Higher Platelet Levels Research Articles

PKCθ-regulated signalling in health and disease.

Protein kinase Cθ (PKCθ) is a key enzyme in T-lymphocytes where it plays an important role in signal transduction downstream of the activated T-cell receptor (TCR) and the CD28 co-stimulatory receptor. Antigenic stimulation of T-cells triggers PKCθ translocation to the centre of the immunological synapse (IS) at the contact site between antigen-specific T-cells and antigen-presenting cells (APCs). The IS-residing PKCθ phosphorylates and activates effector molecules that transduce signals into distinct subcellular compartments and activate the transcription factors, nuclear factor κB (NF-κB), nuclear factor of activated T-cells (NFAT) and activating protein 1 (AP-1), which are essential for the induction of T-cell-mediated responses. Besides its major biological role in T-cells, PKCθ is expressed in several additional cell types and is involved in a variety of distinct physiological and pathological phenomena. For example, PKCθ is expressed at high levels in platelets where it regulates signal transduction from distinct surface receptors, and is required for optimal platelet activation and aggregation, as well as haemostasis. In addition, PKCθ is involved in physiological processes regulating insulin resistance and susceptibility to obesity, and is expressed at high levels in gastrointestinal stromal tumours (GISTs), although the functional importance of PKCθ in these processes and cell types is not fully clear. The present article briefly reviews selected topics relevant to the biological roles of PKCθ in health and disease.

Significance of Platelet and AFP Levels and Liver Function Parameters for HCC Size and Survival

Hepatocellular carcinoma (HCC) is a heterogeneous disease with both tumor and liver factors being involved. To investigate HCC clinical phenotypes and factors related to HCC size. Prospectively-collected HCC patients' data from a large Italian database were arranged according to the maximum tumor diameter (MTD) and divided into tumor size terciles, which were then compared in terms of several common clinical parameters and patients' survival. An higer MTD tercile was significantly associated with increased blood alpha-fetoprotein (AFP), gamma-glutamyl transpeptidase (GGTP), and platelet levels. Patients with higher platelet levels had larger tumors and higher GGTP levels, with lower bilirubin levels. However, patients with the highest AFP levels had larger tumors and higher bilirubin levels, reflecting an aggressive biology. AFP correlation analysis revealed the existence of 2 different groups of patients: those with higher and with lower AFP levels, each with different patient and tumor characteristics. The Cox proportional-hazard model showed that a higher risk of death was correlated with GGTP and bilirubin levels, tumor size and number, and portal vein thrombosis (PVT), but not with AFP or platelet levels. An increased tumor size was associated with increased blood platelet counts, AFP and GGTP levels. Platelet and AFP levels were important indicators of tumor size, but not of survival.

Predictive Value of Proteinuria in Adult Dengue Severity

BackgroundDengue is an important viral infection with different presentations. Predicting disease severity is important in triaging patients requiring hospital care. We aim to study the value of proteinuria in predicting the development of dengue hemorrhagic fever (DHF), utility of urine dipstick test as a rapid prognostic tool.Methodology and principal findingsAdult patients with undifferentiated fever (n = 293) were prospectively enrolled at the Infectious Disease Research Clinic at Tan Tock Seng Hospital, Singapore from January to August 2012. Dengue infection was confirmed in 168 (57%) by dengue RT-PCR or NS1 antigen detection. Dengue cases had median fever duration of 6 days at enrolment. DHF was diagnosed in 34 cases according to the WHO 1997 guideline. Dengue fever (DF) patients were predominantly younger and were mostly seen in the outpatient setting with higher platelet level. Compared to DF, DHF cases had significantly higher peak urine protein creatinine ratio (UPCR) during clinical course (26 vs. 40 mg/mmol; p<0.001). We obtained a UPCR cut-off value of 29 mg/mmol based on maximum AUC in ROC curves of peak UPCR for DF versus DHF, corresponding to 76% sensitivity and 60% specificity. Multivariate analysis with other readily available clinical and laboratory variables increased the AUC to 0.91 with 92% sensitivity and 80% specificity. Neither urine dipstick at initial presentation nor peak urine dipstick value during the entire illness was able to discriminate between DF and DHF.ConclusionsProteinuria measured by a laboratory-based UPCR test may be sensitive and specific in prognosticating adult dengue patients.

