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• W1972320922 abstract "Spontaneous platelet aggregation (SPA) is enhanced in patients with type 2 diabetes. Adiponectin may inhibit platelet aggregation. The aims of the current study were to identify factors associated with in vitro SPA measured by a laser light scattering method and to investigate the effects of short-term glycemic control and adiponectin on SPA. In study 1, we investigated platelet aggregation in 20 healthy control subjects and 82 patients with type 2 diabetes. In study 2, we evaluated the changes of SPA and serum high-molecular-weight (HMW) adiponectin after 2 weeks of improved glycemic control in 20 hospitalized diabetic patients. In study 3, using washed platelets from 10 healthy subjects, in vitro SPA was measured over 15 min in the absence or presence of recombinant adiponectin (20 μg/mL). Platelet aggregation was assessed with a laser light scatter aggregometer that measured the size of platelet aggregates. SPA was defined as formation of small aggregates under constant stirring in the absence of any agonists. The area under the curve was calculated for SPA and also for agonist-induced small, medium, and large aggregates. SPA was increased in diabetic patients compared with control subjects. In diabetic patients, SPA was correlated positively with plasma fibrinogen, fasting plasma glucose, glycated albumin, and high-sensitivity C-reactive protein. A stepwise multivariate analysis showed that plasma fibrinogen was the strongest independent determinant of SPA in diabetic patients. In 20 diabetic patients, SPA decreased significantly after 2 weeks of glycemic control. A significant negative correlation was found between changes of SPA and those of HMW adiponectin during treatment. The in vitro study showed that adiponectin inhibited the spontaneous aggregation of washed platelets. In conclusion, hyperfibrinogenemia and hyperglycemia are associated independently with SPA in patients with type 2 diabetes. SPA is reduced after even short-term improvement of glycemic control and adiponectin also inhibits SPA directly. Spontaneous platelet aggregation (SPA) is enhanced in patients with type 2 diabetes. Adiponectin may inhibit platelet aggregation. The aims of the current study were to identify factors associated with in vitro SPA measured by a laser light scattering method and to investigate the effects of short-term glycemic control and adiponectin on SPA. In study 1, we investigated platelet aggregation in 20 healthy control subjects and 82 patients with type 2 diabetes. In study 2, we evaluated the changes of SPA and serum high-molecular-weight (HMW) adiponectin after 2 weeks of improved glycemic control in 20 hospitalized diabetic patients. In study 3, using washed platelets from 10 healthy subjects, in vitro SPA was measured over 15 min in the absence or presence of recombinant adiponectin (20 μg/mL). Platelet aggregation was assessed with a laser light scatter aggregometer that measured the size of platelet aggregates. SPA was defined as formation of small aggregates under constant stirring in the absence of any agonists. The area under the curve was calculated for SPA and also for agonist-induced small, medium, and large aggregates. SPA was increased in diabetic patients compared with control subjects. In diabetic patients, SPA was correlated positively with plasma fibrinogen, fasting plasma glucose, glycated albumin, and high-sensitivity C-reactive protein. A stepwise multivariate analysis showed that plasma fibrinogen was the strongest independent determinant of SPA in diabetic patients. In 20 diabetic patients, SPA decreased significantly after 2 weeks of glycemic control. A significant negative correlation was found between changes of SPA and those of HMW adiponectin during treatment. The in vitro study showed that adiponectin inhibited the spontaneous aggregation of washed platelets. In conclusion, hyperfibrinogenemia and hyperglycemia are associated independently with SPA in patients with type 2 diabetes. SPA is reduced after even short-term improvement of glycemic control and adiponectin also inhibits SPA directly. Hara K, et al.At a Glance CommentaryBackgroundSeveral studies using a new laser light scatter (LS) method demonstrated that spontaneous platelet aggregation (SPA) in the absence of agonists is enhanced in patients with type 2 diabetes. Adiponectin inhibits the aggregation of platelet-rich plasma induced by some agonists. No reports have been published about the direct effects of adiponectin on SPA.Translational SignificanceIn this article, we show that hyperfibrinogenemia and hyperglycemia are associated independently with SPA in patients with type 2 diabetes. Furthermore, we demonstrate that SPA is reduced after even short-term improvement of glycemic control in diabetic patients and that adiponectin also directly inhibits SPA in vitro. Patients with type 2 diabetes have a high incidence of atherosclerotic cardiovascular disease, which leads to increased morbidity and mortality because of coronary artery disease, stroke, and peripheral arterial disease.1Stamler J. Vaccaro O. Neaton J.D. Wentworth D. Diabetes, other risk factors, and 12-yr cardiovascular mortality for men screened in the Multiple Risk Factor Intervention Trial.Diabetes Care. 1993; 16: 434-444Crossref PubMed Scopus (3573) Google Scholar, 2Haffner S.M. Lehto S. Ronnemaa T. Pyorala K. Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and nondiabetic subjects and without prior myocardial infarction.N Engl J Med. 1998; 339: 229-234Crossref PubMed Scopus (5876) Google Scholar Platelet hyperreactivity may be associated with accelerated atherothrombosis in type 2 diabetes because platelets play a central role in hemostasis, thrombosis, and the pathogenesis of atherosclerosis. Platelets from patients with type 2 diabetes show increased intracellular Ca2+ mobilization and decreased nitric oxide production, resulting in augmented aggregation.3Vinik A.I. Erbas T. Park T.S. Nolan R. Pittenger G.L. Platelet dysfunction in type 2 diabetes.Diabetes Care. 2001; 24: 1476-1485Crossref PubMed Scopus (493) Google Scholar, 4Colwell J.A. Nesto R.W. The platelet in diabetes: focus on prevention of ischemic events.Diabetes Care. 2003; 26: 2181-2188Crossref PubMed Scopus (270) Google Scholar In vitro studies using the conventional optical density method5Born G.V. Aggregation of blood platelets by adenosine diphosphate and its reversal.Nature. 1962; 194: 927-929Crossref PubMed Scopus (3777) Google Scholar demonstrated the enhancement of platelet aggregation in response to several agonists, such as adenosine-5′- diphosphate (ADP) in type 2 diabetics.6Angiolillo D.J. Fernandez-Ortiz A. Bernardo E. et al.Platelet function profiles in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment.Diabetes. 2005; 54: 2430-2435Crossref PubMed Scopus (471) Google Scholar Several recent studies using a new laser light scatter (LS) method demonstrated that spontaneous platelet aggregation (SPA) in the absence of agonists is common in type 2 diabetic patients with nephropathy.7Fukuda K. Ozaki Y. Satoh K. et al.Phosphorylation of myosin light chain in resting platelets from NIDDM patients is enhanced: correlation with spontaneous aggregation.Diabetes. 1997; 46: 488-493Crossref PubMed Google Scholar, 8Iwase E. Tawata M. Aida K. et al.A cross-sectional evaluation of spontaneous platelet aggregation in relation to complications in patients with type II diabetes mellitus.Metabolism. 1998; 47: 699-705Abstract Full Text PDF PubMed Scopus (59) Google Scholar Because the LS method detects much smaller platelet aggregates than the conventional optical density method, it allows us to evaluate small aggregates that form spontaneously in vitro without any agonists.9Ozaki Y. Satoh K. Yatomi Y. et al.Detection of platelet aggregates with a particle counting method using light scattering.Anal Biochem. 1994; 218: 284-294Crossref PubMed Scopus (170) Google Scholar SPA may be associated more strongly with atherosclerotic cardiovascular disease than agonist-induced platelet aggregation8Iwase E. Tawata M. Aida K. et al.A cross-sectional evaluation of spontaneous platelet aggregation in relation to complications in patients with type II diabetes mellitus.Metabolism. 1998; 47: 699-705Abstract Full Text PDF PubMed Scopus (59) Google Scholar because SPA reflects early aggregation that cannot be detected by conventional methods.9Ozaki Y. Satoh K. Yatomi Y. et al.Detection of platelet aggregates with a particle counting method using light scattering.Anal Biochem. 1994; 218: 284-294Crossref PubMed Scopus (170) Google Scholar Adiponectin is a hormone produced by adipocytes that enhances insulin sensitivity and promotes lipid metabolism.10Yamauchi T. Kamon J. Waki H. et al.The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity.Nat Med. 2001; 7: 941-946Crossref PubMed Scopus (4042) Google Scholar Recently, in vitro studies demonstrated that adiponectin inhibits the aggregation of platelet-rich plasma induced by ADP or epinephrine,11Restituto P. Colina I. Varo J.J. Varo N. Adiponectin diminishes platelet aggregation and sCD40L release. Potential role in the metabolic syndrome.Am J Physiol Endocrinol Metab. 2010; 298: E1072-E1077Crossref PubMed Scopus (42) Google Scholar but there have been no reports about the effects of adiponectin or high-molecular weight (HMW) adiponectin on SPA. There have also been few investigations of the factors associated with SPA in patients with type 2 diabetes. Furthermore, there have been no investigations of the changes of SPA in diabetic patients after improvement of glycemic control. To identify factors influencing SPA, we therefore investigated the relationship between SPA and clinical parameters by performing a cross-sectional study in 82 patients with type 2 diabetes. Next, we investigated the changes of SPA prospectively in 20 patients with type 2 diabetes who were admitted to hospital for 2 weeks. Furthermore, we assessed the direct effect of adiponectin on spontaneous aggregation of washed platelets in vitro. The subjects were 20 healthy control subjects and 82 patients with type 2 diabetes. The healthy subjects were recruited from hospital staff. The diabetic patients were referred to the diabetes outpatient clinic at the Dokkyo Medical University Hospital. The diagnosis of type 2 diabetes