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Diabetes is associated with an increased risk of cardiovascular disease. This is partially attributed to an altered activation status of blood platelets in this disease. Previously, alterations have been shown in COX‐1 and protease activated receptor (PAR)‐3 receptor expression in platelets in two animal models of diabetes, there have not been studies which address expression of these proteins in mice with long‐term streptozotocin (STZ)‐induced diabetes. We have also addressed the effect of diabetes on platelet adhesion under flow conditions. With the use of flow cytometry, we have shown that certain markers of platelet basal activation, such as active form of α(II) (b)β(3) and of CD40L were increased in STZ‐induced diabetic mice. Platelets from STZ‐induced diabetic mice were also more reactive when stimulated with PAR‐4 activating peptide as revealed by higher expression of active form of α(II) (b)β(3), membrane‐bound on vWillebrand Factor and binding of exogenous fluorescein isothyanate‐labelled fibrinogen. Expression of COX‐1 and production of thromboxane A (2) in platelets of STZ‐induced diabetic mice were higher than in control animals. We observed no effect of diabetes on ability of platelets to form stable adhesions with fibrinogen in flow conditions. We conclude that although certain similarities exist between patterns of activation of platelets in animal models of diabetes, the differences should also be taken into account.

Background: Fibrin formation and structure may be affected by a plethora of factors, including both genetic and posttranslational modifications, such as glycation, nitration or acetylation. Methods: The present study examines the effect of fibrinogen glycation on fibrin polymerization, measured in fibrinogen concentration-standardized plasma of subjects with type 2 diabetes mellitus (T2DM) and in a solution of human fibrinogen exposed to 30 mM glucose for four days. Results: The fibrin polymerization velocity (Vmax) observed in the T2DM plasma (median 0.0056, IQR 0.0049‒0.0061 AU/s) was significantly lower than in non-diabetic plasma (median 0.0063, IQR 0.0058‒0.0071 AU/s) (p &lt, 0.05). Furthermore, significantly lower Vmax was observed for glucose-treated fibrinogen (Vmax 0.046, IQR 0.022‒0.085 AU/s) compared to control protein incubated with a pure vehicle (Vmax 0.053, IQR 0.034‒0.097 AU/s) (p &lt, 0.05). The same tendency was observed in the fibrinogen samples supplemented with 6 mM glucose just before measurements. It is assumed that glucose may affect the ability of fibrinogen to form a stable clot in T2DM subjects, and that this impairment is likely to influences the outcomes of some diagnostic assays. As the example, the impaired clotting ability of glycated fibrinogen may considerably influence the results of the standard Clauss method, routinely used to determine fibrinogen concentration in plasma. The stoichiometric analysis demonstrated that spontaneous glycation at both the sites with high and low glycation potential clearly dominated in T2DM individuals in all fibrinogen chains.

Blood platelets play a crucial role in the early stages of atherosclerosis development. The process is believed to require firm adhesion of platelets to atherosclerosis-prone sites of the artery. However, little evidence exists regarding whether the blood platelets of individuals with pathological conditions associated with atherosclerosis have higher potential for adhesion. This process is to a large extent dependent on receptors present on the platelet membrane. Therefore, the aim of the presented study was to determine whether blood platelets from diabetic patients have higher capacity of adhesion under flow conditions and how diabetes affects one of the crucial platelet receptors involved in the process of adhesion&mdash, GPIIIa. The study compares the ability of platelets from non-diabetic and diabetic humans to interact with fibrinogen and von Willebrand factor, two proteins found in abundance on an inflamed endothelium, under flow conditions. The activation and reactivity of the blood platelets were also characterized by flow cytometry. Platelets from diabetic patients did not demonstrate enhanced adhesion to either studied protein, although they presented increased basal activation and responsiveness towards low concentrations of agonists. Platelets from diabetic patients were characterized by lower expression of GPIIIa, most likely due to an enhanced formation of platelet-derived microparticles PMPs, as supported by the observation of elevated concentration of this integrin and of GPIIIa-positive PMPs in plasma. We conclude that altered functionality of blood platelets in diabetes does not increase their adhesive potential. Increased glycation and decrease in the amount of GPIIIa on platelets may be partially responsible for this effect. Therefore, higher frequency of interactions of platelets with the endothelium, which is observed in animal models of diabetes, is caused by other factors. A primary cause may be a dysfunctional vascular wall.

Purpose. The goal of this study was to estimate the hierarchical contribution of the most commonly recognized cardiovascular risk factors associated with atherogenesis to activation and reactivity of blood platelets in a group of men and women at ages 60-65.Methods. Socioeconomic and anthropometric data were taken from questionnaires. Blood morphology and biochemistry were measured with standard diagnostic methods. Plasma serum homocysteine was measured by immunochemical method. Plasma concentrations of VCAM, ICAM, total antioxidant status, and total oxidant status were estimated with commercial ELISA kits. Markers of oxidative stress of plasma and platelet proteins (concentrations of protein free thiol and amino groups) and lipids (concentrations of lipid peroxides) and generation of superoxide anion by platelets were measured with colorimetric methods. Platelet reactivity was estimated by impedance aggregometry with arachidonate, collagen, and ADP as agonists. Expression of selectin-P and GPIIb/IIIa on blood platelets was tested by flow cytometry.Results. Platelet aggregation associated significantly negatively with HGB and age and significantly positively with PLT, MPV, PCT, PDW, and P-LCR. When platelet reactivity (“cumulative platelet reactivity_aggregation”) was analyzed in a cumulated manner, the negative association with serum concentration of uric acid (Rs=−0.169,p=0.003) was confirmed. Multivariate analysis revealed that amongst blood morphological parameters, platelet count, plateletcrit, and number of large platelets and uric acid are the most predictive variables for platelet reactivity.Conclusions. The most significant contributors to platelet reactivity in older subjects are platelet morphology, plasma uricaemia, and erythrocyte morphology.

Background Activation of the immune system might affect the severity of depressive episodes as well as response to the antidepressant treatment. The purpose of this study was to investigate whether the occurrence of variant alleles of analyzed SNPs are involved in prevalence and progression of depression. Moreover, selected genes and SNPs have not been investigated in context of the disease severity and treatment. Therefore, six polymorphisms were selected: g.41354391A>G-TGFB1 (rs1800469), g.132484229C>A-IRF (rs2070729), g.186643058A>G-PTGS2 (rs5275), g.186640617C>T-PTGS2 (rs4648308), g.70677994G>A-TGFA (rs2166975) and g.42140549G>T–IKBKB (rs5029748). Methods A total of 360 (180 patients and 180 controls) DNA samples were genotyped using TaqMan probes. Results We observed that A/G of the rs2166975 TGFA, A/C of rs2070729 IRF1 and G/T of rs5029748 IKBKB were associated with an increased risk of depression development while the T/T of rs5029748 IKBKB, T/T of rs4648308 PTGS2 and G/G of rs2166975 TGFA reduced this risk. We also stratified the study group according to gender and found that genotype A/G and allele G of the rs2166975 TGFA, G/T of rs5029748 IKBKB as well as C allele of rs4648308 PTGS2, homozygote A/A and allele A of rs5275 PTGS2 were associated with increased risk of depression development in men while homozygote G/G of rs5275 PTGS2 decreased this risk. Moreover, C/T of rs4648308 PTGS2 and A/G of rs5275 PTGS2 was positively correlated with the risk of the disease occurrence in women. Furthermore, a gene–gene analysis revealed a link between studied polymorphisms and depression. In addition, A/A of rs1800469 TGFB1 was associated with earlier age of onset of the disease while G/G of this SNP increased severity of the depressive episode. Interestingly, A/C of rs2070729 IRF1 and T/T of rs5029748 IKBKB may modulate the effectiveness of selective serotonin reuptake inhibitors therapy. In conclusion, studied SNPs may modulate the risk of occurrence, age of onset, severity of the disease and response to the antidepressant treatment.

The cardiovascular effects of testosterone and dihydrotestosterone are generally attributed to their modulatory action on lipid and glucose metabolism. However, no ex vivo studies suggest that circulating androgen levels influence the activation and reactivity of blood platelets - one of the main components of the haemostasis system directly involved in atherosclerosis. The levels of testosterone, dihydrotestosterone and oestradiol in plasma from men and women aged from 60 to 65 years were measured by LC-MS; the aim was to identify any potential relationships between sex steroid levels and the markers of platelet activation (surface membrane expression of GPII/IIIa complex and P-selectin) and platelet reactivity in response to arachidonate, collagen or ADP, monitored with whole blood aggregometry and flow cytometry. The results of the ex vivo part of the study indicate that the concentrations of testosterone and its reduced form, dihydrotestosterone are significantly negatively associated with platelet activation and reactivity. These observations were confirmed in an in vitro model: testosterone and dihydrotestosterone significantly inhibited platelet aggregation triggered by arachidonate or collagen. Our findings indicate that testosterone and dihydrotestosterone are significant haemostatic steroids with inhibitory action on blood platelets in older people.