Clinical Characteristics of Kawasaki Syndrome and the Risk Factors for Coronary Artery Lesions in China

Kawasaki syndrome (KS) is the leading cause of acquired heart disease in childhood in developed countries. This study was designed to identify the clinical characteristics of a large cohort of KS in China and explore the risk factors for coronary artery lesions. Clinical records of 1370 patients with acute KS were retrospectively reviewed. The clinical features of different age groups were analyzed, and a multivariate logistic regression analysis was performed to identify the risk factors for coronary artery lesions caused by KS. The prevalence of redness at a Bacille Calmette-Guèrin inoculation site was greatest in infants younger than 6 months (18.4%), whereas cervical lymphadenopathy was more frequent in patients older than 60 months (61.5%). Age, sex, therapeutic time, intravenous immunoglobulin dose, platelet count and erythrocyte sedimentation rate were risk factors for coronary artery lesions (P < 0.05). A total fever duration >10 days was a risk factor for coronary artery aneurysms in patients with coronary artery lesions (P < 0.05). KS occurs more frequently in children younger than 5 years, in boys and during the summer months. Redness at a Bacille Calmette-Guèrin inoculation site signals the diagnosis of incomplete KS in infants and young children. Male gender, younger age, intravenous immunoglobulin dose, delayed administration (>10 days), high platelet level and elevated erythrocyte sedimentation rate are predictive for coronary artery lesions, and total fever duration (>10 days) is predictive for coronary artery aneurysms in patients with coronary artery lesions.

Could mean platelet volume among complete blood count parameters be a surrogate marker of metabolic syndrome in pre-pubertal children?

Interest in childhood metabolic syndrome (MetS) has increased substantially due to the increasing prevalence of childhood obesity on a global scale. Early recognition of MetS is critical in order to delay the development of cardiovascular disease (CVD). In this study, we evaluated the relationship between complete blood count (CBC) parameters and MetS among pre-pubertal children which may provide evidence in support of using low cost, readily available clinical haematological parameters for the detection of MetS. A retrospective analysis was carried out on 330 (125 lean vs. 205 overweight) Turkish pre-pubertal children who attend to a paediatric outpatient clinic. Age, gender, puberty, body mass index, CBC parameters, cardiometabolic risk factors including lipid profiles, high sensitive serum reactive protein (hsCRP) and insulin resistance index calculated by homeostasis model assessment (HOMA-IR) were evaluated and compared among lean, overweight children and children with MetS. The mean age of the study population was 7.4 ± 1.9 years. In both gender, the mean values of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were significantly lower and red blood cell (RBC), platelet (PLT) counts were significantly higher in overweight children. Overall, 8.4% (n = 28) of patients met the criteria of MetS. Children with MetS had higher levels of PLT and lower levels of mean platelet volume (MPV). Of all the haematological parameters analysed, PLT was positively, whereas MPV was negatively correlated with MetS in girls. In addition, MPV was inversely correlated with fasting blood glucose, HOMA-IR, low density lipoprotein-cholesterol (LDL-C) and low density lipoprotein-cholesterol/high density lipoprotein-cholesterol (LDL-C/HDL-C) ratio in girls after adjusting for confounding factors. The risk analyses of MetS in terms of MPV quartiles showed that the adjusted OR (95% CI) for the lowest vs. the highest quartile was 7.71 (1.45–40.89) in girls. These data might suggest that MPV could be another feature of MetS in pre-pubertal girls and might be used as a surrogate marker for MetS in clinical settings.