was made according to the new criteria of the American Diabetes Association based on fasting plasma glucose ≥126 mg/dL or hemoglobin A1c ≥ 6.5%.12Diagnosis and classification of diabetes mellitus 2010. Diabetes Care 33:S62-9Google Scholar We excluded patients who were receiving anticoagulants or antiplatelet drugs. Excluded in addition were patients with any history of cardiovascular disease including coronary heart disease (CHD) or stroke. Patients on insulin therapy were excluded also. We also excluded patients with severe infection of the foot or generalized sepsis, or connective tissue disease such as rheumatoid arthritis. Blood was collected into a tube containing 3.2 % sodium citrate after an overnight fast. Platelet-rich plasma (PRP) was prepared by centrifugation of the citrated blood at 150 × g for 10 min at room temperature. Platelet aggregation was measured using PRP at 37°C by laser-light scattering aggregometer PA-20 (Kowa, Tokyo, Japan), which can measure both the size and the number of platelet aggregates.9Ozaki Y. Satoh K. Yatomi Y. et al.Detection of platelet aggregates with a particle counting method using light scattering.Anal Biochem. 1994; 218: 284-294Crossref PubMed Scopus (170) Google Scholar The laser light intensity increases in proportion to the size of the particles in a suspension, and this allows estimation of the size of platelet aggregates,9Ozaki Y. Satoh K. Yatomi Y. et al.Detection of platelet aggregates with a particle counting method using light scattering.Anal Biochem. 1994; 218: 284-294Crossref PubMed Scopus (170) Google Scholar with 25 to 400 mV corresponding to small aggregates (9–25 μm; SA), 400 to 1000 mV representing medium aggregates (25–50 μm; MA), and 1000 to 2048 mV indicating large aggregates (50–70 μm; LA). SPA was defined as the formation of small aggregates in the absence of any agonists despite a constant stirring at a rate of 1000 rpm (Fig 1, D). The area under the curve (AUC) was calculated for SPA. The day-to-day intrasubject coefficients of variation of SPA was 16.2% (1759000 ± 285200) in 1 diabetic patient on 10 separate occasions. As agonists, we used low concentrations of ADP (1.0 and 2.0 μmol/L; Fig 1, A and B) and collagen (1 μg/mL; Fig 1, C). We calculated the AUC for SPA from 0 to 10 min, whereas the AUC for agonist-induced SA, MA, and LA were assessed from 0 to 7 min. Venous blood was obtained between 6 and 7 am. Serum and plasma samples were centrifuged at 2500 rpm for 15 min, and the supernatant was stored at –70°C until use. The plasma concentration of fibrinogen was determined by the Method of Claus, and serum concentration of high-sensitivity C-reactive protein (hs-CRP) was determined by an immunonephelometric assay (N-High-sensitivity CRP; Dade Behring, Deerfield, Ill). The plasma concentration of von Willebrand factor (vWF) was measured with a commercial kit (STA LIA vWF; Behring, Marburg, Germany), and the results were expressed as a percentage of the data obtained with normal pooled plasma. The serum level of HMW adiponectin was measured with our new sandwich enzyme-linked immunosorbent assay that employs a monoclonal antibody targeting human HMW adiponectin,13Nakano Y. Tajima S. Yoshimi A. et al.A novel enzyme-linked immunosorbent assay specific for high-molecular-weight adiponectin.J Lipid Res. 2006; 47: 1572-1582Crossref PubMed Scopus (115) Google Scholar, 14Aso Y. Yamamoto R. Wakabayashi S. et al.Comparison of serum high-molecular weight (HMW) adiponectin with total adiponectin concentrations in type 2 diabetic patients with coronary artery disease using a novel enzyme-linked immunosorbent assay to detect HMW adiponectin.Diabetes. 2006; 55: 1954-1960Crossref PubMed Scopus (236) Google Scholar whereas serum levels of glycated albumin were measured by a commercial enzymatic method (Lucica GA-L kit; Asahi Kasei Pharma, Tokyo, Japan). To assess the glomerular filtration rate (GFR), the estimated GFR (eGFR) was calculated with the Cockcroft-Gault formula.15Cockcroft D.W. Gault M.H. Prediction of creatinine clearance from serum creatinine.Nephron. 1976; 16: 31-41Crossref PubMed Scopus (13039) Google Scholar Urinary albumin excretion (UAE) in a 24-h urine collection specimen was measured with an immunoturbidimetric assay. Twenty consecutive diabetic patients were hospitalized for a 2-week educational program. They were encouraged to reduce total fat intake rather than calories prior to the hospitalization. As the dietary intervention, all patients received a diet that provided 25 kcal of energy per kg of ideal body weight, including 50% carbohydrate, 20% protein, and 30% fat (as percentage of daily energy intake) during the hospitalization. Patients were treated with a sulfonylurea and/or metformin. None of them were treated with thiazolinediones or insulin. During hospitalization, the doses of antidiabetic drugs were fixed without any escalations in the doses of medication from what they received in the outpatient treatment. At the beginning and end of the 2-week period, blood samples were obtained and SPA was measured. We excluded patients who were receiving anticoagulants or anti-platelet drugs. None of the patients had liver disease or severe