Levels In Platelet-poor Plasma Research Articles

Duloxetine protected indomethacin-induced gastric mucosal injury by increasing serotonin-dependent RANTES expression and activating PI3K-AKT-VEGF pathway

Antidepressant duloxetine has been shown protective effect on indomethacin-induced gastric ulcer, which was escorted by inflammation in the gastric mucosa. Cytokines are the principal mediators of inflammation. Thus, by screening the differential expression of cytokines in the gastric mucosa using cytokine array at 3 h after indomethacin exposure, when the gastric ulcer began to format, we found that indomethacin increased cytokines which promoted inflammation responses, whereas duloxetine decreased pro-inflammatory cytokines increased by indomethacin and increased RANTES expression. RANTES was consistently increased by pretreated with both 5 mg/kg and 20 mg/kg duloxetine at 3 h and 6 h after indomethacin exposure in male rats. Selective blockade of RANTES-CCR5 axis by a functional antagonist Met-RANTES or a CCR5 antagonist maraviroc suppressed the protection of duloxetine. Considering the pharmacologic action of duloxetine on reuptake of monoamine neurotransmitters, we examined the serotonin (5-HT), norepinephrine and dopamine contents in the blood and discovered 20 mg/kg duloxetine increased 5-HT levels in platelet-poor plasma, while treatment with 5-HT promoted expression of RANTES in the gastric mucosa and alleviated the indomethacin-induced gastric injury. Furthermore, duloxetine activated PI3K-AKT-VEGF signaling pathway, which was regulated by RANTES-CCR5, and selective inhibitor of VEGF receptor axitinib blocked the prophylactic effect of duloxetine. Furthermore, duloxetine also protected gastric mucosa from indomethacin in female rats, and RANTES was increased by duloxetine after 6 h after indomethacin exposure too. Together, our results identified the role of cytokines, particularly RANTES, and the underlying mechanisms in gastroprotective effect of duloxetine against indomethacin, which advanced our understanding in inflammatory modulation by monoamine-based antidepressants.

Pro-inflammatory sensitization of platelets after a mild course of COVID-19 infection

Abstract Background Coronavirus disease-2019 (COVID-19) is a viral respiratory illness caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) which can lead to an increased risk of thromboembolic events but also complications even after recovery from the disease. Multiple factors may enhance the risk of thrombotic events in COVID-19 patients including excessive inflammatory responses and increased platelet activity. To date there are hardly any data on how platelet function is affected after a mild course of infection. Purpose In this study, we investigated whether platelet function is affected after recovery from mild COVID-19 disease. Besides the response of platelets to platelet stimuli, expression of the transporter protein multidrug resistance protein 4 (MRP4) and one of its substrates, the inflammatory lipid mediator sphingosine-1-phosphate (S1P), were measured. Methods Venous blood samples were collected from patients within 2-15 weeks after recovery from a mild SARS-CoV-2 infection (recovered) and matched individuals (controls) after written informed consent. Participants were invited again at 6-9 months after infection to reexamine platelet function. Parameters of platelet aggregation were determined by light transmission aggregometry (LTA) in platelet-rich plasma (PRP). Expression of platelet activation markers were measured by Flow Cytometry (FACS). In addition, concentrations of S1P were determined by LC-MS/MS and MRP4-expression in platelets via Western blotting. Results Within 2-15 weeks after recovery, no alterations in platelet aggregation were observed after stimulation of PRP with various ADP (1, 2, and 10 µM) or collagen (1 and 2.5 µg/mL) concentrations. However, regarding FACS analysis, platelet surface expression of CD62P and PAC1 from convalescents prior activation by ADP (2 µM) and by CRP-XL (0.125 ng/mL) was significantly upregulated in comparison to samples from controls (p<0.05 for both). Intriguingly, S1P levels in platelet-poor plasma (PPP) from COVID-19 convalescents were significantly elevated compared to controls, i.e. 0.75±0.01 vs 0.65±0.02 nmol/mL, respectively (p<0.001). S1P in PRP and serum samples was not altered. Furthermore, platelet MRP4 expression was significantly elevated in COVID-19 convalescents (p<0.05). These differences in S1P and MRP4 levels were only present after 2-15 weeks of recovery, but not 6-9 months after infection. Conclusions While after a mild course of COVID-19 infection robust platelet functions such as aggregation determined by LTA were not affected, subtle differences in platelet activation markers occurred. Enhanced platelet MRP4 expression, which may correlate with the observed elevated S1P in PPP, are in agreement with a pro-inflammatory sensitization of platelets even after a mild COVID-19 infection. The implications of this observation concerning long-term effects of COVID-19 await further studies.

