Enhanced P-selectin expression and increased soluble CD40 Ligand in patients with Type 1 diabetes mellitus and microangiopathy: evidence for platelet hyperactivity and chronic inflammation

Aims/hypothesis Platelet activation, endothelial dysfunction and inflammation may be involved in early stages of diabetic microangiopathy. We therefore investigated patients with Type 1 diabetes mellitus, without (n=19) and with (n=20) microangiopathy, matched for glycaemic control and duration of disease, and matched with healthy control subjects (n=27).Methods Platelet activation was measured as platelet P-selectin expression using whole blood flow cytometry and as soluble P-selectin by immunoassay. Von Willebrand factor antigen in plasma, serum soluble E-selectin, CD40 ligand (sCD40L) and C-reactive protein (CRP) served as markers for endothelial function and inflammation.Results Thrombin-induced platelet P-selectin expression was enhanced, and soluble P-selectin and sCD40L concentrations were increased in patients with microangiopathy compared with the control subjects (p<0.01 for both) and with patients without microangiopathy (p<0.05 for P-selectin expression and sP-selectin), whereas all three parameters were similar in patients without microangiopathy and in the control subjects. CRP and soluble E-selectin were increased in patients with microangiopathy, compared with the control subjects (p<0.01 and p<0.05), whereas von Willebrand factor did not differ between the groups.Conclusions/interpretation Microangiopathy in Type 1 diabetes is associated with platelet hyperactivity, endothelial dysfunction and low-grade inflammation, indicating an increased risk for cardiovascular disease.Abbreviations sP-selectin Soluble P-selectin - sCD40L soluble CD40 Ligand - CRP C-reactive protein     

Meal-induced platelet activation in Type 2 diabetes mellitus: effects of treatment with repaglinide and glibenclamide.

AIMS: To compare the effects of treatment with repaglinide and glibenclamide on platelet function and endothelial markers in patients with Type 2 diabetes mellitus, before and after a standardized meal. METHODS: Fifteen patients with Type 2 diabetes were investigated on three occasions: at baseline without oral hypoglycaemic drug treatment, and after 6 weeks' treatment with repaglinide or glibenclamide, respectively, in an open randomized cross-over study. Agonist-induced platelet P-selectin expression and platelet aggregation, urinary thromboxane, soluble P-selectin, von Willebrand factor (VWF), soluble E-selectin, intercellular adhesion molecule (ICAM-1) and C-reactive protein (CRP) were measured. In addition, pre-meal data were compared with non-diabetic control subjects (n = 15), matched for sex, age and BMI. RESULTS: Adenosine diphosphate (ADP)-induced platelet P-selectin expression increased post-meal in Type 2 diabetic patients both at baseline and after treatment with repaglinide and glibenclamide (P < 0.01 for all; repeated measures anova). Repaglinide treatment reduced fasting ADP-induced P-selectin expression compared with baseline (P = 0.01), but did not influence meal-induced platelet hyper-reactivity (P = 0.32). No significant anti-platelet effects of glibenclamide treatment were found. Plasma concentrations of VWF and ICAM-1 were elevated in patients with Type 2 diabetes compared with control subjects (P < 0.05 for both) and were reduced during treatment with repaglinide (P < 0.01 for both) but did not change during glibenclamide treatment. CONCLUSIONS: The post-meal state is associated with enhanced platelet reactivity in patients with Type 2 diabetes mellitus. Pre-meal treatment with repaglinide or glibenclamide does not inhibit postprandial platelet activation, but repaglinide treatment is associated with attenuated platelet and endothelial activity in the fasting state.     

Homocysteine-induced inhibition of nitric oxide production in platelets: a study on healthy and diabetic subjects

