2009 Elsevier Inc. All rights reserved. Keywords: Diabetes; Hyperglycemia; Platelets; Aspirin; Flow cytometry
1. Introduction Aspirin [acetylsalicylic acid (ASA)] is the most widely prescribed drug in the world with indications ranging from fever to thrombosis. Since 1997, the American Diabetes Association has recommended that low-dose aspirin be used for both primary and secondary prevention of cardiovascular disease in patients with diabetes (Colwell, 1997). However, several studies conclude that platelets from diabetic patients are less sensitive to aspirin, and these observations are consistent with the reduced effectiveness of ASA in
4 Corresponding author. Tel.: +1 520 626 2329; fax: +1 520 626 4042. E-mail address: [email protected] (P.F. McDonagh). 1056-8727/09/$ – see front matter D 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jdiacomp.2007.06.003
diabetes. Results of the Primary Prevention Project indicate that nondiabetics had a 41% reduction in heart-diseaserelated death with aspirin compared to only 10% in diabetic patients. The study authors suggested that the antiplatelet effects of aspirin in diabetic patients are overwhelmed by aspirin-insensitive mechanisms of platelet activation (Sacco et al., 2003). The term daspirin resistanceT has been used to describe a clinical situation in which a subgroup of patients on therapeutic doses of aspirin experiences thrombotic vascular events. Several hypotheses have been proposed to explain the apparent lack of response of diabetic patients to aspirin. In a laboratory study using the streptozotocin rat model of type 1 diabetes, Watala et al. (2006) concluded that the nonenzymatic glycosylation of biomolecules may compete with aspirin in the attachment to unoccupied
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amino groups on blood platelet proteins. Another possibility is that diabetic platelets may be activated by pathways independent of arachidonic acid (AA). Thromboxane A2 (TXA2) is an eicosanoid produced from AA by the cyclooxygenase 1 (COX1) enzyme. COX1 is irreversibly inhibited by aspirin. TXA2 binds to Gprotein-coupled receptors to generate inside-out signals, leading to activation of the fibrinogen receptor and exocytosis of intracellular granules (Offermanns, 2006). Upon exposure to thrombin, adenosine diphosphate (ADP), or TXA2, the platelet adhesion protein GPIIb–IIIa undergoes a conformational change that allows fibrinogen to bind, promoting aggregation. The adhesion protein P-selectin is stored in the a granules of resting platelets and is expressed on the cell surface upon activation. As a key adhesion molecule, it is involved in platelet–leukocyte conjugate formation. Frelinger et al. (2006) and Zimmermann et al. (2003) report that the expression of both adhesion proteins is inhibited by aspirin. GPIIb–IIIa expression and P-selectin expression are sensitive indicators of platelet activation, which can be evaluated in whole blood by flow cytometry. Previously, we utilized these markers to assess chronic platelet function in type 2 diabetic patients with cardiac conditions. We found a significant chronic increase in P-selectin in the diabetic patients with heart disease compared to their nondiabetic counterparts. We also found a significant chronic increase in GPIIb–IIIa expression in the diabetic patients when the findings were stratified for aspirin usage (McDonagh et al., 2003; Tuttle, Davis-Gorman, Goldman, Copeland, & McDonagh, 2003). The aim of this study was to determine if a more acute stress, in the form of shortterm hyperglycemia, affected the ability of aspirin to attenuate platelet activation. We evaluated two doses of aspirin in vitro, one similar to the dose currently used in clinical practice and a greater dose as a test of a possible dose–response effect.
2. Materials and methods 2.1. Population Eleven healthy volunteers (29F2 years) were recruited after informed consent was obtained. The study was approved by our institutional review board. No subjects had a reported history of diabetes, cardiovascular disease, or platelet abnormalities. No subjects had taken aspirin or any drugs interfering with platelet function within the past 10 days. 2.2. Study design To assess the effect of acute hyperglycemia on platelet adhesion protein expression, we incubated aliquots of whole blood for 60 min at 378C with incremental concentrations of
dextrose (Sigma-Aldrich, St. Louis, MO) to achieve approximate concentrations of 100 (euglycemia), 200, 300, and 600 mg/dl (Keating, Sobel, & Schneider, 2003). Blood glucose concentrations of two and three times normal were evaluated to determine the impact of uncontrolled blood glucose, which may occur in diabetic patients or soon after heart surgery in cardiovascular patients. The unphysiologic blood glucose (six times normal) was tested to determine if this abnormal concentration impaired the sensitivity of platelets to aspirin in a concentration–response manner. Blood glucose concentrations in all samples were measured using an instant-read glucometer (Accu-Chek Active, Roche Diagnostics, Indianapolis, IN), and the results are given in Table 1. To determine if aspirin inhibited the AA-induced expression of platelet adhesion proteins, we incubated the aliquots of euglycemic and hyperglycemic whole blood described above with aspirin (ASA, Sigma-Aldrich) for 10 min at 378C as previously described (Golanski, Nocun, Rozalski, Drygas, & Watala, 2004; Marcus, 1990). Two concentrations of aspirin were chosen, 65 or 200 Ag/ml, based on the earlier reports of Golanski et al. (2004). The final concentration of 65 Ag/ml corresponds to an equivalent dose of 325 mg/day, a dose recommended for diabetic patients. The 200 Ag/ml concentration corresponds to a dose of 1000 mg/day. This larger dose was used to test the hypothesis that more aspirin would override any measured insensitivity to aspirin. 2.3. Flow cytometry of platelet adhesion protein expression The protocol for platelet labeling in whole human blood was described elsewhere (McDonagh et al., 2003). Blood samples were collected in sodium citrate vacutainers (BD Biosciences) after discarding the first 3 ml. The blood was mixed with PerCP-conjugated anti-CD61 mAb (BD Pharmingen) to identify the platelet population of cells, with FITC PAC-1 mAb (BD Pharmingen) to identify the activated configuration of the platelet GPIIb–IIIa complex, and with PE anti-CD62P mAb (BD Pharmingen) to identify P-selectin expression. To examine adhesion protein expression under conditions of platelet activation, samples were stimulated with a final concentration of 1 mM of AA (Chrono-Log, Havertown, PA). AA is a substrate of the Table 1 Expected and measured blood glucose concentrations using an instant-read glucometer
Measured concentration (mg/dl)
Expected concentration of 100 mg/dl
Expected concentration of 200 mg/dl
Expected concentration of 300 mg/dl
84.5F3.3
181.3F4.64
285.3F5.54,y
4 P b .01 when compared to the expected concentration of 100 mg/dl. y P b .01 when compared to the expected concentration of 200 mg/dl.
