- Email: [email protected]
Effects of increased concentrations of glucose on platelet reactivity in healthy subjects and in patients with and without diabetes mellitus
12. Scriver CR, Nowacki PM, Lehvaslaiho H, and the Working Group. Guide-
lines and recommendations for content, structure, and deployment of mutation database: II. Journey in progress. Hum Mutat 2000;15:13–15. 13. Palm T, Graboski S, Hitchcock-DeGregori SE, Greenfield NJ. Diseasecausing mutations in cardiac troponin T: identification of a critical tropomyosinbinding region. Biophys J 2001;81:2827–2837. 14. Anan R, Shono H, Kisanuki A, Arima S, Nakao S, Tanaka H. Patients with familial hypertrophic cardiomyopaty caused by a Phe110Ile missense mutation in the cardiac troponin T gene have variable cardiac morphologies and a favorable prognosis. Circulation 1998;98:391–397. 15. Lin TL, Ichihara S, Yamada Y, Nagasaka T, Ishihara H, Nakashima N, Yokota M. Phenotypic variation of familial hypertrophic cardiomyopathy caused by the Phe110Ile mutation in cardiac troponin T. Cardiology 2000;93:155–162. 16. Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, Seidman CE. ␣-Tropomyosin and cardiac troponin T mutations
cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell 1994;77:701–712. 17. Moolman JC, Corfield VA, Posen B, Posen B, Ngumbela K, Seidman C, Brink PA, Watkins H. Sudden death due to troponin T mutations. J Am Coll Cardiol 1997;29:549 –555. 18. Van Driest SL, Ackerman MJ, Ommen SR, Shakur R, Will ML, Nishimura RA, Tajik AJ, Gersh BJ. Prevalence and severity of “benign” mutations in the beta-myosin heavy chain, cardiac troponin T and alfa-tropomyosin genes in hypertrophic cardiomyopathy. Circulation 2002;106:3085–3090. 19. Ho CY, Lever HM, DeSanctis R, Farver CF, Seidman JG, Seidman CE. Homozygous mutation in cardiac troponin T. Implications for hypertrophic cardiomyopathy. Circulation 2000;102:1950 –1955. 20. Harada K, Yanaga FT, Minakami R, Morimoto S, Ohtsuki I. Functional consequences of the deletion mutation ⌬160Glu in human cardiac troponin T. J Biochem 2000;127:263–268.
Effects of Increased Concentrations of Glucose on Platelet Reactivity in Healthy Subjects and in Patients With and Without Diabetes Mellitus Friederike K. Keating,
MD,
Burton E. Sobel,
Hyperglycemia has been linked to adverse outcomes after myocardial infarction. We characterized the effect of selected concentrations of glucose or mannitol on platelet function in whole blood samples from healthy volunteers and from patients with and without diabetes mellitus. Activation of platelet glycoprotein IIb/IIIa and P-selectin expression was increased similarly after addition of isosmotic concentrations of glucose and mannitol, suggesting that increased osmolarity associated with hyperglycemia increases platelet reactivity. 䊚2003 by Excerpta Medica, Inc. (Am J Cardiol 2003;92:1362–1365)
he extent of hyperglycemia influences both shortand long-term outcomes after acute myocardial T infarction. For patients with diabetes mellitus, poor 1–3
glycemic control is associated with adverse outcomes after coronary bypass surgery.4 We have found that increased platelet reactivity is associated with an increased incidence of ischemic complications after coronary intervention.5 Accordingly, this study was performed to determine whether high concentrations of glucose, per se, alter platelet reactivity. •••
All subjects participating in this study gave written informed consent to participate in protocols approved by the institutional review board of the University of Vermont. Blood samples were obtained from 14 healthy volunteers and from hospitalized patients with (n ⫽ 14) or without (n ⫽ 7) type 2 diabetes mellitus. Healthy volunteers had not taken aspirin or any other antiplatelet agent for at least 10 days before blood was From the Department of Medicine, University of Vermont, Burlington, Vermont. This report was supported in part by grant R01-HL69146 from the National Institutes of Health, Bethesda, Maryland. Dr. Keating’s address is: University of Vermont, Colchester Research Facility, 208 South Park Drive, Colchester, Vermont 05446. E-mail: [email protected]. Manuscript received June 9, 2003; revised manuscript received and accepted August 15, 2003.
