Soluble CD40L Levels Research Articles

Abstract 18686: Association of Obesity Susceptibility Gene Variants With Cardiometabolic Phenotypes in Morbid Obesity

Introduction: Genome-wide association studies (GWAs) have identified several gene variants strongly associated with increased body mass index (BMI) and obesity. However, their association with the cardiometabolic consequences of obesity is unclear. We performed a study of 40 validated variants recently identified in two large metaanalysis GWAS and analyzed their association with related cardiovascular and metabolic traits of severe obesity. Methods: Genotyping was performed in 320 deeply-phenotyped obese subjects (mean BMI 41±9 kg/m2, aged 44±12 years), participating in a standardized weight reduction program. Anthropometric parameters, parameters of glucose/insulin metabolism, oxidative stress and early atherogenesis, adipokines, adhesion molecules, apolipoproteins, cardiac function and structure, intima media thickness and arterial elasticity were assessed standardized after 12h fast. Results: The transmembran protein TMEM18 rs939583 variant was significantly related to several parameters of adiposity (i.e. BMI β-estimate 1/2 risk alleles vs. no risk allele: 3.7±2.2/ 5.5±1.9, p=0.0009), epicardial fat thickness (p=0.023), insulin (p=0.002), fasting glucose (p=0.007), HOMA-IR (p=0.0008), and leptin (p=0.008) levels, systolic (p=0.007) and diastolic (p=0.009) blood pressure, arterial hypertension (frequency in subjects with 0/1/2 risk allele(s): 14/30/46%, p=0.001), and the metabolic syndrome (29/29/48%, p=0.029). Moreover, the BDNF rs10767664 variant was significantly associated with ApoA1, ApoA2, HDL cholesterol and soluble CD40L levels. Conclusion: TMEM18 and BDNF variants increased the risk of metabolic syndrome components, probably through their effect of abdominal obesity. However, several gene variants that have been found to be associated with BMI with small effects in large scale GWAS, did not seriously alter obesity related cardiometabolic traits.

PECAM-1 gene polymorphisms and soluble PECAM-1 level in rheumatoid arthritis and systemic lupus erythematosus patients: any link with clinical atherosclerotic events?

Genetic polymorphisms of platelet endothelial cell adhesion molecule-1 (PECAM-1) were found to play roles in atherosclerotic events. We determined PECAM-1 polymorphisms, soluble PECAM-1, and CD40L levels in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and evaluated their associations with clinical atherosclerotic complications. We included 100 RA patients, 81 SLE patients, and 94 healthy controls. The clinical features about the patients were obtained from medical records. Past cardiovascular complications were recorded. The most frequent gene polymorphisms of PECAM-1 were studied in our genetics laboratory. Soluble PECAM-1 and CD40L levels in serum were determined with ELISA. The frequencies of 373C (rs668) and 1688A (rs12953) alleles were higher in RA patients when compared to controls (p values, 0.028 and 0.016). RA and SLE patients had significantly higher allele frequencies for 2008A (rs1131012) when compared to controls (p values, 0.016 and 0.001). SLE patients had significantly more frequent AA genotype for rs1131012 polymorphism than RA patients and controls (p values, 0.007 and <0.001). Soluble PECAM-1 level was significantly higher in RA patients than in SLE patients and healthy controls (p values <0.001). Atherosclerotic complications were more frequent in SLE patients with AG genotype (rs12953) than those with AA genotype (p = 0.021). SLE patients with CC genotype (rs668) had a significantly lower frequency of atherosclerotic complications than those with CG genotype (p = 0.045). Nevertheless, in multivariate analysis, there was no association between genotype and atherosclerotic complications. We found associations between various PECAM-1 polymorphisms in RA and SLE; PECAM-1 and soluble CD40 ligand (sCD40L) levels were significantly higher in RA patients than in SLE and control groups. PECAM-1 polymorphisms in SLE were protective against atherosclerotic complications.