Aims/hypothesis: The molecular mechanisms involved in the platelet activation observed in hyperhomocysteinemia are not known. We aimed to discover if homocysteine concentrations are associated with abnormal platelet nitric oxide production in healthy and diabetic subjects. Methods: The study cohort included 28 patients with Type I (insulin-dependent) diabetes mellitus, 30 patients with Type II (non-insulin-dependent) diabetes mellitus, and 34 healthy subjects. Homocysteine plasma concentrations were measured by high-performance liquid chromatography. Platelet nitric oxide production was measured using a nitric oxide meter before and after a 3-h incubation with 100 μmol/l homocysteine. Stimulation experiments were done in vitro by the addition of α-thrombin (0.2 U/ml). Results: Basal platelet nitric oxide production was lower in diabetic patients than in healthy subjects. Nitric oxide release was reduced by in vitro homocysteine incubation, being lower in platelets from diabetic patients than in platelets from control subjects. Thrombin increased nitric oxide synthesis in platelets from healthy subjects both in the presence and absence of homocysteine. In diabetic subjects thrombin increased nitric oxide release in the absence of homocysteine. But in the presence of homocysteine the response was reduced. An inverse relation was found between plasma homocysteine levels and basal platelet nitric oxide release in diabetic and healthy subjects. Conclusion/interpretation: Homocysteine could exert its atherogenic action in healthy and diabetic subjects partly by inhibiting platelet nitric oxide production with the subsequent increased platelet activation and aggregation. [Diabetologia (2001) 44: 979–982] Received: 29 November 2000 and in revised form: 4 April 2001     

Platelet and monocyte activation by hyperglycemia and hyperinsulinemia in healthy subjects

Type 2 diabetes mellitus (T2DM) patients have hyperglycemia and hyperinsulinemia and increased risk of atherosclerosis and acute vascular complications. We have reported elevated circulating tissue factor procoagulant activity (TF-PCA) during hyperglycemia (HG) and hyperinsulinemia (HI) in normal subjects. To evaluate the effect of hyperglycemia and hyperinsulinemia on blood cell activation, we assessed platelet CD40L and P-selectin, monocyte tissue factor (TF), and the formation of monocyte-platelet and neutrophil-platelet aggregates. These were assessed in the resting state and following activation with ADP and thrombin (SFLLRN). Healthy individuals were subjected to 24 h of hyperglycemia and hyperinsulinemia, selective hyperglycemia, selective hyperinsulinemia, or normal glucose and insulin. Platelet CD40L expression increased with high glucose/high insulin, selective hyperglycemia and selective hyperinsulinemia. Monocyte-platelet aggregates increased with high glucose/high insulin. Monocyte TF expression increased with high glucose/high insulin and with selective hyperinsulinemia. Upon stimulation with ADP and SFLLRN, monocyte-platelet and neutrophil-platelet aggregates, platelet CD40L and P-selectin, and monocyte TF increased compared to the resting state but was not different between 0 and 24 h, indicating that the responsiveness to those agonists was not altered.Conclusions: Hyperglycemia-hyperinsulinemia in healthy individuals induced platelet activation and monocyte TF expression promoting a procoagulant and proinflammatory state that may contribute to acute vascular events and atherogenesis. Platelet responsiveness to activation with ADP or SFLLRN appears not to be altered by hyperglycemia-hyperinsulinemia.     

Nephropathy in Type 1 diabetes is associated with increased circulating activated platelets and platelet hyperreactivity

Patients with diabetes mellitus (DM) have increased platelet activation compared to non-diabetic controls. Platelet hyperreactivity has been associated with adverse cardiovascular outcomes in Type 2 DM, and with diabetic nephropathy. We investigated the relationship between platelet activation and nephropathy in Type 1 DM. Patients with Type 1 DM and diabetic nephropathy (n = 35), age- and sex-matched Type 1 DM patients with persistent normoalbuminuria (n = 51), and healthy age- and sex-matched controls (n = 30) were studied. Platelet surface P-selectin, platelet surface activated GPIIb/IIIa, monocyte-platelet aggregates (MPAs) and neutrophil-platelet aggregates (NPAs) were measured by whole blood flow cytometry as markers of platelet activation. Platelet reactivity was assessed in response to exogenously added ADP and thrombin receptor activating peptide (TRAP). Platelet surface P-selectin (basal and in response to 0.5 or 20 µM ADP) was higher in nephropathy patients compared with normoalbuminuric patients (P = 0.027), and non-diabetic controls (P = 0.0057). NPAs were higher in nephropathy patients compared to normoalbuminuric patients (P = 0.0088). MPAs were higher in nephropathy patients compared to non-diabetic controls (P = 0.0075). There were no differences between groups in activated GPIIb/IIIa or in response to TRAP at any end-point. More patients with nephropathy received aspirin (71.4%) compared to normoalbuminuric patients (27.4%) (P < 0.0001). Type 1 diabetic nephropathy, as compared with normoalbuminuria, is associated with circulating activated platelets and platelet hyperreactivity to ADP, despite the confounding variable of more nephropathy patients receiving aspirin. This platelet activation is likely to contribute to the known increased risk of cardiovascular events in patients with diabetic nephropathy.     