// Marta Smeda 1 , Anna Kieronska 1 , Bartosz Proniewski 1 , Agnieszka Jasztal 1 , Anna Selmi 1 , Krystyna Wandzel 1 , Agnieszka Zakrzewska 1 , Tomasz Wojcik 1 , Kamil Przyborowski 1 , Katarzyna Derszniak 1 , Marta Stojak 1 , Dawid Kaczor 1 , Elzbieta Buczek 1 , Cezary Watala 2 , Joanna Wietrzyk 3 and Stefan Chlopicki 1, 4 1 Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, Bobrzynskiego 14, Krakow 30-348, Poland 2 Department of Haemostasis and Haemostatic Disorders, Medical University of Lodz, Kosciuszki 4, Lodz 90-419, Poland 3 Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Oncology, Rudolfa Weigla 4, Wroclaw 53-114, Poland 4 Chair of Pharmacology, Jagiellonian University, Medical College, Grzegorzecka 16, Krakow 31-531, Poland Correspondence to: Stefan Chlopicki, email: stefan.chlopicki@jcet.eu Keywords: mouse breast cancer; vascular mimicry; platelet inhibition; aspirin; clopidogrel Received: November 01, 2017 Accepted: February 25, 2018 Published: April 03, 2018 ABSTRACT Platelet inhibition has been considered an effective strategy for combating cancer metastasis and compromising disease malignancy although recent clinical data provided evidence that long-term platelet inhibition might increase incidence of cancer deaths in initially cancer-free patients. In the present study we demonstrated that dual anti-platelet therapy based on aspirin and clopidogrel (ASA+Cl), a routine regiment in cardiovascular patients, when given to cancer-bearing mice injected orthotopically with 4T1 breast cancer cells, promoted progression of the disease and reduced mice survival in association with induction of vascular mimicry (VM) in primary tumour. In contrast, treatment with ASA+Cl or platelet depletion did reduce pulmonary metastasis in mice, if 4T1 cells were injected intravenously. In conclusion, distinct platelet-dependent mechanisms inhibited by ASA+Cl treatment promoted cancer malignancy and VM in the presence of primary tumour and afforded protection against pulmonary metastasis in the absence of primary tumour. In view of our data, long-term inhibition of platelet function by dual anti-platelet therapy (ASA+Cl) might pose a hazard when applied to a patient with undiagnosed and untreated malignant cancer prone to undergo VM.

BACKGROUND There have been occasional reports indicating that plasma concentrations of reduced glutathione (GSH) may be associated in some way with blood glucose. This relationship, however, has not hitherto been explored in the blood plasma of patients with coronary artery disease (CAD). OBJECTIVES The aim of this study was to evaluate potential associations of fasting glycemia and peripheral blood plasma GSH concentrations in CAD-free and CAD-affected subjects. MATERIAL AND METHODS In blood samples obtained from patients with CAD, defined by coronary angiography and/or echocardiography, and from an age-matched control group of patients with a confirmation of no coronary artery occlusion and with no history of cardiovascular events, plasma concentrations of glucose and reduced glutathione were analyzed by routine laboratory diagnostic methods and high performance liquid chromatography (HPLC), respectively. RESULTS The results showed that in the CAD patients, but not in the non-CAD controls, fasting glycemia is negatively associated with plasma levels of GSH (r = -0.328; p = 0.011). Moreover, in the CAD-affected subjects (but not in the controls) the presence of type 2 diabetes mellitus significantly discriminated plasma levels of GSH (rP = -0.125; p = 0.350, between GSH and glucose adjusted for the occurrence of diabetes). CONCLUSIONS The study suggests that GSH may be an important factor contributing to glucose metabolism in CAD patients. Hence, it may be considered a significant therapeutic target in strategies aimed at improving glycemic control in CAD-affected subjects.

Background Vitamin deficiency is frequently associated with inflammatory bowel disease (IBD). Supplementation of vitamins could thus serve as an adjunctive therapy. The present meta-analysis reviews the deficiencies and alterations in serum fat-soluble vitamins (A, D, E, and K) reported in IBD patients. Materials and methods PubMed database search was performed to identify all primary studies up to January 2015 that evaluated the serum concentrations of fat-soluble vitamin levels in IBD patients compared with healthy individuals. We estimated pooled mean differences between groups and estimated their relations with some compounding variables (age, disease duration, C-reactive protein, albumin), using a meta-regression analysis. Results Nineteen case-control studies met selection criteria. In patients with Crohn's disease (CD), vitamin A, D, E, K status was lower than in controls [D=212 μg/L.92; 95% confidence interval (CI), 95.36-330.48 μg/L, P=0.0002; D=6.97 nmol/L, 95% CI, 1.61-12.32 nmol/L, P=0.01; D=4.72 μmol/L, 95% CI, 1.60-7.84 μmol/L, P=0.003; D=1.46 ng/mL, 95% CI, 0.48-2.43 ng/mL, P=0.003, respectively]. Patients with ulcerative colitis had lower levels of vitamin A than controls (D=223.22 μg/L, 95% CI, 44.32-402.12 μg/L, P=0.01). Patients suffering from CD for a longer time had lower levels of vitamins A (95% CI=7.1-67.58 y, P=0.02) and K (95% CI, 0.09-0.71 y, P=0.02). Meta-regression analysis demonstrated statistically significant associations between the levels of inflammatory biomarkers: C-reactive protein (P=0.03, 95% CI, -9.74 to -0.6 mgl/L) and albumin (P=0.0003, 95% CI, 402.76-1361.98 g/dL), and vitamin A status in CD patients. Conclusion Our meta-analysis shows that the levels of fat-soluble vitamins are generally lower in patients with inflammatory bowel diseases and their supplementation is undoubtedly indicated.

Cyclic vomiting syndrome (CVS) is a disorder defined by recurrent, unexplained episodes of severe nausea and vomiting. Our aim was to investigate whether CVS and pathophysiological mechanisms underlying this condition are associated with selected variations in genes encoding the components of the endogenous cannabinoid and opioid systems.This case-control study included 65 patients with CVS-16 male and 49 female, and 1,092 healthy controls-525 male and 567 female from the 1000 Genomes Project. CVS subjects filled out study-specific questionnaires. Single-nucleotide polymorphisms (SNPs) in genes encoding cannabinoid receptors (CNR1 and CNR2), fatty acid amide hydrolase (FAAH) and mu-opioid receptor (OPRM1) were analyzed using the TaqMan SNP genotyping assay. Correlations between SNP's and clinical characteristics of CVS were ascertained.Our study disclosed an increased risk of CVS among individuals with AG and GG genotypes of CNR1 rs806380 (P0.01), whereas the CC genotype of CNR1 rs806368 and AG and GG genotypes of OPRM1 rs1799971 were associated with a decreased risk of CVS (P0.05). In addition, AG and GG genotypes of OPRM1 rs1799971 were correlated with migraine episodes, AG and GG of OPRM1 rs1799971, and CT and CC of CNR1 rs806368 with a family history of migraines (second degree relatives), and CT and CC of CNR1 rs2023239 with a positive response to therapy.Our results show for the first time that the variations in CNR1 and OPRM1 genes are associated with CVS and that different genotypes may contribute to the risk of CVS.

Introduction Recent studies have shown that it may be the concentration of thrombin, which is discriminative in determining of the mechanism of platelet activation via protease activated receptors (PARs). Whether the observed phenomenon of differentiated responses of mouse platelets to various thrombin concentrations in non-diabetic db/+ and diabetic db/db mice depends upon the concerted action of various PARs, remains to be established. Results We found elevated reactivity of platelets, as well as the enhanced PAR-3 expression in response to both the used concentrations of AYPGKF in db/db mice, as compared to db/+ heterozygotes. At low concentration of thrombin platelets from diabetic mice demonstrated hyperreactivity, reflected by higher expression of PAR-3. For higher thrombin concentration, blood platelets from db/db mice appeared hyporeactive, compared to db/+ animals, while no significant differences in PAR-3 expression were observed between diabetic and non-diabetic mice. Conclusions The novel and previously unreported finding resulting from our study is that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low thrombin (when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to higher reactivity of platelets in db/db mice.