1362
©2003 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 92 December 1, 2003
MD,
and David J. Schneider,
MD
obtained. All patients were treated with aspirin (325 mg/day orally) and intravenous unfractionated heparin or low molecular-weight heparin at the time the blood was obtained. No patient had been treated with an antiplatelet agent other than aspirin. Patients were eligible if they had no history of diabetes and a hemoglobin (Hb)A1c level of ⬍6.5%, or a history of type 2 diabetes mellitus and a HbA1c level of ⬎7.5%. HbA1c was determined with the use of a point-of-care instrument (Axis-Shield, Oslo, Norway). Blood was obtained with the use of a 2-syringe technique. For healthy volunteers, the first 3 ml of blood was discarded. For patients with and without diabetes mellitus, the first 3 ml of blood was used to determine blood glucose with the use of an instant-read glucometer (Accu-Chek, Roche Diagnostics, Indianapolis, Indiana) and HbA1c. The next 10 ml of blood was drawn into a separate syringe and anticoagulated with D-phenylalanylL-prolyl-L-arginine chloromethyl ketone, Calbiochem (Calbiochem, San Diego, California) (76 mol/L final concentration) to inhibit coagulation and hence platelet activation during incubation in vitro. Aliquots were incubated for 1 hour in the absence or presence of added glucose or mannitol (25 mmol/L) to identify effects of each on platelet reactivity. Glucose and mannitol were prepared as equimolar stock solutions in Tyrode’s buffer (137 mmol/L sodium chloride, 2.7 mmol/L sodium bicarbonate, 0.36 mmol/L monobasic sodium phosphate, 2 mmol/L calcium chloride 2, and 4 mmol/L magnesium chloride 2, pH 7.35). Platelet activation was subsequently assessed as previously described.6 Flow cytometric analysis was performed with the use of a Coulter EPICS Elite flow cytometer (Hialeah, Florida). The population of platelets was identified on the basis of particle size and association with a peridinin chlorophyll protein-conjugated antibody to glycoprotein (GP) IIIa (CD61; Becton Dickinson, San Jose, California). Fluorescein isothiocyanateconjugated fibrinogen was added to assess the activation 0002-9149/03/$–see front matter doi:10.1016/j.amjcard.2003.08.032
FIGURE 1. Platelet reactivity in blood from healthy subjects. Blood from 6 volunteers was incubated for 60 minutes with glucose (0 [control], 500 mg/dl [27.5 mmol/L], or 1,000 mg/dl [55 mmol/L]) before stimulation with ADP. (A) Flow cytometry was performed to determine the percentage of platelets capable of binding fibrinogen as a marker for GP IIb/IIIa activation in the absence of an agonist and in response to 0.2 mol/L ADP. *p <0.05 for trend by repeated-measures analysis of variance. (B) Flow cytometry was performed to determine the percentage of platelets expressing P-selectin (CD62) as a marker of platelet reactivity in the absence of an agonist and in response to 0.2 mol/L ADP. *p <0.05 for trend by repeated-measures analysis of variance, and p <0.05 for all concentrations of glucose compared with controls by paired t test.
of GP IIb/IIIa, and a phycoerythrin-conjugated antibody to P-selectin (CD62, Becton Dickinson) was used for assessment of platelet surface P-selectin expression. A threshold for activation was determined by assessing the association of control ligands, Fluorescein isothiocya-
FIGURE 2. Platelet P-selectin expression in unstimulated platelets and in platelets exposed to 0.2 mol/L ADP in blood from 8 healthy subjects to which 25 mmol/L of glucose or mannitol was added. Platelets were incubated for 60 minutes before assessment of platelet function by flow cytometry. (A) GP IIb/IIIa activation (percentage of platelets capable of binding fibrinogen) in the absence of an agonist and in response to 0.2 mol/L ADP. (B) P-selectin expression (percentage of platelets expressing Pselectin) in the absence of an agonist and in response to 0.2 mol/L ADP. *p <0.05 for comparison with controls. Mannitol and glucose augmented platelet reactivity to a similar extent.
nate-conjugated albumin and nonimmune immunoglobulin-phycoerythrin with platelets. Results are expressed as mean ⫾ SE unless otherwise noted. Effects of selected concentrations of glucose on platelets from healthy subjects were compared with the use of analysis of variance. Effects of glucose and mannitol on platelets from healthy subjects and effects of glucose on platelets from patients with or BRIEF REPORTS
1363
TABLE 1 Clinical Characteristics of Patients With and Without Diabetes Mellitus (DM) No DM (n ⫽ 7)
DM (n ⫽ 14)
5/2 63 ⫾ 13 5 0 2 4/3 104 ⫾ 30 6.0 ⫾ 0.3
10/4 67 ⫾ 14 10 2 2 8/6 227 ⫾ 66 8.7 ⫾ 1.1
Variable Men/women Age (mean ⫾ SD yrs) Acute coronary syndrome Heart failure Other* Heparin/enoxaparin Glucose (mean ⫾ SD) HbA1c (mean ⫾ SD)
*Other diagnoses were abdominal aortic aneurysm repair (n ⫽ 1), atrial fibrillation (n ⫽ 1) in the No-DM group, and chronic obstructive pulmonary disease (n ⫽ 1), and atrial fibrillation (n ⫽ 1) in the DM group.