Shedding of soluble glycoprotein VI is neither affected by animal-derived antibeta-2-glycoprotein 1 antibodies nor IgG fractions from patients with systemic lupus erythematosus.

: Antibeta-2-glycoprotein 1 (antiβ2GP1) antibodies are associated with increased risk of thrombosis in patients with systemic lupus erythematosus (SLE). The specific effect(s) of antiβ2GP1 antibodies on platelets are unclear. Platelet activation in response to antiplatelet antibodies has been shown to induce shedding of the ectodomain of the platelet collagen receptor, glycoprotein VI (GPVI), releasing soluble GPVI (sGPVI). The aim of this study was to therefore determine whether antiβ2GP1 antibodies, and/or purified IgG fractions, from patients with SLE shed sGPVI from platelets. We determined sGPVI levels in platelet poor plasma from SLE patients with/without antiβ2GP1 antibodies (n = 37), as well as in platelet-rich plasma from healthy donors treated with either SLE-derived IgG fractions containing antiβ2GP1, animal-derived antiβ2GP1, or isotype control antibodies. Levels of sGPVI were higher in three SLE-derived platelet poor plasma with antiβ2GP1 antibodies (103.52 ± 12.32 ng/ml) compared with those without (28.11 ± 12.73 ng/ml). Neither SLE-derived IgG fractions containing antiβ2GP1 antibodies, nor animal-derived antiβ2GP1 antibodies induced significant shedding of sGPVI from healthy donor platelets compared with isotype controls. These results suggest that antiβ2GP1 antibodies do not affect shedding of sGPVI, and therefore collagen-mediated platelet signalling pathways. The shedding activity in SLE patients may be due to factors other than antiβ2GP1 antibodies, for example, metalloproteinases.

Abstract 12834: Increased Plasma Serotonin and Circulating microRNA-1914 Reflect Oxidative Stress and Play Crucial Roles in Atherosclerosis

Introduction: Serotonin, synthesized from enterochromaffin cells, modulates vascular tonus and thrombus formation, and mediates development of atherosclerotic plaque. Oxidative stress is involved in vascular inflammation and progression of atherosclerosis. Hypothesis: We hypothesized that plasma serotonin plays a crucial role in increasing oxidative stress and aimed to identify the circulating microRNA(s) associated with serotonin ratio for coronary artery disease (CAD). Methods: Blood was sampled from 120 outpatients with cardiovascular risk factors but without a history of previous cardiovascular events. Serotonin levels both in platelet poor plasma (PPP) and whole blood (WB) were measured with HPLC, and the ratio of serotonin in PPP to that in WB (PPP/WB) was used as an index of platelet activation. For assessment of oxidative stress, serum levels of derivative reactive oxygen metabolites (d-ROM) were measured. Total RNA was isolated from subjects who were scheduled for coronary angiography with suspicion of CAD and a microRNA array analysis was performed. The expression levels of microRNAs were scaled by global normalization. Results: Serotonin levels in both PPP and PPP/WB ratio were significantly correlated with the d-ROM levels. Among enrolled patients, 25 subjects were diagnosed as having CAD. The levels of the PPP/WB ratio of serotonin, d-ROM, and CRP were significantly higher in subjects with CAD than in those without CAD. Logistic regression analysis performed with endpoint of having CAD revealed that the PPP/WB ratio of serotonin was independently associated with CAD. In subjects with CAD, 5 increased microRNAs and 30 decreased microRNAs were identified among more than 2000 microRNAs as compared to those without CAD. Among candidate microRNAs, expression of microRNA-1914 showed significant inverse correlation with PPP/WB ratio (r=-0.61, P<0.05) and d-ROM levels (r=-0.63, P<0.05). Conclusions: A significant association among platelet activation assessed by PPP and PPP/WB ratio and oxidative stress assessed by d-ROM, was detected in individuals with cardiovascular risk factors. Targeting serotonin-oxidative stress axis, and circulating microRNA-1914 may serve as novel strategy for diagnosis of atherosclerotic CAD.