renal impairment. PRP was collected from 10 healthy subjects (5 women and 5 men) and washed platelets were isolated from the PRP by centrifugation at 850 g for 15 min. The platelet pellet was resuspended in modified Tyrode’s buffer (137.5 mmol/L NaCl, 12 mmol/L NaHCO3, 0.4 mmol/L NaH2PO4 · 2H2O, 2.7 mmol/L KCl, 1 mmol/L MgCl2 · 6H2O, 5.5 mmol/L dextrose, pH 7.4) and the platelet concentration was adjusted to 2.0 × 108/L in the buffer. Then the occurrence of SPA was measured in the absence or presence of 20 μg/mL recombinant adiponectin (a molecular weight of 28 kDa; Biovender Laboratory Medicine, Inc., Modrice, Czech Republic) over 15 min. All subjects gave informed consent to these studies, and they were approved by the hospital ethics committee. Data are presented as the mean ± standard deviation (SD) or the median and interquartile range. Normally distributed data were analyzed by the Student paired t test or an unpaired t test. Nonparametric data were compared between groups by Wilcoxon’s matched-pairs test or the Mann-Whitney U test. Correlations were determined by linear regression analysis or stepwise multivariate analysis. The differences in prevalence between groups were assessed by the chi-squared test. Logarithmic transformation of the data for hs-CRP and UAE was done to normalize the distribution and allow the use of parametric tests. In all analyses, a P value of less than 0.05 was accepted as indicating statistical significance. The clinical characteristics of the control subjects and the diabetic patients, including platelet aggregation, are shown in Table I. SPA was significantly greater in the diabetic patients than in the control subjects (5.73 [3.29, 10.7] × 106 vs 3.25 [1.88, 5.95)] × 106, P = 0.0136). In contrast, there was no significant difference of agonist-induced platelet aggregation between control subjects and diabetic patients.Table IDemographic, clinical, and laboratory data in healthy control subjects and diabetic patientsControlDiabetesP valueN (F/M)20 (9/11)82 (30/52)0.4874Age (years)51.9 ± 7.354.5± 11.00.2975BMI (kg/m2)21.8 ± 2.525.0 ± 4.00.0009Duration of diabetes (years)—7.0 (3.8, 15.0)FPG (mg/dL)88.7 ± 8.7168.7 ± 52.2<0.0001HbA1c (%)5.10 ± 0.509.55 ± 1.70<0.0001LDL cholesterol (mg/dL)112.1 ± 20.6118.2 ± 29.60.3659HDL cholesterol (mg/dL)60.3 ± 13.947.6 ± 13.60.0002Triglyceride (mg/dL)68.50 (58.5, 125.0)147.0 (108.0, 198.0)<0.0001eGFR (mL/min)90.9 ± 19.6113.1 ± 39.60.0171Hematocrit (%)42.4 ± 3.542.7 ± 4.10.7938Platelet counts (×105/μL)24.6 ± 4.120.6 ± 6.90.0168HMW adiponectin (μg/mL)—3.0 (1.7, 5.4)SPA (×106)3.25 (1.88, 5.95)5.73 (3.29, 10.7)0.0136ADP 1.0 μmol/L Small aggregates (×106)26.4 (15.6, 39.8)20.4 (10.6, 39.1)0.3370 Medium aggregates (×106)7.39 (1.91, 25.7)3.92 (1.93, 13.2)0.0729 Large aggregates (×106)1.64 (0.12, 13.3)0.48 (0.08, 7.13)0.3018ADP 2.0 μmol/L Small aggregates (×106)24.4 (13.4, 29.0)27.3 (15.2, 43.4)0.3726 Medium aggregates (×106)16.6 (9.25, 18.6)13.7 (7.58, 21.9)0.0729 Large aggregates (×106)29.8 (21.1, 35.5)30.2 (20.2, 38.8)0.5388Collagen 1.0 μg/mL Small aggregates (×106)11.3 (8.08, 16.0)9.02 (7.51, 10.7)0.0547 Medium aggregates (×106)9.22 (7.46, 10.5)8.90 (6.76, 10.6)0.9751 Large aggregates (×106)30.6 (21.1, 36.2)30.2 (20.2, 38.8)0.6981Data are the mean ± SD or the median and inter-quartile ranges.Abbreviations: BMI, body mass index; NS, not significant. Open table in a new tab Data are the mean ± SD or the median and inter-quartile ranges. Abbreviations: BMI, body mass index; NS, not significant. In the patients with type 2diabetes, simple linear regression analysis showed a positive correlation of SPA with age, fasting plasma glucose (FPG), glycated albumin, fibrinogen, and hs-CRP, whereas a negative correlation was found with eGFR and hematocrit (Table II). To identify the independent factors for SPA, we performed stepwise regression with forward selection that included all significant variables. According to the model that explained 71.3% of the variation of SPA, fibrinogen, GA, hs-CRP, and hematocrit were independent determinants of SPA in patients with type 2 diabetes (Table II).Table IIUnivariate and multivariate analyses of relationships between SPA and clinical parameters in patients with type 2 diabetesVariableUnivariate analysisMultivariate analysisrP valueβP valueAge (years)0.33090.0040NSBMI−0.17550.1347NEDiabetes duration (years)0.18730.1100NEFPG (mg/dL)0.35540.0015NSGlycated albumin (%)0.35280.00220.3870.001HbA1c (%)0.07090.5454NEHOMA-IR0.05290.6545NELDL cholesterol (mg/dL)0.07200.9428NETriglyceride (mg/dL)−0.07040.5482NEHDL cholesterol (mg/dL)0.03780.7496NEeGFR (mL/min)−0.27120.0185NSUAE (log10 mg/24 h)0.08090.4931NEAlbumin (g/dL)−0.13880.2315NEHematocrit (%)0.30690.0078−0.2770.022Platelet counts (×105/μl)0.06450.5822NEHMW adiponectin (μg/mL)0.18090.1177NEFibrinogen (mg/dL)0.5470<0.00010.480<0.001vWF (%)0.22730.0498NEhs-CRP (log10 ng/mL)0.35400.00070.3000.013R20.509Abbreviations: BMI, body mass index; NS, not significant.Note: β indicates partial coefficient; and NE indicates that the value does not enter the final model. Open table in a new tab Abbreviations: BMI, body mass index; NS, not significant. Note: β indicates partial coefficient; and