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COX1 enzyme, which is irreversibly inhibited by aspirin. Flow cytometric data acquisition was performed using a FACSCalibur device (Becton Dickinson) after performing an instrument quality control procedure with BD CaliBRITE 3 beads. FACS data analysis was performed using the CellQuest Pro Software. From each sample, the percent positive (%Pos) cells and the mean channel of fluorescence (MCF) were obtained. The results were then expressed as total fluorescence intensity (TFI), where TFI=(%Pos/ 100)MCF. As previously described, TFI (units: %Channel) takes into account both the %Pos cells and MCF (McDonagh et al., 2003). To illustrate how the blood samples were evaluated using FACS, we show representative histograms for the expression of activated platelet GPIIb–IIIa (PAC-1) in Fig. 1. Fig. 1A represents the platelet adhesion protein expression in euglycemic blood before (2) and after (3) stimulation with AA. As can be seen, AA caused a marked rightward shift from (2) to (3), indicating increased PAC-1 expression.
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Fig. 2. The effect of hyperglycemia on inhibition of PAC-1 expression by aspirin. The inhibition of GPIIb–IIIa (PAC-1) expression in AA-stimulated platelets (1 mM) as a function of dextrose concentration (100, 200, 300, and 600 mg/dl). Two aspirin concentrations were evaluated in vitro: 65 and 200 Ag/ml (equivalent to in vivo doses of 325 and 1000 mg/day, respectively). The results are expressed as the percent inhibition of PAC-1 expression when the blood was incubated with aspirin compared to the same blood without aspirin. Data were compared using a two-way repeated measures ANOVA and a Holm–Sidak test. *Pb.01, aspirin versus no aspirin; #Pb.005, 200 mg/dl versus 100 mg/dl; zPb.01, 600 mg/dl versus 200 mg/dl.
Fig. 1B represents AA-stimulated platelets in euglycemic blood before (1) and after (2) incubation with 65 Ag/ml aspirin (equivalent dose of 325 mg/day). In this case, a downward and leftward shift is indicative of an inhibition of platelet PAC-1 expression by aspirin. Fig. 1C represents AA-stimulated platelets after preincubation with ASA (equivalent dose of 325 mg) in euglycemic (1) and hyperglycemic (2) blood. The inhibition of PAC-1 expression by aspirin was less pronounced in the hyperglycemic blood compared with euglycemic blood. 2.4. Statistical analysis
Fig. 1. Flow cytometry histograms representing platelet PAC-1 expression. (A) Negative control (1), baseline expression of platelet PAC-1 (2), and expression after stimulation with AA (1 mM) (3). There was an increase of PAC-1 expression due to AA stimulation, as demonstrated by a shift of the histogram from (2) to (3). (B) AA-stimulated platelets in euglycemic blood before (1) and after (2) in vitro incubation with 65 Ag/ml aspirin (equivalent to an in vivo dose of 325 mg/day). A leftward and downward shift of the histogram from (1) to (2) indicates that aspirin inhibited the expression of platelet PAC-1. (C) AA-stimulated platelets after incubation with ASA in euglycemic (100 mg/dl) (1) and hyperglycemic (300 mg/dl) (2) blood. Aspirin inhibition of PAC-1 expression was reduced in the hyperglycemic blood.
Data were expressed as meanFstandard error of the mean. Platelet adhesion molecule expressions before and after activation with AA were compared using a paired t test. Comparison between blood glucose concentrations was made using a t test or a Mann–Whitney Rank Sum Test. The effects of the different blood glucose concentrations and aspirin doses on platelet receptor expression and the interaction between blood glucose and aspirin were evaluated using a two-way repeated measures ANOVA. If the RMANOVA was significant, a Holm–Sidak test was performed (Sigma Stat 3.5). Pb.05 was considered statistically significant.
3. Results 1.
In euglycemic blood, AA markedly increases both platelet GPIIb–IIIa expression and P-selectin expression. Prior incubation with aspirin inhibits human platelet adhesion protein expression.
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2.
In the euglycemic blood, the baseline expression of platelet GPIIb–IIIa and P-selectin adhesion receptors was 59.6F8.2 and 4.4F0.7 TFI, respectively. AA is the COX1 substrate for synthesis for thromboxane, a potent platelet activator. After incubating whole blood with AA, a marked increase in platelet adhesion protein expression was observed. Platelet GPIIb–IIIa expression increased to 319.6F42.7 TFI ( P=.002) and platelet P-selectin expression increased to 179.5F38.5 TFI ( Pb.001). When the blood was pretreated in vitro with 65 Ag/ml ASA (equivalent to 325 mg in vivo dose), we observed a significant inhibition of AA-stimulated platelet adhesion protein expression for both GPIIb–IIIa (36.5%, Fig. 2) and Pselectin (81%, Fig. 3; Pb.005). Increasing the ASA dose to 200 Ag/ml (equivalent to a 1000-mg in vivo dose) also significantly inhibited AA-stimulated platelet expression for both markers ( Pb.005); however, no statistical difference was observed between the two doses. Acute hyperglycemia reduced the sensitivity of human platelets to aspirin. The inhibition of GPIIb–IIIa (PAC-1) expression by aspirin (65 Ag/ml) was impaired by hyperglycemia. When blood glucose concentration was twice normal (200 mg/dl), the inhibitory effect of ASA on PAC-1 expression was reduced by 84% ( Pb.005; Fig. 2). There was no further significant change in ASA inhibition between the 200- and 300-mg/dl glucose concentrations (82.5%, P=NS; Fig. 2). For P-selectin, there was an obvious but statistically insignificant 48% reduction of inhibition at 200 mg/dl plasma
glucose (Fig. 3). When the blood glucose concentration was three times normal (300 mg/dl), there was a 64% reduction of inhibition, which was not statistically significant (Fig. 3). When the blood glucose concentration was increased to 600 mg/dl, the inhibitory effect of aspirin was completely eliminated. The negative values of inhibition in both Figs. 2 and 3 indicate that, at 600 mg/dl glucose, the adhesion protein expression was actually increased in the presence of ASA. 3. Increasing the ASA dose attenuates the effect of stress hyperglycemia on platelet PAC-1 expression but not platelet P-selectin expression. The inhibition of both GPIIb–IIIa and P-selectin platelet expression by ASA at the increased concentration of 200 Ag/ml was also impaired by hyperglycemia in a dose-dependent manner. However, at 200 mg/dl plasma glucose, the increased ASA concentration partially reversed the effects of hyperglycemia on platelet GPIIb–IIIa expression when compared to the 65-Ag/ml concentration of aspirin ( Pb.01, Fig. 2). Also, the complete abolition of inhibition, observed at 600 mg/ml glucose, was somewhat blunted by increased ASA. There was no observed improvement in P-selectin inhibition with the increased ASA dose at any glucose concentration (Fig. 3). 4. The effects of glucose and aspirin concentrations on adhesion protein expression were interdependent. The inhibitory effect of aspirin was found to depend on the different blood glucose concentrations. A statistically significant interaction between glucose concentration and aspirin concentration was found ( Pb.001 for PAC-1 and P=.004 for P-selectin).