The antioxidant properties of melatonin can be successfully used to reduce the effects of oxidative stress caused by homocysteine. The beneficial actions of melatonin are mainly due to its ability to inhibit the generation of the hydroxyl radical during the oxidation of homocysteine. Melatonin protects endothelial cells, neurons, and glia against the action of oxygen radicals generated by homocysteine and prevents the structural changes in cells that lead to impaired contractility of blood vessels and neuronal degeneration. It can be, therefore, assumed that the results obtained in experiments performed mainly in the in vitro models and occasionally in animal models may clear the way to clinical applications of melatonin in patients with hyperhomocysteinemia, who exhibit a higher risk of developing neurodegenerative diseases (e.g., Parkinson’s disease or Alzheimer’s disease) and cardiovascular diseases of atherothrombotic etiology. However, the results that have been obtained so far are scarce and have seldom been performed on advanced in vivo models. All findings predominately originate from the use of in vitro models and the scarcity of clinical evidence is huge. Thus, this mini-review should be considered as a summary of the outcomes of the initial research in the field concerning the use of melatonin as a possibly efficient attenuator of oxidative stress induced by homocysteine.

Aims The high glucose concentration observed in diabetic patients is a recognized factor of mitochondrial damage in various cell types. Its impact on mitochondrial bioenergetics in blood platelets remains largely vague. The aim of the study was to determine how the metabolism of carbohydrates, which has been impaired by streptozotocin-induced diabetes may affect the functioning of platelet mitochondria. Materials and methods Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Platelet mitochondrial respiratory capacity was monitored as oxygen consumption (high-resolution respirometry). Mitochondrial membrane potential was assessed using a fluorescent probe, JC-1. Activation of circulating platelets was monitored by flow cytometry measuring of the expressions of CD61 and CD62P on a blood platelet surface. To determine mitochondrial protein density in platelets, Western Blot technique was used. Key findings The results indicate significantly elevated mitochondria mass, increased mitochondrial membrane potential (ΔΨm) and enhanced respiration in STZ-diabetic animals, although the respiration control ratios appear to remain unchanged. Higher ΔΨm and elevated mitochondrial respiration were closely related to the excessive activation of circulating platelets in diabetic animals. Significance Long-term diabetes can result in increased mitochondrial mass and may lead to hyperpolarization of blood platelet mitochondrial membrane. These alterations may be a potential underlying cause of abnormal platelet functioning in diabetes mellitus and hence, a potential target for antiplatelet therapies in diabetes.

The aim of the study was to assess the responsiveness of blood platelets to acetylsalicylic acid (ASA) in patients following coronary artery bypass grafting (CABG) surgery with relation to oxidative and antioxidative plasma status. The study included 37 patients treated with the CABG procedure. During the first 24 h after CABG patients were given 300 mg of ASA with the following dose of 150 mg daily. The blood was collected before the procedure and 10 days after. Whole blood platelet aggregation induced with arachidonic acid, collagen and adenosine diphosphate (ADP) was performed together with whole blood generation of thromboxane B2 (TxB2). Oxidative stress was measured before and 10 days after CABG with total oxidative plasma status (TOS) and total antioxidative status of the plasma (TAS). TOS/TAS index was calculated. We observed a significant increase in the TOS and TOS/TAS index and ADP-induced aggregation 10 days after CABG in comparison with its level before operation. There was a significant decrease in the arachidonic acid-induced aggregation and serum TxB2 level. Patients with ADP-induced and collagen-induced aggregation in the upper quartile had significantly higher TOS and TOS/TAS index before (ADP) and after the operation (ADP and collagen). There were 19 patients (51%) with high on aspirin platelet reactivity after CABG who had also higher TOS and TOS/TAS index and lower TAS value in comparison with aspirin responders. Despite ASA use, increased oxidative stress after CABG can overcome its antiplatelet effect and increase platelet activation through other pathways.

In diabetes-related states of chronic hyperglycaemia elevated concentrations of glucose may alter the functioning of platelet enzymes involved in arachidonic acid metabolism, including prostaglandin H2 synthase (cyclooxygenase) (PGHS, COX). Therefore, the principal aim of this study was to assess the effects of experimental chronic hyperglycaemia on platelet PGHS-1 (COX-1) expression and activity. Blood platelet activation and reactivity were assessed in Sprague-Dawley rats with the 5-month streptozotocin (STZ) diabetes. The PGHS-1 abundance in platelets was evaluated with flow cytometry and Western blotting, while its activity monitored using a high resolution respirometry and the peroxidase fluorescent assay. The production of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in platelets were assayed immunoenzymatically. Circulating platelets from diabetic were characterised by increased size, elevated 'priming' and altered reactivity, compared to non-diabetic animals. Both Western blot analysis and flow cytometry revealed significantly elevated expressions of platelet PGHS-1 in STZ-diabetic rats (p < 0.05). We also observed significantly elevated platelet PGHS-1-related arachidonic acid metabolism in diabetic vs. non-diabetic animals, with the use of polarographic (p < 0.05) and total activity assay (p < 0.001). Such increases were accompanied by the elevated production of PGE2 (p < 0.001) and TXB2 (p < 0.05) in diabetic animals. The increased PGHS-1-dependent oxygen consumption and the total activity of PGHS-1 in diabetic animals remained very significant (p < 0.001) also upon adjusting for blood platelet PGHS-1 abundance. Therefore, our results further contribute to the explanation of the increased metabolism of arachidonic acid observed in diabetes.

Several lines of evidence suggest that cyclooxygenase-2 (COX-2) activity can have a beneficial role in the maintenance of vascular tone of the blood vessels in diabetes. Specifically, the increased production of prostacyclin (PGI2) and prostaglandin E2 (PGE2), mediated by COX-2, has been suggested to compensate for decreased synthesis of nitric oxide (NO). The study investigates whether inhibition of COX-2 may reduce the coronary flow in diabetic animals and may also lead to decreased synthesis of prostaglandins. Mice aged 18-20 weeks were used for the study: those with leptin receptor deficiency (db/db) served as a model of diabetes while heterozygous (db/+) mice served as controls. Coronary flow was measured by the Langendorff method, and prostaglandin synthesis by myocardia was assayed in heart perfusates. COX-2 inhibition was found to reduce basal coronary flow in db/db mice but had no effect in db/+ mice. Secretion of PGE2 was found to be higher in db/db mice, while prostacyclin synthesis did not differ. COX-2 inhibition decreased production of both prostaglandins to similar levels in both groups. The use of ONO-1301, a specific agonist for the prostacyclin receptor revealed that vasodilating responses mediated by the receptor were impaired in db/db mice. The expression levels of the receptor in cardiac tissue did not differ between the groups. It is concluded that the increased COX-2 contribution to vasodilation in diabetic animals appears to be partially a result of increased COX-2-dependent synthesis of PGE2 and also may be caused by impaired vasodilation mediated by the prostacyclin receptor.

Currently, there are several animal models of diabetes mellitus and hypertension, but relatively little is known about blood platelet function in these models. The aim of this work was to characterise and compare platelet reactivity and activation in db/db mice (mouse model of diabetes) and mice receiving L-NAME (model of chronic inhibition of NO synthesis), using various platelet function assays. We found higher platelet activation (circulating resting platelets) in db/db mice than in db/+heterozygotes, as evidenced by elevated expressions of CD62P and CD40L and a lower expression of CD42b. The expression of COX-1 was significantly increased, and the phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser(157) significantly reduced in platelets from db/db mice. Similarly, we observed platelet hyperreactivity in db/db mice following the in vitro responses to 20μg/ml collagen (reflected by increased expressions of CD62P and CD40L, and reduced CD42b), 20μM ADP (reduced CD42b) and lower concentrations of thrombin (0.025 U/ml) (increased CD62P, JON/A, bound vWF, and bound fibrinogen). Otherwise, platelet hyporeactivity was revealed for higher thrombin (0.25 U/ml) (reduced CD62P and bound vWF), while hyperreactivity occurred for CD40L and bound Fg in db/db mice compared to non-diabetic control, db/+. Plasma levels of sCD40L, but not of sCD62P, were increased in db/db mice; also plasma TXB2 concentrations were over 3.5-fold higher in this group than in the heterozygous db/+mice (P0.01). In contrast, in the mice administered with L-NAME, no statistical differences in expressions of platelet activation markers were found between mice supplemented with L-NAME and controls. Likewise, the TXB2 level did not differ between L-NAME mice and controls, but L-NAME mice had significantly higher plasma levels of sCD62P and sCD40L than controls. In conclusion, these two studied models differ in the overall picture of blood platelet activation and reactivity, as they demonstrated opposite time sequence patterns of platelet activation in circulating blood. More generally, our study provides another argument for the opinion that multiparametric analysis of platelet function offers a much better tool for investigation and minimizes the likelihood of artefacts.