without diabetes were compared with paired Student’s t tests. A p value ⬍0.05 was considered significant. Blood from 6 healthy volunteers was incubated in the absence (control) or presence of glucose (27.5 mmol/L [500 mg/dl] or 55 mmol/L [1000 mg/dl]). Platelet reactivity increased with increasing glucose concentration in all subjects. Both the capacity to bind fibrinogen and P-selectin expression were increased in the presence of added glucose (Figure 1). In the presence of 27.5 mmol/L glucose, the capacity to bind fibrinogen in response to 0.2 mol/L adenosine diphosphate (ADP) increased from 24 ⫾ 4% to 30 ⫾ 4% (n ⫽ 6, p ⬍0.05), and the surface expression of P-selectin increased from 10 ⫾ 2% to 15 ⫾ 2% (n ⫽ 6, p ⬍0.05). To determine whether the effect of glucose was attributable to osmolarity, blood from 8 healthy volunteers was incubated with vehicle alone (control), glucose (25 mmol/L), or mannitol (25 mmol/L). Isoosmolar concentrations of either glucose or mannitol had a similar effect, potentiating the activation of GP IIb/IIIa and P-selectin expression in response to ADP (0.2 mol/L; Figure 2). To determine whether the effect of glucose on platelet function was similar in blood from patients with and without diabetes, blood was obtained from hospitalized patients with (n ⫽ 14) and without (n ⫽ 7) diabetes mellitus. Clinical characteristics of the patients are listed in Table 1. The effect of 25 mmol/L glucose on platelet function is depicted in Figure 3. The capacity of platelets to bind fibrinogen and express P-selectin in response to 0.2 mol/L ADP was greater after incubation with glucose in samples from those with and without diabetes. The magnitude of the impact of additional glucose on expression of P-selectin and activation of GP IIb/IIIa was similar in patients with and without diabetes. •••
Exposure of platelets from healthy patients and from patients with and without diabetes to high concentrations of glucose for a relatively short interval (1 hour) increased platelet reactivity. The effect of glucose on P-selectin expression was more pronounced than the effect on activation of GP IIb/IIIa. Additional 1364 THE AMERICAN JOURNAL OF CARDIOLOGY姞
VOL. 92
FIGURE 3. Platelet reactivity in blood from patients with (n ⴝ 14) or without (n ⴝ 7) diabetes mellitus (DM). Platelets were incubated for 60 minutes before assessment of platelet function by flow cytometry. (A) GP IIb/IIIa activation (percentage of platelets capable of binding fibrinogen) in response to 0.2 mol/L ADP. (B) P-selectin expression (percentage of platelets expressing Pselectin) in response to 0.2 mol/L ADP. *p <0.05 for comparison with controls (no added glucose).
experiments in which platelets were exposed to isosmolar concentrations of the metabolically inactive agent mannitol led to a similar augmentation of platelet reactivity. The results with mannitol suggest that osmotic effects of glucose are an important mechanism by which hyperglycemia increases platelet reactivity and activity. These results are also consistent with our previous results demonstrating that exposure of platelets to increased concentrations of fibrinogen increased agonist-stimulated P-selectin expression.7 Accordingly, the present results demonstrate that exposure of platelets to increased osmolarity increases the propensity of platelets to aggregate and degranuDECEMBER 1, 2003
late, and may therefore promote thromboembolic complications. The effects of the addition of glucose to blood in vitro are consistent with those seen in vivo in patients with hyperosmolar hyperglycemia. In a retrospective study, mortality associated with this condition was 14.6%. The principal cause of death was cerebral and peripheral arterial thrombosis in 25% of these patients.8 A small prospective study of 15 patients with nonketotic and ketotic diabetic coma reported 2 of 3 deaths attributable to disseminated intravascular coagulation.9 Results of previous studies have implicated diabetes as a disorder that can alter platelet function.10 Proposed contributors to altered platelet function in diabetes are a change in membrane fluidity,11,12 altered responsiveness to platelet-activating factor (a product of lipid metabolism),13 or the generation of oxygen-centered free radicals.14 Some investigators have suggested that concentrations of glucose seen in patients with diabetes influence calcium uptake by platelets15 and reduce nitric oxide synthesis.16 Although we did not detect a significant difference in platelet reactivity in the absence of added glucose between patients with and without diabetes, the small sample size and interindividual variability