Analysis Of Platelet Proteome Research Articles

Variations in Blood Platelet Proteome and Transcriptome Revealed Altered Expression of Transgelin-2 in Acute Coronary Syndrome Patients.

Proteomic analyses based on mass spectrometry provide a powerful tool for the simultaneous identification of proteins and their signatures. Disorders detection at the molecular level delivers an immense impact for a better understanding of the pathogenesis and etiology of various diseases. Acute coronary syndrome (ACS) refers to a group of heart diseases generally associated with rupture of an atherosclerotic plaque and partial or complete thrombotic obstruction of the blood flow in the infarct-related coronary artery. The essential role in the pathogenesis of ACS is related to the abnormal, pathological activation of blood platelets. The multifactorial and complex character of ACS indicates the need to explain the molecular mechanisms responsible for thrombosis. In our study, we performed screening and comparative analysis of platelet proteome from ACS patients and healthy donors. Two-dimensional fluorescence difference gel electrophoresis and nanoscale liquid chromatography coupled to tandem mass spectrometry showed altered expressions of six proteins (i.e., vinculin, transgelin-2, fibrinogen β and γ chains, apolipoprotein a1, and tubulin β), with the overlapping increased expression at the mRNA level for transgelin-2. Dysregulation in protein expression identified in our study may be associated with an increased risk of thrombotic events, correlated with a higher aggregability of blood platelets and induced shape change, thus explaining the phenomenon of the hyperreactivity of blood platelets in ACS.

Platelet-Derived Drug Targets and Biomarkers of Ischemic Stroke-The First Dynamic Human LC-MS Proteomic Study.

(1) Objective: The aim of this dynamic LC-MS (liquid chromatography and mass spectrometry) human platelet proteomic study was to identify the potential proteins candidates for biomarkers of acute ischemic stroke (AIS), their changes during the acute phase of stroke and to define potential novel drug targets. (2) Methods: A total of 32 patients (18–80 years old) were investigated that presented symptoms of AIS lasting less than 24 h from the onset, confirmed by neurological examination and/or new cerebral ischemia visualized in the CT (computed-tomography) scans. The analysis of platelet proteome was performed using LC-MS at baseline, and then on the third and seventh day from the onset of symptoms. The control group was demographically matched without any clinical signs of acute brain injury. (3) Results: The differences between platelets, at 24 h after first symptoms of stroke subjects and the control group included: β-amyloid A4 and amyloid-like protein 2, coactosin-like protein, thymidine phosphorylase 4 (TYMP-4), interferon regulatory factor 7 (IRF7), vitamin K-dependent protein S, histone proteins (H2A type 1 and 1-A, H2A types 2B and J, H2Av, -z, and -x), and platelet basic protein. The dynamic changes in the platelet protein concentration involved thrombospondin-1, thrombospondin-2, filamin A, B, and C. (4) Conclusions: This is the first human dynamic LC-MS proteomic study that differentiates platelet proteome in the acute phase of ischemic stroke in time series and compares the results with healthy controls. Identified proteins may be considered as future markers of ischemic stroke or therapeutic drug targets. Thymidine phosphorylase 4 (TYMP-4) holds promise as an interesting drug target in the management or prevention of ischemic stroke.

The Integrin Activating Protein Kindlin-3 Is Cleaved in Human Platelets during ST-Elevation Myocardial Infarction.