NE indicates that the value does not enter the final model. Next, we divided the patients into 2 groups without and with hyperfibrinogenemia because stepwise analysis showed that the fibrinogen level had the strongest independent influence on SPA. Hyperfibrinogenemia was defined as a plasma fibrinogen level ≥ 350 mg/dL, in keeping with most previous studies.16Ernst E. Resch K.L. Fibrinogen as a cardiovascular risk factor: a meta-analysis and review of the literature.Ann Intern Med. 1993; 118: 956-963Crossref PubMed Scopus (998) Google Scholar As shown in Table III, the patients with hyperfibrinogenemia were older than those without it. Low-density lipoprotein (LDL) cholesterol and hs-CRP levels were higher in patients with hyperfibrinogenemia, whereas eGFR was lower. SPA, MA, and LA induced by 1.0 μmol/L ADP, LA induced by 2.0 μmol/L ADP, and MA induced by 1.0 μg collagen were all significantly greater in the patients with hyperfibrinogenemia than in those without hyperfibrinogenemia.Table IIIDemographic, clinical, and laboratory data in diabetic patients with and without hyperfibrinogenemiaNo hyperfibrinogenemiaHyperfibrinogenemiaP valueN (F/M)54 (17/37)28 (13/15)0.1827Age (years)52.4 ± 11.659.3±8.40.0063BMI (kg/m2)25.4 ± 3.924.1±4.20.1826Duration of diabetes (years)6.0 (3.0, 11.5)14.0 (5.0, 18.5)0.292FPG (mg/dL)163.5 ± 42.4179.0±67.20.2046HbA1c (%)9.37 ± 1.619.91±1.840.1654HOMA-IR2.92 ± 1.503.36±1.740.2539LDL cholesterol (mg/dL)113.5 ± 29.5127.4±28.10.420HDL cholesterol (mg/dL)46.5 ± 12.749.6±15.40.3280Triglyceride (mg/dL)146.0 (101.5, 200.5)148.0 (111.0, 194.5)0.8209eGFR (mL/min)122.5 ± 42.094.8±27.90.0023UAE (mg/g.Cr)9.0 (4.5, 23.5)12.5 (7.0, 51.3)0.1330Hematocrit (%)43.0 ± 4.042.0±4.30.2978Platelet counts (×105/μl)20.0 ± 6.822.7±5.70.0750HMW adiponectin (μg/mL)2.90(1.90, 4.00)3.25 (1.50, 8.20)0.4504hs-CRP (mg/L)0.05 (0.0, 0.12)0.11 (0.05, 0.27)0.0149SPA (×106)4.75 (2.93, 8.10)11.4 (3.47, 22.3)0.0190ADP 1.0 μmol/L Small aggregates (×106)18.9 (10.6, 32.5)25.1 (11.3, 43.3)0.5926 Medium aggregates (×106)1.33 (0.08, 6.62)8.12 (0.78, 19.2)0.0234 Large aggregates (×106)0.22 (0.04, 1.35)2.69 (0.25, 15.7)0.0202ADP 2.0 μmol/L Small aggregates (×106)28.5 (15.6, 42.9)25.3 (13.1, 46.4)0.4805 Medium aggregates (×106)12.7 (7.07, 19.9)18.0 (8.45, 23.8)0.1697 Large aggregates (×106)9.85 (1.73, 30.4)32.7 (15.2, 45.4)0.0284Collagen 1.0 μg/mL Small aggregates (×106)8.97 (7.44, 10.8)9.61 (7.77, 10.6)0.7836 Medium aggregates (×106)8.50 (6.03, 10.0)9.83 (8.76, 11.7)0.0413 Large aggregates (×106)27.9 (14.1, 38.5)33.8 (25.2, 39.7)0.1997Hypertension (n; %)11 (20.3)13 (46.4)0.0139Statin use (n; %)11 (20.3)6 (21.4)0.9108Current smoking (n; %)25 (46.2)14 (50.0)0.7501SU/Met (n)44/2525/12Data are the mean ± SD or the median and interquartile ranges.Abbreviations: Met, metformin; NS, not significant; SU, sulfonylurea. Open table in a new tab Data are the mean ± SD or the median and interquartile ranges. Abbreviations: Met, metformin; NS, not significant; SU, sulfonylurea. Next, we investigated changes of SPA and agonist-induced platelet aggregation in 20 patients with type 2 diabetes after the 2-week hospital education program. As shown in Table IV, body weight and BMI decreased significantly after 2 weeks. FPG also showed a significant decrease, as did LDL cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride concentrations. Moreover, SPA decreased significantly after 2 weeks (Fig 2). However, we found no significant changes of agonist-induced platelet aggregation. The decrease of SPA after 2 weeks showed a significant negative correlation with the changes of serum HMW adiponectin (Fig 3). However, no significant correlation was observed between the change of SPA and that of fasting plasma glucose (r = 0.063, P = 0.7906), triglycerides, or HDL-cholesterol (data not shown).Table IVClinical characteristic and laboratory data before and after 2-week diet therapy in patients with type 2 diabetesBeforeAfterP valueN (F/M)20 (5/15)20 (5/15)Age (years)53.9 ± 7.9—Duration of diabetes (years)8.5 (5.0, 14.5)—Body weight (kg)74.3 ± 10.472.3 ± 9.8<0.0001BMI (kg/m2)27.9 ± 3.326.8 ± 3.0<0.0001FPG (mg/dL)167.0 ± 39.7113.3 ± 26.3<0.0001HbA1c (%)9.18 ± 1.03—Glycated albumin (%)26.1 ± 4.1—LDL cholesterol (mg/dL)114.9 ± 25.8103.0 ± 25.50.0475HDL cholesterol (mg/dL)43.8 ± 8.339.6 ± 8.60.0076Triglyceride (mg/dL)186.5 (137.0, 235.0)97.0 (80.0, 129.5)0.0002HMW adiponectin (μg/mL)3.15 (2.15, 6.15)2.70 (1.50, 7.50)0.9569SPA (×106)6.02 (5.41, 9.78)5.47 (2.88, 7.14)0.0073ADP 1.0 μmol/L Small aggregates (×106)32.5 (20.4, 41.2)31.8 (20.5, 40.4)0.8265 Medium aggregates (×106)6.69 (0.52, 18.2)2.17 (0.87, 9.35)0.7315 Large aggregates (×106)0.95 (0.18, 6.05)0.33 (0.05, 1.53)0.8875ADP 2.0 μmol/L Small aggregates (×106)27.8 (14.8, 47.8)19.2 (7.55, 38.0)0.5529 Medium aggregates (×106)16.3 (5.70, 24.1)15.5 (6.96, 25.1)0.6949 Large aggregates (×106)22.3 (2.77, 44.2)23.6 (17.1, 42.9)0.4752Collagen 1.0 μg/mL Small aggregates (×106)9.11 (7.03, 11.2)8.24 (7.73, 10.0)0.6833 Medium aggregates (×106)8.19 (5.04, 10.0)9.17 (5.29, 10.7)0.8301 Large aggregates (×106)25.7 (7.46, 36.6)30.4 (8.17, 40.9)0.5077Hypertension (n; %)8 (40%)—Statin use (n; %)5 (25%)—SU use/Met use (n)19/14—Data