in platelet reactivity preclude accurate comparison. Platelet reactivity was enhanced after the addition of glucose to blood from patients with and without diabetes. Therefore, our data suggest that direct effects of glucose are evident with respect to altering platelet reactivity. The results of Sakamoto and colleagues17 are consistent with those of the present report. They found that the formation of platelet microaggregates increased equal to the concentration of glucose during acute hyperglycemia. In conclusion, we observed changes in the threshold for the activation of GP IIb/IIIa and for the induction of ␣-granule degranulation as a result of changes in osmolarity, an effect independent of intraplatelet metabolism. This may explain, in part, why a hyperglycemic procoagulant state occurs with diabetes18,19 because of the powerful influence on thrombin generation of even subtle changes in properties of platelet membranes.20
1. Malmberg K, Norhammar A, Wedel H, Ryden L. Glycometabolic state at
admission: important risk marker of mortality in conventionally treated patients with diabetes mellitus and acute myocardial infarction: long-term results from the Diabetes and Insulin-Glucose Infusion in Acute Myocardial Infarction (DIGAMI) study. Circulation 1999;99:2626 –2632. 2. Fava S, Aquilina O, Azzopardi J, Agius Muscat H, Fenech FF. The prognostic value of blood glucose in diabetic patients with acute myocardial infarction. Diabetic Med 1996;13:80 –83. 3. Wahab NN, Cowden EA, Pearce NJ, Gardner MJ, Merry H, Cox JL. Is blood glucose an independent predictor of mortality in acute myocardial infarction in the thrombolytic era? J Am Coll Cardiol 2002;40:1748 –1754. 4. McAlister FA, Man J, Bistritz L, Amad H, Tandon P. Diabetes and coronary artery bypass surgery: an examination of perioperative glycemic control and outcomes. Diabetes Care 2003;26:1518 –1524. 5. Kabbani SS, Watkins MW, Ashikaga T, Terrien EF, Holoch PA, Sobel BE, Schneider DJ. Platelet reactivity characterized prospectively: a determinant of outcome 90 days after percutaneous coronary intervention. Circulation 2001;10: 181–186. 6. Schneider DJ, Tracy PB, Mann KG, Sobel BE. Differential effects of anticoagulants on the activation of platelets ex vivo. Circulation 1997;96:2877–2883. 7. Schneider DJ, Taatjes DJ, Howard DB, Sobel BE. Increased reactivity of platelets induced by fibrinogen independent of its binding to the IIb-IIIa surface glycoprotein: a potential contributor to cardiovascular risk. J Am Coll Cardiol 1999;33:261–266. 8. Hamblin PS, Topliss DJ, Chosich N, Lording DW, Stockigt JR. Deaths associated with diabetic ketoacidosis and hyperosmolar coma: 1973–1988. Med J Aust 1989;151:439 –444. 9. Paton RC. Haemostatic changes in diabetic coma. Diabetologia 1981;21:172– 177. 10. Sagel J, Colwell JA, Crook L, Laimins M. Increased platelet aggregation in early diabetes mellitus. Ann Intern Med 1975;82:733–738. 11. Winocour PD, Bryszewska M, Watala C, Rand ML, Epand RM, KinloughRathbone RL, Packham MA, Mustard JF. Reduced membrane fluidity in platelets from diabetic patients. Diabetes 1990;39:241–244. 12. Winocour PD, Watala C, Perry DW, Kinlough-Rathbone RL. Decreased platelet membrane fluidity due to glycation or acetylation of membrane proteins. Thromb Haemost 1992;68:577–582. 13. Shukla SD, Paul A, Klachko DM. Hypersensitivity of diabetic human platelets to platelet activating factor. Thromb Res 1992;66:239 –246. 14. Leoncini G, Signorello MG, Piana A, Carrubba M, Armani U. Hyperactivity and increased hydrogen peroxide formation in platelets of NIDDM patients. Thromb Res 1997;86:153–160. 15. 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 – H1489. 16. Giugliano D, Marfella R, Coppola L, Verrazzo G, Acampora R, Giunta R, Nappo F, Lucarelli C, D’Onofrio F. Vascular effects of acute hyperglycemia in humans are reversed by L-arginine. Evidence for reduced availability of nitric oxide during hyperglycemia. Circulation 1997;95:1783–1790. 17. Sakamoto T, Ogawa H, Kawano H, Hirai N, Miyamoto S, Takazoe K, Soejima H, Kugiyama K, Yoshimura M, Yasue H. Rapid change of platelet aggregability in acute hyperglycemia. Detection by a novel laser-light scattering method. Thromb Haemost 2000;83:475–479. 18. Lebovitz HE. Effect of the postprandial state on nontraditional risk factors. Am J Cardiol 2001;88:20H–25H. 19. Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfunction in type 2 diabetes. Diabetes Care 2001;24:1476 –1485. 20. Swords NA, Tracy PB, Mann KG. Intact platelet membranes, not plateletreleased microvesicles, support the procoagulant activity of adherent platelets. Arterioscler Thromb 1993;13:1613–1622.