Rac1 regulates platelet shedding of CD40L in abdominal sepsis

Matrix metalloproteinase-9 (MMP-9) regulates platelet shedding of CD40L in abdominal sepsis. However, the signaling mechanisms controlling sepsis-induced shedding of CD40L from activated platelets remain elusive. Rac1 has been reported to regulate diverse functions in platelets; we hypothesized herein that Rac1 might regulate platelet shedding of CD40L in sepsis. The specific Rac1 inhibitor NSC23766 (N6-[2-[[4-(diethylamino)-1-methylbutyl] amino]-6-methyl-4-pyrimidinyl]-2 methyl-4, 6-quinolinediamine trihydrochloride) was administered to mice undergoing cecal ligation and puncture (CLP). Levels of CD40L and MMP-9 in plasma, platelets, and neutrophils were determined by use of ELISA, western blot, and confocal microscopy. Platelet depletion abolished the CLP-induced increase in plasma levels of CD40L. Rac1 activity was significantly increased in platelets from septic animals. Administration of NSC23766 abolished the CLP-induced enhancement of soluble CD40L levels in the plasma. Moreover, Rac1 inhibition completely inhibited proteinase-activated receptor-4-induced surface mobilization and secretion of CD40L in isolated platelets. CLP significantly increased plasma levels of MMP-9 and Rac1 activity in neutrophils. Treatment with NSC23766 markedly attenuated MMP-9 levels in the plasma from septic mice. In addition, Rac1 inhibition abolished chemokine-induced secretion of MMP-9 from isolated neutrophils. Finally, platelet shedding of CD40L was significantly reduced in response to stimulation with supernatants from activated MMP-9-deficient neutrophils compared with supernatants from wild-type neutrophils, indicating a direct role of neutrophil-derived MMP-9 in regulating platelet shedding of CD40L. Our novel data suggest that sepsis-induced platelet shedding of CD40L is dependent on Rac1 signaling. Rac1 controls surface mobilization of CD40L on activated platelets and MMP-9 secretion from neutrophils. Thus, our findings indicate that targeting Rac1 signaling might be a useful way to control pathologic elevations of CD40L in the systemic circulation in abdominal sepsis.

Maintenance of virologic efficacy and decrease in levels of β2-microglobulin, soluble CD40L and soluble CD14 after switching previously treated HIV-infected patients to an NRTI-sparing dual therapy

Novel strategies are necessary to decrease inflammatory parameters in successfully treated HIV-infected patients. Our aim was to evaluate the maintenance of viral suppression and potential changes in inflammatory, immune-activation and coagulation biomarkers in virologically suppressed HIV-infected patients switched to a nucleoside reverse transcriptase inhibitor-sparing (NRTI) and maraviroc (MVC)-containing combined antiretroviral therapy (cART). Fifty-eight HIV-infected patients were observed after their treatment regimens were changed to MVC 150mg/once daily plus ritonavir-boosted protease inhibitor therapy. Activation-, inflammation- and coagulation-associated biomarkers and mitochondrial (mt)DNA were analyzed after a median of 24weeks of follow-up. We observed that after changing to an NRTI-sparing regimen, 96.6% of HIV-patients on viral suppressive cART maintained viral suppression and their CD4+ T cell counts did not change significantly (median of 31weeks of follow-up). This cART switch reduced soluble CD40 ligand (p=0.002), beta-2 microglobulin (p=0.025), and soluble CD14 (p=0.009) in patients with higher baseline levels of these inflammation biomarkers after a median of 24weeks of follow-up. The results of our study show that changing to NRTI-sparing dual therapy decreased the levels of inflammatory biomarkers and maintained the immune-virologic efficacy. The potential benefits of this regimen warrant further investigation to uncover the association of this therapy with the potential decrease in the morbidity and mortality of HIV-infected patients from non-AIDS-defining illnesses.

The aspirin resistance and platelets` receptors expression in chronic heart failure patients with type 2 diabetes mellitus