4. Discussion
Fig. 3. The effect of hyperglycemia on inhibition of P-selectin expression by aspirin. The inhibition of P-selectin expression in AA-stimulated platelets (1 mM) as a function of dextrose concentration (100, 200, 300, and 600 mg/dl). Two aspirin concentrations were evaluated in vitro, 65 and 200 Ag/ml (equivalent to in vivo doses of 325 and 1000 mg/day, respectively). The results are expressed as the percent inhibition of P-selectin expression when the blood was incubated with aspirin compared to the same blood without aspirin. Data were compared using a two-way repeated measures ANOVA and a Holm–Sidak test. *Pb.01, aspirin versus no aspirin.
The first comprehensive study to demonstrate the efficacy of antiplatelet therapy in preventing cardiovascular events was reported by the Antiplatelet Trialists’ Collaboration. Low-dose aspirin (75–325 mg) was found to be equal to or better than any other form of antiplatelet therapy, including high-dose aspirin (Antithrombotic Trialists’ Collaboration, 2002). Because diabetic patients have a two- to fourfold increase in the risk of dying from complications of cardiovascular disease, the use of aspirin is highly recommended. Prolonged aspirin therapy in patients with diabetes mellitus is associated with up to a 15% reduction in cardiovascular events and a 35% reduction of myocardial infarction. However, diabetic patients have been also occasionally reported to be less sensitive to aspirin, and such observations remain consistent with the reduced drug effectiveness noted in this disease. Results of the Primary Prevention Project indicated that nondiabetics had a 41% reduction in heart-disease-related death with aspirin versus
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10% in diabetic patients. The study authors suggested that the antiplatelet effects of aspirin in diabetic patients are overwhelmed by aspirin-insensitive mechanisms of platelet activation (Sacco et al., 2003). The term daspirin resistanceT has been used to describe a clinical situation in which a subgroup of patients on therapeutic doses of aspirin experience thrombotic vascular events. The concept of dclinical aspirin resistance,T or rather treatment failure, is based on the unlikely assumption that atherothrombosis can be controlled by using a single pharmaceutical agent. Several hypotheses have been proposed to explain the reduced response to aspirin by diabetic patients. Apart from noncompliance to treatment, which may be problematic, diabetic platelets are in a chronic activated state hypersensitive to agonists even at subthreshold concentrations (McDonagh et al., 2003). These platelets may become exhausted and consumed sooner, thus contributing to accelerated thrombopoiesis and release of fresh hyperactive platelets. Another theory to explain why diabetic patients are daspirin resistantT was proposed by Watala et al. In a series of studies, these investigators found that both glucose and aspirin compete to either glycosylate or acetylate unoccupied amino groups on platelet proteins. They suggest that occupation of these protein sites by aspirin modulates platelet activation and aggregation (Watala et al., 2004, 2005, 2006). This theory is attractive, but it is unclear if it can be applied to the current study. The nonenzymatic glycosylation studies of Watala et al. involved extended periods (days to weeks) of platelet incubation under hyperglycemic conditions. In our study, the blood samples were incubated with glucose for 60 min at 378C. If only 1 h for glycosylation is sufficient time to interfere with aspirin acetylation, then the theory of Watala et al. may apply to short-term or stress hyperglycemia as well as chronic hyperglycemia. Both PAC-1 (the activated form of GPIIb–IIIa) and P-selectin adhesion proteins are sensitive surface markers of platelet activation, which can be evaluated in samples of whole blood by flow cytometry. Previously, we reported a chronic expression of PAC-1 and P-selectin receptors in patients with type 2 diabetes and heart disease. Also, we found a significant reduction in PAC-1 expression in those diabetic patients with heart disease who were taking aspirin (McDonagh et al., 2003; Tuttle et al., 2003). In this study, we tested the hypothesis that in vitro, stress hyperglycemia would impair the inhibition of these adhesion proteins by aspirin in a concentration-dependent manner. In euglycemic blood, an ASA concentration of 65 Ag/ml (equivalent to an in vivo daily dose of 325 mg) was found to be as equally effective as a concentration of 200 Ag/ml, although a 100% inhibition of both platelet receptors was not achieved. This finding agrees with a report from Frelinger et al. (2006) who also measured PAC-1 and P-selectin expression by flow cytometry in in vitro aspirin-treated normal subjects. Using indomethacin (a COX1 and COX2 nonspecific inhibitor) instead of aspirin followed by activation with AA, they failed to block AA-induced platelet activation. These
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findings suggest that residual AA-induced platelet activation occurs via a COX1- and COX2-independent pathway. Both diabetic patients and surgical coronary artery patients have been reported to be less sensitive to aspirin. Platelets from a type 2 diabetic animal model (Watala et al., 2006) and type 2 diabetic patients (Watala et al., 2004) have reduced sensitivity to aspirin because of increased protein glycation decreasing aspirin-mediated protein acetylation (Watala et al., 2005) and increased blood plasma hydrolysis of the drug (Gresner et al., 2006). In patients undergoing coronary artery bypass grafting (CABG), hyperglycemia occurs during the perioperative period, including nondiabetic patients (Doenst et al., 2005), and it has been reported that aspirin resistance occurs within the first 10 days after surgery (Golanski et al., 2005; Zimmermann et al., 2003). Until this study, no link has been reported between perioperative hyperglycemia and the incidence of aspirin resistance. Stress hyperglycemia, that is, short-term elevated glucose concentration, has been reported to be responsible for increased cardiovascular morbidity and mortality in diabetic and CABG patients (Capes, Hunt, Malmberg, & Gerstin Hertzel, 2000; Doenst et al., 2005). In this study, we demonstrated that the inhibitory effect of aspirin, at two doses, depends on the blood glucose concentration. Keating et al. (2003) reported an increase in platelet GPIIb–IIIa and Pselectin expression with in vitro incremental concentrations of glucose (500 and 1000 mg/dl) in healthy subjects not taking aspirin. We found, under more clinically relevant hyperglycemic conditions, that the inhibitory effect of aspirin was impaired. Platelet adhesion and activation are initiated by multiple processes. Specific receptors are expressed on the platelet membrane for a variety of activators including thrombin, ADP, and TXA2. These activators are produced or released from the platelet when adhesion has been initiated. They commonly act via heterometric guanine nucleotide-binding proteins (or G proteins). TXA2 is a potent activator of platelets via its specific TXA2 receptor (or TP). The action of TXA2 is locally restricted because of its short life. TP couples to Gq and G12/G13, resulting in outside-in signaling, which includes activation of protein kinase C and elevation of free cytoplasmic [Ca2+]. In addition, inside-out signaling leads to activation of the intracellular conformation of GPIIb–IIIa and platelet degranulation (dense and a granules; Offermanns, 2006). These pathways should be inhibited by aspirin due to the irreversible blockade of TXA2 synthesis. In this study, whole blood was treated with aspirin after incubation with glucose. Thus, blocking the AA (or TXA2) pathway with aspirin, even at the elevated dose tested, was not sufficient to fully inhibit both platelet adhesion receptors. From our previous study, diabetic platelets are chronically activated and capable of an enhanced response to an acute stimulus, such as platelet activating factor (McDonagh et al., 2003; Tuttle et al., 2003). Other investigators have also observed a priming or enhanced response with ADP and thrombin (Keating et al., 2003; Winocour, 1994). ADP activates