BRIEF REPORTS
1365
lines and recommendations for content, structure, and deployment of mutation database: II. Journey in progress. Hum Mutat 2000;15:13–15. 13. Palm T, Graboski S, Hitchcock-DeGregori SE, Greenfield NJ. Diseasecausing mutations in cardiac troponin T: identification of a critical tropomyosinbinding region. Biophys J 2001;81:2827–2837. 14. Anan R, Shono H, Kisanuki A, Arima S, Nakao S, Tanaka H. Patients with familial hypertrophic cardiomyopaty caused by a Phe110Ile missense mutation in the cardiac troponin T gene have variable cardiac morphologies and a favorable prognosis. Circulation 1998;98:391–397. 15. Lin TL, Ichihara S, Yamada Y, Nagasaka T, Ishihara H, Nakashima N, Yokota M. Phenotypic variation of familial hypertrophic cardiomyopathy caused by the Phe110Ile mutation in cardiac troponin T. Cardiology 2000;93:155–162. 16. Thierfelder L, Watkins H, MacRae C, Lamas R, McKenna W, Vosberg HP, Seidman JG, Seidman CE. ␣-Tropomyosin and cardiac troponin T mutations
cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell 1994;77:701–712. 17. Moolman JC, Corfield VA, Posen B, Posen B, Ngumbela K, Seidman C, Brink PA, Watkins H. Sudden death due to troponin T mutations. J Am Coll Cardiol 1997;29:549 –555. 18. Van Driest SL, Ackerman MJ, Ommen SR, Shakur R, Will ML, Nishimura RA, Tajik AJ, Gersh BJ. Prevalence and severity of “benign” mutations in the beta-myosin heavy chain, cardiac troponin T and alfa-tropomyosin genes in hypertrophic cardiomyopathy. Circulation 2002;106:3085–3090. 19. Ho CY, Lever HM, DeSanctis R, Farver CF, Seidman JG, Seidman CE. Homozygous mutation in cardiac troponin T. Implications for hypertrophic cardiomyopathy. Circulation 2000;102:1950 –1955. 20. Harada K, Yanaga FT, Minakami R, Morimoto S, Ohtsuki I. Functional consequences of the deletion mutation ⌬160Glu in human cardiac troponin T. J Biochem 2000;127:263–268.
Effects of Increased Concentrations of Glucose on Platelet Reactivity in Healthy Subjects and in Patients With and Without Diabetes Mellitus Friederike K. Keating,
MD,
Burton E. Sobel,
Hyperglycemia has been linked to adverse outcomes after myocardial infarction. We characterized the effect of selected concentrations of glucose or mannitol on platelet function in whole blood samples from healthy volunteers and from patients with and without diabetes mellitus. Activation of platelet glycoprotein IIb/IIIa and P-selectin expression was increased similarly after addition of isosmotic concentrations of glucose and mannitol, suggesting that increased osmolarity associated with hyperglycemia increases platelet reactivity. 䊚2003 by Excerpta Medica, Inc. (Am J Cardiol 2003;92:1362–1365)
he extent of hyperglycemia influences both shortand long-term outcomes after acute myocardial T infarction. For patients with diabetes mellitus, poor 1–3
glycemic control is associated with adverse outcomes after coronary bypass surgery.4 We have found that increased platelet reactivity is associated with an increased incidence of ischemic complications after coronary intervention.5 Accordingly, this study was performed to determine whether high concentrations of glucose, per se, alter platelet reactivity. •••
All subjects participating in this study gave written informed consent to participate in protocols approved by the institutional review board of the University of Vermont. Blood samples were obtained from 14 healthy volunteers and from hospitalized patients with (n ⫽ 14) or without (n ⫽ 7) type 2 diabetes mellitus. Healthy volunteers had not taken aspirin or any other antiplatelet agent for at least 10 days before blood was From the Department of Medicine, University of Vermont, Burlington, Vermont. This report was supported in part by grant R01-HL69146 from the National Institutes of Health, Bethesda, Maryland. Dr. Keating’s address is: University of Vermont, Colchester Research Facility, 208 South Park Drive, Colchester, Vermont 05446. E-mail: [email protected]. Manuscript received June 9, 2003; revised manuscript received and accepted August 15, 2003.