Hepatocellular carcinoma diameter and blood platelet levels in a large cohort of Chinese patients in Taiwan.

e15157 Background: Cirrhosis is often associated with thrombocytopenia and predisposes to HCC. Platelets are known to produce several HCC growth factors. Methods: A cohort of 4,139 Chinese HCC patients from Taiwan was interrogated for baseline clinical, tumor and liver function characteristics in relation to blood platelet counts. Results: Trichotomization of the cohort by tumor size after log-linearization, revealed significant relationships of tumor size to blood platelet, AFP and GGTP levels. Patients with higher platelets had larger tumors, more tumor nodules and higher AFP and GGTP levels than those with lower platelet levels. 80% of patients with larger tumors had normal platelet counts, compared to 50% of patients with smaller tumors. Analysis of the cohort according to platelet sub-sets, revealed significant relationships of platelet counts to tumor size and AFP levels. By Spearman’s correlation coefficient, platelet counts correlated moderately with tumor size (rho=0.411), but weakly with tumor numbers, PVT, AST or AFP levels. Sub-set analysis of the cohort according to platelet levels showed that 38% of patients had thrombocytopenia and they had the smallest tumors, lowest tumor numbers, lowest percent with PVT and lowest AFP levels. Conclusions: There was a significant correlation between HCC size and blood platelet counts; patients with larger tumors had higher platelet levels.

Microparticles in atrial fibrillation: A link between cell activation or apoptosis, tissue remodelling and thrombogenicity

Microparticles (MPs) are small membrane vesicles that are shed from virtually all cells in response to stress. Widely described in atherothrombotic diseases, recent data suggest a role for circulating MPs in the hypercoagulable state associated with supraventricular tachyarrhythmia. During atrial fibrillation, several mechanisms, such as high ventricular heart rate, low or oscillatory shear stress, stretch, hypoxia, inflammation and oxidative stress, are potent inducers of apoptotic cell death, which leads to the shedding of procoagulant MPs within the vasculature. As key regulators of cell-cell cross-talk and important mediators of inflammatory, thrombogenic and proteolytic pathways, MPs directly or indirectly contribute to the amplification loops involved in atrial fibrillation. Because high levels of platelets and endothelial-derived MPs are identified during stroke and are associated with infarct size and clinical outcome, they are proposed to be a potent marker of ischaemic risk. During pulmonary vein isolation, the additional increases of platelet and leukocyte MP levels suggest the extent of tissue damage and reflect a transient activation of the coagulation cascade that could favour ischaemic stroke. Conversely, the observed decreases of several apoptotic markers some months after the restoration of sinus rhythm suggest that the extent of apoptotic processes is reversible and might enable restoration of haemostasis. In this review, we will summarise the current evidence supporting the roles of apoptosis and cell activation in the development of the prothrombotic state observed in atrial fibrillation, with a particular focus on procoagulant MPs.

Phenotypic Categorization and Profiles of Small and Large Hepatocellular Carcinomas

We used a database of 4139 Taiwanese HCC patients to take a new approach (Network Phenotyping Strategy) to HCC subset identification. Individual parameters for liver function tests, complete blood count, portal vein thrombosis, AFP levels and clinical demographics of age, gender, hepatitis or alcohol consumption, were considered within the whole context of complete relationships, being networked with all other parameter levels in the entire cohort. We identified 4 multi-parameter patterns for one tumor phenotype of patients and a separate 5 multi-parameter patterns to characterize another tumor phenotype of patterns. The 2 subgroups were quite different in their clinical profiles. The means of the tumor mass distributions in these phenotype subgroups were significantly different, one being associated with larger (L) and the other with smaller (S) tumor masses. These significant differences were seen systematically throughout the tumor mass distributions. Essential and common clinical components of L-phenotype patterns included simultaneously high blood levels of AFP and platelets plus presence of portal vein thrombosis. S included higher levels of liver inflammatory parameters. The 2 different parameter patterns of L and S subgroups suggest different mechanisms; L, possibly involving tumor-driven processes and S more associated with liver inflammatory processes.