Introduction. Chronic heart failure (CHF) is a complex clinical syndrome associated with high mortality rates. The presence of type 2 diabetes mellitus (DM) is an independent unfavourable prognostic factor in heart failure patients. Chronic heart failure and type 2 diabetes mellitus are closely inter-related conditions through different pathological mechanisms, including platelet activation. The presence of DM in CHF patients can dramatically increase platelet dysfunction. The problem is much complicated by high level of aspirin resistance in diabetic patients. The prevalence of aspirin resistance in CHF with DM has never been studied.The aim of our research was to estimate the prevalence of aspirin resistance in heart failure patients with type 2 diabetes mellitus and to clarify platelets` abnormalities according to glycemic control status.Materials and methods. The criteria for study inclusion were: a more than 1-year documented history of CHF due to coronary artery disease, sinus rhythm, type 2 diabetes mellitus, no change in medication for &gt; 3 months, daily aspirin use 100 mg &gt; 6 months. All patients had previous history of myocardial infarction. Exclusion criteria were: type 1 DM, patients with type 2 DM on insulin therapy and/or thiazolidinediones, major bleedings in anamnesis, chronic renal failure, chronic obstructive pulmonary disease, autoimmune diseases, significant liver failure, cancer. We studied 65 patients with heart failure and type 2 diabetes mellitus (mean age 62±8 years). The control population comprised 35 heart failure patients without diabetes mellitus (mean age 60±9 years). Platelet function was evaluated using standard light transmission aggregometry. Aspirin resistance was defined by known criteria: ≥ 70% ADP- and ≥ 40% epinephrine-induced aggregation. Measurements of CD41, CD41a, CD61 and CD62P were carried out by fluorescent microscopy study. The plasma levels of soluble P-selectin and soluble CD40L were measured by ELISA assays.Results. Age, sex, severity of CHF, left ventricular ejection fraction (LVEF), estimated glomerular filtration rate and cardiovascular treatment were comparable between three groups. Hypertension rate was slightly frequent in patients with CHF and decompensated DM. The patients with CHF and decompensated DM had higher fasting plasma glucose, HbA1cand HOMA-IR levels. The duration of the diabetes was similar. Glucose lowering treatment on admission was comparable between groups. The rate of aspirin resistance was tend to be higher in patients with CHF and DM (by ADP-induced aggregation: 23.1% in diabetic patients vs. 14.3% in non-diabetic patients, P=0.43; by epinephrine-induced aggregation: 36.9% in diabetic patients vs. 31.4% in non-diabetic patients, P=0.66), but the difference was not statistically significant. The compensation of DM didn’t influence on aspirin resistance rate also. The compensation of DM didn’t influence on aspirin resistance rate also. No significant changes in platelets’ parameters were observed between CHF patients without DM and patients with CHF and compensated DM. In contrast, the patients with CHF and decompensated DM presented higher platelet surface CD62P, CD41, CD41a and CD61 expression in comparison to CHF patients without DM. The patients with CHF and HbA1c of more than 7 % showed also higher plasma P-selectin and higher platelet surface CD41 and CD61 expression compared with CHF patients with HbA1c of less than 7 %. Plasma sP-selectin correlated positively with FPG, insulin, C-peptide and HOMA-IR. Platelet surface P-selectin correlated only with C-peptide. Both IIb and IIIa glycoprotein receptors correlated with HbA1c. Conclusions. The rate of aspirin resistance varies from 23 to 37% in patients with chronic heart failure and type 2 diabetes mellitus depending on tests used. Platelet reactivity is associated with glycemic control status in heart failure diabetic patients on long-term aspirin therapy.

B cells from relapsing remitting multiple sclerosis patients modulate T cell behavior in a neuroantigen-specific manner (HUM1P.322)

Abstract The role of B cells in the pathoetiology of relapsing remitting multiple sclerosis is not well understood. B cells may contribute to MS by antibody secretion, cytokine production and antigen presentation. Antigen-specific B cells are highly efficient antigen presenting cells in the context of their cognate antigens. Since antibodies from multiple sclerosis patients bind to neuroantigens we hypothesized that neuroantigen specific memory B cells promote disease in multiple sclerosis patients by serving as antigen presenting cells for auto-reactive T cells. We observed that B cells from a subset of multiple sclerosis patients induce naïve and memory T cell proliferation in the presence of recombinant neuroantigens, myelin oligodendrocyte glycoprotein and myelin basic protein, but not MOG 35-55 peptide, in B-T cell co-cultures. Similarly, in the presence of neuroantigen proteins cells from multiple sclerosis patients secreted greater levels of IFN-γ, IL-6, -17A, -17F, -21, -22, -31, -33, TNF-α and soluble CD40L than healthy donors, while levels of IL-4 and IL-25 were similar. These data support a role for human B cells as antigen presenting cells in multiple sclerosis and highlight the role of B cells in the context of autoimmune disease beyond antibody production.