1362
©2003 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 92 December 1, 2003
MD,
and David J. Schneider,
MD
obtained. All patients were treated with aspirin (325 mg/day orally) and intravenous unfractionated heparin or low molecular-weight heparin at the time the blood was obtained. No patient had been treated with an antiplatelet agent other than aspirin. Patients were eligible if they had no history of diabetes and a hemoglobin (Hb)A1c level of ⬍6.5%, or a history of type 2 diabetes mellitus and a HbA1c level of ⬎7.5%. HbA1c was determined with the use of a point-of-care instrument (Axis-Shield, Oslo, Norway). Blood was obtained with the use of a 2-syringe technique. For healthy volunteers, the first 3 ml of blood was discarded. For patients with and without diabetes mellitus, the first 3 ml of blood was used to determine blood glucose with the use of an instant-read glucometer (Accu-Chek, Roche Diagnostics, Indianapolis, Indiana) and HbA1c. The next 10 ml of blood was drawn into a separate syringe and anticoagulated with D-phenylalanylL-prolyl-L-arginine chloromethyl ketone, Calbiochem (Calbiochem, San Diego, California) (76 mol/L final concentration) to inhibit coagulation and hence platelet activation during incubation in vitro. Aliquots were incubated for 1 hour in the absence or presence of added glucose or mannitol (25 mmol/L) to identify effects of each on platelet reactivity. Glucose and mannitol were prepared as equimolar stock solutions in Tyrode’s buffer (137 mmol/L sodium chloride, 2.7 mmol/L sodium bicarbonate, 0.36 mmol/L monobasic sodium phosphate, 2 mmol/L calcium chloride 2, and 4 mmol/L magnesium chloride 2, pH 7.35). Platelet activation was subsequently assessed as previously described.6 Flow cytometric analysis was performed with the use of a Coulter EPICS Elite flow cytometer (Hialeah, Florida). The population of platelets was identified on the basis of particle size and association with a peridinin chlorophyll protein-conjugated antibody to glycoprotein (GP) IIIa (CD61; Becton Dickinson, San Jose, California). Fluorescein isothiocyanateconjugated fibrinogen was added to assess the activation 0002-9149/03/$–see front matter doi:10.1016/j.amjcard.2003.08.032
FIGURE 1. Platelet reactivity in blood from healthy subjects. Blood from 6 volunteers was incubated for 60 minutes with glucose (0 [control], 500 mg/dl [27.5 mmol/L], or 1,000 mg/dl [55 mmol/L]) before stimulation with ADP. (A) Flow cytometry was performed to determine the percentage of platelets capable of binding fibrinogen as a marker for GP IIb/IIIa activation in the absence of an agonist and in response to 0.2 mol/L ADP. *p <0.05 for trend by repeated-measures analysis of variance. (B) Flow cytometry was performed to determine the percentage of platelets expressing P-selectin (CD62) as a marker of platelet reactivity in the absence of an agonist and in response to 0.2 mol/L ADP. *p <0.05 for trend by repeated-measures analysis of variance, and p <0.05 for all concentrations of glucose compared with controls by paired t test.
of GP IIb/IIIa, and a phycoerythrin-conjugated antibody to P-selectin (CD62, Becton Dickinson) was used for assessment of platelet surface P-selectin expression. A threshold for activation was determined by assessing the association of control ligands, Fluorescein isothiocya-
FIGURE 2. Platelet P-selectin expression in unstimulated platelets and in platelets exposed to 0.2 mol/L ADP in blood from 8 healthy subjects to which 25 mmol/L of glucose or mannitol was added. Platelets were incubated for 60 minutes before assessment of platelet function by flow cytometry. (A) GP IIb/IIIa activation (percentage of platelets capable of binding fibrinogen) in the absence of an agonist and in response to 0.2 mol/L ADP. (B) P-selectin expression (percentage of platelets expressing Pselectin) in the absence of an agonist and in response to 0.2 mol/L ADP. *p <0.05 for comparison with controls. Mannitol and glucose augmented platelet reactivity to a similar extent.
nate-conjugated albumin and nonimmune immunoglobulin-phycoerythrin with platelets. Results are expressed as mean ⫾ SE unless otherwise noted. Effects of selected concentrations of glucose on platelets from healthy subjects were compared with the use of analysis of variance. Effects of glucose and mannitol on platelets from healthy subjects and effects of glucose on platelets from patients with or BRIEF REPORTS
1363
TABLE 1 Clinical Characteristics of Patients With and Without Diabetes Mellitus (DM) No DM (n ⫽ 7)
DM (n ⫽ 14)
5/2 63 ⫾ 13 5 0 2 4/3 104 ⫾ 30 6.0 ⫾ 0.3
10/4 67 ⫾ 14 10 2 2 8/6 227 ⫾ 66 8.7 ⫾ 1.1
Variable Men/women Age (mean ⫾ SD yrs) Acute coronary syndrome Heart failure Other* Heparin/enoxaparin Glucose (mean ⫾ SD) HbA1c (mean ⫾ SD)
*Other diagnoses were abdominal aortic aneurysm repair (n ⫽ 1), atrial fibrillation (n ⫽ 1) in the No-DM group, and chronic obstructive pulmonary disease (n ⫽ 1), and atrial fibrillation (n ⫽ 1) in the DM group.
