Platelets Of Diabetic Patients Research Articles

Syzygium cumini Extract Decrease Adenosine Deaminase, 5′Nucleotidase Activities and Oxidative Damage in Platelets of Diabetic Patients

Diabetes mellitus, a chronic metabolic disorder, has assumed epidemic proportions and its long-term complications can have devastating consequences. The oxidative stress in diabetes was greatly increased due to prolonged exposure to hyperglycemia and impairment of oxidant/antioxidant equilibrium. Syzygium cumini is being widely used to treat diabetes by the traditional practitioners over many centuries. Adenosine deaminase (ADA) and 5′-Nucleotidase (5′NT) are enzymes of purine nucleoside metabolism that play an important role in the regulation of adenosine (Ado) levels. In this study, we investigated the effect of Syzygium cumini aqueous leaves extract (ASc) on ADA and 5′NT activities and on parameters of oxidative stress under in vitro conditions, using platelets of patients with Type 2 diabetes mellitus. Platelet-Rich Plasma (PRP) was assayed by ADA, 5′NT, Catalase (CAT), Superoxide Dismutase (SOD) activities and Thiobarbituric acid reactive substances (TBARS) levels. We observed that ADA, 5′NT activities and TBARS levels were significantly higher when compared to the control group, and ASc (100 and 200 µg/mL) prevented these effects. Our study demonstrates that ASc was able to remove oxidant species generated in diabetic conditions and modulates in the Ado levels. Then, ASc may promote a compensatory response in platelet function, improving the susceptibility-induced by the diabetes mellitus.

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.

Alterations of Adenine Nucleotide Metabolism and Function of Blood Platelets in Patients With Diabetes

Increased activity of blood platelets contributes to vascular complications in patients with diabetes. The aim of this work was to investigate whether persisting hyperglycemia in diabetic patients generates excessive accumulation of ATP/ADP, which may underlie platelet hyperactivity. Platelet ATP and ADP levels, thiobarbituric acid-reactive species synthesis, and aggregation of platelets from patients with diabetes were 18-82% higher than in platelets from healthy participants. In patients with diabetes, platelet stimulation with thrombin caused about two times greater release of ATP and ADP than in the healthy group while decreasing intraplatelet nucleotide content to similar levels in both groups. This indicates that the increased content of adenylate nucleotides in the releasable pool in the platelets of diabetic patients does not affect their level in metabolic cytoplasmic/mitochondrial compartments. Significant correlations between platelet ATP levels and plasma fructosamine, as well as between platelet ATP/ADP and platelet activities, have been found in diabetic patients. In conclusion, chronic hyperglycemia-evoked elevations of ATP/ADP levels and release from blood platelets of patients with diabetes may be important factors underlying platelet hyperactivity in the course of the disease.

Elevated release of sCD40L from platelets of diabetic patients by thrombin, glucose and advanced glycation end products

The pro-inflammatory CD40/CD40L dyad participates in atherogenesis. Plasma levels of the soluble ligand (sCD40L) predict cardiovascular events and are elevated in diabetic patients. This study compared CD40/CD40L surface expression on platelets and T lymphocytes of diabetic and control subjects, and tested whether glucose and advanced glycation end products (AGEs) stimulate sCD40L release. Constitutive and inducible surface expression of CD40/CD40L on platelets or T lymphocytes did not differ between diabetic patients (n = 9) and controls (n = 13). Platelets from diabetic patients contained higher intracellular CD40L than controls (p < 0.05) and thrombin stimulated greater platelet sCD40L release in diabetic patients (15.11 +/- 16.77 ng/ml) compared to controls (3.64 +/- 2.03 ng/ml; p < 0.05). Glucose and AGEs induced platelet sCD40L release and CD40L expression in mouse megakaryocytes. This study demonstrates elevated CD40L content and inducible release from platelets of diabetic patients, and identifies glucose and AGEs as potential triggers of expression and release accounting for the elevated sCD40L plasma levels in these patients.

P2Y12 receptors play a significant role in the development of platelet microaggregation in patients with diabetes.