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platelets via its receptors P2Y1 and P2Y12, coupled with Gq and Gi proteins. Activation of the G protein receptors results in platelet degranulation and intracellular activation of GPIIb–IIIa (Offermanns, 2006). Clopidogrel is known to incompletely block ADP-induced platelet activation via the P2Y12 receptor (Patrono, Coller, FitzGerald, Hirsh, & Roth, 2004). Frelinger et al. (2006) demonstrated that patients treated with both clopidogrel and aspirin had significantly reduced AA-induced platelet activation compared to patients receiving aspirin alone. This result highlights an ADPdependent pathway of residual AA-induced platelet activation in aspirin-treated patients. However, the inhibition was partial, perhaps due to an incomplete inhibition of the ADP pathway by clopidogrel or, again, due to other activation pathways. In conclusion, we found that the expression of two platelet adhesion proteins, involved in aggregation and cell– cell interactions, is dependent on both the dose of aspirin and the concentration of blood glucose. Under the conditions of acute or chronic stress, it is known that inflammation contributes to a procoagulant condition, which may affect platelet function (Tuttle, Davis-Gorman, Goldman, Copeland, & McDonagh, 2004). Our results suggest that stress hyperglycemia, observed in diabetes and after onpump heart surgery, may also explain, in part, the transient lack of response of patients to aspirin. Acknowledgments This work was supported by NIH HLB 58859 and The Hudson/Lovaas Endowment Fund. References Antithrombotic Trialists’ Collabroation. (2002). Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. British Medical Journal, 324, 71 – 86. Capes, S. E., Hunt, D., Malmberg, K., & Gerstin Hertzel, C. (2000). Stress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: A systematic overview. Lancet, 355, 773 – 778. Colwell, J. A. (1997). Aspirin therapy in diabetes. Diabetes Care, 20, 1767 – 1771. Doenst, T., Wijeysundera, D., Karkouti, K., Zechner, C., Maganti, M., Rao, V., & Borger, M. A. (2005). Hyperglycemia during cardiopulmonary bypass is an independent risk factor for mortality in patients undergoing cardiac surgery. Journal of Thoracic and Cardiovascular Surgery, 130, 1144 – 1150. Frelinger, A. L., Furman, M. I., Linden, M. D., Li, Y., Fox, M. L., Barnard, M. R., & Michelson, A. D. (2006). Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclo-
oxygenase-1- and cyclooxygenase-2-independent pathway: A 700patient study of aspirin resistance. Circulation, 113, 2888 – 2896. Golanski, J., Chlopicki, S., Golanski, R., Gresner, P., Iwaszkiewicz, A., & Watala, C. (2005). Resistance to aspirin in patients after coronary artery bypass grafting is transient. Impact on the monitoring of aspirin antiplatelet therapy. Therapeutic Drug Monitoring, 27, 484 – 490. Golanski, J., Nocun, M., Rozalski, M., Drygas, W., & Watala, C. (2004). An in vitro model for the detection of reduced platelet sensitivity to acetylsalicylic acid. Blood Coagulation and Fibrinolysis, 15, 187 – 195. Gresner, P., Dolnik, M., Waczulikova, I., Bryszewska, M., Sikurova, L., & Watala, C. (2006). Increased blood plasma hydrolysis of acetylsalicylic acid in type 2 diabetic patients: A role of plasma esterases. Biochimica et Biophysica Acta, 1760, 207 – 215. Keating, F. K., Sobel, B. E., & Schneider, D. J. (2003). Effects of increased concentrations of glucose on platelet reactivity in healthy subjects and in patients with and without diabetes mellitus. American Journal of Cardiology, 92, 1362 – 1365. Marcus, A. J. (1990). Eicosanoid interactions between platelets, endothelial cells, and neutrophils. Methods in Enzymology, 187, 585 – 599. McDonagh, P. F., Hokama, J. Y., Gale, S. C., Logan, J. J., Davis-Gorman, G., Goldman, S., & Copeland, J. G. (2003). Chronic expression of platelet adhesion proteins is associated with severe ischemic heart disease in type 2 diabetic patients. Chronic platelet activation in diabetic patients. Journal of Diabetes and its Complications, 17, 269 – 278. Offermanns, S. (2006). Activation of platelet function through G proteincoupled receptors. Circulation Research, 99, 1293 – 1304. Patrono, C., Coller, B., FitzGerald, G. A., Hirsh, J., & Roth, G. (2004). Platelet-active drugs: The relationships among dose, effectiveness, and side effects: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest, 126 (3 Suppl.), 234S – 264S. Sacco, M., Pellegrini, F., Roncaglioni, M. C., Avanzini, F., Tognoni, G., on behalf of the PPP Collaborative Group. (2003). Primary prevention of cardiovascular events with low-dose aspirin and vitamin E in type 2 diabetic patients. Diabetes Care, 26, 3264 – 3272. Tuttle, H., Davis-Gorman, G., Goldman, S., Copeland, J., & McDonagh, P. (2003). Platelet–neutrophil conjugate formation is increased in diabetic women with cardiovascular disease. Cardiovascular Diabetology, 2, 12. Tuttle, H. A., Davis-Gorman, G., Goldman, S., Copeland, J. G., & McDonagh, P. F. (2004). Proinflammatory cytokines are increased in type 2 diabetic women with cardiovascular disease. Journal of Diabetes and its Complications, 18, 343 – 351. Watala, C., Golanski, J., Pluta, J., Boncler, M., Rozalski, M., Luzak, B., Kropiwnicka, A., & Drzewoski, J. (2004). Reduced sensitivity of platelets from type 2 diabetic patients to acetylsalicylic acid (aspirin)— Its relation to metabolic control. Thrombosis Research, 113, 101 – 113. Watala, C., Pluta, J., Golanski, J., Rozalski, M., Czyz, M., Trojanowski, Z., & Drzewoski, J. (2005). Increased protein glycation in diabetes mellitus is associated with decreased aspirin-mediated protein acetylation and reduced sensitivity of blood platelets to aspirin. Journal of Molecular Medicine, 83, 148 – 158. Watala, C., Ulicna, O., Golanski, J., Nocun, M., Waczulı´kova´, I., Markuszewski, L., & Drzewoski, J. (2006). High glucose contributes to aspirin insensitivity in streptozotocin-diabetic rats: A multiparametric aggregation study. Blood Coagulation and Fibrinolysis, 17, 113 – 124. Winocour, P. D. (1994). Platelets, vascular disease, and diabetes mellitus. Canadian Journal of Physiology and Pharmacology, 72, 295 – 303. Zimmermann, N., Wenk, A., Kim, U., Kienzle, P., Weber, A. -A., Gams, E., Schrfr, K., & Hohlfeld, T. (2003). Functional and biochemical evaluation of platelet aspirin resistance after coronary artery bypass surgery. Circulation, 108, 542 – 547.