without diabetes were compared with paired Student’s t tests. A p value ⬍0.05 was considered significant. Blood from 6 healthy volunteers was incubated in the absence (control) or presence of glucose (27.5 mmol/L [500 mg/dl] or 55 mmol/L [1000 mg/dl]). Platelet reactivity increased with increasing glucose concentration in all subjects. Both the capacity to bind fibrinogen and P-selectin expression were increased in the presence of added glucose (Figure 1). In the presence of 27.5 mmol/L glucose, the capacity to bind fibrinogen in response to 0.2 mol/L adenosine diphosphate (ADP) increased from 24 ⫾ 4% to 30 ⫾ 4% (n ⫽ 6, p ⬍0.05), and the surface expression of P-selectin increased from 10 ⫾ 2% to 15 ⫾ 2% (n ⫽ 6, p ⬍0.05). To determine whether the effect of glucose was attributable to osmolarity, blood from 8 healthy volunteers was incubated with vehicle alone (control), glucose (25 mmol/L), or mannitol (25 mmol/L). Isoosmolar concentrations of either glucose or mannitol had a similar effect, potentiating the activation of GP IIb/IIIa and P-selectin expression in response to ADP (0.2 mol/L; Figure 2). To determine whether the effect of glucose on platelet function was similar in blood from patients with and without diabetes, blood was obtained from hospitalized patients with (n ⫽ 14) and without (n ⫽ 7) diabetes mellitus. Clinical characteristics of the patients are listed in Table 1. The effect of 25 mmol/L glucose on platelet function is depicted in Figure 3. The capacity of platelets to bind fibrinogen and express P-selectin in response to 0.2 mol/L ADP was greater after incubation with glucose in samples from those with and without diabetes. The magnitude of the impact of additional glucose on expression of P-selectin and activation of GP IIb/IIIa was similar in patients with and without diabetes. •••
Exposure of platelets from healthy patients and from patients with and without diabetes to high concentrations of glucose for a relatively short interval (1 hour) increased platelet reactivity. The effect of glucose on P-selectin expression was more pronounced than the effect on activation of GP IIb/IIIa. Additional 1364 THE AMERICAN JOURNAL OF CARDIOLOGY姞
VOL. 92
FIGURE 3. Platelet reactivity in blood from patients with (n ⴝ 14) or without (n ⴝ 7) diabetes mellitus (DM). Platelets were incubated for 60 minutes before assessment of platelet function by flow cytometry. (A) GP IIb/IIIa activation (percentage of platelets capable of binding fibrinogen) in response to 0.2 mol/L ADP. (B) P-selectin expression (percentage of platelets expressing Pselectin) in response to 0.2 mol/L ADP. *p <0.05 for comparison with controls (no added glucose).
experiments in which platelets were exposed to isosmolar concentrations of the metabolically inactive agent mannitol led to a similar augmentation of platelet reactivity. The results with mannitol suggest that osmotic effects of glucose are an important mechanism by which hyperglycemia increases platelet reactivity and activity. These results are also consistent with our previous results demonstrating that exposure of platelets to increased concentrations of fibrinogen increased agonist-stimulated P-selectin expression.7 Accordingly, the present results demonstrate that exposure of platelets to increased osmolarity increases the propensity of platelets to aggregate and degranuDECEMBER 1, 2003
late, and may therefore promote thromboembolic complications. The effects of the addition of glucose to blood in vitro are consistent with those seen in vivo in patients with hyperosmolar hyperglycemia. In a retrospective study, mortality associated with this condition was 14.6%. The principal cause of death was cerebral and peripheral arterial thrombosis in 25% of these patients.8 A small prospective study of 15 patients with nonketotic and ketotic diabetic coma reported 2 of 3 deaths attributable to disseminated intravascular coagulation.9 Results of previous studies have implicated diabetes as a disorder that can alter platelet function.10 Proposed contributors to altered platelet function in diabetes are a change in membrane fluidity,11,12 altered responsiveness to platelet-activating factor (a product of lipid metabolism),13 or the generation of oxygen-centered free radicals.14 Some investigators have suggested that concentrations of glucose seen in patients with diabetes influence calcium uptake by platelets15 and reduce nitric oxide synthesis.16 Although we did not detect a significant difference in platelet reactivity in the absence of added glucose between patients with and without diabetes, the small sample size and interindividual variability in platelet reactivity preclude