Platelet activation in diabetic cardiovascular autonomic neuropathy.

AIMS: Platelet activation is known to be associated with arrhythmic effects in myocardial ischaemia. The present study attempts to clarify whether diabetic cardiovascular autonomic neuropathy (CAN) is associated with intravascular platelet activation. METHODS: Platelet activation was assessed by flow cytometry analysis in 30 patients with Type 1 diabetes mellitus screened for diabetic complications. Fifteen patients showed evidence of CAN as assessed by a battery of standard cardiovascular autonomic reflex tests. Fifteen patients without CAN were then selected as a matched control group. Platelet activation was assessed by flow cytometric detection of activation-dependent platelet membrane antigens (P-selectin (CD62), thrombospondin, lysosomal GP53 (CD63) and ligand-induced binding site-1 of GPIIb/IIIa (LIBS-1)). RESULTS: Significantly more activated platelets were detected in the patients with CAN showing 20.9% (coefficient of variation (CV) 44%) CD63+ (vs. 17.2% (CV 19%) in controls, P < or = 0.05), 6.4% (CV 87%) CD62+ (vs. 4.1% (CV 37%), P < or = 0.05), and 6.7% (CV 55%) thrombospondin+ (vs. 4.6% (CV 39%), P < or = 0.01) platelets, respectively. LIBS-1 on platelets was not significantly different between patients with and without CAN. No correlation was found between glucose metabolism and platelet activation. CONCLUSIONS: Cardiovascular autonomic neuropathy is associated with platelet activation in Type 1 diabetes mellitus. The high platelet activation may reflect an increased prothrombotic state in diabetic cardiovascular autonomic dysfunction.     

Leucocyte Na+/H+ antiport activity in Type 1 (insulin-dependent) diabetic patients with nephropathy

Summary The development of proteinuria in Type 1 (insulin-dependent) diabetic patients may depend on predisposition to essential hypertension in addition to poor glycaemic control. Previous work has shown increased leucocyte Na^–/H⁺ antiport activity in essential hypertension and increased erythrocyte Li⁺/Na⁺ exchange in Type 1 diabetic patients with proteinuria. To test whether susceptibility to nephropathy in Type 1 diabetes was linked to abnormalities of leucocyte Na⁺/H⁺ antiport activity, we measured the intracellular pH and kinetics of the Na⁺/H⁺ antiport in 19 Type 1 diabetic subjects with, and 15 diabetic subjects without albuminuria and compared them to 25 matched normal control subjects. Intracellular pH (mean ±SD 7.59 ± 0.14) and maximal transport capacity of the antiport (V_max 87.7 ±24.9 mmol· 1^–1·min^–1) were higher in diabetic subjects with albuminuria compared to normotensive control subjects (pH 7.44±0.09; V_max 55.6±10.3 mmol·1^–1min^–1; p <0.001 for both), similar to the defect described in essential hypertension. These differences were not seen in diabetic subjects with normal urinary albumin/creatinine ratios (pH 7.46 ±0.09; V_max 61.0 ±13.6mmol·1^–1min^–1). Buffering characteristics of the leucocytes at different pH in the Type 1 diabetic subjects with albuminuria differed from normal control subjects and diabetic subjects with normal urinary albumin/creatinine ratios. We conclude that increased leucocyte Na⁺/H⁺ antiport activity, a known marker of essential hypertension, is usually associated with nephropathy in Type 1 diabetes.     