Strict heart rate control attenuates prothrombotic state and platelet activity in patients with non-valvular permanent atrial fibrillation

The underlying mechanisms of increased risk of thrombo-embolism in atrial fibrillation (AF) are not completely understood; however, substantial evidence supports that AF is associated with a prothrombotic state. Accordingly, we hypothesized that strict rate control could attenuate platelet activity and thrombotic state in patients with non-valvular AF. Seventy-five patients with non-valvular AF were divided into 2 groups based on heart rate: (1) normal ventricular rate (n = 34, 18 female) and (2) high ventricular rate (n = 39). Thirty-three sex- and age-matched subjects in sinus rhythm were included as control. Thirty patients with high ventricular rate (16 female) were successfully followed. Markers of platelet function were measured at baseline and repeated 1-month after adequate rate control in high ventricular rate group. Serum fibrinogen levels were significantly higher in AF patients with high ventricular rate than that in controls. Mean platelet volume, soluble CD40L and β-Thromboglobulin were significantly higher in AF patients with high ventricular rate than those in both AF patients with normal ventricular rate and controls. Soluble CD40L and β-Thromboglobulin were significantly higher in AF patients with normal ventricular rate than those in controls. One-month after adequate rate control, serum fibrinogen, soluble CD40L and β-Thromboglobulin levels significantly decreased (from 2.26 ± 1.02, 85.01 ± 37.05, 3.10 ± 0.90 to 1.55 ± 1.08, 66.34 ± 33.72, 2.71 ± 0.53; p < 0.001, p = 0.002, p = 0.03, respectively) in high ventricular rate group. AF patients with high ventricular rate had increased indices of platelet activity and thrombotic state. Furthermore, strict rate control significantly decreased indices of thrombotic state and platelet activity in those patients.

Total adiponectin is inversely associated with platelet activation and CHA₂DS₂-VASc score in anticoagulated patients with atrial fibrillation.

Background. Adiponectin (APN) possesses anti-inflammatory and antiatherogenic effects. Atrial fibrillation (AF) is burdened by enhanced systemic inflammation and platelet activation, as documented by increased blood levels of soluble CD40L (sCD40L). The interplay between APN and platelet activation in AF is still undefined. Materials and Methods. Circulating levels of APN and sCD40L were measured in 257 anticoagulated nonvalvular AF patients. Exclusion criteria were as follows: prosthetic heart valves, cardiac revascularization in the previous year, severe cognitive impairment, chronic infectious or autoimmune diseases, and active cancer. Results. Mean age was 72.9 (±8.7) years and 41.6% were female. Serum APN and plasmatic sCD40L were inversely correlated (R −0.626, P < 0.001). A progressive increase of sCD40L across tertiles of CHA2DS2-VASc score was observed (rS 0.473, P < 0.001), whilst APN was inversely correlated (rS −0.463, P < 0.001). A multivariable linear regression analysis showed that CHA2DS2-VASc score (B −0.227, P < 0.001) and sCD40L (B −0.524, P < 0.001) correlated to APN. Conclusions. AF patients at high risk of stroke disclose low and high levels of APN and sCD40L, respectively, suggesting a role for APN if it favors platelet activation in vivo in this clinical setting. Enhancing APN levels may be a future goal to reduce the risk of vascular outcomes in AF patients.

Inactivation of the immune receptor CD40 attenuates the development of cardiac hypertrophy in angiotensin-II induced hypertensive heart disease

Emerging evidence indicates that inflammatory activation is a crucial pathway in disease progression during HF. The co-stimulatory dyad CD40-CD40L is involved in pro-inflammatory activation of a wide range of immune and non-immune cells and plasma levels of soluble CD40L in HF patients are raised. Here we investigated whether CD40-CD40L interactions attenuate cardiac hypertrophy in hypertensive heart disease. CD40 knockout and wild type C57Bl6-J mice were treated with angiotensin II (AngII, 2.5 mg/kg/day) for 4 weeks. AngII-induced hypertension led to a significant increase in cardiac mass in Wt mice (+31%, P<0.01), but not in CD40 knockout mice (+10%, N.S.). Comparable changes were observed in diastolic wall thickness (echocardiography) and cardiomyocyte cross sectional area. To determine which CD40-TRAF interactions were involved, the effect of mutations in the intracellular TRAF binding domains of CD40 was investigated. Relative to mice with intact downstream CD40 signaling, disruption of TRAF2/3/5, TRAF6 or both binding sites was associated with reduced cardiac hypertrophy (cardiac mass -24% for CD40-TΔ2, -28% for CD40-TΔ6 and -26% in CD40-TΔ2/6; p<0.05). Reduced hypertrophy was not associated with a decline in immune cell infiltration (CD45+ cells) or collagen content. Although the exact mechanism remains to be elucidated the present findings indicate a role for the CD40 receptor in the development of cardiac hypertrophy.