The distribution of circulating microRNA and their relation to coronary disease

Background: MicroRNAs (miRNAs) are small RNAs that regulate gene expression by suppressing protein translation and may influence RNA expression. MicroRNAs are detected in extracellular locations such as plasma; however, the extent of miRNA expression in plasma its relation to cardiovascular disease is not clear and many clinical studies have utilized array-based platforms with poor reproducibility. Methods and Results: Initially, to define distribution of miRNA in human blood; whole blood, platelets, mononuclear cells, plasma, and serum from 5 normal individuals were screened for 852 miRNAs using high-throughput micro-fluidic quantitative RT-PCR (qRT-PCR). In total; 609, 448, 658, 147, and 178 miRNAs were found to be expressed in moderate to high levels in whole blood, platelets, mononuclear cells, plasma, and serum, respectively, with some miRNAs uniquely expressed. To determine the cardiovascular relevance of blood miRNA expression, plasma miRNA (n=852) levels were measured in 83 patients presenting for cardiac catheterization. Eight plasma miRNAs were found to have over 2-fold increased expression in patients with significant coronary disease (≥70% stenosis) as compared to those with minimal coronary disease (less than 70% stenosis) or normal coronary arteries. Expression of miR-494, miR-490-3p, and miR-769-3p were found to have significantly different levels of expression. Using a multivariable regression model including cardiovascular risk factors and medications, hsa-miR-769-3p was found to be significantly correlated with the presence of significant coronary atherosclerosis. Conclusions: This study utilized a superior high-throughput qRT-PCR based method and found that miRNAs are found to be widely expressed in human blood with differences expressed between cellular and extracellular fractions. Importantly, specific miRNAs from circulating plasma are associated with the presence of significant coronary disease.

Strategies for reducing the incidence of skin complications in newborns treated with whole-body hypothermia

Objective: To present the results of a strategy designed to reduce the incidence of skin complications in newborns with hypoxic–ischemic encephalopathy treated with moderate whole-body hypothermia. Design: Retrospective study. Setting: Neonatal Intensive Care Unit (NICU). Patients: Thirty-nine neonates cooled in the considered period. Intervention: Starting from January 2008, for neonates treated with moderate whole-body hypothermia (33.5 °C), the cooling system was set in “automatic servo-controlled mode (ACM)”, where the temperature of the circulating water could vary between 4 °C and 42 °C. Starting from January 2009, cooling blankets were used in another type of automatic mode, the “gradient variable mode (GVM)”, where the circulating water was maintained at a specific pre-set gradient towards the patient’s body temperature, and a specific nursing protocol (NP) was adopted. Measurements and main results: Two of the eleven newborns treated with the “ACM” exhibited skin complications compatible with subcutaneous fat necrosis (SFN). None of the twenty-eight newborns treated with the “GVM” exhibited skin complications. A comparison of the biochemical and hematological data between these two groups revealed that newborns treated after the adopting of a NP and the “GVM” showed lower serum protein C and calcium levels, and higher platelet levels. Conclusions: Our data suggest that newborns undergoing therapeutic cooling may benefit from a specific NP and correct cooling unit setting. Should further studies confirm our data, this nursing approach could be easily adopted.

Comparison of the Laboratory Data Between Kawasaki Disease and Enterovirus After Intravenous Immunoglobulin Treatment

Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. The laboratory findings before and after intravenous immunoglobulin (IVIG) in KD have been discussed, but the characteristics of IVIG therapy still are unclear. This study aimed to compare laboratory data from patients with KD and enterovirus (EV) infection to evaluate the differences after IVIG therapy. The study enrolled 171 KD patients and 38 EV patients treated with a single dose of IVIG from 2003 to 2010. Laboratory data including total white blood cell counts (WBC) and hemoglobin (Hb), platelet, segment, lymphocyte, eosinophil, and monocyte levels were analyzed. Compared with the KD patients, the EV patients had higher Hb, lymphocyte, and monocyte levels and lower eosinophil levels before IVIG treatment (p < 0.05). After IVIG treatment, the KD patients had lower Hb and segment levels but higher platelet, lymphocyte, and eosinophil levels than the EV patients (p < 0.05). In the KD patients, the platelet, eosinophil, and monocyte levels increased after IVIG treatment, whereas Hb, WBC, and segment levels decreased significantly (p < 0.001). In the EV patients, eosinophil levels increased after IVIG treatment, whereas WBC and Hb levels decreased significantly (p < 0.05). The study results provide evidence that eosinophilia may be related to IVIG therapy in KD and EV patients. The KD patients had higher eosinophil levels both before and after IVIG therapy than the EV patients, which may have been due to the inflammatory mechanism of KD. The KD patients had higher platelet levels than the EV patients, suggesting that platelets are involved in the inflammatory response to KD.