Mutation analysis of suppressor of cytokine signalling 3, a candidate gene in Type 1 diabetes and insulin sensitivity

Aims/hypothesis Beta cell loss in Type 1 and Type 2 diabetes mellitus may result from apoptosis and necrosis induced by inflammatory mediators. The suppressor of cytokine signalling (SOCS)-3 is a natural inhibitor of cytokine signalling and also influences insulin signalling. SOCS3 could therefore be a candidate gene in the development of Type 1 and Type 2 diabetes mellitus.Methods Mutation analysis of the SOCS3 gene was performed in 21 patients with Type 1 diabetes mellitus and in seven healthy subjects. An identified promoter variant was examined in (i) 250 families with Type 1 diabetic family members (1097 individuals); (ii) 212 glucose-tolerant first-degree relatives of Type 2 diabetic patients; and (iii) 370 population-based young, healthy subjects who were unrelated.Results Three mutations were identified in the promoter region, but none in the coding region or the 3UTR. Two of the three mutations had allele frequencies below 1% whereas the C –920A substitution had a minor allele frequency of 8%. In the group of young healthy subjects the insulin sensitivity index was higher among homozygous carriers of the A-allele than among heterozygous and wild-type subjects (p=0.027, uncorrected). The same trend was found in the group of first-degree relatives of Type 2 diabetic patients. No association or linkage was found to Type 1 diabetes mellitus.Conclusions/interpretation Homozygosity for the A-allele of the C –920A promoter polymorphism of the SOCS3 gene may be associated with increased whole-body insulin sensitivity, but deserves further investigation.Abbreviations AIR acute insulin response - AP2 activator protein 2 - GH growth hormone - SI Insulin sensitivity index - Sib-TDT Sib Transmission Disequilibrium Test - SNP single nucleotide polymorphism - SOCS suppressor of cytokine signalling - SSCP single-strand conformational polymorphism     

Increased platelet,leukocyte, and coagulation activation in primary myelofibrosis

Platelet and leukocyte activation has been demonstrated in polycythemia vera (PV) and essential thrombocythemia (ET), but such information is limited in primary myelofibrosis (PMF). Platelet, leukocyte, endothelial, and coagulation activation status was assessed in 26 PMF patients and compared with data from 22 age- and sex-matched healthy individuals. Study included flow cytometry assessment of platelet P-selectin expression [at baseline and after adenosine diphosphate (ADP), thrombin and arachidonic acid stimulation], platelet–neutrophil and platelet–monocyte complexes, and CD11b expression in neutrophils and monocytes. Additionally, soluble P-selectin, sCD40L, tissue factor, thrombomodulin, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer were measured by enzyme-linked immunosorbent assays. The above parameters were correlated with the patients’ clinical data and presence of the JAK2 V617F mutation. Compared with controls, PMF patients had increased baseline platelet activation, as shown by significantly higher levels of soluble and platelet P-selectin expression, and also higher percentages of platelet–monocyte complexes. Neutrophil and monocyte CD11b expression was significantly higher in patients with the JAK2 mutation than in those with wild-type allele or the controls. Endothelial and coagulation activation, as demonstrated by increased plasma levels of thrombomodulin and F1 + 2, was also found in PMF, with patients with the JAK2 mutation showing significantly higher values of F1 + 2 than those with wild-type allele. In conclusion, PMF patients have platelet, leukocyte, endothelial, and coagulation activation similar to that in PV and ET. CD11b overexpression and F1 + 2 are correlated with the presence of the JAK2 mutation.     

Clinically apparent atherosclerotic disease in diabetes is associated with an increase in platelet microparticle levels.