Inhibition of platelet GPIIb–IIIa and P-selectin expression by aspirin is impaired by stress hyperglycemia Alexandre Le Guyadera,b, Garrett Pachecoa, Norma Seavera, Grace Davis-Gormana, Jack Copelanda, Paul F. McDonagha,4 a
Cardiovascular and Thoracic Surgery and the Sarver Heart Center, University of Arizona Health Sciences Center, Tucson, AZ 85724, USA b Thoracic and Cardiovascular Surgery Department, Dupuytren University Hospital, University of Limoges, 87042 Limoges, France Received 14 March 2007; received in revised form 24 May 2007; accepted 1 June 2007
Abstract Increased aspirin resistance may contribute to the increase in thrombotic events observed in patients with type 2 diabetes. In this study, we examined if acute exposure to increased plasma glucose impaired the inhibitory effects of aspirin on platelet activation. Whole-blood samples were incubated with 100 (euglycemia), 200, 300, and 600 mg/dl glucose followed by incubation with aspirin [acetylsalicylic acid (ASA)]. Using flow cytometry, GPIIb–IIIa and P-selectin were analyzed in unstimulated and arachidonic acid (AA)-stimulated platelets. In euglycemic blood, AA caused a significant increase in platelet GPIIb–IIIa expression [unstimulated: 59.5F8.2 total fluorescence intensity (TFI), AA stimulated: 319.6F42.7 TFI, P=.002] and P-selectin (4.4F0.7 and 179.5F38.5 TFI, Pb.001). In vitro, ASA significantly inhibited both GPIIb–IIIa expression (36.5%) and P-selectin expression (81%; Pb.005). However, increased blood glucose (200 mg/dl) significantly impaired the inhibitory effect of ASA (84% for GPIIb–IIIa, Pb.005; 48% for P-selectin, P=NS). Increasing glucose to 600 mg/dl completely overwhelmed the inhibitory effect of ASA. A statistically significant interaction between glucose concentration and ASA dose was found ( Pb.001 for GPIIb–IIIa and P=.004 for P-selectin). In vitro, concentration-dependent stress hyperglycemia significantly impaired the inhibitory effects of aspirin on human platelet GPIIb–IIIa and P-selectin expression. Under acute hyperglycemic conditions, the effectiveness of ASA to inhibit platelets via the AA-activation pathway may be significantly reduced. D 2009 Elsevier Inc. All rights reserved. Keywords: Diabetes; Hyperglycemia; Platelets; Aspirin; Flow cytometry
1. Introduction Aspirin [acetylsalicylic acid (ASA)] is the most widely prescribed drug in the world with indications ranging from fever to thrombosis. Since 1997, the American Diabetes Association has recommended that low-dose aspirin be used for both primary and secondary prevention of cardiovascular disease in patients with diabetes (Colwell, 1997). However, several studies conclude that platelets from diabetic patients are less sensitive to aspirin, and these observations are consistent with the reduced effectiveness of ASA in
4 Corresponding author. Tel.: +1 520 626 2329; fax: +1 520 626 4042. E-mail address: [email protected] (P.F. McDonagh). 1056-8727/09/$ – see front matter D 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jdiacomp.2007.06.003
diabetes. Results of the Primary Prevention Project indicate that nondiabetics had a 41% reduction in heart-diseaserelated death with aspirin compared to only 10% in diabetic patients. The study authors suggested that the antiplatelet effects of aspirin in diabetic patients are overwhelmed by aspirin-insensitive mechanisms of platelet activation (Sacco et al., 2003). The term daspirin resistanceT has been used to describe a clinical situation in which a subgroup of patients on therapeutic doses of aspirin experiences thrombotic vascular events. Several hypotheses have been proposed to explain the apparent lack of response of diabetic patients to aspirin. In a laboratory study using the streptozotocin rat model of type 1 diabetes, Watala et al. (2006) concluded that the nonenzymatic glycosylation of biomolecules may compete with aspirin in the attachment to unoccupied
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amino groups on blood platelet proteins. Another possibility is that diabetic platelets may be activated by pathways independent of arachidonic acid (AA). Thromboxane A2 (TXA2) is an eicosanoid produced from AA by the cyclooxygenase 1 (COX1) enzyme. COX1 is irreversibly inhibited by aspirin. TXA2 binds to Gprotein-coupled receptors to generate inside-out signals, leading to activation of the fibrinogen receptor and exocytosis of intracellular granules (Offermanns, 2006). Upon exposure to thrombin, adenosine diphosphate (ADP), or TXA2, the platelet adhesion protein GPIIb–IIIa undergoes a conformational change that allows fibrinogen to bind, promoting aggregation. The adhesion protein P-selectin is stored in the a granules of resting platelets and is expressed on the cell surface upon activation. As a key adhesion molecule, it is involved in platelet–leukocyte conjugate formation. Frelinger et al. (2006) and Zimmermann et al. (2003) report that the expression of both adhesion proteins is inhibited by aspirin. GPIIb–IIIa expression and P-selectin expression are sensitive indicators of platelet activation, which can be evaluated in whole blood by flow cytometry. Previously, we utilized these markers to assess chronic platelet function in type 2 diabetic patients with cardiac conditions. We found a significant chronic increase in P-selectin in the diabetic patients with heart disease compared to their nondiabetic counterparts. We also found a significant chronic increase in GPIIb–IIIa expression in the diabetic patients when the findings were stratified for aspirin usage (McDonagh et al., 2003; Tuttle, Davis-Gorman, Goldman, Copeland, & McDonagh, 2003). The aim of this study was to determine if a more acute stress, in the form of shortterm hyperglycemia, affected the ability of aspirin to attenuate platelet activation. We evaluated two doses of aspirin in vitro, one similar to the dose currently used in clinical practice and a greater dose as a test of a possible dose–response effect.