are the mean±SD or the median and inter-quartile ranges.Abbreviations: BMI, body mass index; Met, metformin; NS, not significant; SU, sulfonylurea. Open table in a new tab Fig 3Correlation between changes of SPA and serum HMW adiponectin after 2 weeks of hospitalization in 20 patients with type 2 diabetes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Data are the mean±SD or the median and inter-quartile ranges. Abbreviations: BMI, body mass index; Met, metformin; NS, not significant; SU, sulfonylurea. In vitro SPA decreased significantly when washed platelets were incubated with 20 μg/mL recombinant adiponectin compared with incubation without adiponectin (4.30 [3.71, 5.66] × 106 vs 7.10 [5.70, 9.68)] × 106, P = 0.0115; Fig 4) The current study demonstrated that the AUC for SPA detected by laser LS method was greater in diabetic patients than in control subjects; this finding is in agreement with the results of previous studies.7Fukuda K. Ozaki Y. Satoh K. et al.Phosphorylation of myosin light chain in resting platelets from NIDDM patients is enhanced: correlation with spontaneous aggregation.Diabetes. 1997; 46: 488-493Crossref PubMed Google Scholar, 8Iwase E. Tawata M. Aida K. et al.A cross-sectional evaluation of spontaneous platelet aggregation in relation to complications in patients with type II diabetes mellitus.Metabolism. 1998; 47: 699-705Abstract Full Text PDF PubMed Scopus (59) Google Scholar In contrast, we found no significant difference of agonist-induced platelet aggregation between the control subjects and diabetic patients, suggesting that SPA is a more sensitive measure of platelet hyperaggregability than agonist-induced platelet aggregation in patients with type 2 diabetes. SPA is considered to represent the first phase of platelet aggregation and is followed by the formation of medium and large platelet aggregates.9Ozaki Y. Satoh K. Yatomi Y. et al.Detection of platelet aggregates with a particle counting method using light scattering.Anal Biochem. 1994; 218: 284-294Crossref PubMed Scopus (170) Google Scholar, 17Eto K. Takeshita S. Ochiai M. et al.Platelet aggregation in acute coronary syndromes: use of a new aggregometer with laser light scattering to assess platelet aggregability.Cardiovasc Res. 1998; 40: 223-229Crossref PubMed Scopus (64) Google Scholar Large aggregates are associated with the second step of aggregation, by which it becomes irreversible.17Eto K. Takeshita S. Ochiai M. et al.Platelet aggregation in acute coronary syndromes: use of a new aggregometer with laser light scattering to assess platelet aggregability.Cardiovasc Res. 1998; 40: 223-229Crossref PubMed Scopus (64) Google Scholar Thus, SPA reflects a slight increase of platelet aggregability and initial (primary) aggregation contributing to thrombus formation, which cannot be detected by the conventional optical density method.9Ozaki Y. Satoh K. Yatomi Y. et al.Detection of platelet aggregates with a particle counting method using light scattering.Anal Biochem. 1994; 218: 284-294Crossref PubMed Scopus (170) Google Scholar Measurement of SPA may allow the early detection of thrombus formation in high-risk patients. However, it remains unclear why SPA is enhanced in patients with type 2 diabetes. A previous study showed a relationship between SPA and albuminuria in patients with type 2 diabetes.18Araki S. Matsuno H. Haneda M. et al.Correlation between albuminuria and spontaneous platelet microaggregate formation in type 2 diabetic patients.Diabetes Care. 2009; 32: 2062-2067Crossref PubMed Scopus (14) Google Scholar In the current study, linear regression analysis showed that the AUC of SPA was correlated positively with age, fasting plasma glucose, glycated albumin, hs-CRP, and plasma fibrinogen. However, we found no significant correlation between SPA and urinary albumin excretion, suggesting that albuminuria is not important for the formation of spontaneous platelet aggregates in diabetic patients. By a stepwise multivariate analysis, we found that fibrinogen was the strongest independent determinant of SPA in patients with type 2 diabetes. In addition, SPA was increased in diabetic patients with hyperfibrinogenemia compared with those without it. Fibrinogen mediates platelet aggregation by binding to glycoprotein IIb/IIIa and is associated with irreversible aggregation.19Varga-Szabo D. Pleines I. Nieswandt B. Cell adhesion mechanisms in platelets.Arterioscler Thromb Vasc Biol. 2008; 28: 403-412Crossref PubMed Scopus (446) Google Scholar In fact, MA and LA induced by 1 μmol/L ADP, LA induced by 2.0 μmol/L ADP, and MA induced by 1.0 μg/mL collagen were all significantly greater in patients with hyperfibrinogenemia than in those without it, which suggests that fibrinogen is involved in irreversible platelet aggregation. Plasma fibrinogen levels are reported to be increased in type 2 diabetes, especially in patients with diabetic nephropathy.20Ganda O.P. Arkin C.F. Hyperfibrinogenemia. An important risk factor for vascular complications in diabetes.Diabetes Care. 1992; 15: 1245-1250Crossref PubMed Scopus (155) Google Scholar Taken together, it seems that an elevated plasma fibrinogen levels play a significant role in SPA (initial platelet aggregation) as well as irreversible aggregation in patients with type 2 diabetes. The current study demonstrated that the serum level of hs-CRP was an independent determinant of SPA in patients with type 2 diabetes. A previous study revealed that CRP was an independent predictor of platelet aggregation in the setting of acute coronary syndromes.21Modica A. Karlsson F. Mooe T. Platelet aggregation and aspirin non-responsiveness increase when an acute coronary syndrome is complicated by an infection.J Thromb Haemost. 