BACKGROUND: The commonest cause of mortality in patients with Type 2 diabetes is atherothrombosis, which can be related to abnormalities in the coagulation and fibrinolytic pathways, as well as in platelet function. Platelet microparticles (PMPs) may contribute to the prothrombotic state and may promote the progression of atherosclerosis. We hypothesized that PMPs are elevated in Type 2 diabetes and that patients with Type 2 diabetes and clinically apparent atherosclerosis would have the highest levels. Similarly, we hypothesized that soluble plasma P-selectin (sPsel) and CD40L (both molecules which are released by activated platelets), as well as %CD62P (P-selectin) and %CD63 positivity on platelets quantified by flow cytometry, would be highest in patients with Type 2 diabetes and clinically apparent atherosclerotic disease, and might be correlated to PMP levels. METHODS: Venous blood was obtained from 21 Type 2 diabetic patients without atherosclerotic complications, 18 diabetic patients with clinically apparent atherosclerotic disease and 21 non-diabetic control subjects. PMPs, as well as %CD62P and %CD63 positivity on platelets, were quantified by flow cytometry. sPsel and CD40L were measured using ELISA. RESULTS: Patients with Type 2 diabetes and clinically apparent atherosclerotic disease had the highest PMP (P=0.045) and sPsel (P=0.046) levels, compared with patients without complications (who had intermediate PMP levels) and control subjects. Control subjects had the lowest CD40L levels (P<0.001) when compared with patients with Type 2 diabetes, with no difference in sCD40L levels between the two diabetic subgroups. %CD62P and %CD63 positivity did not differ between the groups. PMP levels correlated with %CD62P positivity (P=0.026) but not to %CD63 positivity (P=0.089), sCD40L (P=0.407) or sP-sel (P=0.163); sCD40L levels did not correlate with any other marker of platelet activation. CONCLUSION: PMPs are elevated in Type 2 diabetes. In addition, patients with clinically apparent atherosclerosis had the highest levels of PMPs and sPsel. Thus, PMPs may be a marker of symptomatic atherosclerotic vascular disease in Type 2 diabetes, and may both represent a useful risk stratification tool as well as a novel therapeutic target for anti-thrombotic drugs.     

Increased intracellular calcium mobilization in platelets from patients with Type 2 (non-insulin-dependent) diabetes mellitus

Summary Enhanced platelet functions have been reported in patients with diabetes mellitus. Our recent study demonstrated that phosphoinositide turnover is increased in platelets from diabetic patients. In the present study, we evaluated the abnormality in platelet intracellular calcium mobilization in patients with Type 2 (non-insulin-dependent) diabetes mellitus using fura-2, a fluorescent calcium indicator. Washed platelets were prepared from six diabetic patients with increased platelet aggregation rates (DM-A group), seven diabetic patients with normal platelet aggregation rates (DM-B group), and eight age-matched healthy control subjects. The basal intracellular free calcium concentrations in platelets were similar among the three groups. Thrombin (0.025–0.1 U/ml) induced a dose-dependent increase in intracellular calcium in both the presence and the absence of extracellular calcium. This increase in the presence of extracellular calcium, which depends on calcium influx and release, was significantly higher in the DM-A group than in the DM-B and control groups. However, there was no significant difference between the control group and the DM-B group. In the absence of extracellular calcium, thrombin-induced calcium increase, which depends only on calcium release, was also significantly enhanced in the DM-A group. Furthermore, the calcium increase stimulated by platelet-activating factor (10 nmol/l) with and without extracellular calcium was significantly higher in the DM-A group than in the other groups. Additionally, calcium ionophore A23187 (100 nmol/l) caused a significantly higher calcium increase in the DM-A group with extracellular calcium, while the calcium increase without extracellular calcium showed no significant difference among the three groups. These observations suggest that enhanced intracellular calcium mobilization due to increased calcium influx and release may be closely related to platelet hyperfunctions in diabetes mellitus.     

Possible mechanisms of the altered platelet volume distribution in type 2 diabetes: does increased platelet activation contribute to platelet size heterogeneity?

A direct consequence of increased platelet sensitivity in diabetes mellitus might be augmented release of platelet granule contents, which, in turn, may lead to the formation of a platelet volume gradient, increased platelet turnover and reduced survival of platelets from diabetic individuals. In this study we addressed the question whether diabetes-induced and lipid fluidity-mediated changes in platelet receptor exposure and accessibility might be part of a general mechanism underlying the increased rate of platelet ageing and reduced platelet survival in diabetes. Diabetic individuals showed higher numbers of platelets of extreme dimensions: very small platelets and larger platelets were more frequent compared to controls ( P(chi(2))< 0.03). The shifts in platelet volume distributions were paralleled by decreased expression of the alpha subunit of glycoprotein Ib (by up to 17%, P < 0.01) in platelet membranes from diabetic patients, increased expression of P-selectin in thrombin-stimulated diabetic platelets (P< 0.02), an increased number of platelet microparticles in diabetic individuals (P< 0.05 or P< 0.03 for resting or stimulated platelets, respectively), and reduced platelet membrane fluidity (by 5.2 +/- 0.6%, P< 0.01). We suggest that the distinct bimodality of platelet distribution in diabetic patients might arise from accelerated thrombopoiesis in diabetic subjects, and this is supported by the demonstration of elevated fractions of reticulated (rich with residual RNA) platelets in diabetic patients (14.6 +/- 5.6% vs 8.1 + 2.1% p(u) < 0.025). Overall, our results point to a fluidity-mediated platelet hypersensitivity and accelerated rate of platelet production in subjects with type 2 diabetes mellitus, which results in a greater number of very large and hypersensitive younger platelets and a more abundant fraction of small exhausted platelets.     