accurate comparison. Platelet reactivity was enhanced after the addition of glucose to blood from patients with and without diabetes. Therefore, our data suggest that direct effects of glucose are evident with respect to altering platelet reactivity. The results of Sakamoto and colleagues17 are consistent with those of the present report. They found that the formation of platelet microaggregates increased equal to the concentration of glucose during acute hyperglycemia. In conclusion, we observed changes in the threshold for the activation of GP IIb/IIIa and for the induction of ␣-granule degranulation as a result of changes in osmolarity, an effect independent of intraplatelet metabolism. This may explain, in part, why a hyperglycemic procoagulant state occurs with diabetes18,19 because of the powerful influence on thrombin generation of even subtle changes in properties of platelet membranes.20
1. Malmberg K, Norhammar A, Wedel H, Ryden L. Glycometabolic state at
admission: important risk marker of mortality in conventionally treated patients with diabetes mellitus and acute myocardial infarction: long-term results from the Diabetes and Insulin-Glucose Infusion in Acute Myocardial Infarction (DIGAMI) study. Circulation 1999;99:2626 –2632. 2. Fava S, Aquilina O, Azzopardi J, Agius Muscat H, Fenech FF. The prognostic value of blood glucose in diabetic patients with acute myocardial infarction. Diabetic Med 1996;13:80 –83. 3. Wahab NN, Cowden EA, Pearce NJ, Gardner MJ, Merry H, Cox JL. Is blood glucose an independent predictor of mortality in acute myocardial infarction in the thrombolytic era? J Am Coll Cardiol 2002;40:1748 –1754. 4. McAlister FA, Man J, Bistritz L, Amad H, Tandon P. Diabetes and coronary artery bypass surgery: an examination of perioperative glycemic control and outcomes. Diabetes Care 2003;26:1518 –1524. 5. Kabbani SS, Watkins MW, Ashikaga T, Terrien EF, Holoch PA, Sobel BE, Schneider DJ. Platelet reactivity characterized prospectively: a determinant of outcome 90 days after percutaneous coronary intervention. Circulation 2001;10: 181–186. 6. Schneider DJ, Tracy PB, Mann KG, Sobel BE. Differential effects of anticoagulants on the activation of platelets ex vivo. Circulation 1997;96:2877–2883. 7. Schneider DJ, Taatjes DJ, Howard DB, Sobel BE. Increased reactivity of platelets induced by fibrinogen independent of its binding to the IIb-IIIa surface glycoprotein: a potential contributor to cardiovascular risk. J Am Coll Cardiol 1999;33:261–266. 8. Hamblin PS, Topliss DJ, Chosich N, Lording DW, Stockigt JR. Deaths associated with diabetic ketoacidosis and hyperosmolar coma: 1973–1988. Med J Aust 1989;151:439 –444. 9. Paton RC. Haemostatic changes in diabetic coma. Diabetologia 1981;21:172– 177. 10. Sagel J, Colwell JA, Crook L, Laimins M. Increased platelet aggregation in early diabetes mellitus. Ann Intern Med 1975;82:733–738. 11. Winocour PD, Bryszewska M, Watala C, Rand ML, Epand RM, KinloughRathbone RL, Packham MA, Mustard JF. Reduced membrane fluidity in platelets from diabetic patients. Diabetes 1990;39:241–244. 12. Winocour PD, Watala C, Perry DW, Kinlough-Rathbone RL. Decreased platelet membrane fluidity due to glycation or acetylation of membrane proteins. Thromb Haemost 1992;68:577–582. 13. Shukla SD, Paul A, Klachko DM. Hypersensitivity of diabetic human platelets to platelet activating factor. Thromb Res 1992;66:239 –246. 14. Leoncini G, Signorello MG, Piana A, Carrubba M, Armani U. Hyperactivity and increased hydrogen peroxide formation in platelets of NIDDM patients. Thromb Res 1997;86:153–160. 15. 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 – H1489. 16. Giugliano D, Marfella R, Coppola L, Verrazzo G, Acampora R, Giunta R, Nappo F, Lucarelli C, D’Onofrio F. Vascular effects of acute hyperglycemia in humans are reversed by L-arginine. Evidence for reduced availability of nitric oxide during hyperglycemia. Circulation 1997;95:1783–1790. 17. Sakamoto T, Ogawa H, Kawano H, Hirai N, Miyamoto S, Takazoe K, Soejima H, Kugiyama K, Yoshimura M, Yasue H. Rapid change of platelet aggregability in acute hyperglycemia. Detection by a novel laser-light scattering method. Thromb Haemost 2000;83:475–479. 18. Lebovitz HE. Effect of the postprandial state on nontraditional risk factors. Am J Cardiol 2001;88:20H–25H. 19. Vinik AI, Erbas T, Park TS, Nolan R, Pittenger GL. Platelet dysfunction in type 2 diabetes. Diabetes Care 2001;24:1476 –1485. 20. Swords NA, Tracy PB, Mann KG. Intact platelet membranes, not plateletreleased microvesicles, support the procoagulant activity of adherent platelets. Arterioscler Thromb 1993;13:1613–1622.
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