INTRODUCTION: Poor antioxidant protection of cardiomyocytes due to cardiac ischemia and low serum levels of reduced glutathione (GSH) may be associated with enhanced risk of coronary restenosis after primary percutaneous coronary intervention (pPCI). OBJECTIVES: The aim of this study was to investigate whether preprocedural serum reduced GSH, reflecting the antioxidant status, may be predictive of major adverse cardiac events (MACE) in patients with acute coronary syndrome (ACS) treated with pPCI. PATIENTS AND METHODS: Preprocedural serum GSH level was evaluated in 141 patients with ACS treated with pPCI with bare-metal stent (BMS) deployment. During a 15-month follow-up, 30 patients (mean age, 61 ±10 years) experienced a MACE. The remaining 111 subjects constituted the non-MACE group (mean age, 63 ±10 years). RESULTS: The MACE group had significantly lower GSH levels compared with the non-MACE group (P 2.26; 2.09-2.99 μmol/l; P

Strenuous physical exercise leads to platelet activation that is normally counterbalanced by the production of endothelium-derived anti-platelet mediators, including prostacyclin (PGI2) and nitric oxide (NO). However, in the case of endothelial dysfunction, e.g. in atherosclerosis, there exists an increased risk for intravascular thrombosis during exercise that might be due to an impairment in endothelial anti-platelet mechanisms. In the present work, we evaluated platelet activation at rest and following a single bout of strenuous treadmill exercise in female ApoE/LDLR-/- mice with early (3-month-old) and advanced (7-month-old) atherosclerosis compared to female age-matched WT mice. In sedentary and post-exercise groups of animals, we analyzed TXB2 generation and the expression of platelet activation markers in the whole blood ex vivo assay. We also measured pre- and post-exercise plasma concentration of 6-keto-PGF1α, nitrite/nitrate, lipid profile, and blood cell count. Sedentary 3- and 7-month-old ApoE/LDLR-/- mice displayed significantly higher activation of platelets compared to age-matched wild-type (WT) mice, as evidenced by increased TXB2 production, expression of P-selectin, and activation of GPIIb/IIIa receptors, as well as increased fibrinogen and von Willebrand factor (vWf) binding. Interestingly, in ApoE/LDLR-/- but not in WT mice, strenuous exercise partially inhibited TXB2 production, the expression of activated GPIIb/IIIa receptors, and fibrinogen binding, with no effect on the P-selectin expression and vWf binding. Post-exercise down-regulation of the activated GPIIb/IIIa receptor expression and fibrinogen binding was not significantly different between 3- and 7-month-old ApoE/LDLR-/- mice; however, only 7-month-old ApoE/LDLR-/- mice showed lower TXB2 production after exercise. In female 4-6-month-old ApoE/LDLR-/- but not in WT mice, an elevated pre- and post-exercise plasma concentration of 6-keto-PGF1α was observed. In turn, the pre- and post-exercise plasma concentrations of nitrite (NO2-) and nitrate (NO3-) were decreased in ApoE/LDLR-/- as compared to that in age-matched WT mice. In conclusion, we demonstrated overactivation of platelets in ApoE/LDLR-/- as compared to WT mice. However, platelet activation in ApoE/LDLR-/- mice was not further increased by strenuous exercise, but was instead attenuated, a phenomenon not observed in WT mice. This phenomenon could be linked to compensatory up-regulation of PGI2-dependent anti-platelet mechanisms in ApoE/LDLR-/- mice.

INTRODUCTION Recently, the responder status to clopidogrel therapy has been observed to change over time. OBJECTIVES The aim of the study was to investigate changes in the responder status to clopidogrel therapy over time with the use of 4 platelet function tests (light transmission aggregometry [LTA], multiple electrode aggregometry [MEA], vasodilator-stimulated phosphoprotein (VASP) phosphorylation, and INNOVANCE® PFA P2Y assays [PFA]) in patients after percutaneous coronary intervention (PCI). We also compared the results of these tests to determine the most reliable method. PATIENTS AND METHODS The study included 35 patients after PCI, receiving acetylsalicylic acid (75 mg/d) and clopidogrel (75 mg/d). The control group included 50 healthy volunteers. Platelet function was measured at 3 different time points (4 ±2 days after PCI, and then after 6 and 12 weeks). RESULTS The responder status to clopidogrel changed in 5 patients (14%) as shown by MEA; in 7 patients (20%), by LTA and PFA; and in 13 patients (37%), by VASP. The Cohen's κ coefficient showed a moderate or poor agreement between the tests. The strongest agreement was between MEA and PFA (80%; κ = 0.46, P = 0.003), PFA and LTA (82%; κ = 0.41, P = 0.004), and MEA and LTA (80%; κ = 0.36, P = 0.008). The κ coefficient for all comparisons with VASP was less than 0.30. CONCLUSIONS Changes in the responder status over time are present for all platelet function tests, but a large discrepancy between the tests does not allow a careful assessment of this phenomenon. The tests showed only moderate agreement (in relation to one another and to time points), which significantly limits their interchangeable use in clinical practice.

1-Methylnicotinamide (MNA) is a primary metabolite of nicotinamide recently proven to cause systemic increase in PGI(2) plasma levels in an unknown mechanism. Our present study was aimed at verifying whether the increased production of PGI(2), a vasodilating prostanoid, in response to MNA, its metabolite N-methyl-2-pyridone-5-carboxamide (Met2PY), and nicotinamide may be reproduced under in vitro conditions. Since prostacyclin is a vasodilating prostanoid, we also performed the functional tests in the ex vivo model of coronary vascular bed perfusion to evaluate the vasoactive properties of those compounds. We did not observe any significant effect of the tested drugs on either PGI(2) or PGE(2) secretion in our in vitro model. Nicotinamide at the concentrations of 10 and 100 μmol/l and 100 μmol/l Met2PY slightly but significantly increased coronary flow in rat heart. These increases, however, remained very low when compared to that induced by the reference compound, bradykinin (100 nmol/l). Perfusion of rat hearts with Met2PY in the presence of 50 μmol/l indomethacin resulted in decreased coronary flow, which proves that the effect is cyclooxygenase dependent. We conclude that MNA metabolites should be more carefully addressed in reference to pro-prostacyclin activity and that systemic mechanism of MNA-induced PGI(2) production needs further clarification.

Objectives This study was focused on the monitoring how the anti-inflammatory substance, N(1)-methylnicotinamide (MNA), could influence oxidation and glycooxidation stress markers in rats under conditions of streptozotocin (STZ)-induced diabetes mellitus. Methods Diabetes mellitus was induced in 60 male Wistar rats by intraperitoneal injection of STZ and after 7 days diabetic animals were allocated to five groups according to the dose of MNA administered for 7 weeks. The degree of DNA damage in lymphocytes, as well as advanced glycation endproducts (AGEs), protein carbonyls, lipid peroxides, and total antioxidant capacity (TEAC) in plasma were measured. Results Glycation damage to proteins (represented by AGEs level) was significantly increased in all diabetic groups compared to untreated non-diabetic animals. MNA did not affect TEAC of plasma in any group of diabetic rats. Supplementation of diabetic rats with MNA at the dose of 200 mg/kg resulted in decreased protein carbonyls (from 0.0818 ± 0.0091 to 0.0558 ± 0.0044 nmol/mg proteins; P < 0.05, n = 15) and DNA oxidation, reflected by the levels of 8-oxoG (0.6302 ± 0.085 vs. 0.9213 ± 0.108 8-oxoG/10(6) G; P < 0.05, n = 15), compared to untreated diabetic animals. Discussion Our results demonstrated that MNA at suitable concentrations could influence oxidative modifications of proteins and DNA.

In relation to the interplay between C-reactive protein (CRP) and its various ligands, including extracellular matrix proteins or plasma proteins, herein we compared the effectiveness of modified CRP (mCRP) in modulation of platelet function under different experimental conditions. mCRP (100 μg/ml) was significantly more effective in stimulation of platelet activation when measured in suspensions of isolated platelets than in whole blood (fraction of CD62-positive platelets was 73.4 ± 5.3 vs. 0.8 ± 1.0% in isolated platelets and 7.0 ± 1.8 vs. 1.3 ± 0.4% in whole blood). Platelet adhesion to fibrinogen was almost seven-fold higher in suspensions of isolated platelets compared to platelet-rich plasma (PRP) (P < 0.005). Furthermore, mCRP enhanced platelet aggregation in a whole blood but it had no effect in PRP. The effectiveness of mCRP in stimulation of platelet response in plasma has been associated with the proportions of gamma globulin and albumin in human serum (rp = 0.78, P < 0.0001 for gamma globulin and Rp = -0.52, P = 0.02 for albumin concentrations; for albumin/gamma globulin ratio rp = -0.72, P < 0.0005). Such associations have been further confirmed by experiments showing that mCRP interacts with some immunoglobulins. Taken together, the modulation of platelet function by mCRP seems to be strongly determined by the presence of the gamma globulin fraction in platelet milieu.