2. Materials and methods 2.1. Population Eleven healthy volunteers (29F2 years) were recruited after informed consent was obtained. The study was approved by our institutional review board. No subjects had a reported history of diabetes, cardiovascular disease, or platelet abnormalities. No subjects had taken aspirin or any drugs interfering with platelet function within the past 10 days. 2.2. Study design To assess the effect of acute hyperglycemia on platelet adhesion protein expression, we incubated aliquots of whole blood for 60 min at 378C with incremental concentrations of
dextrose (Sigma-Aldrich, St. Louis, MO) to achieve approximate concentrations of 100 (euglycemia), 200, 300, and 600 mg/dl (Keating, Sobel, & Schneider, 2003). Blood glucose concentrations of two and three times normal were evaluated to determine the impact of uncontrolled blood glucose, which may occur in diabetic patients or soon after heart surgery in cardiovascular patients. The unphysiologic blood glucose (six times normal) was tested to determine if this abnormal concentration impaired the sensitivity of platelets to aspirin in a concentration–response manner. Blood glucose concentrations in all samples were measured using an instant-read glucometer (Accu-Chek Active, Roche Diagnostics, Indianapolis, IN), and the results are given in Table 1. To determine if aspirin inhibited the AA-induced expression of platelet adhesion proteins, we incubated the aliquots of euglycemic and hyperglycemic whole blood described above with aspirin (ASA, Sigma-Aldrich) for 10 min at 378C as previously described (Golanski, Nocun, Rozalski, Drygas, & Watala, 2004; Marcus, 1990). Two concentrations of aspirin were chosen, 65 or 200 Ag/ml, based on the earlier reports of Golanski et al. (2004). The final concentration of 65 Ag/ml corresponds to an equivalent dose of 325 mg/day, a dose recommended for diabetic patients. The 200 Ag/ml concentration corresponds to a dose of 1000 mg/day. This larger dose was used to test the hypothesis that more aspirin would override any measured insensitivity to aspirin. 2.3. Flow cytometry of platelet adhesion protein expression The protocol for platelet labeling in whole human blood was described elsewhere (McDonagh et al., 2003). Blood samples were collected in sodium citrate vacutainers (BD Biosciences) after discarding the first 3 ml. The blood was mixed with PerCP-conjugated anti-CD61 mAb (BD Pharmingen) to identify the platelet population of cells, with FITC PAC-1 mAb (BD Pharmingen) to identify the activated configuration of the platelet GPIIb–IIIa complex, and with PE anti-CD62P mAb (BD Pharmingen) to identify P-selectin expression. To examine adhesion protein expression under conditions of platelet activation, samples were stimulated with a final concentration of 1 mM of AA (Chrono-Log, Havertown, PA). AA is a substrate of the Table 1 Expected and measured blood glucose concentrations using an instant-read glucometer
Measured concentration (mg/dl)
Expected concentration of 100 mg/dl
Expected concentration of 200 mg/dl
Expected concentration of 300 mg/dl
84.5F3.3
181.3F4.64
285.3F5.54,y
4 P b .01 when compared to the expected concentration of 100 mg/dl. y P b .01 when compared to the expected concentration of 200 mg/dl.
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COX1 enzyme, which is irreversibly inhibited by aspirin. Flow cytometric data acquisition was performed using a FACSCalibur device (Becton Dickinson) after performing an instrument quality control procedure with BD CaliBRITE 3 beads. FACS data analysis was performed using the CellQuest Pro Software. From each sample, the percent positive (%Pos) cells and the mean channel of fluorescence (MCF) were obtained. The results were then expressed as total fluorescence intensity (TFI), where TFI=(%Pos/ 100)MCF. As previously described, TFI (units: %Channel) takes into account both the %Pos cells and MCF (McDonagh et al., 2003). To illustrate how the blood samples were evaluated using FACS, we show representative histograms for the expression of activated platelet GPIIb–IIIa (PAC-1) in Fig. 1. Fig. 1A represents the platelet adhesion protein expression in euglycemic blood before (2) and after (3) stimulation with AA. As can be seen, AA caused a marked rightward shift from (2) to (3), indicating increased PAC-1 expression.
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Fig. 2. The effect of hyperglycemia on inhibition of PAC-1 expression by aspirin. The inhibition of GPIIb–IIIa (PAC-1) expression in AA-stimulated platelets (1 mM) as a function of dextrose concentration (100, 200, 300, and 600 mg/dl). Two aspirin concentrations were evaluated in vitro: 65 and 200 Ag/ml (equivalent to in vivo doses of 325 and 1000 mg/day, respectively). The results are expressed as the percent inhibition of PAC-1 expression when the blood was incubated with aspirin compared to the same blood without aspirin. Data were compared using a two-way repeated measures ANOVA and a Holm–Sidak test. *Pb.01, aspirin versus no aspirin; #Pb.005, 200 mg/dl versus 100 mg/dl; zPb.01, 600 mg/dl versus 200 mg/dl.
Fig. 1B represents AA-stimulated platelets in euglycemic blood before (1) and after (2) incubation with 65 Ag/ml aspirin (equivalent dose of 325 mg/day). In this case, a downward and leftward shift is indicative of an inhibition of platelet PAC-1 expression by aspirin. Fig. 1C represents AA-stimulated platelets after preincubation with ASA (equivalent dose of 325 mg) in euglycemic (1) and hyperglycemic (2) blood. The inhibition of PAC-1 expression by aspirin was less pronounced in the hyperglycemic blood compared with euglycemic blood. 2.4. Statistical analysis
Fig. 1. Flow cytometry histograms representing platelet PAC-1 expression. (A) Negative control (1), baseline expression of platelet PAC-1 (2), and expression after stimulation with AA (1 mM) (3). There was an increase of PAC-1 expression due to AA stimulation, as demonstrated by a shift of the histogram from (2) to (3). (B) AA-stimulated platelets in euglycemic blood before (1) and after (2) in vitro incubation with 65 Ag/ml aspirin (equivalent to an in vivo dose of 325 mg/day). A leftward and downward shift of the histogram from (1) to (2) indicates that aspirin inhibited the expression of platelet PAC-1. (C) AA-stimulated platelets after incubation with ASA in euglycemic (100 mg/dl) (1) and hyperglycemic (300 mg/dl) (2) blood. Aspirin inhibition of PAC-1 expression was reduced in the hyperglycemic blood.