Ninety-eight diabetic patients (type 2) were studied together with 24 healthy normotensive controls. Microaggregates (particle scale, <25 microm) of platelets were detected by a laser scattering system. Microaggregates in the control group showed a time-dependent reversible change; however, they existed continuously in 82 of 98 diabetic patients. When platelets of diabetics were stimulated by a shear stress alone without ADP, 74 also showed spontaneous and irreversible microaggregates even though they were not observed in all control subjects. In control subjects, microaggregates were inhibited by MRS2279 (a P2Y1 antagonist), but not AR-C69931MX (a P2Y12 antagonist). However, AR-C69931MX prevented irreversible microaggregates in diabetic patients. When either aspirin or ticlopidine was administered to diabetic patients with irreversible microaggregates, both drugs significantly decreased microaggregates induced by a low dose of ADP. Ticlopidine additionally reduced the microaggregates induced by shear stress alone. In conclusion, microaggregates of platelets via P2Y12 receptors could play a key role in the hypersensitivity of platelets in diabetic patients, and the measurement of microaggregation could be a useful marker to estimate of thrombogenesis. These findings present a possible new means for patients with diabetes to prevent ischemic events.

Inhibitory effect of pyridyloxy- or phenoxylphenoxyalkanate derivatives on rat lens aldose reductase and rat platelet aggregation.

The therapeutic potential of aldose reductase inhibitors for the prevention of the secondary complications of diabetes has been extensively reported. On the other hand, the hyperaggregability of platelets in diabetic patients has also been reported as a cause of chronic diabetic complications. The purpose of this study was to develop new compounds with these dual effects from pyridyloxy- or phenoxylphenoxyalkanate synthesized derivatives and examine the effect of their structure-activity relationships on the inhibition of rat lens aldose reductase (RLAR) as well as on platelet aggregation. 2-[4-(2,6-dichloro-3-methyl-phenoxy)-3-nitro-phenoxy]-propionic acid (3) exhibited the most potent inhibitory effect (IC(50) = 3.0 +/- 0.21 microM), comparable to tetramethylene glutaric acid (IC(50) = 6.1 +/- 0.2 microM), which is used as a positive control on RLAR, and showed potent inhibitory activities on rat platelet aggregation induced by ADP and collagen (IC(50) = 0.093 +/- 0.01 and 0.032 +/- 0.01 microM, respectively) comparable with aspirin (IC(50) = 0.15 +/- 0.05 and 0.047 +/- 0.01 microM, respectively), used as a positive control.

Important triad in cardiovascular medicine: diabetes, coronary intervention, and platelet glycoprotein IIb/IIIa receptor blockade.

Diabetes mellitus is a potent risk factor for greater prevalence, complexity, and complications of cardiovascular disease, and diabetics thus constitute a sizable proportion of patients for whom coronary revascularization is required. However, both percutaneous and surgical revascularization techniques are associated with particular challenges in this population. Diabetics typically have an increased likelihood of restenosis and repeat revascularization procedures after stenting compared with their non-diabetic counterparts.1 Trends toward higher death rates in diabetics have also been observed over 1 year or longer after stenting,1–4 and patients with diabetes thus remain one of the few groups for whom percutaneous coronary intervention (PCI) does not yet provide survival benefit commensurate with that of coronary artery bypass surgery.5–7 Diabetes mellitus is also a predictor of elevated mortality risk and accelerated disease progression after surgical revascularization.5,8,9 The processes leading to adverse prognosis after revascularization in diabetics are likely multifactorial and relate to widespread atheroma, small vessel vasculopathy, impaired collateral formation, exuberant neointimal hyperplasia, and adverse remodeling.10,11 There is also evidence that hypercoagulability contributes to accelerated atherosclerosis and exaggerated injury response after revascularization in diabetics.11–13 Diabetes is associated with increased levels of circulating fibrinogen, thrombin, and factor VII, and with diminished plasma fibrinolytic and antithrombin III activity. Moreover, platelets in diabetic patients are increased in size, are more frequently activated, and exhibit greater adhesiveness, thromboxane synthesis, glycoprotein (GP) IIb/IIIa expression, and mitogenic activity. Adjunctive antiplatelet therapy with GP IIb/IIIa blockade reduces the incidence of acute ischemic events by as much as 35% to 50% among the broad population of patients undergoing elective or urgent PCI,14 and evidence supporting long-term suppression of mortality with at least one of these inhibitors, abciximab, has been accrued as well.3,15,16 The benefits of GP IIb/IIIa inhibitors in this setting seem to be complementary …

Relationship between metabolic glycaemic control and platelet content of glutathione and its related enzymes, in insulin-dependent diabetes mellitus