Polymorphic configurations of the coagulation factor VII gene (F7) are associated with plasma levels of FVII antigen (FVII:Ag) and FVII coagulant activity (FVII:C). Our aim was to determine whether F7 polymorphisms influence risk of ischemic stroke in young adults. One hundred and fifty survivors of ischemic stroke before the age of 45 and an equal number of age and sex-matched controls were genotyped for five F7 polymorphisms: the -A670C transversion, -323 decanucleotide insertion (P + 10), the number (which varies between five and eight) of a 37 base pair repeat polymorphisms in intron 7 (IVS7), amino acid substitution R353Q, and +154AA insertion. 353Q, P + 10 and +154AA were demonstrated to associate with significantly decreased plasma FVII:Ag, whereas -670C and IVS7 seven or higher were associated with a tendency towards increased plasma FVII:Ag. The former three polymorphisms were significantly more common in control individuals than in patients, whereas the latter two were significantly more common in patients than in control individuals. The multiple logistic regression analysis revealed that two F7 polymorphisms, -670C and IVS7 seven or higher, are independent risk factors for ischemic stroke in young adult patients.

SummaryThe study was aimed at the evaluation of blood platelet activation and reactivity in patients in the convalescent phase of stroke (n=58) and controls matched in respect to risk factors of vascular pathology (n=55). Both groups were treated daily with acetylsalicylic acid (ASA), 150 mg/day. Using flow cytometry, the expressions of P-selectin and the active GP IIb/IIIa receptor, as well as the fraction of platelet-derived microparticles (PMPs) and total platelet aggregates (Ag), were evaluated in non-stimulated platelets and in platelets stimulated in vitro by thrombin, thrombin receptor activating peptide (TRAP) or ADP. The expression of P-selectin in non-stimulated platelets was found to be significantly (p=0.04) lower in stroke patients. In parallel, these patients manifested a significantly (p=0.0008) higher proportion of PMPs and a lowered (p=0.003) proportion of Ag, as compared to the controls. In the stroke patients the increased expressions of P-selectin and active GP IIb/IIIa in TRAP- or ADP-activated cells were less pronounced (pNote: The results of this study were presented in part during a poster session at the XVIII European Stroke Conference in Stockholm, 26–29 May 2009.

Purpose. There is increasing evidence that adventitial inflammation may participate in atherosclerosis development. The aim of this study was to investigate which atherosclerotic risk factors correlated with carotid adventitial thickness (AT) and to compare them with those associated with carotid intima-media thickness (IMT). We also set out to test the hypothesis that there is a significant correlation between IMT and AT in the carotid arteries. Methods. The far carotid artery wall IMT and AT were measured by high-resolution B-mode ultrasound in 128 persons (mean, 65 ± 8 years). A number of conventional and novel, clinically and laboratory-derived risk factors were assessed. Results. Significant correlation (r = 0.35, p < 0.0001) was demonstrated between the IMT and AT. The stepwise forward multiple regression analysis revealed correlations between IMT and leukocyte count, C-reactive protein level, and hypertension, whereas the Chlamydia (C.) pneumoniae IgA antibodies and fibrinogen levels, gender, and smoking correlated merely with AT. The homocysteine/methionine ratio correlated with both IMT and AT. Conclusion. The association between IMT and AT may reflect an interaction between intimal, medial, and adventitial pathology. Different risk factors are associated with the increased AT or IMT. It is possible that inflammation and some chronic infections, such as those induced by C.pneumoniae, could have a marked influence on adventitial cell proliferation. © 2009 Wiley Periodicals, Inc. J Clin Ultrasound, 2009

1-Methylnicotinamide (MNA), a major endogenous metabolite of nicotinamide, possesses anti-thrombotic and anti-inflammatory activity, and reverses endothelial dysfunction. In the present work, we investigated whether such a vasoprotective profile of MNA activity affords anti-diabetic action in rats. Diabetes was induced by streptozotocin (STZ) in Sprague-Dawley rats. Eight weeks after STZ injection in untreated or MNAtreated rats (100 mg kg –1 daily), development of diabetes (plasma concentrations of fasting and non-fasting glucose, HbA 1c , peptide C), development of oxidant stress (lipid peroxidation, carbonylation of plasma proteins), as well as NO-dependent endothelial function in aorta, coronary and mesenteric vessels were analyzed. Finally, the effect of chronic treatment with MNAon long-term survival of diabetic rats was determined. Chronic treatment withMNAprofoundly lowered fasting glucose concentrations in plasma, displayed mild effects on plasma HbA 1c and peptide C concentrations, while having no effects on non-fasting glucose. On the other hand, MNAtreatment considerably lowered lipid peroxidation, protein carbonylation, completely prevented impairment of endothelium-dependent vasodilatation in the aorta that was mediated entirely by NO, but failed to affect endothelial function in resistant vessels, which was mediated only partially by NO. Most importantly, chronic treatment with MNAprolonged the long-term survival of diabetic rats. In conclusion, MNA displayed a significant anti-diabetic effect that may be linked to its vasoprotective activity.

For nearly a decade poly(amidoamine) (PAMAM) dendrimers G4 were claimed unnegligible cytotoxic agents. Here we monitored whether in vivo cytotoxic effect of PAMAM G4 (0.5 micromol kg(-1) day(-1)) may be compromised by its ameliorating effect on severe hyperglycaemia in chronic streptozotocin-diabetic Wistar rats. PAMAM G4 significantly reduced the 60-day overall survival in long-term experimental diabetes: treated animals were 6.7 times more likely to die than control animals (p

Udgivelsesdato: 2009-Jan-27 Oral antiplatelet drugs are a cornerstone of modern pharmacotherapy in cardiovascular atherothrombotic diseases. The efficacy of acetylsalicylic acid (ASA, aspirin) and clopidogrel in decreasing the risk of adverse events in coronary heart disease patients has been well established in the past 20 years. Despite chronic oral antiplatelet therapy, a number of atherothombotic events continue to occur. In recent years, a number of reports in the literature have shown possible relationships between residual platelet activity, as measured with a variety of laboratory tests, and clinical outcome, raising the possibility that 'resistance' to oral antiplatelet drugs may underlie many such clinical adverse events. The present position paper, conveyed within a group of clinical cardiologists with expertise in thrombosis appointed by the Section of Cardiovascular Interventions of the Polish Cardiac Society, has been further elaborated and endorsed by the Working Group on Thrombosis of the European Society of Cardiology. It aims at summarizing the main findings in this complex area, issuing opinions in cases of high controversy, and fostering future research in this area to obtain reliable laboratory and clinical data for the resolution of the many problems still open.

The protective effect of Pycnogenol ® against cardiovascular diseases was clearly demonstrated. Nevertheless, little is known about its antithrombotic effect, especially in diabetes associated with enhanced thromboxane synthesis leading to severe vascular complications. Therefore, the main purpose of our study was to evaluate the effect of long-term Pycnogenol ® intake on synthesis of prothrombotic thromboxane A 2 (TXA 2 ) in animal model of insulin-dependent diabetes. The levels of main plasma TXA 2 metabolite, thromboxane B 2 (TXB 2 ), were assessed by enzyme-linked immunosorbent assay. Diabetes was induced in Wistar male rats by single injection of streptozotocin, resulting after 8 weeks in significant body weight reduction, increased plasma glucose concentrations, and decreased plasma C-peptide levels, compared to non-diabetic animals. There was no significant reduction of plasma glucose concentrations after Pycnogenol ® ingestion. It was found, however, that daily administration of either Pycnogenol ® (5 mg/kg b.wt.) or acetylsalicylic acid (ASA, 10 mg/kg b.wt.) significantly reduced plasma TXB 2 concentrations, and this inhibitory effect was higher in the latter case. Nonetheless, simultaneous administration of Pycnogenol ® and ASA did not improve effectiveness of ASA-mediated decrease in TXB 2 generation. The results of the present study suggest that Pycnogenol ® might have a beneficial antithrombotic effect when administered alone or as a supplementation of standard antiplatelet therapy in diabetic patients.