2007; 5: 507-511Crossref PubMed Scopus (43) Google Scholar Chronic inflammation plays a role in the pathogenesis of atherosclerosis.22Ross R. Atherosclerosis–an inflammatory disease.N Engl J Med. 1999; 340: 115-126Crossref PubMed Scopus (19127) Google Scholar Increased blood levels of inflammatory markers, especially hs-CRP, have been associated with CHD mortality in the general population23Ridker P.M. Hennekens C.H. Buring J.E. Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women.N Engl J Med. 2000; 342: 836-843Crossref PubMed Scopus (5011) Google Scholar and with a poor prognosis among patients with acute coronary syndromes.24Ridker P.M. Cannon C.P. Morrow D. et al.Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction 22 (PROVE IT-TIMI 22) Investigators. C-reactive protein levels and outcomes after statin therapy.N Engl J Med. 2005; 352: 20-28Crossref PubMed Scopus (1960) Google Scholar The increase of serum hs-CRP may be associated with platelet hyperaggregability, leading to accelerated atherothombosis, but the mechanisms responsible for the relation between serum hs-CRP and SPA in type 2 diabetes remain unclear. One possible explanation is that CRP may impair endothelial cells production of nitric oxide and prostacyclin, which are potent inhibitors of platelet aggregation, leading to platelet hyperaggregabilty.25Devaraj S. Singh U. Jialal I. The evolving role of C-reactive protein in atherothrombosis.Clin Chem. 2009; 55: 229-238Crossref PubMed Scopus (165) Google Scholar Another possibility is that modified forms of CRP (eg, monomeric CRP), may induce platelet aggregation directly, although native CRP inhibits platelet aggregation.26Khreiss T. József L. Potempa L.A. Filep J.G. Opposing effects of C-reactive protein isoforms on shear-induced neutrophil-platelet adhesion and neutrophil aggregation in whole blood.Circulation. 2004; 110: 2713-2720Crossref PubMed Scopus (92) Google Scholar The current study demonstrated that fasting plasma glucose and glycated albumin (GA), but not hemoglobin A1c (HbA1c), were correlated positively with SPA in diabetic patients. Similarly, Toshima et al27Toshima H. Sugihara H. Hamano H. et al.Spontaneous platelet aggregation in normal subject assessed by a laser light scattering method: an attempt at standardization.Platelets. 2008; 19: 293-299Crossref PubMed Scopus (9) Google Scholar reported that plasma glucose is an independent determinant of SPA in 167 healthy subjects. Furthermore, multivariate analysis demonstrated that GA was an independent determinant of SPA in our diabetic patients. Serum GA levels are more short-term indicator of glycemic control than HbA1c levels because the half-life of albumin in serum is approximately 12–19 days.28Roohk H.V. Zaidi A.R. A review of glycated albumin as an intermediate glycation index for controlling diabetes.J Diabetes Sci Technol. 2008; 2: 1114-1121Crossref PubMed Scopus (129) Google Scholar Taken together, these results suggest that acute hyperglycemia may be associated more strongly with an increase of SPA than chronic hyperglycemia. In contrast, GA itself may promote SPA in diabetic patients because in vitro studies have shown that platelet aggregation is enhanced in the presence of glycated albumin compared with nonglycated albumin.29Rubenstein D.A. Yin W. Glycated albumin modulates platelet susceptibility to flow induced activation and aggregation.Platelets. 2009; 20: 206-215Crossref PubMed Scopus (42) Google Scholar However, there have been no previous reports about the short-term effects of improved glycemic control on SPA in diabetic patients, and the current study is the first to investigate changes of SPA prospectively after short-term improved glycemic control treatment in patients with type 2 diabetes. We found that SPA was decreased significantly after 2 weeks of hospitalization for education in 20 patients with type 2 diabetes, whereas no significant changes of agonist-induced platelet aggregation were noted. We confirmed also that fasting plasma glucose was decreased significantly after 2 weeks of hospitalization. These results suggest that hyperglycemia itself may influence SPA because it was reduced by even 2 weeks of improved glycemic control. However, the current study has a limitation on measures of improved glycemic control after 2 weeks of hospitalization. It would be ideal to measure serum GA again at the end of 2- week inpatient treatment. Interestingly, the current study showed a negative correlation between the changes of SPA and serum HMW adiponectin after hospitalization in our diabetic patients. Furthermore, the in vitro study demonstrated that the formation of