The relationship between glycaemic metabolic control and intracellular concentration of reduced glutathione (GSH) and related enzymes GSH-peroxidase (GSH-Px), GSH-reductase (GSH-Red), GSH-transferase (GSH-Tr), glucose-6-P-dehydrogenase (G6PDH), and thioltransferase (TT) in patients with insulin-dependent diabetes mellitus (IDDM) is controversial. Choosing platelets as cell model (as commonly done in previous studies), the aim of this study was to relate the platelet content of GSH and related enzymes to glycaemic metabolic control, expressed as glycated haemoglobin (HbA1c), as well as to presence of retinopathy and nephropathy in 114 IDDM patients. As compared to controls, both GSH and GSH-Red (geometric means (95% CI)) were significantly increased in platelets of diabetic patients: 3.3 (0.7–9.6) vs. 2.4 (0.8–7.6) mmol 10 −9 platelets; P=0.01 for GSH, and 30.6 (14.7–61.6) vs. 22.2 (8.7–52.2) mU 10 −9 platelets, P=0.0002 for GSH-Red, and TT activity was marginally decreased in the IDDM group ( P=0.06). While no clear relationship was present between GSH-related enzymes and HbA1c, a trend was present toward a non-linear relation between HbA1c and GSH, being significantly related by a parabolic curve ( P=0.002). As compared to patients with normoalbuminuria ( n=88), diabetic patients with increased urinary albumin excretion rate ( n=26) had a significant decrease in platelet TT concentration (3.2 (0.9–6.7) vs. 5.1 (1.9–18.7) mU 10 −9 platelets; P=0.0002), whereas retinopathy was not associated to modifications in GSH or in the enzymatic pattern. In summary: (a) platelet GSH and GSH-Red are increased in IDDM, while other enzymes are unmodified; (b) GSH seems to be related to metabolic control according to non-linear parabolic curve; (c) presence of increased albuminuria is associated to a selective decrease in platelet TT content.

Purine metabolites and malondialdehyde in platelets of diabetic patients

The concentration of some of the purine nucleotides and their metabolites together with that of malondialdehyde (MDA) have been measured in resting and stimulated platelets of type 1 and type 2 diabetic patients. While control platelets show a net decrease of guanosine triphosphate (GTP) (3.1 vs. 2.3 nmol per 10 9 platelets) and guanosine diphosphate (GDP) (3.0 vs. 2.0 nmol per 10 9 platelets) and a significant increase of adenosine (0.04 vs. 0.55 nmol per 10 9 platelets) with platelet stimulation, platelets of type 1 and type 2 diabetic patients have a lesser change of these metabolites (GTP, 2.6 vs. 2.4; GDP, 2.3 vs. 2.4; adenosine, 0.04 vs. 0.30 ( P<0.05 vs. control) nmol per 10 9 platelets in type 1 diabetics; GTP, 2.4 vs. 2.7; GDP, 2.4 vs. 2.1; adenosine, 0.08 vs. 0.32 ( P<0.05 vs. control) nmol per 10 9 platelets in type 2 diabetics). These results indicate that the change (stimulated minus resting) of GTP, GDP and adenosine in diabetic platelets is significantly different from that of controls ( P<0.001). Moreover, the amount of MDA produced during platelet activation seems to be lower than controls only in type 2 diabetes (1.81 vs. 2.86 nmol per 10 9 platelets, P<0.05). These results seem to indicate that a difference in the pattern of platelet nucleotides could be an important feature even in well-controlled diabetes, while MDA is probably modified only in association with the late vascular complications of diabetes.

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.

Does reduced membrane lipid fluidity underlie the altered thrombin-induced expression of integrin αIIbβ3and PADGEM-140 in membranes of platelets from diabetic juveniles?