Platelet shedding of CD40L is regulated by matrix metalloproteinase‐9 in abdominal sepsis

Platelet-derived CD40L is known to regulate neutrophil recruitment and lung damage in sepsis. However, the mechanism regulating shedding of CD40L from activated platelets is not known. We hypothesized that matrix metalloproteinase (MMP)-9 might cleave surface-expressed CD40L and regulate pulmonary accumulation of neutrophils in sepsis. Abdominal sepsis was induced by cecal ligation and puncture (CLP) in wild-type and MMP-9-deficient mice. Edema formation, CXC chemokine levels, myeloperoxidase levels, neutrophils in the lung and plasma levels of CD40L and MMP-9 were quantified. CLP increased plasma levels of MMP-9 but not MMP-2. The CLP-induced decrease in platelet surface CD40L and increase in soluble CD40L levels were significantly attenuated in MMP-9 gene-deficient mice. Moreover, pulmonary myeloperoxidase (MPO) activity and neutrophil infiltration in the alveolar space, as well as edema formation and lung injury, were markedly decreased in septic mice lacking MMP-9. Invitro studies revealed that inhibition of MMP-9 decreased platelet shedding of CD40L. Moreover, recombinant MMP-9 was capable of cleaving surface-expressed CD40L on activated platelets. In human studies, plasma levels of MMP-9 were significantly increased in patients with septic shock as compared with healthy controls, although MMP-9 levels did not correlate with organ injury score. Our novel data propose a role of MMP-9 in regulating platelet-dependent infiltration of neutrophils and tissue damage in septic lung injury by controlling CD40L shedding from platelets. We conclude that targeting MMP-9 may be a useful strategy to limit acute lung injury in abdominal sepsis.

Assessment of serum levels of soluble CD40L in Egyptian children and adolescents with type 1 diabetes mellitus: Relationship to microalbuminuria and glycemic control

Context:Soluble CD40 ligand (sCD40L) is known to be elevated in different clinical situations including hypercholesterolemia, acute coronary syndromes, and type 2 diabetes mellitus (T2DM), Data about the relationship between type 1 diabetes mellitus (T1DM) and sCD40L is limited. In addition, the potential role ofsCD40Lin the pathogenesis of vascular complications in children and adolescents with T1DM is to be clarified. Hence, the study aimed at assessment of sCD40L levels in children and adolescents with T1DM and correlation of these levels with glycemic control and microalbuminuria.Settings and Design:Cross-sectional controlled study.Materials and Methods:The study was performed in the Pediatric Endocrinology and Diabetes Unit, Assuit University Children Hospital, Assiut, Egypt. It included 70 children and adolescents with T1DM (mean age 14. 76 ± 2.21 years). Cases were further subdivided into 43 cases with normoalbuminuria and 27 cases with microalbuminuria according to presence or absence or microalbuminuria in fresh urine samples. Twentyfive healthy subjects, age- and sex-matched were included as control group (mean age = 13.62 ± 2.11 years). Studied cases were subjected to medical history, clinical examination, and laboratory assessment of fasting blood glucose (FBG), lipid profile, glycosylated hemoglobin (HbA1c), and sCD40L were performed.Results:Mean HbA1c and sCD40L were significantly higher in diabetic children (n = 70) compared to control (n = 25) (P < 0.001 for each). Mean HbA1c and sCD40L levels were significantly higher in microalbuminuric cases (n = 27) compared to normoalbuminuric cases (n = 43) (P < 0.05 and <0.01, respectively). We also observed a significant positive correlation between sCD40L levels and the age, diabetes duration, HbA1c, and urinary albumin creatinine ratio.Conclusions:The high serum sCD40L levels in children and adolescents with T1DM particularly in those with microalbminuria and its positive correlation with diabetes duration, urinary albumin excretion, and glycemic control may reflect the role of sCD40L in diabetic vasculopathy in the pediatric age group. Moreover, measurement of serum sCD40L levels in poorly controlled patients would help to identify those at high risk of developing nephropathy.