SPA was significantly decreased by incubation of washed platelets with recombinant adiponectin at a concentration of 20 μg/mL, which is a relatively high but physiologic concentration in humans. These results suggest that adiponectin directly inhibits SPA. In adiponectin knockout mice, platelet aggregation is enhanced by low concentrations of ADP.30Kato H. Kashiwagi H. Shiraga M. et al.Adiponectin acts as an endogenous antithrombotic factor.Arterioscler Thromb Vasc Biol. 2006; 26: 224-230Crossref PubMed Scopus (168) Google Scholar A recent in vitro study also demonstrated that full-length adiponectin inhibits aggregation of human platelet induced by ADP or epinephrine in a dose-dependent manner.11Restituto P. Colina I. Varo J.J. Varo N. Adiponectin diminishes platelet aggregation and sCD40L release. Potential role in the metabolic syndrome.Am J Physiol Endocrinol Metab. 2010; 298: E1072-E1077Crossref PubMed Scopus (42) Google Scholar In contrast, globular adiponectin (a product of proteolytic cleavage product) induces platelet activation via the collagen receptor glycoprotein VI.31Riba R. Patel B. Aburima A. Naseem K.M. Globular adiponectin increases cGMP formation in blood platelets independently of nitric oxide.J Thromb Haemost. 2008; 6: 2121-2131Crossref PubMed Scopus (15) Google Scholar Adiponectin undergoes posttranslational modification before its release from adipocytes to yield various multimeric forms: a trimer (low-molecular-weight adiponectin), a hexamer (trimer-dimer) of medium molecular weight, and a larger multimeric form with an HMW.32Pajvani U.B. Hawkins M. Combs T.P. et al.Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity.J Biol Chem. 2004; 279: 2152-2162Crossref Scopus (1010) Google Scholar These different forms of adiponectin may have distinct cellular and biological activities. We showed here that a change of serum HMW adiponectin may be associated with a decrease of SPA after improved glycemic control in type 2 diabetics and that incubation with full-length recombinant adiponectin inhibited SPA in washed platelets. Unlike Restituto et al,11Restituto P. Colina I. Varo J.J. Varo N. Adiponectin diminishes platelet aggregation and sCD40L release. Potential role in the metabolic syndrome.Am J Physiol Endocrinol Metab. 2010; 298: E1072-E1077Crossref PubMed Scopus (42) Google Scholar we employed washed platelets, and thus we evaluated the direct effect of recombinant adiponectin on platelet aggregation without any influence of native adiponectin in the PRP samples. Among the several isoforms of adiponectin, HMW adiponectin may be the strongest inhibitor of platelet aggregation. However, a future in vitro study will be required to investigate the direct effect of HMW adiponectin on the aggregation of washed platelets. The current study showed that SPA was decreased significantly in 20 diabetic patients after 2 weeks of hospitalization for education, whereas no significant changes of serum HMW adiponectin were noted (Table IV). However, we found a negative correlation between the changes of SPA and serum HMW adiponectin after hospitalization in our diabetic patients, suggesting that the greater increase in serum HMW adiponectin was associated with the greater reduction in SPA. We speculate that some factors other than HMW adiponectin may be associated with the decrease in SPA after 2 weeks of hospitalization because the negative correlation between the changes of SPA and serum HMW adiponectin was weak, although significant. In conclusion, SPA was shown to be enhanced in patients with type 2 diabetes compared with control subjects by measurement with a laser-light scattering aggregometer. The measurement of SPA seems to be a sensitive and useful marker for early detection of thrombus formation. Hyperfibrinogenemia and hyperglycemia are associated independently with SPA in type 2 diabetics. SPA was reduced after even short-term improvement of glycemic control, and adiponectin inhibited SPA directly by washed platelets in vitro. We wish to thank Professor T. Inukai at our department for reviewing this manuscript, Drs K. Morita and K. Takebayashi for their helpful suggestions, and our colleagues for assistance with this work. Does the fat cell have something to say to the platelet about keeping thrombosis in check in diabetes?Translational ResearchVol. 159Issue 1PreviewDiabetes mellitus, especially type 2 diabetes, is associated with accelerated atherosclerosis, resulting in an increased risk of myocardial infarction, stroke, and peripheral vascular disease.1 Patients with type 2 diabetes show multiple pathophysiologic abnormalities, including a proinflammatory state, increased production of highly reactive oxidation products, dyslipidemia, abnormally functioning endothelium, aberrant cellular signal transduction, glycation of structural proteins, and abnormal platelet function. Full-Text PDF" @default.
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• W1972320922 title "Spontaneous platelet aggregation evaluated by laser light scatter in patients with type 2 diabetes: Effects of short-term improved glycemic control and adiponectin" @default.
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