The study was designed to assess blood platelet sensitivity to acetylsalicylic acid and its associations with dyslipidaemia and inflammation in coronary artery disease patients. Platelet non-responsiveness to aspirin is associated with an increased risk of serious cardiovascular events. Several environmental and hereditary factors are reportedly involved in sub-optimal acetylsalicylic acid response. Forty-five coronary artery disease patients and 45 non-coronary artery disease controls received acetylsalicylic acid at a daily dose of 75-150 mg. Controls were examined twice: on the day of entering the study and 10 days later. Urinary 11-dehydrothromboxane B2 was assessed as the marker of platelet thromboxane generation. Aggregation was studied in platelet-rich plasma using turbidimetric aggregometry with collagen and arachidonic acid. Fifty to seventy percent of coronary artery disease patients showed an extent of collagen-induced aggregation above the upper quartile of the reference range compared with 8-15% in controls (P

The effect of chronic hyperglycaemia on blood platelet response to acetylsalicylic acid was studied in rats with experimental diabetes. Platelet aggregation was determined in non-diabetic and streptozotocin-diabetic rats treated orally with 4 or 40 mg aspirin (ASA)/kg per day (for 8 weeks from the eighth day of diabetes) using whole blood impedance aggregometry with arachidonic acid or ADP as platelet agonists. The dose-dependent effect of ASA 'therapy' on ADP-agonized platelets was significant only in non-diabetic animals, while in diabetic rats both doses were ineffective in reducing ADP-stimulated platelet aggregation. ASA-mediated increased acetylation of platelet proteins favoured reduced platelet aggregation and slower platelet disaggregation (P r < 0.025 or less). Interestingly, however, the occupation of platelet protein-free amino groups was significantly higher in control rats compared with diabetic rats (P < 0.001), pointing out that proteins of platelets in non-diabetic animals were more vulnerable for the ASA-induced acetylation. We conclude that chronic hyperglycaemia interferes with preventive effects of ASA on platelet reactivity. Our data validate the suggestion that the relationship between aspirin ineffectiveness and poor metabolic control, first revealed in humans, concerns also other animals' platelets and holds regardless of the model or type of diabetes.

The aim of the present study was to assess the responsiveness of blood platelets to aspirin in patients following coronary artery bypass grafting (CABG) surgery. Aspirin was administered following CABG in 24 operated patients (aged 63.2 +/- 6.3 years). Platelet function was monitored on the 10th postoperative day (A) and 1 month after CABG (B) with the use of whole-blood aggregometry (WBEA) and PFA-100 closure time (PFA-100 CTCEPI). Normal platelet response to aspirin was defined by 3 criteria: the complete inhibition of WBEA induced by arachidonic acid (0.5 mmol/L), partial inhibition of collagen (1 microg/mL)-induced aggregation (WBEA14 Omega), and prolongation of PFA-100 CTCEPI (150 seconds) ("good responders"). Depending on whether 0, 1, 2, or 3 of these 3 criteria were fulfilled, patients were classified as "nonresponders," "weak responders," "incomplete responders," or "good responders," respectively. On the 10th postoperative day, there were 3 good responders, 6 incomplete responders, 11 weak responders, and 4 nonresponders among 24 patients. In contrast, 1 month after CABG within the same group of 24 patients there were 18 good responders, 5 incomplete responders, and 1 weak responder. Using a new methodology to assess impaired platelet responsiveness to aspirin ex vivo, we describe here the transient nature of "aspirin resistance" following CABG. These results indicate the necessity for the prolonged monitoring of the antiplatelet effectiveness of aspirin in the postoperative period after CABG.

Blood platelets play a crucial role in physiological haemostasis and in pathology of prothrombotic states, including atherosclerosis. In this paper, we review major factors underlying altered platelet reactivity, with special attention paid to abnormalities in platelet function in people with diabetes mellitus (DM). The overall picture of platelet abnormalities in DM, including altered adhesion and aggregation, is hypersensitivity of diabetic platelets to agonists. "Primed" diabetic platelets respond more frequently even to subthreshold stimuli, sooner become exhausted, consumed and finally hyposensitive, thus contributing to accelerated thrombopoiesis and release of 'fresh' hyperreactive platelets. In diabetes disturbed carbohydrate and lipid metabolism may lead to physicochemical changes in cell membrane dynamics, and consequently result in altered exposure of surface membrane receptors. These phenomena, together with increased fibrinogen binding, prostanoid metabolism, phosphoinositide turnover and calcium mobilisation often present in diabetic patients, contribute to enhanced risk of small vessel occlusions and accelerated development of atherothrombotic disease of coronary, cerebral and other vessels in diabetes. As platelet hypersensitivity in DM makes a major contribution to enhanced risk of thromboembolic macroangiopathy, and consequently enhanced morbidity and mortality, it validates use of antiplatelet agents in diabetic individuals. Platelet hyperreactivity may be cured with various antiplatelet drugs to a considerably large extent notwithstanding, evidence gathered from clinical and experimental surveys shows that this approach may not always be equally efficient in people with diabetes. Observations from clinical studies rather support the use of multifactorial strategy under such circumstances, like a combined therapy of aspirin plus either purinoreceptor blocker or GPIIb-IIIa antagonist.

Activated blood platelets play crucial role in restenosis due to their fundamental significance in thrombus formation. Therefore, platelets are attractive targets for the inhibition with a variety of antagonists. In this study, we present direct evidence that GR144053F [non-peptide antagonist of glycoprotein IIb-IIIa complex (GPIIb-IIIa)] inhibits activation and degranulation of human platelets, and opposes the action of aurintricarboxylic acid (ATA), the antagonist of von Willebrand factor, which augments platelet secretion. The effects of both drugs on platelet function were monitored by using various instrumental methods. Platelet-rich plasma and whole-blood aggregation was measured by using ADP and collagen as agonists. Platelet degranulation was assessed based on the expression of surface membrane activation markers: P-selectin, glycoprotein Ib, and activated GPIIb-IIIa complex. Measurements of closure time with platelet function analyzer PFA-100™ enabled us to reason on primary hemostatic capacity and reflected both aggregability and adhesiveness. GR144053F markedly reduced primary hemostatic platelet response ( ic 50 = 114.0 ± 9.6 nM) under conditions that closely mimicked natural blood flow in circulation, and inhibited aggregation in platelet-rich plasma ( ic 50 = 17.7 ± 7.0 nM). It was equally potent inhibitor of platelet activation, degranulation, fibrinogen binding, platelet consumption, and aggregate formation. Also, ATA was efficient in inhibition of platelet aggregation and adhesion (by up to 50% at 100 μM), but the combined action of both drugs on primary haemostatic capacity was not additive. GR144053F suppressed the activating effects of ATA on platelet degranulation and secretion. Overall, our data indicate that GR144053F is not only the efficient blocker of fibrinogen binding to GPIIb-IIIa, but also hampers platelet degranulation and may attenuate the activating effects of ATA.

Thrombospondin (TSP), which is secreted from α-granules of activated platelets, binds to its surface receptor (CD36) in the presence of Ca 2+ . Objectives: We monitored how the modulation of intraplatelet Ca 2 affects TSP binding to CD36 on platelets from healthy donors and patients with type 2 diabetes mellitus. We also aimed to verify whether the impaired Ca 2+ mobilisation in diabetes influences TSP binding upon the pharmacological modulation of calcium transport. Methods: Whole blood cytometry was used to monitor TSP release/binding and CD36 presentation in platelets from 28 type 2 patients and 33 healthy donors. Results: No significant changes in TSP and CD36 levels were revealed between the groups in circulating platelets and TRAP-, collagen- or thrombin-activated platelets. In healthy donors, 1 μM thapsigargin (TG) elevated the TRAP-activated TSP binding (by up to 50%, p

Abnormal platelet function has been hypothesised to play a role in the haemostatic abnormalities in cyanotic congenital heart disease (CCHD) patients. Using whole blood flow cytometry we found that platelets from cyanotic patients were hyperreactive and we related such hyperreactivity directly to young age, unoperated state, high haematocrit, reduced saturation with oxygen and low platelet count. Circulating platelets from CCHD children showed significantly enhanced P-selectin expression (P0.004) and remained more reactive to 0.2 IU/ml thrombin, 1-8 microM TRAP and 2-4 microM ADP (P0.04), especially in younger (0-3-year-olds) patients. Such a platelet 'priming' largely concerned CCHD children who were not subjected to modified Blalock-Taussig shunts in the past (non-MBTS). Only non-MBTS cyanotic children, but not MBTS-operated patients, showed significantly higher platelet reactivity compared to controls in response to ADP or 1 microM TRAP with respect to P-selectin expression (p0.05) and in response to all examined agonists with respect to GPIb expression (P0.045). The enhanced P-selection expression in MBTS-operated CCHD children and reduced GPIb expression in non-MBTS patients, especially in younger patients, were positively associated with the occurrence of the polymorphic variant Pl(A2) of platelet membrane glycoprotein IIIa gene. Altered blood morphology parameters (elevated RBC, Hb, Hct and MCHC, for all P0.0005) in CCHD children correlated with the enhanced degranulation of circulating blood platelets and their hyperreactivity in response to some agonists (P0.05). Overall, our data encourage the reasoning that circulating platelets are remarkably hyperreactive in non-MBTS cyanotic children, which are at higher risk to often encounter platelets activation in circulation. It seems unlikely that the apparently unchanged platelet reactivity in MBTS-operated children is due to the advantageous effects of the shunt, since these patients showed neither altered haematological parameters nor improved oxygen carrying capacity. Otherwise, it may rather result from more frequent episodes of platelet degranulation and preactivation in the past, and/or post-operative enhanced platelet consumption.