Effects of outdoor air pollutants on platelet activation in people with type 2 diabetes

Exposure to air pollution is associated with increased morbidity and mortality from cardiovascular disease. We hypothesized that increases in exposure to ambient air pollution are associated with platelet activation and formation of circulating tissue factor-expressing microparticles. We studied 19 subjects with type 2 diabetes, without clinical evidence of cardiovascular disease, who had previously participated in a human clinical study of exposure to ultrafine particles (UFP). Blood was obtained for measurements of platelet activation following an overnight stay in the Clinical Research Center, prior to each of their two pre-exposure visits. Air pollution and meteorological data, including UFP counts, were analyzed for the 5 days prior to the subjects’ arrival at the Clinical Research Center. Contrary to expectations, increases in UFP were associated with decreases in surface expression of platelet activation markers. The number of platelet-leukocyte conjugates decreased by −80 (95% confidence interval (CI) −123 to −37, p = 0.001) on the first lag day (20–44 h prior to the blood draw) and by −85 (CI −139 to −31, p = 0.005) on combined lag days 1 to 5, per interquartile range (IQR) increase in UFP particle number (2482). However, levels of soluble CD40L increased 104 (CI 3 to 205, p = 0.04) pg/ml per IQR increase in UFP on lag day 1, a finding consistent with prior platelet activation. We speculate that, in people with diabetes, exposure to UFP activates circulating platelets within hours of exposure, followed by an increase in soluble CD40L and a rebound reduction in circulating platelet surface markers.

Periodontopathogens induce expression of CD40L on human platelets via TLR2 and TLR4

IntroductionThe outstanding importance of (soluble) CD40L to cardiovascular disease (CVD) is becoming increasingly apparent as CD40L is an important mediator of thrombotic and inflammatory processes. Platelets are the main source for CD40 ligand, linking platelet stimulatory events to inflammation and adverse adaptive immune responses. Periodontitis represents a chronic dental infection by distinct gram negative bacteria that is associated with an increased risk for CVD. However, the effects of periodontopathogens on CD40L expression by platelets have not been determined. Material and MethodsEffects of periodontopathogens A. actinomycetemcomitans Y and P. gingivalis on the expression of CD40L were determined and the underlying receptors and pathways were investigated. 26 patients with periodontitis and 19 controls were included in the clinical part of this study. ResultsPeriodontopathogens directly induce surface expression of CD40L in human platelets. This activation depends on plasma factors like CD14 and involves TLR2 and TLR4 but not FcγRII. Inhibition of PI3K and PLC completely abolishes bacteria-induced surface expression of CD40L. TLR2 and TLR4 agonists, for example, are also able to induce expression and release of CD40L in human platelets.In patients with periodontitis, plasma levels of soluble CD40L are elevated and positivity for P. gingivalis is associated with a statistical significant increase of soluble CD40L. ConclusionsOur data indicate an involvement of periodontopathogens in increased plasma levels of soluble CD40L in periodontitis and therefore provide a novel link between periodontitis and increased risk for CVD.

Impact of Statins on the Coagulation Status of Type 2 Diabetes Patients Evaluated by a Novel Thrombin-Generation Assay