In diabetic patients, where the membrane lipid microviscosity of blood platelets is altered, the availability of platelet membrane receptors may change concomitantly. Platelet hypersensitivity in diabetic subjects was previously hypothesized to result from the nonenzymatic glycosylation-induced loss in platelet membrane fluidity. In our present study juvenile type 1 diabetic subjects were compared with their relevant controls with respect to thrombin-stimulated platelet activation in relation to glycation-induced impairments of platelet membrane dynamics. Our results indicate that: (a) the mean steady-state fluorescence polarization (p) of both 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-anilino-8-naphthalenesulphonate (ANS) in membranes from diabetic subjects were significantly greater than for control subjects, thus indicating reduced membrane lipid fluidity in diabetic platelets in various membrane regions; (b) the significantly higher [(3)H]NaBH(4) reduction, indicating the increased attachment of glucose to protein amino groups, was attributed to the proteins extracted from diabetic platelet membranes; (c) CD62-positive resting platelets were not significantly more abundant in diabetic patients; (d) basically, unaltered amounts of PADGEM-140 membrane antigen (CD62) copies were detected in resting diabetic platelets; (e) significantly higher numbers of membrane glycoprotein β(3) were found in diabetic platelets; (f) thrombin-induced elevations in the expression of CD61 (β(3)) and CD62 (PADGEM-140) occurred to much higher extent in platelets of diabetic patients, thus pointing to more profound activation of diabetic platelets by thrombin; (g) the total amounts of platelet membrane glycoprotein β(3) was significantly reduced in platelet lysates from diabetic subjects. We conclude that glycation-induced rigidization of platelet membranes might hypersensitize diabetic platelets to aggregating agents by rendering platelet membrane receptors more exposed to the external environment. Thus, thrombin may bind more efficiently to the exposed glycoprotein receptors (due to glycation) in diabetic platelets. Such excessive exposure and displacements toward the external environment might favour the accelerated shedding of some membrane proteins in diabetic platelets. We further suggest that their subsequent replacements would render platelet intrinsic storage pools exhausted and thus, might explain the diminished total amount of β(3) found in platelets of diabetic patients.

Expression of Adhesion Molecules on the Surface of Activated Platelets is Diminished by PGI2-analogues and an NO (EDRF)-Donor: A Comparison Between Platelets of Healthy and Diabetic Subjects

Adhesion molecules such as P-selectin (CD 62), glycoprotein (CP) 53 (CD63) and thrombospondin play a decisive role in the thrombogenic transformation of platelets. Here we present evidence obtained using flow cytometric analysis that the PGI(2)-mimetics iloprost and taprostene, and an NO (EDRF)donor (SIN-1) are able to inhibit the expression of P-selectin, GP 53 and thrombospondin on human platelets activated by submaximal concentrations of thrombin. Since the half-maximal concentrations for inhibition of antigen expression (0.15 nM for iloprost, 3.0-5.3 nM for taprostene) are much lower than for activation of adenylate cyclase (1.4 nM for iloprost and 29.4 nM for taprostene) our data suggest that the occupation of a small number of PGI(2)-receptors is sufficient to inhibit the thrombogenic transformation and that spare PGI(2)-receptors are present on human platelets. In diabetes, the EC(50) for inhibition of expression of platelet antigens is shifted to higher concentrations suggesting that platelets from type 1 diabetic patients are partly resistant to PGI(2). Since the dose dependent increase in c-AMP by iloprost is not changed and intraplatelet c-AMP is elevated in platelets of diabetic patients, we assume that steps in the activation cascade subsequent to activation of adenylate cyclase are disturbed in diabetes.

The in vitro thromboxane A2 production by platelets of diabetic patients is normal at physiological [Ca 2+] o: [formula omitted], M. Cattaneo, A. Ghidoni ∗, P.M. Mannucci. A. Bianchi Bonomi Hemophilia and Thrombosis Center and 1st Clinical Medicine ,IRCCS Maggiore Hospital, University of Milan

The in vitro thromboxane A2 production by platelets of diabetic patients is normal at physiological [Ca 2+] o: [formula omitted], M. Cattaneo, A. Ghidoni ∗, P.M. Mannucci. A. Bianchi Bonomi Hemophilia and Thrombosis Center and 1st Clinical Medicine ,IRCCS Maggiore Hospital, University of Milan

The In Vitro Production of Thromboxane B2 by Platelets of Diabetic Patients Is Normal at Physiological Concentrations of lonized Calcium