Possible mechanisms of the altered platelet volume distribution in type 2 diabetes: does increased platelet activation contribute to platelet size heterogeneity?

A direct consequence of increased platelet sensitivity in diabetes mellitus might be augmented release of platelet granule contents, which, in turn, may lead to the formation of a platelet volume gradient, increased platelet turnover and reduced survival of platelets from diabetic individuals. In this study we addressed the question whether diabetes-induced and lipid fluidity-mediated changes in platelet receptor exposure and accessibility might be part of a general mechanism underlying the increased rate of platelet ageing and reduced platelet survival in diabetes. Diabetic individuals showed higher numbers of platelets of extreme dimensions: very small platelets and larger platelets were more frequent compared to controls ( Px2< 0.03). The shifts in platelet volume distributions were paralleled by decreased expression of the alpha subunit of glycoprotein Ib (by up to 17%, P < 0.01) in platelet membranes from diabetic patients, increased expression of P-selectin in thrombin-stimulated diabetic platelets (P< 0.02), an increased number of platelet microparticles in diabetic individuals (P< 0.05 or P< 0.03 for resting or stimulated platelets, respectively), and reduced platelet membrane fluidity (by 5.2 +/- 0.6%, P< 0.01). We suggest that the distinct bimodality of platelet distribution in diabetic patients might arise from accelerated thrombopoiesis in diabetic subjects, and this is supported by the demonstration of elevated fractions of reticulated (rich with residual RNA) platelets in diabetic patients (14.6 +/- 5.6% vs 8.1 + 2.1% pu < 0.025). Overall, our results point to a fluidity-mediated platelet hypersensitivity and accelerated rate of platelet production in subjects with type 2 diabetes mellitus, which results in a greater number of very large and hypersensitive younger platelets and a more abundant fraction of small exhausted platelets.     

Elevated serum placental isoferritin in newly diagnosed Type 1 (insulin-dependent) diabetes mellitus. A possible marker for identification of high risk subjects

Summary Placental isoferritin is produced by activated T lymphocytes and may, therefore, be considered as a manifestation of T cell involvement. Placental isoferritin is measured using CM-H-9 monoclonal antibody which binds exclusively to placental isoferritin. Placental isoferritin has been determined in the serum of 80 patients with Type I (insulin-dependent) diabetes mellitus, 100 healthy first degree relatives and 81 healthy children. Serum levels which were measured in Type 1 diabetic patients, (24,0–140 U/ml; median and range) were significantly higher than those of family members (0,0–73; median and range; p<0.0001) and normal control subjects (0,0–48; median and range; p<0.0001). Using 0–10 U/ml as the upper limit of normal, it was found that 31 of 50 (62%) of Type 1 diabetic patients, 25 of 100 (25%) family members and 7 of 81 (8.6%) healthy control subjects had abnormal placental isoferritin levels. Islet cell antibodies were positive in 31 of 44 tested diabetic patients and, in 8 of 71 tested family members, and among them 54.8% and 50% respectively also had elevated placental isoferritin levels. However, no statistically significant correlation was found between islet cell antibodies and placental isoferritin levels. Treatment of Type 1 diabetic patients with insulin was accompanied by a significant decrease (p<0.002) of serum placental isoferritin within 2–4 weeks of treatment. It is noteworthy that placental isoferritin was below detection in 34 of 35 Type 2 (non-insulin-dependent) diabetic patients. Our findings suggest that placental isoferritin may be a marker of T cell involvement in autoimmune diabetes and that it could be used to identify high risk healthy first degree relatives.     