Dyslipidemia is common in type 2 diabetes (T2D) and contributes to cardiovascular disease (CVD) by exacerbating atherosclerosis and hypercoagulability. Statins can stabilize atherosclerotic plaque and reduce prothrombotic status. In the present study we aimed to evaluate the coagulation activity and the effect of statins on procoagulant state of T2D patients using a novel activated protein C (APC)-dependent thrombin-generation assay. Procoagulant status (by HemosIL ThromboPath (ThP) assay) and in vivo platelet activation (by plasma soluble (s)CD40L levels) were analyzed in a retrospective, cross-sectional study of 198 patients with long-standing T2D and 198 controls. Procoagulant status of T2D patients was enhanced when compared to control subjects (p < 0.0001). Similarly, sCD40L levels were increased in T2D (p < 0.0001). When testing ThP as the dependent variable in a multivariate regression model, sCD40L (p < 0.0001) and statin treatment (p = 0.019) were independent predictors of the procoagulant state of T2D patients. Subgroup analysis showed a significant improvement of coagulability in T2D patients on statins (p = 0.012). The use of a standardized, easy-to-run, and commercially available APC-dependent thrombin-generation assay detected the presence of a procoagulant status in a large series of patients with long-standing T2D and demonstrated a significant impact of statins in the coagulation status of patients with T2D.

Platelet activation in chronic cocaine users: Effect of short term abstinence

Cocaine abuse increases the risk of cardiac and cerebrovascular events, such as myocardial infarction and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation has been observed. The aim of this study was to investigate the existence of platelet activation and the effect of short-term abstinence in chronic cocaine consumers. We studied 23 cocaine dependent individuals (aged 20–54 years) who met DSM-IV criteria for cocaine dependence and 20 controls. Samples were obtained at baseline, within 72 h of last drug exposure and after 4 weeks of controlled abstinence. Monocyte-platelet aggregates (MPA) were measured by flow cytometry. Plasma levels of soluble CD40L (sCD40L), Neutrophil-Activating Peptide-2 (NAP-2) and regulated on activation normal T cells expressed and secreted (RANTES) were determined by ELISA. Levels of MPA, sCD40L, NAP-2 and RANTES were significantly higher (all p < 0.05) in cocaine addicts compared to controls at baseline. All the parameters returned to values similar to the control group after 4-weeks’ abstinence. Levels of sCD40L and RANTES were associated with an index of intensity of drug consumption (p < 0.02). Our results demonstrate that cocaine use induces platelet activation which is a prominent finding after recent consumption. The persistence over time of this condition may contribute not only to acute thrombotic complications but also to the development of early-onset atherosclerotic process observed in cocaine abusers.

Aspirin treatment influences platelet-related inflammatory biomarkers in healthy individuals but not in acute stroke patients

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 TxB 2, 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 TxB 2 and platelet aggregation under AA stimulation were lower than before treatment (P < 0.001). 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.

Chronic hyper-reactivity of platelets resulting in enhanced monocyte recruitment in patients after ischaemic stroke

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.

Relationship Between Plasma Inflammatory Markers and Platelet Aggregation in Patients With Clopidogrel Resistance After Angioplasty

We evaluated the relationship between plasma inflammation markers and clopidogrel resistance in patients after stent implantation. The plasma levels of C-reactive protein (CRP), P-selectin, platelet soluble CD40 ligand (sCD40L), interleukin 6 (IL-6) and platelet aggregation were measured in 352 patients undergoing percutaneous coronary intervention (PCI) at baseline and after 6 months. The plasma levels of CRP, P-selectin, sCD40L, IL-6 was higher in 65 (18.5%) patients with clopidogrel resistance than in those with normal responsiveness at 6 months after PCI. There was a significant positive correlation between soluble CD40L levels and platelet aggregation (r = .28, P < .05). Diabetes (DM) and sCD40L level were independent predictors for unresponsiveness after stent implantation according to stepwise multivariate analyses. The hazard ratio (HR) for sCD40L level was 3.02 (95% CI = 1.28 to 3.25; P = .036) and for DM 2.53 (95% CI = 1.28 to 6.55, P = .03). We conclude that sCD40L and DM may influence clopidogrel resistance.

Activation of platelets and cellular coagulation in cerebral small-vessel disease