Serotonin in peripheral blood reflects oxidative stress and plays a crucial role in atherosclerosis: Novel insights toward holistic anti-atherothrombotic strategy

We enrolled 132 outpatients with cardiovascular risk factors to evaluate the serotonin levels in platelet-poor plasma (PPP) and whole blood (WB). PPP serotonin levels and PPP/WB serotonin ratio were significantly correlated with levels of oxidative stress measured by derivative reactive oxygen metabolites (d-ROM). Twenty-five subjects were revealed to have stable coronary artery disease (CAD), and the levels CRP, d-ROM, and PPP/WB serotonin ratio were significantly higher in subjects with CAD than in those without CAD. Logistic regression analysis performed with the endpoint of having CAD revealed that the PPP/WB serotonin ratio was independently associated with CAD (odds ratio 3.37, 95% confidence interval 1.04–10.9, P = 0.04). Receiver operating characteristic (ROC) curve analyses to discriminate subjects with CAD from those without CAD indicated that combining PPP/WB serotonin ratio and d-ROM improved diagnostic utility. Targeting the serotonin–oxidative stress axis as part of a holistic anti-atherothrombotic strategy could be beneficial for patients with atherosclerosis.

Increased Soluble GPVI Levels in Cirrhosis: Evidence for Collagen Induced Platelet Activation In Vivo

BackgroundCirrhosis is a consequence of prolonged inflammation arising from chronic liver disease of different aetiologies. It is characterised by tissue fibrosis, the deposition of collagen-rich extracellular matrix tissue within the liver. Glycoprotein VI (GPVI) is platelet-specific collagen receptor that is shed from the platelet surface in a metalloproteinase-dependent manner in response to GPVI ligation. The shed extracellular region of GPVI can be detected in plasma and used as a measure of GPVI-dependent platelet activation in vivo. Several lines of evidence suggest that GPVI-dependent platelet activation occurs in cirrhosis. Platelets have been shown to accumulate at sites of collagen-rich fibrotic tissue. Circulating levels of collagen are increased in cirrhosis. Collagen-induced platelet aggregation responses are reduced in vitro with cirrhosis. Based on these results, we hypothesised that soluble GPVI (sGPVI) levels are increased in patients with cirrhosis. As such, the aim of this study was to quantify sGPVI levels in patients with cirrhosis and compare to healthy controls.MethodsCompensated cirrhotic patients were recruited at the Mater Misericordiae University Hospital, Dublin, Ireland. The diagnosis of cirrhosis was based on clinical examination, blood tests, and radiological examination (nodular surface, larger right lobe, coarse echotexture). Exclusion criteria were decompensated cirrhosis, recent thrombotic events, and antiplatelet and/or anticoagulant therapies. Healthy controls were recruited at the Royal College of Surgeons in Ireland and Beaumont Hospital, Dublin, Ireland. Blood samples were collected into vacutainers containing 3.2 % sodium citrate as anticoagulant. sGPVI levels in platelet poor plasma were measured using an in house custom ELISA.Results57 patients with mixed aetiology cirrhosis and 55 healthy controls were recruited. In the patient group, 42% of patients had alcoholic liver disease (ALD), 30% had hepatitis C (HCV), 7% had non alcoholic fatty liver disease (NAFLD), 5% had Hepatitis B (HBV), 5% had autoimmune hepatitis (AIH), 5% had cryptogenic liver disease, 4% had hereditary haemochromatosis (HH), and 2% had primary biliary cholangitis (PBC). sGPVI levels were significantly increased in patients with cirrhosis (5.8 ± 0.6 ng/ml, n = 57) compared to healthy controls (3.2 ± 0.4 ng/ml, n = 55, p < 0.0001). There was no significant difference between sGPVI levels in AIH (4 ± 1 ng/ml, n = 3), ALD (5.6 ± 1 ng/ml, n = 24), cryptogenic (12 ± 5 ng/ml, n = 3), HBV (3.1 ± 1 ng/ml, n = 3), HCV (5 ± 0.6 ng/ml), or NAFLD (5.3 ± 1.1 ng/ml, n = 4). sGPVI levels did not correlate with platelet count (r = 0.12, p = 0.3) or parameters of liver cell function (albumin, bilirubin, prothrombin time, and liver stiffness measurements).ConclusionsGPVI levels are significantly increased in patients with mixed aetiology cirrhosis. This indicates collagen induced platelet activation is occurring in vivo and suggests the presence of an underlying coagulopathy in patients with cirrhosis. DisclosuresNí Áinle:Actelion Pharma: Research Funding; Leo Pharma: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