Data were expressed as meanFstandard error of the mean. Platelet adhesion molecule expressions before and after activation with AA were compared using a paired t test. Comparison between blood glucose concentrations was made using a t test or a Mann–Whitney Rank Sum Test. The effects of the different blood glucose concentrations and aspirin doses on platelet receptor expression and the interaction between blood glucose and aspirin were evaluated using a two-way repeated measures ANOVA. If the RMANOVA was significant, a Holm–Sidak test was performed (Sigma Stat 3.5). Pb.05 was considered statistically significant.
3. Results 1.
In euglycemic blood, AA markedly increases both platelet GPIIb–IIIa expression and P-selectin expression. Prior incubation with aspirin inhibits human platelet adhesion protein expression.
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2.
In the euglycemic blood, the baseline expression of platelet GPIIb–IIIa and P-selectin adhesion receptors was 59.6F8.2 and 4.4F0.7 TFI, respectively. AA is the COX1 substrate for synthesis for thromboxane, a potent platelet activator. After incubating whole blood with AA, a marked increase in platelet adhesion protein expression was observed. Platelet GPIIb–IIIa expression increased to 319.6F42.7 TFI ( P=.002) and platelet P-selectin expression increased to 179.5F38.5 TFI ( Pb.001). When the blood was pretreated in vitro with 65 Ag/ml ASA (equivalent to 325 mg in vivo dose), we observed a significant inhibition of AA-stimulated platelet adhesion protein expression for both GPIIb–IIIa (36.5%, Fig. 2) and Pselectin (81%, Fig. 3; Pb.005). Increasing the ASA dose to 200 Ag/ml (equivalent to a 1000-mg in vivo dose) also significantly inhibited AA-stimulated platelet expression for both markers ( Pb.005); however, no statistical difference was observed between the two doses. Acute hyperglycemia reduced the sensitivity of human platelets to aspirin. The inhibition of GPIIb–IIIa (PAC-1) expression by aspirin (65 Ag/ml) was impaired by hyperglycemia. When blood glucose concentration was twice normal (200 mg/dl), the inhibitory effect of ASA on PAC-1 expression was reduced by 84% ( Pb.005; Fig. 2). There was no further significant change in ASA inhibition between the 200- and 300-mg/dl glucose concentrations (82.5%, P=NS; Fig. 2). For P-selectin, there was an obvious but statistically insignificant 48% reduction of inhibition at 200 mg/dl plasma
glucose (Fig. 3). When the blood glucose concentration was three times normal (300 mg/dl), there was a 64% reduction of inhibition, which was not statistically significant (Fig. 3). When the blood glucose concentration was increased to 600 mg/dl, the inhibitory effect of aspirin was completely eliminated. The negative values of inhibition in both Figs. 2 and 3 indicate that, at 600 mg/dl glucose, the adhesion protein expression was actually increased in the presence of ASA. 3. Increasing the ASA dose attenuates the effect of stress hyperglycemia on platelet PAC-1 expression but not platelet P-selectin expression. The inhibition of both GPIIb–IIIa and P-selectin platelet expression by ASA at the increased concentration of 200 Ag/ml was also impaired by hyperglycemia in a dose-dependent manner. However, at 200 mg/dl plasma glucose, the increased ASA concentration partially reversed the effects of hyperglycemia on platelet GPIIb–IIIa expression when compared to the 65-Ag/ml concentration of aspirin ( Pb.01, Fig. 2). Also, the complete abolition of inhibition, observed at 600 mg/ml glucose, was somewhat blunted by increased ASA. There was no observed improvement in P-selectin inhibition with the increased ASA dose at any glucose concentration (Fig. 3). 4. The effects of glucose and aspirin concentrations on adhesion protein expression were interdependent. The inhibitory effect of aspirin was found to depend on the different blood glucose concentrations. A statistically significant interaction between glucose concentration and aspirin concentration was found ( Pb.001 for PAC-1 and P=.004 for P-selectin).
4. Discussion
Fig. 3. The effect of hyperglycemia on inhibition of P-selectin expression by aspirin. The inhibition of P-selectin expression in AA-stimulated platelets (1 mM) as a function of dextrose concentration (100, 200, 300, and 600 mg/dl). Two aspirin concentrations were evaluated in vitro, 65 and 200 Ag/ml (equivalent to in vivo doses of 325 and 1000 mg/day, respectively). The results are expressed as the percent inhibition of P-selectin expression when the blood was incubated with aspirin compared to the same blood without aspirin. Data were compared using a two-way repeated measures ANOVA and a Holm–Sidak test. *Pb.01, aspirin versus no aspirin.
The first comprehensive study to demonstrate the efficacy of antiplatelet therapy in preventing cardiovascular events was reported by the Antiplatelet Trialists’ Collaboration. Low-dose aspirin (75–325 mg) was found to be equal to or better than any other form of antiplatelet therapy, including high-dose aspirin (Antithrombotic Trialists’ Collaboration, 2002). Because diabetic patients have a two- to fourfold increase in the risk of dying from complications of cardiovascular disease, the use of aspirin is highly recommended. Prolonged aspirin therapy in patients with diabetes mellitus is associated with up to a 15% reduction in cardiovascular events and a 35% reduction of myocardial infarction. However, diabetic patients have been also occasionally reported to be less sensitive to aspirin, and such observations remain consistent with the reduced drug effectiveness noted in this disease. Results of the Primary Prevention Project indicated that nondiabetics had a 41% reduction in heart-disease-related death with aspirin versus
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10% in diabetic patients. The study authors suggested that the antiplatelet effects of aspirin in diabetic patients are overwhelmed by aspirin-insensitive mechanisms of platelet activation (Sacco et al., 2003). The term daspirin resistanceT has been used to describe a clinical situation in which a subgroup of patients on therapeutic doses of aspirin experience thrombotic vascular events. The concept of dclinical aspirin resistance,T or rather treatment failure, is based on the unlikely assumption that atherothrombosis can be controlled by using a single pharmaceutical agent. Several hypotheses have been proposed to explain the reduced response to aspirin by diabetic patients. Apart from noncompliance to treatment, which may be problematic, diabetic platelets are in a chronic activated state hypersensitive to agonists even at subthreshold concentrations (McDonagh et al., 2003). These platelets may become exhausted and consumed sooner, thus contributing to accelerated thrombopoiesis and release of fresh hyperactive platelets. Another theory to explain why diabetic patients are daspirin resistantT was proposed by Watala et al. In a series of studies, these investigators found that both glucose and aspirin compete to either glycosylate or acetylate unoccupied amino groups on platelet proteins. They suggest that occupation of these protein sites by aspirin modulates platelet activation and aggregation (Watala et al., 2004, 2005, 2006). This theory is attractive, but it is unclear if it can be applied to the current study. The nonenzymatic glycosylation studies of Watala et al. involved extended periods (days to weeks) of platelet incubation under hyperglycemic conditions. In our study, the blood samples were incubated with glucose for 60 min at 378C. If only 1 h for glycosylation is sufficient time to interfere with aspirin acetylation, then the theory of Watala et al. may apply to short-term or stress hyperglycemia as well as chronic hyperglycemia. Both PAC-1 (the activated form of GPIIb–IIIa) and P-selectin adhesion proteins are sensitive surface markers of platelet activation, which can be evaluated in samples of whole blood by flow cytometry. Previously, we reported a chronic expression of PAC-1 and P-selectin receptors in patients with type 2 diabetes and heart disease. Also, we found a significant reduction in PAC-1 expression in those diabetic patients with heart disease who were taking aspirin (McDonagh et al., 2003; Tuttle et al., 2003). In this study, we tested the hypothesis that in vitro, stress hyperglycemia would impair the inhibition of these adhesion proteins by aspirin in a concentration-dependent manner. In euglycemic blood, an ASA concentration of 65 Ag/ml (equivalent to an in vivo daily dose of 325 mg) was found to be as equally effective as a concentration of 200 Ag/ml, although a 100% inhibition of both platelet receptors was not achieved. This finding agrees with a report from Frelinger et al. (2006) who also measured PAC-1 and P-selectin expression by flow cytometry in in vitro aspirin-treated normal subjects. Using indomethacin (a COX1 and COX2 nonspecific inhibitor) instead of aspirin followed by activation with AA, they failed to block AA-induced platelet activation. These
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findings suggest that residual AA-induced platelet activation occurs via a COX1- and COX2-independent pathway. Both diabetic patients and surgical coronary artery patients have been reported to be less sensitive to aspirin. Platelets from a type 2 diabetic animal model (Watala et al., 2006) and type 2 diabetic patients (Watala et al., 2004) have reduced sensitivity to aspirin because of increased protein glycation decreasing aspirin-mediated protein acetylation (Watala et al., 2005) and increased blood plasma hydrolysis of the drug (Gresner et al., 2006). In patients undergoing coronary artery bypass grafting (CABG), hyperglycemia occurs during the perioperative period, including nondiabetic patients (Doenst et al., 2005), and it has been reported that aspirin resistance occurs within the first 10 days after surgery (Golanski et al., 2005; Zimmermann et al., 2003). Until this study, no link has been reported between perioperative hyperglycemia and the incidence of aspirin resistance. Stress hyperglycemia, that is, short-term elevated glucose concentration, has been reported to be responsible for increased cardiovascular morbidity and mortality in diabetic and CABG patients (Capes, Hunt, Malmberg, & Gerstin Hertzel, 2000; Doenst et al., 2005). In this study, we demonstrated that the inhibitory effect of aspirin, at two doses, depends on the blood glucose concentration. Keating et al. (2003) reported an increase in platelet GPIIb–IIIa and Pselectin expression with in vitro incremental concentrations of glucose (500 and 1000 mg/dl) in healthy subjects not taking aspirin. We found, under more clinically relevant hyperglycemic conditions, that the inhibitory effect of aspirin was impaired. Platelet adhesion and activation are initiated by multiple processes. Specific receptors are expressed on the platelet membrane for a variety of activators including thrombin, ADP, and TXA2. These activators are produced or released from the platelet when adhesion has been initiated. They commonly act via heterometric guanine nucleotide-binding proteins (or G proteins). TXA2 is a potent activator of platelets via its specific TXA2 receptor (or TP). The action of TXA2 is locally restricted because of its short life. TP couples to Gq and G12/G13, resulting in outside-in signaling, which includes activation of protein kinase C and elevation of free cytoplasmic [Ca2+]. In addition, inside-out signaling leads to activation of the intracellular conformation of GPIIb–IIIa and platelet degranulation (dense and a granules; Offermanns, 2006). These pathways should be inhibited by aspirin due to the irreversible blockade of TXA2 synthesis. In this study, whole blood was treated with aspirin after incubation with glucose. Thus, blocking the AA (or TXA2) pathway with aspirin, even at the elevated dose tested, was not sufficient to fully inhibit both platelet adhesion receptors. From our previous study, diabetic platelets are chronically activated and capable of an enhanced response to an acute stimulus, such as platelet activating factor (McDonagh et al., 2003; Tuttle et al., 2003). Other investigators have also observed a priming or enhanced response with ADP and thrombin (Keating et al., 2003; Winocour, 1994). ADP activates
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platelets via its receptors P2Y1 and P2Y12, coupled with Gq and Gi proteins. Activation of the G protein receptors results in platelet degranulation and intracellular activation of GPIIb–IIIa (Offermanns, 2006). Clopidogrel is known to incompletely block ADP-induced platelet activation via the P2Y12 receptor (Patrono, Coller, FitzGerald, Hirsh, & Roth, 2004). Frelinger et al. (2006) demonstrated that patients treated with both clopidogrel and aspirin had significantly reduced AA-induced platelet activation compared to patients receiving aspirin alone. This result highlights an ADPdependent pathway of residual AA-induced platelet activation in aspirin-treated patients. However, the inhibition was partial, perhaps due to an incomplete inhibition of the ADP pathway by clopidogrel or, again, due to other activation pathways. In conclusion, we found that the expression of two platelet adhesion proteins, involved in aggregation and cell– cell interactions, is dependent on both the dose of aspirin and the concentration of blood glucose. Under the conditions of acute or chronic stress, it is known that inflammation contributes to a procoagulant condition, which may affect platelet function (Tuttle, Davis-Gorman, Goldman, Copeland, & McDonagh, 2004). Our results suggest that stress hyperglycemia, observed in diabetes and after onpump heart surgery, may also explain, in part, the transient lack of response of patients to aspirin. Acknowledgments This work was supported by NIH HLB 58859 and The Hudson/Lovaas Endowment Fund. References Antithrombotic Trialists’ Collabroation. (2002). Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. British Medical Journal, 324, 71 – 86. Capes, S. E., Hunt, D., Malmberg, K., & Gerstin Hertzel, C. (2000). Stress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: A systematic overview. Lancet, 355, 773 – 778. Colwell, J. A. (1997). Aspirin therapy in diabetes. Diabetes Care, 20, 1767 – 1771. Doenst, T., Wijeysundera, D., Karkouti, K., Zechner, C., Maganti, M., Rao, V., & Borger, M. A. (2005). Hyperglycemia during cardiopulmonary bypass is an independent risk factor for mortality in patients undergoing cardiac surgery. Journal of Thoracic and Cardiovascular Surgery, 130, 1144 – 1150. Frelinger, A. L., Furman, M. I., Linden, M. D., Li, Y., Fox, M. L., Barnard, M. R., & Michelson, A. D. (2006). Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclo-
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