Kindlins are important proteins for integrin signaling and regulation of the cytoskeleton, but we know little about their precise function and regulation in platelets during acute ischemic events. In this work, we investigated kindlin-3 protein levels in platelets isolated from patients with ST-elevation myocardial infarction (STEMI) compared to patients with non-ischemic chest pain. Platelets from twelve patients with STEMI and twelve patients with non-ischemic chest pain were isolated and analyzed for kindlin-3 protein levels and intracellular localization by immunoblotting and two-dimensional gel electrophoresis. Platelet proteome analysis by two-dimensional gel electrophoresis and protein sequencing identified kindlin-3 as a protein that is cleaved in platelets from patients with myocardial infarction. Kindlin-3 full-length protein was significantly decreased in patients with STEMI compared to patients with non-ischemic chest pain (1.0 ± 0.2 versus 0.28 ± 0.2, p < 0.05) by immunoblotting. Kindlin-3 showed a differential distribution and was primarily cleaved in the cytosolic and membrane compartment of platelets in myocardial infarction. Platelet activation with thrombin alone did not affect kindlin-3 protein levels. The present study demonstrates that kindlin-3 protein levels become significantly reduced in platelets of patients with myocardial infarction compared to controls. The results suggest that kindlin-3 cleavage in platelets is associated with the ischemic event of myocardial infarction.

Platelet proteome reveals specific proteins associated with platelet activation and the hypercoagulable state in \u03b2-thalassmia/HbE patients

A hypercoagulable state leading to a high risk of a thrombotic event is one of the most common complications observed in β-thalassemia/HbE disease, particularly in patients who have undergone a splenectomy. However, the hypercoagulable state, as well as the molecular mechanism of this aspect of the pathogenesis of β-thalassemia/HbE, remains poorly understood. To address this issue, fifteen non-splenectomized β-thalassemia/HbE patients, 8 splenectomized β-thalassemia/HbE patients and 20 healthy volunteers were recruited to this study. Platelet activation and hypercoagulable parameters including levels of CD62P and prothrombin fragment 1 + 2 were analyzed by flow cytometry and ELISA, respectively. A proteomic analysis was conducted to compare the platelet proteome between patients and normal subjects, and the results were validated by western blot analysis. The β-thalassemia/HbE patients showed significantly higher levels of CD62P and prothrombin fragment 1 + 2 than normal subjects. The levels of platelet activation and hypercoagulation found in patients were strongly associated with splenectomy status. The platelet proteome analysis revealed 19 differential spots which were identified to be 19 platelet proteins, which included 10 cytoskeleton proteins, thrombin generation related proteins, and antioxidant enzymes. Our findings highlight markers of coagulation activation and molecular pathways known to be associated with the pathogenesis of platelet activation, the hypercoagulable state, and consequently with the thrombosis observed in β-thalassemia/HbE patients.

Platelet Carbonic Anhydrase II, a Forgotten Enzyme, May Be Responsible for Aspirin Resistance.

Background Thromboembolic events constitute a major health problem, despite the steadily expanding arsenal of antiplatelet drugs. Hence, there is still a need to optimize the antiplatelet therapy. Objectives The aim of our study was to verify a hypothesis that there are no differences in platelet proteome between two groups of healthy people representing different acetylsalicylic acid (aspirin) responses as assessed by the liquid chromatography/mass spectrometry (LC/MS) technique. Patients/Methods A total of 61 healthy volunteers were recruited for the study. Physical examination and blood collection were followed by platelet-rich plasma aggregation assays and platelet separation for proteomic LC/MS analysis. Arachidonic acid- (AA-) induced aggregation (in the presence of aspirin) allowed to divide study participants into two groups aspirin-resistant (AR) and aspirin-sensitive (AS) ones. Subsequently, platelet proteome was compared in groups using the LC/MS analysis. Results The LC/MS analysis of platelet proteome between groups revealed that out of all identified proteins, the only discriminatory protein, affecting aspirin responsiveness, is platelet carbonic anhydrase II (CA II). Conclusions CA II is a platelet function modulator and should be taken into consideration as a cardiovascular event risk factor or therapeutic target.