Blood serotonin levels in autism spectrum disorder: A systematic review and meta-analysis

Elevated blood serotonin (5-HT) levels were the first biomarker identified in autism research. Many studies have contrasted blood 5-HT levels in autistic patients and controls, but different measurement protocols, technologies, and biomaterials have been used through the years. We performed a systematic review and meta-analysis to provide an overall estimate of effect size and between-study heterogeneity, while verifying whether and to what extent different methodological approaches influence the strength of this association. Our literature search strategy identified 551 papers, from which 22 studies providing patient and control blood 5-HT values were selected for meta-analysis. Significantly higher 5-HT levels in autistic patients compared to controls were recorded both in whole blood (WB) [O.R.=4.6; (3.1–5.2); P=1.0×10−12], and in platelet-rich plasma (PRP) [O.R.=2.6 (1.8–3.9); P=2.7×10−7]. Predictably, studies measuring 5-HT levels in platelet-poor plasma (PPP) yielded no significant group difference [O.R.=0.54 (0.2–2–0); P=0.36]. Altogether, elevated 5-HT blood levels were recorded in 28.3% in WB and 22.5% in PRP samples of autistic individuals, as reported in 15 and 4 studies, respectively. Studies employing HPLC vs fluorometric assays yield similar cumulative effect sizes, but the former display much lower variability. In summary, despite some limitations mainly due to small study sample sizes, our results significantly reinforce the reliability of elevated 5-HT blood levels as a biomarker in ASD, providing practical indications potentially useful for its inclusion in multi-marker diagnostic panels for clinical use.

Decreased TSP-1 following percutaneous coronary intervention is associated with major adverse cardiac events in ST-elevation myocardial infarction

TSP-1 is a vasoconstrictive protein, which is released from both endothelium and cardiomyocytes during ischemia and promotes platelet aggregation and adhesion to subendothelial layers in atherosclerotic lesions. During myocardial ischemia and reperfusion, TSP-1 disturbs local microcirculation by disrupting both NO-signaling as well as VEGF-pathways by activation of CD47 and CD36. Furthermore, activation of TGF-ß might induce excessive fibrosis after infarction. It was assumed that TSP-1 is washed out after successful coronary reperfusion. In this study, we examined circulating TSP-1 post emergency PCI as a risk factor for major adverse cardiac events after STEMI with and without ventricular fibrillation. TSP-1 levels in platelet poor plasma were measured in 54 patients after ST-elevation myocardial infarction. Major adverse cardiac events were monitored for 426 days. Patients with decreased TSP levels after coronary stenting showed a significantly higher risk for MACE than patient with higher TSP levels (TSP-1[d0]: n = 46, no MACE = 16.38 ± 1.98 ug/mL vs. MACE 7.11 ± 1.54 ug/mL; p = 0.003). Kaplan-Meyer-analysis for MACE showed a better outcome above 10 ug/mL (p = 0.02). For MACE later than 3 months post-STEMI, the corresponding Kaplan-Meier-analysis yielded a p-value of 0.01. The number needed to diagnose for late MACE was 2.158. Low plasma levels of TSP1 after PCI are associated with MACE. Due to its procoagulant effects and dysregulation of microvascular tone, adequately powered prospective studies are warranted to test the impact of TSP-1 on cardiac microcirculation, endothelial function and remodeling. TSP-1 might serve as a new diagnostic and therapeutic approach in cardiovascular disease.