The contribution of platelets to the pathogenesis and progression of vascular complications in diabetes is supported by several studies. In general, platelets obtained from diabetic subjects show increased adhesiveness and an exaggerated aggregation, both spontaneous and in response to stimulating agents. The causes for this activation are multifold: altered exposure and/or abundance of glycoprotein receptors for agonists and adhesive proteins on the platelet surface, increased binding of fibrinogen, decreased membrane fluidity, altered platelet metabolism and changes in intraplatelet signalling pathways. The altered biophysical state of platelet membrane components in diabetes mellitus may be one of the major determinants of platelet hypersensitivity and hyperfunction and may contribute to impairments in various metabolic pathways, like intensified calcium mobilisation and accentuated thromboxane synthesis and release. Activated platelets interact with other cells, such as endothelial cells and leukocytes as well with the coagulation system in the process of atherosclerosis. Some studies indicated that platelet dysfunction was especially apparent in diabetic subjects with macro- or microangiopathy, while others showed that it may be related to the presence of diabetes mellitus per se. Several pharmaceutical compounds have been developed for the inhibition of platelet activation. However, aspirin treatment is cheap and effective, and aspirin remains to be the drug of choice for diabetic patients. It should be prescribed widely for patients who are at high risk of cardiovascular events.

This study examined the significance of selected parameters of primary haemostasis to discriminate between relatives of children with insulin-dependent diabetes mellitus (IDDM). Platelet function, including markers of spontaneous and agonist-induced platelet activation (CD62), platelet consumption (microparticles) and clumping (aggregates), as well as selected parameters of the fibrinolytic system (t-PA and PAI-1), were studied in IDDM children (n = 45), their parents (n = 65), siblings (n = 17) and unrelated healthy controls (n = 51). The fraction of activated platelets circulating in whole blood amounted to 4.3±2.1% in IDDM children, and significantly exceeded the level found in parents (1.3±0.7%, P < 0.002), siblings (1.2±1.0%, P < 0.002), and controls (1.2±0.6%, P < 0.002). Furthermore, an enhanced formation of platelet microparticles was observed in the IDDM group, both in resting platelets and also when platelets were stimulated with thrombin. Significantly decreased total PAI-1 occurred in IDDM chi...

The spontaneous anticoagulant-dependent platelet activation in vitro may potentially interfere with the determination of haemostatic parameters. The effects of various blood anticoagulants on platelet activation were monitored using flow cytometry. Regardless of a blood anticoagulant used (EDTAK 2 , heparin, citrate or PPACK), platelet activation began immediately after blood withdrawal and was most pronounced in the EDTAK 2 -anticoagulated blood samples. The progressing expression of GMP-140 antigen was accompanied by the enhanced abundance of the subunit β 3 of the platelet membrane integrin α IIb β 3 without parallel changes in the fluorescence attributed to the complex form of the integrin α IIb β 3 . The increased expression of GMP-140 was paralleled by the enhanced platelet clumping in the samples anticoagulated with either EDTAK 2 or heparin, and the raised platelet microparticles in blood withdrawn into citrate. The EDTAK 2 -induced platelet activation was markedly reduced by methyl 2,5-dihydroxycinnamate, tyrosine kinase inhibitor. The influence of disodium EDTA on platelet membrane dynamics closely mimicked the alterations induced upon the interaction of fibrinogen with platelet GPIIb-IIIa. Thus, the EDTAK 2 -induced platelet activation might result from an interference with platelet membrane protein structure and conformation and possibly relate to an “unspecific” trigerring of a signal transduction pathway. Overall, EDTAK 2 and heparin appeared the least suitable anticoagulants, particularly with the regard to the expression of GMP-140 antigen. The failure to recognize the importance of a spontaneous anticoagulant-induced platelet activation may result in misdiagnoses during the monitoring of coagulation parameters.

The increased nonenzymatic glycosylation of platelet membrane proteins has been suggested to underlie platelet hypersensitivity in diabetes and the relationship of this to the reduced membrane lipid fluidity has been reported. As the modulation in membrane fluidity may determine the degree of accessibility of membrane receptors, the consequent alterations in membrane lipid-protein interactions in diabetes mellitus may also underlie the differentiated effects of various thrombotic and fibrinolytic agents on platelet membrane lipid bilayer. In the present study we employed electron paramagnetic resonance and fluorescence spectroscopy to explore the ligand-induced platelet membrane fluidity changes in diabetic state, i.e. under conditions when the membrane architecture is considerably altered. The yield of the excimer formation of pyrenemaleimide (PM), which depends directly upon the collisional rate and distances between molecules, was elevated in diabetic platelet membranes, thus pointing to the occurrence of some constraints in the structure/conformation of platelet membrane proteins in diabetes mellitus. Such an immobilization of PM was accompanied by the significant elevation in membrane protein glycation in diabetic platelets. The effects of various interacting ligands on platelet membrane fluidity were significantly lower in diabetic platelets, and the differences were much more distinct at the lower depths of a lipid bilayer. Nevertheless, the alterations in membrane lipid fluidity observed upon the interaction of a given ligand occurred with an approximately equal frequency in control and diabetic platelets. Moreover, the probability that these alterations were less profound in diabetic platelets was the same for all types of ligands studied. In diabetic patients the interaction of RGDS and tissue-type plasminogen activator with platelet membranes resulted in much smaller reductions of the h +1 /h 0 parameters in 5-DOXYL-Ste acid-labelled platelets, thus indicating a lesser rigidization of membrane lipid bilayer in diabetes. Likewise, the fluidizing effect of both fibrinogen itself and fibrinogen-derived peptides containing γ-chain carboxy-terminal sequence H-12-V was less pronounced in diabetic platelet membranes. Overall, much hampered effects on a lipid bilayer dynamics in diabetic platelets might have resulted most likely from the altered exposure of platelet membrane proteins and the considerable rearrangements in the lipid-protein interactions in the rigidized membranes of diabetic platelets.

The incomplete inhibition of platelet function by acetylsalicylic acid (ASA), despite the patients are receiving therapeutic doses of the drug ('aspirin-resistance'), is caused by numbers of risk factors. In this study we verified the idea that plasma homocysteine (Hcy) contributes to 'aspirin-resistance' in patients with coronary artery disease (CAD) and with or without type 2 diabetes mellitus (T2DM). A cross-designed randomized controlled intervention study has been performed (126 CAD pts incl. 26 with T2DM) to determine whether increasing ASA dose from 75mg to 150mg daily may result in the increased antiplatelet effect, in the course of four-week treatment. Platelet response to collagen (coll) or arachidonic acid (AA) was monitored with whole blood aggregometry, plasma thromboxane (Tx), and Hcy levels were determined immunochemically. The ASA-mediated reductions in platelet response to coll (by 12±3%) or AA (by 10±3%) and in plasma Tx (by 20±9%; p

The role of cyclooxygenase-2 (COX-2) in restoring the functions of impaired endothelium is attracting considerable attention, notably the function of COX-2-derived vasodilatory prostaglandins is disputed in the context of the regulation function in the impaired vascular beds. We have examined the hypothesis that COX-2 activity contributes more to vasodilation in hyperglycemic animals than in healthy counterparts, and that COX-2 derived vasodilatory prostaglandins (PGI(2) and PGE(2)) are responsible for this effect. Using the Langendorff heart perfusion system, the effects of COX-2 inhibition were monitored on both basal and bradykinin-induced coronary flows in Sprague-Dawley rats given 8-week streptozotocin-induced diabetes and in age-matched controls (n=15). Secretions of PGI(2) and PGE(2), both total and the COX-2 dependent pools, have also been compared. The selective COX-2 inhibitor, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398), had no effect on coronary flow in the diabetic group of animals. Thus, the compensatory role of COX-2 in regulation of vascular tone in experimental diabetes found in other experimental models was not confirmed. However, COX-2 activity significantly contributed to PGI(2) synthesis in healthy rats, with prostacyclin secretion being two-fold decreased by NS-398. Contrary to our hypothesis, neither prostacyclin nor PGE(2) production differed between the experimental groups under the basal conditions. Bradykinin had no effect on the secretion of PGI(2) in either group, but increased PGE(2) synthesis in healthy animals, although not in the streptozotocin group. PGE(2) production in response to bradykinin was COX-2-dependent in control animals. We conclude that, in rats with 8-week streptozotocin-induced diabetes, the activity of COX-2 in coronary vasculature is not significantly enhanced.