Platelet Proteome Analysis Reveals Integrin-dependent Aggregation Defects in Patients with Myelodysplastic Syndromes

Bleeding complications are a significant clinical problem in patients with myelodysplastic syndromes even at sufficient platelet counts (>50,000/μl). However, the underlying pathology of this hemorrhagic diathesis is still unknown. Here, we analyzed the platelet proteome of patients with myelodysplastic syndromes by quantitative two-dimensional difference gel electrophoresis followed by mass spectrometric protein identification. Proteins identified with lower concentrations, such as Talin-1, Vinculin, Myosin-9, Filmain-A, and Actin play critical roles in integrin αIIbβ3 signaling and thus platelet aggregation. Despite normal agonist receptor expression, calcium flux, and granule release upon activation, the activation capacity of integrin αIIbβ3 was diminished in myelodysplastic syndrome platelets. Förster resonance energy transfer analysis showed a reduced co-localization of Talin-1 to the integrin's β3-subunit, which is required for receptor activation and fibrinogen binding. In addition, platelet spreading on immobilized fibrinogen was incomplete, and platelet aggregation assays confirmed a general defect in integrin-dependent platelet aggregation in patients with myelodysplastic syndromes. Our data provide novel aspects on the molecular pathology of impaired platelet function in myelodysplastic syndromes and suggest a mechanism of defective integrin αIIbβ3 signaling that may contribute to the hemorrhagic diathesis observed in these patients.

Comparative platelet proteome analysis reveals an increase of monoamine oxidase-B protein expression in Alzheimer's disease but not in non-demented Parkinson's disease patients

Monoamine oxidase-B (Mao-B) catalysing the breakdown of the neurotransmitter dopamine, is known to be involved in the pathophysiology of Parkinson's (PD) and Alzheimer's disease (AD). Increased brain Mao-B activity is associated with AD. This alteration can also be seen in platelets, albeit the cause has hitherto remained elusive. To gain a deeper understanding of the etiology of AD, the platelet proteome was characterised, (2D DIGE pH6-9, including Mao-B) from 150 individuals: 34 AD, 13 vascular dementia, 15 non-demented PD patients, 49 matched controls, 18 oldest old and 21 young individuals. One significant change was noted after applying false discovery rate with the upregulation of the Mao-B expression (30% adjusted P value<0.001; effect size 1.31) in AD compared to age- and sex-matched controls. In contrast, Mao-B levels were unchanged in PD to matched controls. Western blot and mRNA analyses verified these findings. Moreover, Mao-B concentration correlated with age in the cognitive healthy individuals (r=0.53; P<0.001) and PD patients but not in those suffering from AD (r=-0.03; P=0.874). Mao-B activity correlated with the increased Mao-B protein expression in AD (r=0.81; P=0.016). We suggest that Mao-B platelet protein level may serve as a biomarker for age-related dementia, especially AD.

Proteomics in the Study of Qualitative Platelet Defects: Validation of the Approach in the Gray Platelet Syndrome and Quebec Platelet Disorder.

Qualitative platelet defects are a heterogeneous group of disorders characterized by abnormal platelet function. Currently available laboratory assays lack sensitivity and specificity for detecting Storage Pool and Platelet Secretion defects. The Gray Platelet Syndrome (GPS) is an autosomal recessive disorder characterized by a decrease or absence of alpha granule contents. The Quebec Platelet Disorder (QPD) results from proteolysis of alpha granule proteins due to increased amounts of platelet urokinase. A decrease or deficiency of multimerin is characteristic. Proteomics is a rapidly developing field with great promise in the study of pathophysiology and biomarker development. Recent advances have made measurement of relative protein abundance in multiple samples feasible. We used one such approach, “isotope Tagging for Relative and Absolute Quantitation” (iTRAQ) to characterize the platelet proteome of healthy volunteers and patients with GPS and QPD. iTRAQ allows analysis of up to 4 different samples simultaneously, and differences in protein abundance as low as 20% are reliably detected. The platelet proteomes of 4 healthy volunteers were analyzed. Platelet proteome analysis of study patients was as follows;a.2 GPS patients compared to 2 controls;b.2 heterozygous GPS patients compared to a homozygote and a control; andc.3 QPD patients compared to a control.For comparison, platelet proteins were grouped by location into 3 groups - receptor, alpha granule and cytoskeletal proteins. The results are shown in the figure. Protein abundance in healthy volunteers was very consistent, with inter-individual variability ≤ 20%. Distribution of receptor and cytoskeletal proteins in GPS patients mirrored the controls, but alpha granule proteins were significantly decreased as expected. Comparison of homozygous to heterozygous GPS platelets showed this to be true only in the homozygotes, again, as expected (figure inset). Variable decreases in alpha granule proteins were observed in QPD patients but this was less profound. Multimerin was detected in QPD patients, but at levels significantly lower than controls. While urokinase was not detected in QPD platelets in this study, it was detected in a parallel study evaluating platelet releasates. The iTRAQ technique was reproducible and yielded consistent results, but currently is a relatively expensive and time-consuming approach. In summary, our results suggest that the platelet proteome is tightly regulated and very stable in healthy individuals. A significant decrease in alpha granule proteins was noted in patients with GPS but this was less remarkable in the QPD group, probably because peptides resulting from urokinase-mediated proteolysis are still labeled and quantified. [Display omitted]