Raised serum apolipoprotein (a) in active diabetic retinopathy

Summary Progressive capillary occlusion often leads to severe retinopathy within 15–20 years of the onset of Type 1 (insulin-dependent) diabetes mellitus. Lipoprotein (a), a complex formed by apolipoprotein (a), apo B-100 and lipids, is considered an independent, genetically determined, predictor of cardiovascular disease. It may have antifibrinolytic properties in view of its similarity to plasminogen. To test the hypothesis that circulating lipoprotein (a) is associated with the process that leads to clinically active diabetic retinopathy, we measured the circulating levels of apolipoprotein (a) (which are strictly correlated with those of lipoprotein (a)) in two groups of patients with Type 1 diabetes of at least 15 years duration: 25 with active retinopathy and 27 without clinically detectable retinal lesions. Thirty-eight healthy subjects of the same age and sex served as controls. Serum apolipoprotein (a) was higher in the patients with active retinopathy (36(2-193) U/dl, geometric mean and range) than in those without clinically detectable retinal lesion (17(1–160)) and the control subjects (14(0–115)), p < 0.01 in both cases. The distribution of apolipoprotein (a) levels was skewed to the left, as expected, in the patients without clinically evident retinal lesions and the control groups, but there was a bimodal trend of distribution among those with active retinopathy. The levels of glycated haemoglobin were similar in the two groups of diabetic patients, and no significant differences were found for total and HDL cholesterol, triglycerides or apolipoproteins A1 and B between them and the control subjects. These preliminary results suggest that serum apolipoprotein (a) is elevated in patients with active retinopathy. The role of this lipoprotein as a predictor or a pathogenic effector of diabetic retinopathy, or both deserves further investigation.     

Effects of hyperglycemia and hyperinsulinemia on the tissue factor pathway of blood coagulation

Tissue factor (TF) is the primary initiator of blood coagulation. Circulating TF procoagulant activity (TF-PCA) is associated with blood cells and microparticles and is elevated in patients with type 2 diabetes mellitus. Combined hyperinsulinemia and hyperglycemia and to a lesser degree selective hyperinsulinemia for 24 hours in healthy volunteers increased circulating TF-PCA, monocyte TF surface expression and mRNA, plasma thrombin generation, and coagulation factors VII and VIII activities, suggesting that the coagulation system had been activated. In addition, platelet CD40L and platelet-monocyte aggregates increased, indicating platelet activation. Somatostatin abolished these changes. We conclude that hyperinsulinemia, but particularly the combination of hyperinsulinemia and hyperglycemia, creates a prothrombotic state and may, in addition, be proinflammatory and proatherogenic by virtue of the actions of CD40L and TF.     

Soluble P-selectin levels in diabetes mellitus patients with coronary artery disease

Type 2 (non-insulin-dependent) diabetes is associated with a marked increase in the risk of coronary heart disease. Platelets play a significant role in coronary artery disease. Soluble P-selectin is an index of platelets activation. In this study, Soluble P-selectin levels were measured by ELISA in the peripheral blood of 55 diabetic patients with coronary artery disease [21 acute myocardial infarction (AMI), 20 with unstable angina (UA), 14 with stable angina (SA)], 20 patients with diabetes mellitus without coronary artery disease (DM without), and 10 healthy controls.Soluble P-selectin level was significantly higher in patients with AMI (M+/-SD; 239.3+/-13.0 ng/ml), than those with UA (141.5+/-15.2 ng/ml), SA (92.1+/-7.7 ng/ml), DM without (89.8+/-7.1 ng/ml), and healthy control (86.1+/-4.5 ng/ml) (P < 0.001). In patients with US, sP-selectin was found to be significantly elevated as compared to the SA, DM without and control group. sP-selectin was not significantly different in DM without as compared to healthy controls. The sP-selectin levels was correlated to the duration of diabetes mellitus(R=0.33, P=0.03 ). Moreover, sP-selectin level was significantly higher in AMI patients with recurrent anginal attack as compared to that in those with single attack Multivariate analysis revealed that sP-selectin level at presentation had high adverse influence on coronary artery insult compared to healthy LDL cholesterol level, and the degree of hypertension. IN CONCLUSION: Plasma levels of soluble P-selectin were increased in patients with AMI, and UA compared to patients with SA and normal controls. Measurement of soluble P-selectin may be helpful marker of impending coronary artery insult in diabetic patients.     