We hypothesized that BBB is impaired in rat model of streptozotocin-induced diabetes and can be sealed by poly(amido)amine dendrimers G4.0 (PAMAM G4), which reveal anti-glycation activity. The BBB permeabilization was monitored in rats with the 60-day streptozotocin-diabetes and non-diabetic animals, using three fluorescent dyes (given intraperitoneally) differing in molecular weight: fluorescein, fluorescein isothiocyanate (FITC)–dextran and Evans blue. All animals were administered for 2 months with either PAMAM G4 dendrimer or placebo . The fluorescence intensities of the injected fluorescent markers were recorded in the homogenates of selected brain regions. The highest accumulations of the used fluorescent dyes were observed for fluorescein, predominantly in thalamus, hippocampus, frontal cortex, striatum and cerebellum. FITC-dextran leaked to much smaller extent, however, higher permeabilization for FITC–dextran was revealed in pons–medulla oblongata, frontal and parietal cortex of diabetic compared to control animals. Evans blue leaked very slowly into striatum and pons–medulla oblongata in diabetic rats. The treatment of diabetic animals with PAMAM G4 significantly reduced blood glucose concentration and hallmarks of late diabetic complications, compared to non-treated diabetic animals. PAMAM G4 significantly reduced diabetes-induced permeabilization of BBB, which remained in line with the reduced blood glucose and the amelioration of the biochemical hallmarks of severe hyperglycaemia.

We estimated membrane fluidity by the (ESR) spectroscopy and the expression of membrane P-selectin and glycoproteins GP Ib alpha and GP IIb/IIIa by flow cytometry in platelets (plts) from 12 AD sufferers. In AD patients membrane fluidity was significantly increased at two different depths (p < 0.05 or less). Platelet reactivity was significantly decreased, as reflected by reduced expression of GP Ib alpha in the resting (p < 0.0001) and activated (p < 0.005) platelets, as well as the expression of P-selectin and beta 3 subunit of GP IIb/IIIa in activated platelets (p < 0.0001; p < 0.04).

SummaryLeptin is an adipokine that in vitro enhances agonist-induced platelet aggregation and adipokine expression. Hyperleptinaemia represents a risk factor for cardiovascular disease. We conducted a prospective evaluation of the potential link between blood platelet activation and plasma leptin levels in post-stroke patients. Using five-colour flow cyto-metry, the platelet surface expression of CD40L, CD62P, the subpopulations of monocyte-platelet aggregates and platelet-derived microparticles (PMPs) as well as the plasma leptin, soluble leptin receptor (sOb-R), leptin/sOb-R ratio, the plasma adiponectin, and leptin/adiponectin ratio were assessed in 98 stroke patients on the first (V0), 10th (V1) and 90th (V2) day after stroke and once in 78 age-, gender- and vascular risk factor-matched disease controls. We demonstrated that at V0 leptin resistance, defined as leptin/sOb-R ratio, was higher than in the controls [1.1 (0.5–1.8 vs. 0.5 (0.2–1.1); p=0.02]. After adjustment according to the factors which influence platelet activation, we confirmed the relationship between percentage of circulating PMPs and plasma leptin level (B=0.18; p=0.02) or the leptin/sOb-R ratio (B=0.23; p=0.02) in normal-weight subjects in the acute phase of stroke. No correlation could be demonstrated between the adipokine parameters and the percentage of monocyte-platelet aggregates or expression of platelet pro-inflammatory glycoproteins. In conclusion, formation of PMPs on the first day following an ischaemic stroke shows a positive correlation with leptin levels and with resistance to leptin. Leptin level does not seem to affect the expression of platelet surface proinflammatory glycoproteins.

Although the platelet activation profile after stroke is a well-known issue, the platelet reactivity assessed prospectively after ischaemic stroke still remains equivocal. The aim of this study was to evaluate the reactivity of platelets in response to stimulation with thrombin receptor-activating peptide (TRAP) at 1, 10 and 90 days after ischaemic stroke and to compare it with results obtained in control groups. We determined the increment in surface expression of CD62P, CD40L and monocyte- and granulocyte-platelet aggregate formation using five-colour flow cytometry in 86 subjects after an ischaemic event, in 62 disease controls, and in 38 healthy volunteers. We assessed the plasma levels of CD62P and CD40L soluble forms. In patients after stroke a significantly lower increment in CD62P surface expression (p 0.01) and higher increments in both CD40L platelet surface expression (p 0.01) and monocyte-platelet aggregate percentage (p 0.01) were found at every studied time point, as compared with the control groups. Plasma levels of soluble CD62P (sCD62P) and soluble CD40L (sCD40L) were increased in stroke subjects in both the acute and the subacute phase of the stroke and they dropped to levels observed in controls at day 90 after the ischaemic incident. In all studied groups a positive correlation was noted between plasma levels of sCD62P and sCD40L. In conclusion, while at 3-month follow-up the levels of soluble forms normalize in stroke patients, the profile of platelet reactivity in response to activation with TRAP differs from that observed in the controls despite the secondary stroke prevention.

SummaryThe white blood cell count and mean platelet volume determined shortly after the symptom onset are known as independent predictors for clinical outcome after stroke. In the present study we sought to evaluate the prognostic value of platelet-derived inflammatory biomarkers measured prospectively after an ischaemic event. Using five-colour flow cytometry, the platelet surface expression of CD40L, CD62P and subpopulations of leukocyte-platelet aggregates were assessed in 93 stroke patients on the first (V0), 10th (V1) and 90th (V2) day after stroke, and once in 65 disease controls. The clinical outcome was evaluated using the Scandinavian Stroke Scale (SSS) and modified Rankin Scale (mRS) at the same time points as blood sampling and 24 months after the event. Patients with either CD40L surface expression or the percentage of monocyte-platelet aggregates (M-plt) in the third tertile (T3) at V0 had a significantly lower score on the SSS at V1. Patients with the percentage M-plt at V0 higher than the median value of M-plt in controls were at increased risk of SSS < 40 at V1 (odds ratio: 2.6; 95% confidence interval [CI]: 1.4 – 8.7; p=0.006). Patients with the percentage of M-plt in T3 at V0 showed progressive decline in survival (hazard ratio [HR]: 1.6; 95% CI: 1.1–1.9; p=0.02) and a significantly higher number of recurrent vascular events (HR: 2.64; 95% CI: 1.3–3.2; p=0.02) when compared to the first tertile. In conclusion, increased levels of M-plt could be a predictive marker for both early outcome and long-term prognosis while increased CD40L was correlated with worse clinical outcome.The preliminary results of this study were presented in part during a poster session at the 62nd Annual Meeting of the American Academy of Neurology in Toronto, 7–17 April 2010.

article i nfo Objectives: Platelet-leukocyte aggregation is believed to contribute to acute thrombotic events. While the effect of aspirin on platelet-to-platelet aggregation is well established, the impact of the drug on pro- inflammatory platelet function remains equivocal. Thus we investigated the effect of aspirin on selected platelet-related inflammatory biomarkers in both acute ischaemic stroke patients and healthy volunteers. Methods: Using five-colour flow cytometry the platelet surface expression of CD62P and CD40L and subpopulations of leukocyte-platelet aggregates were assessed in 63 acute stroke patients and 40 healthy volunteers at baseline and after a 10-day period of aspirin intake at a daily dose of 150 mg. Simultaneously the plasma levels of soluble CD62P and CD40L, serum level of TxB2, and whole blood impedance platelet aggregation under arachidonic acid (AA) stimulation were investigated. Results: No differences in values of studied platelet-related inflammatory biomarkers in both resting platelets and those activated with TRAP after 10-day treatment with aspirin were confirmed in stroke subjects. In healthy individuals the resting platelet expression of CD62P, plasma level of soluble CD62P and percentage of circulating monocyte-platelet aggregates were lower after the aspirin intake period (P=0.009; P=0.04; P=0.004, respectively). In both studied groups serum level of TxB2 and platelet aggregation under AA stimulation were lower than before treatment (Pb0.001). Conclusion: Despite effective inhibition of COX-1-dependent platelet aggregation, aspirin does not influence the platelet α-granule-derived inflammatory mediators and monocyte-platelet aggregation in acute stroke subjects, although it does in healthy individuals.