Donor-Graft Compatibility Tests in Pig-to-Primate Xenotransplantation Model: Serum Versus Plasma in Real-Time Cell Analyzer Trials

Introduction Various strategies have been designed to assess in vitro donor-graft compatibility in pig-to-primate xenotransplantation models. Most of them are based on a cytolysis assessment by exposing donor tissue to host serum with investigations by flow cytometry, and photocolorimetric levels. The aim of this study was to analyze the difference in cytolysis produced by sera and plasma obtained using various anticoagulants, or containing high versus low levels of platelets. Methods The cytolysis trials were performed using an xCELLigence real-time cell analyzer (RTCA) in a cell model involving transgenic pig fibroblasts exposed to sera (S) or plasma obtained using EDTA, Li-heparin, or Na-heparin in combination with plasma containing high versus low content of platelets. Samples were obtained from two baboons and five volunteer human donors. Evolution of fibroblast cell growth was assessed by RTCA as the cell index (CI). After 9 hours of growth, cells were exposed to 20 μL of each sample. The minimum CI (CImin), time to CImin (TCImin), and time to reach the CI observed before compound addition (Trec) were recorded for each microwell. Results The lowest CImin, highest TCImin, and Trec observed for EDTA plasma showed significant differences from other samples ( P < .001). Discussion On the basis of this study, using the RTCA assay, heparinized plasma produced complement inhibition and with undervaluation of the cytolysis reaction. EDTA plasma produced total death of most of cultures. The most accurate sample matrix seems to be serum.

Membrane array–based differential profiling of platelets during storage for 52 miRNAs associated with apoptosis

Enucleated platelets (PLTs) utilize posttranscriptional gene (mRNA) regulation (PTGR) for their normal morphologic and physiologic functions, which are altered in their ex vivo storage, also collectively referred to as storage lesions. While cellular micro-RNAs (miRNAs) play a significant role in posttranscriptional gene (mRNA) regulation by binding to their target mRNAs, comprehensive analysis of apoptosis-associated miRNAs and global changes in their profiles during PLT storage have not been evaluated to date. In this report room temperature-stored PLTs of Days 0, 2, and 9 were analyzed by differential profiling for 52 apoptosis-associated human miRNAs. After total RNA extraction from the samples, a membrane array-based miRNA analysis was carried out. Prediction of target genes was performed by bioinformatics-based approaches. Our analysis revealed that during storage, Let-7a, -7c, -7e, -7f, -7g, and -7i miRNA profiles of the PLTs were barely detectable, while levels of miR-150, -151, -152, -184, -188, -196a, -197, and -202 remained at high levels in PLTs. The rest of the miRNA levels were in between. However, two miRNAs, Let-7b and miR-16, distinctly demonstrated an increasing trend while miR-7 and miR-145 showed a decreasing profile during PLT storage. For these four miRNAs, we also identified their potential target mRNAs. Overall, these results confirm the fact that miRNAs do exist in PLTs, and among 52 apoptosis-specific miRNAs studied, only a few selected miRNAs did perturb during PLT storage. Future experimental evaluation of these miRNA-target mRNA interactions will provide new insights into the molecular mechanisms of PLT storage-associated lesions.

MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort

AbstractActivating mutations of MPL exon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET), but their prevalence and clinical significance are unclear. Here we demonstrate that MPL mutations outside exon 10 are uncommon in platelet cDNA and identify 4 different exon 10 mutations in granulocyte DNA from a retrospective cohort of 200 patients with ET or IMF. Allele-specific polymerase chain reaction was then used to genotype 776 samples from patients with ET entered into the PT-1 studies. MPL mutations were identified in 8.5% of JAK2 V617F− patients and a single V617F+ patient. Patients carrying the W515K allele had a significantly higher allele burden than did those with the W515L allele, suggesting a functional difference between the 2 variants. Compared with V617F+ ET patients, those with MPL mutations displayed lower hemoglobin and higher platelet levels at diagnosis, higher serum erythropoietin levels, endogenous megakaryocytic but not erythroid colony growth, and reduced bone marrow erythroid and overall cellularity. Compared with V617F− patients, those with MPL mutations were older with reduced bone marrow cellularity but could not be identified as a discrete clinicopathologic subgroup. MPL mutations lacked prognostic significance with respect to thrombosis, major hemorrhage, myelofibrotic transformation or survival.

Platelet-released supernatants enhance hematopoietic stem cell proliferation in vitro

Peripheral blood stem cell transplantation (PBSCT) is used to reconstitute normal hematopoiesis after myeloablative chemotherapy. The hematopoietic stem cells are collected from the blood by apheresis machines using density gradient centrifugation. Because of density similarities the grafts contain high levels of leukocytes and platelets that release various mediators into the grafts. The collected hematopoietic stem cells are therefore exposed to platelet released mediators including platelet-derived growth factor, transforming growth factor-β, vascular endothelial growth factor and platelet factor-4. To investigate whether platelet activation and secretion can affect hematopoietic stem cells during PBSCT, we cultured (i) normal cord blood stem cells and (ii) mobilized peripheral blood stem cells from autografts together with the total secretion product of thrombin activated platelets (i.e. platelet released supernatants). Platelet released supernatants enhanced the cell proliferation of both peripheral blood stem cell (PBSC) autograft and normal cord blood CD34+ cells. Our study shows that platelet secretion in PBSCT autografts affect the hematopoietic stem cell function and possibly thereby the hematopoietic reconstitution after PBSCT.

Effects of Feeding Periods of High Cholesterol and Saturated Fat Diet on Blood Biochemistry and Hydroxyproline Fractions in Rabbits

Hypercholesterolemia and hypertriglyceridemia are considered as important risk factors during the atherosclerotic process. The aim of the present investigation was to study the total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), high density lipoprotein (HDL), triglyceride (TG), platelet levels and hydroxyproline fractions during the pathogenesis of atherosclerosis. For this purpose, twenty five 12-weeks, New Zealand white male rabbits, were purchased, individually caged, and divided into either control group or cholesterol-fed group. The control group (n = 10) was fed 100 g/day of normal diet, ORC-4 (Oriental Yeast Co. Ltd., Tokyo, Japan) for a period of 15 weeks. The cholesterol-fed group (n = 15) was fed a high cholesterol and saturated fat diet of ORC-4 containing 1% cholesterol plus 1% olive oil (100 g/day) for periods of 5 (group 1), 10 (group 2) and 15 (group 3) weeks. Blood sample from each animal was taken at the end of the experimental period for the biochemical analysis. The results of the present study showed that TC, LDLC, TG, HDLC and platelets were significantly (P < 0.01) increased in cholesterol-fed rabbits as compared with control rabbits. The serum hydroxyproline (Hyp) in rabbits belonging to group 1 showed no significant alteration when compared to control group. Group 2 rabbits showed a significant increase of 103% (P < 0.01) and 100% (P < 0.001) in free and protein-bound hydroxyproline fractions respectively when compared to control rabbits. However, there was no significant change in peptide-bound and total serum hydroxyproline levels as compared to the control group (P > 0.05). There was no significant (P > 0.05) decrease of free serum hydroxyproline in group 3 rabbits when compared to control rabbits. On the other hand, group 3 rabbits showed a significant increase in peptide-bound and protein-bound Hyp by 517% (P < 0.05) and 100% (P < 0.01) respectively when compared to control rabbits. However, total serum Hyp in group 3 rabbits showed no significant (P > 0.05) change when compared to control rabbits. These results suggest that feeding rabbits high cholesterol and saturated fat diet for feeding periods of 5, 10 and 15 weeks induced significant change in TC, LDLC, HDL, TG, platelet levels and various Hyp fractions in serum without any significant change in the total Hyp content.