Daily variations of plasma serotonin levels in 2-year-old horses

Abstract Serotonin (5-HT) is a neurotransmitter implicated in the regulation of circadian rhythms. Synthesis and release of 5-HT are subjected to daily fluctuations, but there are no data on plasma serotonin levels in 2-year-old horses. Aim of this study was to investigate the daily variations in 5-HT levels in platelet-poor plasma from 2-year-old horses, so as to understand whether plasma 5-HT could be considered as an additional variable to assess the natural adaptability of horses to training. The research was carried out on 6 clinically healthy crossbred mares. Data showed higher levels of platelet-poor plasma 5-HT at 5 p.m. ( P P P

Correlation between platelet gelsolin levels and different types of coronary heart disease

Gelsolin is an important cytoskeletal protein of platelets and studies have shown a close relationship between gelsolin and cardiovascular disease. However, the role of gelsolin in the development of coronary heart disease (CHD) is unclear. In this study, we record the distribution of gelsolin in human platelets and plasma and its association with different types of CHD. This study included 114 cases, with 33 stable angina pectoris (SAP) cases, 81 acute coronary syndrome (ACS) cases—composed of 39 unstable angina pectoris (UAP) and 42 acute myocardial infarction (AMI) cases, and 31 healthy control participants. Gelsolin concentration in platelet rich plasma (PRP) and platelet poor plasma (PPP), actin filament (F-actin) and Gc-globulin of PPP were determined by enzyme-linked immunoadsorbent assay (ELISA). The fluorescence intensity of CD62p and cytoplasmic calcium ([Ca2+]i) in human platelets measured by flow cytometry. We also used turbidimetry to detect the platelet aggregation rate (PAR). We analyzed the correlation between platelet gelsolin concentration and CD62p or plasma F-actin levels among each different patient group. Compared with the control group, the gelsolin level in PRP of UAP and AMI groups increased significantly (P<0.01), while the gelsolin level in PPP of all the three patient groups decreased markedly (P<0.01), and the CD62p, PAR, [Ca2+]i of platelets, F-actin and Gc-globulin of the UAP and AMI groups increased significantly (P<0.01). Compared with the SAP group, the gelsolin level in PRP, the PAR, [Ca2+]i of platelets and CD62p of other two groups increased significantly (P<0.01), F-actin of the AMI group increased markedly (P<0.01). Platelet cytoskeleton protein dynamics vary among the different types of CHD. Platelet gelsolin levels are markedly increased and accompanied by increased platelet activity, F-actin and [Ca2+]i of ACS patients, while gelsolin levels in PPP are markedly lower. Abnormally increased platelet gelsolin levels show high positive correlation with the level of platelet activity. Therefore, platelet gelsolin might be a novel molecular marker and/or a new potential therapeutic target of anti-platelet therapy of ACS.

RANTES/CCL5 and Risk for Coronary Events: Results from the MONICA/KORA Augsburg Case-Cohort, Athero-Express and CARDIoGRAM Studies

BackgroundThe chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events.Methods and FindingsWe conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events (HR [95% CI] for increasing RANTES tertiles 1.0, 1.03 [0.75–1.42] and 1.11 [0.81–1.54]). None of six CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study (>22,000 cases, >60,000 controls), none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years).ConclusionsHigh RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii) CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies.

Correlation between platelet gelsolin level and different types of coronary heart disease

PurposeIn this study, we observe the distribution of gelsolin in human platelet and plasma and its association with different types of coronary heart disease (CHD).MethodsOne hundred and fourteen cases were...