Acute hyperglycemia increases soluble P-selectin in male patients with mild diabetes mellitus.

The aim of this study was to examine if acute hyperglycemia (an oral glucose tolerance test) activates platelet function, endothelial cells or thrombin generation in diabetic patients and healthy controls. Eleven males with mild type II diabetes mellitus and 11 healthy male volunteers, matched for age and body mass index, were investigated before and after the glucose load. Soluble P-selectin, von Willebrand factor antigen and markers of thrombin generation in plasma were determined by immunoassays, and platelet P-selectin expression (unstimulated and agonist-stimulated) by flow cytometry in whole blood. Acute hyperglycemia elevated plasma soluble P-selectin from 32.5 to 50.9 ng/ml in the diabetic group (P = 0.05) but not in the controls (from 27.3 to 28.8 ng/ml; P = 0.6). Also, soluble P-selectin levels were higher in patients with diabetes than in healthy controls during hyperglycemia, but not in the fasting state. Adenosine diphosphate- and thrombin-induced platelet P-selectin expression was slightly, but significantly, decreased by the glucose load, whereas platelet P-selectin expression in unstimulated samples was not affected. Plasma levels of von Willebrand factor and thrombin generation were similar in patients and controls, and were not altered by hyperglycemia. In conclusion, we found that acute hyperglycemia elevates soluble P-selectin in plasma in males with mild type II diabetes mellitus. Our observation of unaltered plasma levels of the endothelial marker von Willebrand factor is in agreement with platelets being the main source of P-selectin released into plasma following hyperglycemia. Thus, platelets in individuals with type II diabetes may be more susceptible to hyperglycemia than platelets in non-diabetic individuals.     

Elevated concentrations of soluble adhesion molecules and large platelets in diabetic patients: are they markers of vascular disease and diabetic nephropathy?

P-selectin, E-selectin, and mean platelet volume are markers associated with platelet reactivity that have been demonstrated to be increased in diabetes. We were particularly interested to see if there was a difference in mean platelet volume and selectins between diabetics and nondiabetics, and in diabetics with and without nephropathy, and whether there was a correlation between mean platelet volume and selectins. One hundred and fourteen diabetic patients and 31 healthy controls were investigated. Plasma levels of P-selectin and E-selectin were higher in the diabetic group than in controls (P = .001 and P = .007, respectively) and in diabetic patients with proteinuria than in patients without proteinuria (P = .002 and P = .004, respectively). Protein excretion was lower in patients with low mean platelet volume values (P = .004). In conclusion, elevated platelet volume and high selectin values may play a role in the development of vasculopathies and complications in diabetes mellitus. Further studies are needed to prove these results.     

Plasma histamine concentrations are elevated in patients with diabetes mellitus and peripheral vascular disease

Previous work has shown that plasma and tissue concentrations of histamine are elevated in rats with experimental diabetes mellitus and that leucocytes and platelets from patients with peripheral vascular disease have a higher histamine content than those from controls. In the present study, we have measured: (a) plasma histamine concentrations; (b) leucocyte and platelet histidine decarboxylase (the enzyme responsible for the biosynthesis of histamine) in patients with diabetes mellitus (Types I and II) and peripheral vascular disease; and (c) platelet and leucocyte histamine content. Plasma histamine concentration was significantly higher in patients with diabetes and peripheral vascular disease respectively than that in age-matched controls. Leucocyte histidine decarboxylase activity in diabetic and peripheral vascular disease patients was similar to that in controls, while platelets had no histidine decarboxylase activity. The leucocyte and platelet content of histamine were greater in patients with peripheral vascular disease than those in controls, but they were not altered in diabetic patients. There was no correlation between plasma histamine concentration, leucocyte and platelet histamine content, and histidine decarboxylase activity. We conclude that plasma histamine is elevated in diabetics and in patients with peripheral vascular disease and that platelet and leucocyte histamine content is increased in the latter. This increase in platelet and leucocyte histamine content is not due to an increase in histidine decarboxylase activity of these cells. The increase in plasma and cellular histamine content may contribute to the pathogenesis of increased endothelial permeability in diabetes and to the pathogenesis of intimal damage in atherosclerosis.