Clinical Significance of MPL Mutations in Essential Thrombocythemia: Analysis of the PT-1 Cohort.

Activating mutations in MPL, the thrombopoietin receptor, were originally described in idiopathic myelofibrosis (IMF) and subsequently in a small minority of patients with essential thrombocythemia (ET). The prevalence and clinical significance of MPL mutations in ET has not been fully characterised. Using prospectively acquired diagnostic and follow-up data from the large MRC PT-1 study, we describe the clinical characteristics, complications and laboratory features of these mutations. Sequencing the entire MPL cDNA from 19 JAK2 wild-type ET patients did not reveal any mutations outside exon 10. Sequencing MPL exon 10 DNA in 88 ET and 112 IMF patients revealed MPLW515L (n=11), MPLW515K (n=3) and MPLS505N (n=2) mutations. The MPLS505N mutation, previously associated with inherited thrombocythemia, was clearly an acquired mutation in both patients (1 ET and 1 IMF). Sensitive PCR-based methods were then developed for all three mutant MPL alleles and used to genotype 776 patients with ET from the PT-1 trial. MPL mutations were detected in 4% of all patients, comprising 9% of JAK2 wild-type patients. MPL mutant patients were older at diagnosis than both JAK2 wild-type (p=0.0001) and JAK2V617F patients (p=0.09). Compared to JAK2V617F patients, MPL mutant patients had lower hemoglobin and higher platelet levels at diagnosis (p=0.0001 and p=0.006 respectively). There were no differences in diagnostic blood counts between MPL mutant and JAK2 wild-type patients. Diagnostic trephine histology for JAK2V617F (n=168), JAK2 wild-type (n=130) and MPL mutant patients (n=13) was reviewed by three independent histopathologists blinded to mutation status. MPL mutant samples were less cellular than JAK2V617F and JAK2 wild-type samples (p=0.0001 and p=0.003 respectively). Compared to both JAK2V617F and JAK2 wild-type samples, MPL mutant samples had lower erythroid cellularity (p=0.0006 and p=0.005 respectively) and granulocyte cellularity (p=0.006 and p=0.05 respectively). There were no differences in megakaryocyte cellularity or reticulin grade between the three groups. MPL mutant patients had an increased risk of venous thrombosis after trial entry when compared to JAK2 wild-type patients (p=0.02); the rate of venous thrombosis in the MPL mutant group was comparable to the JAK2V617F group. The mutant allele burden (assessed quantitatively by pyrosequencing) was significantly higher in patients with MPLW515K compared to patients with MPLW515L mutations (p=0.0001), suggesting biological differences in the transforming abilities of the two mutations. In colony assays using patient mononuclear cells the MPLW515L mutation was associated with cytokine-independent growth of megakaryocyte colonies but not with erythropoietin-independent erythroid colony growth (4/4 patients). Of 48 MPL mutant patients, only one also carried the JAK2V617F mutation by allele-specific PCR. Taken together, our results describe the prevalence, clinical features and laboratory manifestations of MPL mutations in the context of a large prospective study of ET.

Influence of platelets on the healing of dry eye of patients treated with topical eye drops of autologous Platelet‐Rich Plasma (PRP)