Serotonin levels in platelet-poor plasma and whole blood in people with type 2 diabetes with chronic kidney disease

Aims Patients with diabetes mellitus (DM) are prone to atherosclerosis. Atherosclerosis activates platelets; activated platelets release serotonin, and therefore, evaluation of serotonin levels in blood could be a valuable biomarker for future risk of cardiovascular events. Methods Plasma serotonin levels obtained from patients with DM complicated with chronic kidney disease were measured using HPLC and were compared to serotonin levels of healthy control subjects. Patients with DM were classified into 2 subgroups of mildly (group 1) and moderately/severely (group 2) impaired renal function. Results Serotonin concentration in platelet-poor plasma for group 1 was significantly higher than that of healthy control subjects ( p < 0.01), and was significantly higher than that of patients from group 2 ( p < 0.05). The concentration of serotonin in whole blood for group 2 patients was significantly lower than that measured from healthy control subjects ( p < 0.01). The ratio of the plasma to whole blood level was significantly elevated in both groups 1 and 2 compared with healthy controls ( p < 0.01). Conclusions Our results indicate that platelets are activated to release serotonin into plasma in diabetic patients with mildly impaired renal function. When renal damage is advanced, platelets are over-activated to release serotonin.

Serotonin Levels in Platelet-Poor Plasma and Whole Blood from Healthy Subjects: Relationship with Lipid Markers and Coronary Heart Disease Risk Score

The serotonin level in platelet-poor plasma (PPP) is an important clinical marker for monitoring platelet activation accompanying vascular endothelial injury. We previously developed an HPLC method that combines column-switching with a post-column reaction to measure serotonin in PPP. This study determined serotonin levels in healthy volunteers and evaluated the possible relationship of serotonin levels with lipid markers and Framingham 10-year risk scores (FRS) for coronary heart disease. Serotonin was isolated from samples using our HPLC method and converted into a fluorescent derivative with benzylamine for specific and sensitive detection. Lipid fractionation was also performed by HPLC. FRS was calculated using the following: age, sex, LDL cholesterol, HDL cholesterol, blood pressure, and smoking and diabetes status. The PPP serotonin to whole blood serotonin ratio (PPP/WB) and the PPP serotonin level correlated significantly with HDL cholesterol levels: r= 0.187, p< 0.05 and r= 0.184, p< 0.05, respectively. The PPP/WB ratio also correlated significantly with FRS (r= 0.176, p< 0.05). The reference range (95% confidence interval) of PPP and WB serotonin levels in healthy subjects were 0.86-16.96 and 385-1319 nmol/L, respectively. Significant positive correlations between the serotonin PPP/WB ratio and FRS suggest that the PPP/WB ratio can serve as a biomarker for estimating atherosclerotic cardiovascular risk.

Improved Regulatory T Cell Activity in Patients with Chronic Immune Thrombocytopenia Purpura Treated with Thrombopoietic Agents.

Abstract 684Immune thrombocytopenic purpura (ITP) is an autoimmune, autoantibody-mediated disease with accelerated platelet destruction and impaired platelet production resulting in. thrombocytopenia and bleeding due to breakdown of immune tolerance. We and others have reported impaired regulatory CD4+CD25hi T cells (Treg) suppressive function in ITP patients. Clinical trials using thrombopoietic agents to stimulate platelet production have shown favorable outcomes in ITP patients with increased platelet counts and reduced bleeding. Two such agents, romiplostim (Nplate) and eltrombopag (Promacta), were recently licensed in the USA and elsewhere. Information on the immune responses of patients treated with these drugs pertaining to prognosis and response to treatment are lacking. We therefore studied the immunological profile of ITP patients with an average platelet counts of 21×109/L before treatment (n=6) and those who had been more than 3 months on treatment (total n=12, average platelet count 133 ×109/L) with either Nplate (n=5), eltrombopag (n=2) or an investigational thrombopoietic agent AKR-501 (n=5). We found no statistically significant differences in the Treg frequencies (Foxp3+CD25hi in the CD4+ population) of patients pre- and on-treatment (2.4± 0.6% versus 2.9± 0.5, p=0.5). However, the Treg activity as measured by suppression of proliferation of autologous CD4+CD25 cells at 1:1 and 1:4 ratios of Tregs : CD4+CD25 cells was significantly improved in patients on treatment compared to the pre-treatment group (at 1:1 ratio, 60% on-treatment versus 41% pre-treatment, p=0.001 and at 1:4 ratio, 44% versus 24%, p=0.04) and comparable to the overall Treg activity of controls (p=0.9). [Display omitted] Improved Treg function correlated with reduction in IL-2-producing Th cells (Pearsons r=0.6, p=0.01), and patients on treatment had a 30% reduced frequency single positive type 1 IFN-g-producing Th cells (p=0.04). Circulatory levels of pro-inflammatory sCD40L in platelet poor plasma (PPP) were decreased in patients on treatment versus pre-treatment (0.54 ± 0.05 ng/ml versus 0.89 ± 0.13ng/ml, p=0.02) and comparable to controls (p=0.9). TGF-b levels in PPP strongly correlated with improved platelet counts (Pearsons r=0.8, p<0.0001) and were increased in patients following treatment (2177 ± 258 ng/ml versus 1083 ± 127 ng/ml p=0.004). In summary, our data indicates that treatment with thrombopoietic agents results in improved immune regulation by Tregs which may be responsible for the observed dampening of the pro-inflammatory Th1-associated responses. The concomitant increase in TGF-b levels and decrease in sCD40L levels, both of which are platelet-derived, are consistent with a rise in platelet counts in patients on treatment. This raises the interesting possibility that platelets in patients on treatment may play a role in improving Treg function either directly through cell-cell interactions or indirectly by release of TGF-b. In conclusion, our findings suggest that thrombopoietic agents are not immunologically inert but rather have profound effects to restore immune tolerance. Disclosures:Bussel:Genzyme: Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai, Inc: Research Funding; Sysmex: Research Funding; Scienta: Speakers Bureau; Shionogi: Membership on an entity's Board of Directors or advisory committees.