Platelets of patients with diabetes and no evidence of macroangiopathy produce normal amounts of thromboxane (Tx) B2 in vivo, whereas they usually show increased production in vitro. Since in vitro studies have been usually performed in citrated PRP, we tested the hypothesis that the discrepancy between in vivo and in vitro studies is due to the low concentration of plasma ionized calcium ([Ca2+]o) that is present in citrated PRP. In fact, low [Ca2+]o artifactually potentiates the platelet TxB2 production in vitro. Forty patients with diabetes mellitus and 37 matched controls were studied. Blood was anticoagulated with citrate, the thrombin inhibitor D-phenylalanyl-l-prolyl-l-chloromethylketone (PPACK) or both anticoagulants. Platelet aggregation, release of 14C-serotonin and TxB2 production were induced in platelet rich plasma (PRP) by several agonists. The following results were obtained: i) Citrated PRP: Arachidonic acid induced aggregation (p < 0.01) and TxB2 production (p < 0.02) were significantly greater in patients than in controls. No statistically significant differences were found with other agonists. ii) PPACK PRP: No statistically significant difference was found between diabetic platelets and controls. iii) PPACK plus citrate PRP: The results were not different from those obtained with citrate alone. Therefore, our results show that diabetic platelets produce normal amounts of TxB2 in vitro when the [Ca2+]o is physiological.

The in vitro thromboxane A2 production by platelets of diabetic patients is normal at physiological [Ca 2+] : C.R. Falcon, M. Cattaneo, A. Ghidoni ∗, P.M. Mannucci. A. Bianchi Bonomi Hemophilia and Thrombosis Center and ∗1st Clinical Medicine. University of Milan, Italy

The in vitro thromboxane A2 production by platelets of diabetic patients is normal at physiological [Ca 2+] : C.R. Falcon, M. Cattaneo, A. Ghidoni ∗, P.M. Mannucci. A. Bianchi Bonomi Hemophilia and Thrombosis Center and ∗1st Clinical Medicine. University of Milan, Italy

The modification of peroxidative processes and phospholipid metabolism in platelets of diabetic patients induced by indobufen

The modification of peroxidative processes and phospholipid metabolism in platelets of diabetic patients induced by indobufen

Alterations of phospholipid asymmetry in the membrane of spontaneously aggregated platelets in diabetes

The changes of asymmetric distribution of anionic phospholipids of human platelets in diabetic patients were studied by fluorescent and freeze fracture cytochemistry, using merocyanine 540 (MC 540) and polymyxin B (P×B) as specific markers. The membrane anionic phospholipids were detected with P×B, a membrane nonpermeant probe, used either in native form for freeze fracture electron microscopy or as dansylated or iodinated derivative for fluorescence microscopy or gamma counting, respectively. MC 540 is a naturally fluorescent probe which reportedly inserts into less packed hilayer domains. Both in platelet rich plasma and in washed platelets obtained from diabetic patients, some small platelet aggregates were observed, their number being generally dependent on the level of hyperglycemia. In contrast with single platelets, the aggregated ones bind P×B as revealed by all assay methods. The fluorescence microscopic studies with dansyl P×B and MC 540 displayed a strong binding of the fluorescent markers to aggregated platelets. The electron microscopic examination of freeze fracture replicas showed the appearance of characteristic PxB-induced deformations in the plasmalemma of aggregated platelets. The gamma counting of 125I-P×B incubated samples indicates significant differences on the platelets of diabetic patients as compared to those obtained from healthy subjects. Our data provide evidence that in diabetic patients, the spontaneous aggregated platelets are a result of the appearance of the anionic phospholipids in the outer half of plasmalemma. These changes may enhance the procoagulant activity and should represent a determinant of activated platelet recognition and their removal from circulation by splenic macrophages.

Studies on Platelets in Diabetic Patients

Studies on Platelets in Diabetic Patients

Studies on Platelets in Diabetic Patients

Studies on Platelets in Diabetic Patients

On the detection of the activated platelets in diabetic patients with vascular complications

In order to detect the activated platelets, plasma beta-thromboglobulin (β-TG) levels and nucleotide pools in platelets were determined in blood samples from 34 diabetic patients with various vascular complications i. e. retinopathy, nephropathy, ischemic heart disease, cerebral thrombosis and obstruction of arteries in the extremities.β-TG levels were measured using RIA kit (Radiochemical Center, Amersham). Higher plasma, β-TG levels than 50ng/ml were found in seven patients (21%).Nucleotide pools in platelets were determined with high pressure liquid chromatography. ATP and ADP levels in platelets were decreased in 47% and 35 of the patients, respectively. ATP/ADP ratio in those cases was ranged between 0.9 and 2.8. Amount of GTP in platelets was elevated in 3 cases and decreasedd in 2 cases. All three cases whose plasma β-TG levels elevated more than 100ng/ml showed decrease of both ATP and ADP in the platelets.Measurement of nucleotide pools in platelets is a more sensitive method to detect the evidence of platelet activation in patients with arteriosclerosis.