Abstract Purpose: To study the influence of high levels of platelets on patients suffering from dry eye and treated successfully with autologous PRP. Methods: Platelet enrichment on autologous PRP of 16 patients (age 65±14) suffering from moderate to severe dry eye was evaluated and different dry eye parameters (symptoms, BCVA, fluorescein staining, impression cytology) were correlated with this value. 7 patients had lower that 1.3 fold platelet enrichment on their autologous PRP (low platelets (PLTs) group) and 9 had more that 1.75 fold platelet enrichment (high PLTs group). Results: On the low PLTs group (7 patients), 50% of them showed a significant improvement of symptoms, 45% showed more than 1 line of BCVA improvement and 35% showed full disappearance of fluorescein staining. On the high PLTs group (9 patients), 78% of patient showed a significant improvement of symptoms, out of 10 patients evaluated for BCVA, 50% of them gained more that 2 lines of vision and 71% experienced a full disappearance of fluorescein staining. Conclusions: Autologous PRP has been shown to be very effective on dry eye treatment. On this study we show that high levels of platelets present on this biological product play an important role for the healing of dry eye. Other components of autologous PRP such as growth factors and vitamins should also help for improving these patients symptoms and main clinical signs.

Can serotonin transporter genotype predict serotonergic function, chronicity, and severity of drinking?

Serotonin transporter (5-HTT) activity is greater in carriers of the long (L) vs. short (S) alleles of the 5-HTT-linked polymorphic region (5′-HTTLPR) among healthy control subjects but not alcohol-dependent adults. In 198 alcoholics, we determined the relationship between current or lifetime drinking and platelet 5-HTT function and density among allelic variants of the 5′-HTTLPR. SS subjects were younger than L-carriers (LL and LS) ( p < 0.0085) and had fewer years of lifetime drinking. For L-carriers, the mean of B max for paroxetine binding, but not V max for serotonin (5-HT) uptake, was lower than that for SS subjects ( p < 0.05). More L-carriers than their SS counterparts had V max for 5-HT uptake below 200 nmol/10 7 platelets-min ( p < 0.05) and B max for paroxetine binding below 600 nmol/mg protein ( p < 0.06). Current drinking (drinks per day during the past 14 days) correlated positively with K m and V max of platelet 5-HT uptake ( p < 0.05) and negatively with B max, but not K d, of paroxetine binding ( p < 0.05) for L-carriers alone. Years of lifetime drinking correlated negatively with K m and V max of platelet 5-HT uptake ( p < 0.05) and B max, but not K d, of paroxetine binding ( p < 0.05) for L-carriers alone. Among L-carriers alone, there were higher levels of platelet 5-HT uptake and lower levels of platelet paroxetine binding with increased drinking, and more lifetime drinking was associated with modestly lower levels of 5-HT uptake and paroxetine binding. Thus, 5-HTT expression varies with current and lifetime drinking in L-carriers alone.

Quantification of platelet composition in experimental venous thrombosis by real-time polymerase chain reaction

Introduction Platelets play a key role in thrombus formation. Determination of the platelet component in a thrombus provides pathophysiological insights to the thrombotic event and aids in selecting an appropriate therapeutic intervention. In this study a sensitive and reliable method to characterize the cellular components of experimental thrombi was developed using real-time polymerase chain reaction (PCR). Methods and results Vena cava thrombosis was induced by either oxidative injury to topical FeCl 2 (FeCl 2-VT) or stenosis-limited blood flow and a hypotonic pressure stress (stasis-VT) in rats. High levels of platelets were identified in the thrombus containing vessels by real-time PCR analysis of target gene amplification using the 2 − ΔΔCT values by normalizing the data with gene expression in naive vessels and with a housekeeping gene, ribosomal protein L32. By this analysis, the levels of PF-4 (as a platelet marker) mRNA were significantly higher in FeCl 2-VT (2 − ΔΔCT = 7.8) than in stasis-VT (2 − ΔΔCT = 4.2, p < 0.05). Enhanced platelet enrichment in FeCl 2-VT was also confirmed qualitatively by scanning electronic microscopic analysis. In addition, real-time PCR using a panel of genes representing vascular injury, inflammation and thrombosis showed marked induction (2 − ΔΔCT > 5) in MCP-1, IL-1β, iNOS and P-selectin mRNA expression in both models. Conclusions These data demonstrate the utility of real-time PCR to quantitate platelets and other cell components in vascular thrombosis, which may facilitate the characterization and thus therapeutic intervention of a particular thrombotic event in both preclinical animal models and clinical conditions.