Thrombin-generating capacity in patients with von Willebrand's disease

on Willebrand's disease (VWD) is the most common hereditary bleeding disorder. Its severity can be classified on the basis of von Willebrand factor (VWF) and factor VIII (FVIII) plasma levels and according to the clinical relevance of bleeding episodes. However, patients with very low VWF activity may exhibit a mild bleeding tendency. The basis for this heterogeneous clinical expression of the deficit is still poorly understood. We investigated the relationship between thrombin generation and levels of factor VIII, VWF and clinical bleeding tendency. Thrombin generation was measured in platelet-rich (PRP) and platelet-poor plasma (PPP) from 53 patients with VWD. We observed a statistically significant higher risk of bleeding in patients with a low thrombin peak in PRP (OR=14.5; 95% CI=5-41.3). Similar results were found in PPP (OR=8.71; 95% CI=3.4-22.3). Two parameters of the thrombin generation curve, peak height and thrombin generation speed (slope), correlated significantly with VWF:RCo and FVIII levels both in PPP and in PRP. Regression analysis showed that thrombin generation was mainly dependent on plasma FVIII activity. Our results suggest that the thrombin generation test, in combination with routine FVIII and VWF measurements, could be of interest in the assessment of the individual bleeding risk in patients with VWD.

Increased Serum Vascular Endothelial Growth Factor Following Major Surgical Injury

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. We evaluated the changes in serum levels of VEGF following major surgical trauma and postoperative inflammatory complications. The serum concentration of VEGF was measured with enzyme-linked immunosorbent assay in 41 patients with esophageal cancer who underwent right transthoracic esophagectomy with extensive lymphadenectomy and in 13 patients with gallstones who underwent less-invasive laparoscopic cholecystectomy for comparison. Serum and plasma samples were obtained before the operation and on postoperative days (PODs) 1, 7, 14, 21, and 28. The changes in serum VEGF levels were compared among groups categorized by age, sex, blood loss volume during operation, amount of transfusion, pathological stage of the tumor, and postoperative inflammatory complications. The correlation between serum VEGF levels and inflammatory factors, such as peripheral blood cell count, interleukin-6 (IL-6), C-reactive protein (CRP), and severity of postoperative inflammatory complications, was also investigated. Furthermore, because platelets are a potential source of serum VEGF, platelet-poor plasma (PPP) was prepared from plasma samples, and the VEGF concentration in PPP was measured to compare with those in sera. Serum VEGF levels increased significantly postoperatively. After reaching maximal levels on POD 14, VEGF levels gradually decreased until POD 28. The increase in the tranthoracic esophagectomy group was approximately twice that in laparoscopic cholecystectomy group on POD 14. Serum VEGF levels were not correlated with sex, age, blood loss, amount of transfusion, or tumor stage. However, serum VEGF levels were significantly higher in patients with postoperative inflammatory lung complications than in patients without such complications, and the maximal level of serum VEGF correlated with the severity of postoperative lung complications. However, there were no significant correlations between the increase in the level of serum VEGF and that of serum IL-6 or CRP. The increase of platelet counts in the peripheral blood correlated with that of the serum VEGF level, and VEGF levels in PPP were significantly lower than those in sera. Serum VEGF levels increased in the angiogenesis phase of wound healing following major surgical injury. Platelets are a potential source of increased serum VEGF levels, whereas inflammatory lung complications might also be related to increased serum VEGF levels.