Platelets and monocytes play a pivotal role in the initiation and progression of large-vessel atherosclerosis. An up-regulation of various platelet and coagulation activation markers has been described in cardiovascular diseases and in patients with acute cerebral ischemia. In the present study the role of platelets and cellular coagulation activation in cerebral small-vessel disease (cSVD) was assessed. In 24 patients with cSVD but without established large-vessel disease, whole blood samples were obtained. Patients were divided into three subgroups (Fazekas 1, 2 and 3) according to extent of cSVD based on morphological magnetic resonance imaging criteria. Surface expression of CD40L and CD62P on platelets, tissue-factor exposition on monocytes and platelet-monocyte aggregates were measured with flow cytometry. Plasma levels of soluble CD40L, interleukin (IL)-6 and IL-7 were assessed by ELISA. Patients with cSVD show a significantly elevated expression of platelet CD40L (P < 0.001) and CD62P (P < 0.023), significantly elevated amounts of platelet-monocyte aggregates (P < 0.004), a significantly enhanced tissue-factor exposition on monocytes (P < 0.019) and significantly lower plasma levels of IL-7 compared to 10 healthy controls. However, this platelet and monocyte activation did not correlate with the severity of cSVD. Patients with cSVD show an up-regulation of the platelet CD40L and CD62P system and an activation of cellular coagulation which might contribute to the initiation and progression of cSVD.

Effect of Short-Term Abstinence on Cocaine-Induced Endothelial Dysfunction and Platelet Activation

AbstractAbstract 4205 Background.Cocaine abuse is associated with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation and accelerated atherosclerosis are prominent findings. We have previously shown (Pereira et al. J Thromb Haemost 2009; 7[suppl 2]: 232a) that chronic cocaine use is associated with markers of endothelial dysfunction and platelet activation in subjects studied after recent consumption. It has been suggested that many of the adverse effects of cocaine on the vessel wall are due to its acute effects related to the sympathomimetic properties of the drug. However, from a pathogenic standpoint, we hypothesized that important pathologic consequences, such as early onset atherosclerosis and regional brain perfusion, defects, are not solely explained by the acute vasomotor actions of cocaine. Objectives.The main aim of this work was to investigate the effect of short-term abstinence on markers of endothelial injury and platelet activation in chronic cocaine consumers. methods. We studied 23 cocaine dependent individuals (aged 19–52years mean age 30 years) who met DSM-IV criteria for cocaine dependence, seeking treatment for cocaine abuse and 25 healthy controls (aged 20–49 years, mean age 31 years). Samples were obtained at admission within 72 hours of drug exposure and after 4 weeks of strict, controlled abstinence in a rehabilitation clinic.Endothelial cell damage was assessed by enumerating circulating endothelial cells (CECs) and plasma levels of soluble markers: soluble intercellular adhesion molecule (sICAM); monocyte chemoattractant protein (MCP-1), von Willebrand factor (VWF) and high-sensitivity C reactive protein (hs-CRP). Plasma levels of soluble CD40L (sCD40L), NAP-2 and RANTES were determined to demonstrate platelet activation in vivo. Results.Markers of endothelial cell damage/activation and platelet activation, were significantly higher in cocaine dependents individuals after recent consumption (baseline) as compared with the controls. The variations observed after 4 weeks drug withdrawal (post-abstinence) are shown in Table 1:CECs/mLsICAM (ng/mL)MCP-1 (ng/mL)VWF (%)sCD40L (ng/mL)NAP-2 (ng/mL)RANTES (ng/mL)Baseline62 ± 18*346 ± 140*154 ± 80*94 ± 21*1.7 ± 11.1*113 ± 53*4.1 ± 2.6*Abstinence44 ± 15**257 ± 180**166 ± 170**93 ± 22**0.98 ± 0.5**98 ± 40**2.4 ± 1.1**Controls8 ± 14219 ± 14061 ± 2678 ± 160.83 ± 0.879 ± 462.2 ± 1.6P *vs Ctrls **vs Ctrls<0.00010.00520.0030.0480.0160.0260.0070.00070.220.0040.0450.3710.1060.75 Conclusions.Our results demonstrate that cocaine use is associated to endothelial dysfunction and platelet activation which are prominent findings after recent consumption. Evidence of endothelial cell activation/damage is still present after 4 weeks of strict and controlled abstinence when markers of platelet activation returned to their baseline levels. Taken together, these observations suggest that cocaine induced-endothelial cell damage is maintained independent of the acute effect of the drug on the blood vessels. The persistence of this condition may play a role in long-term ischemic complications associated with cocaine abuse such as early onset atherosclerosis and regional brain perfusion defects. Further studies on the mechanisms underlying cocaine-induced endothelial dysfunction might provide novel strategies to improve endothelial function as part of the treatment in recently abstinent cocaine addicts. Disclosures:No relevant conflicts of interest to declare.