Increased Plasma Transforming Growth Factor‐β1 in Migraine

Migraine is characterized by the peripheral and central sensitization of pain perceptive neural systems, and neurogenic inflammation is a key step in the development of migraine headache. We focused on transforming growth factor-beta1 (TGF-beta1), which is a multifunctional proinflammatory cytokine. To address the possibility of TGF-beta1 involvement in migraine, we investigated the plasma level of TGF-beta1 in patients with migraine headache during headache-free periods. Sixty-eight subjects with migraine participated: 23 with migraine with aura (MWA) and 45 without aura (MWoA). We recruited 58 healthy subjects without headache as controls. In addition, we examined 12 subjects with episodic tension-type headache. Platelet poor plasma (PPP) was obtained from subjects during headache free-periods. TGF-beta1 levels in PPP were determined by enzyme-linked immunosorbent assay. The TGF-beta1 level in PPP was 2.62*+/- 0.23 (mean +/- SE) ng/mL in migraine, 2.08 +/- 0.20 ng/mL in tension-type headache, and 1.80 +/- 0.09 ng/mL in controls (P= .007, ANOVA; *P < .01, post hoc tests vs. the controls). TGF-beta1 in PPP was significantly increased in patients with migraine during headache-free periods. TGF-beta1 may play some role in the development of migraine headache.

Low Platelet-Poor Plasma Levels of Serotonin in Adult Autistic Patients

Background: Hyperserotonemia has been reported in about a third of autistic patients. However, most studies have examined whole blood levels of serotonin (5-HT), the vast majority of which is found in platelets. The aim of this study was to determine 5-HT levels in platelet-poor plasma (PPP) in a group of adult patients with autism. Methods: Levels of PPP 5-HT were compared between 10 adult drug-free autistic patients and 12 healthy controls. The Ritvo-Freeman Real-Life Rating Scale and the Overt Aggression Scale (OAS) were administered to the autistic group as a measure of symptom severity. Results: Significantly lower PPP 5-HT levels were observed in the autistic group as compared to the controls (p = 0.03). In addition, PPP 5-HT levels were inversely correlated with OAS scores among subjects with autism (r = –0.64, p < 0.05). Conclusion: PPP 5-HT (‘free’) levels appear to be low in autistic patients and may play a role in the pathophysiology and symptomatology of the disorder.

High-performance liquid chromatographic method with column-switching and post-column reaction for determination of serotonin levels in platelet-poor plasma

Objectives: Measurement of serotonin concentrations in blood is considered to be important for the clinical studies of diseases involving vascular endothelial injury. We have developed a high-performance liquid chromatographic (HPLC) method for measurement of serotonin level in platelet-poor plasma (PPP). Methods: Venous blood samples were collected using vacuum tubes containing ethylenediaminetetraacetic acid (EDTA) dipotassium salt; the optimum concentration in blood was 7.4 mmol/l. Serotonin in samples was separated by HPLC with a column-switching system, and was specifically converted into a fluorescent derivative with benzylamine for convenient detection. Results: The between-day assay coefficient of variation for a serotonin level (5.1 nmol/l) in PPP was 6.2%. The mean concentration of serotonin in PPP in healthy subjects was 5.7 ± 3.0 nmol/l. The serotonin levels in PPP in patients with ischemic heart disease were significantly higher than those in healthy subjects ( P = 0.001). Conclusions: The developed method had sufficient performance to determine serotonin levels in PPP from patients with ischemic heart disease.