Evaluation of Critical Genetic Variations in Idiopathic Recurrent Miscarriages Among South Indian Women- A Genomic and Proteomic Approach

Genomic and proteomic analysis of females with idiopathic recurrent early pregnancy loss (REPL) to find the unknown etiological factors involved. A case-control based study approach. Well-defined idiopathic REPL subjects (n=145) as well as control females (n=84) were included for the genotyping studies. For studying platelet proteome profiles, 25 cases and 10 controls were included. Polymorphisms in detoxification genes (NAT1, NAT2, CYP1A1, CYP2D6, SULT1A1, CYP17, CYP19, aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor), vasoregulatory genes (eNOS, VEGF, anandamide hydrolase) and genes involved in blood homeostasis (Prothrombin, Factor V Leiden) were analyzed and their significance was estimated using a two tailed Fisher’s P-value at 95% significance level. Haplotype analysis of the unphased data using expectation-maximization was done wherever necessary. Platelet proteome analysis was carried out after isolation and solublization of platelets. Two dimensional gel electrophoresis using 3-10 range IPG strips and 12% SDS-PAGE was used for the separation of the proteins. Polymorphisms in CYP1A1 (C4887A and T6235C) showed significant association with REPL as evidenced by logistic regression and haplotype analysis. Novel variants were observed in CYP2D6, anandamide hydrolase, eNOS and were found to be associated with REPL. Apart from these results, present study revealed the lack of association between REPL with other variants pressing the need to consider the ethnic background in epidemiological studies. Proteome analysis of platelets revealed a differential expression pattern in three proteins with approximate molecular masses of 65kDa, 35kDa and 20kDa which were exclusively present in REPL females. Present study revealed certain novel and already reported polymorphisms in a multitude of genes to be associated with the risk of REPL. A role of genetic factor in REPL is further strengthened by the proteome analysis of platelets.

Towards complete analysis of the platelet proteome.

Platelets exert a crucial function in haemostasis, wound repair, and the formation of vascular plugs, underlying thrombotic diseases such as stroke and myocardial infarction. Analysis of platelet biochemistry is largely dependent on protein analysis as platelets are anucleated cells providing little analytical target for DNA or RNA based strategies. Here we present data from our analysis of the human platelet proteome, the entire set of proteins building a platelet at a given point in time. Proteins were separated by two-dimensional electrophoresis (2-DE) using broad and narrow range pH gradients in the isoelectric focusing step. Consequently, a high-resolution 2-DE proteome map has been generated that comprises approximately 2300 different protein features. From the 536 protein features detected in the 4-5 pI range 284 features were identified by electrospray ionisation time of flight tandem mass spectrometry. These 284 proteins originate from 123 different open reading frames. This includes the five human proteins KIAA0193, KIAA0573, KIAA0830, WUGSC:H_DJ0777O23 protein, and cytokine receptor related protein 4, all isolated for the first time. The data are discussed with regard to proteome characteristics, protein function, and the high prevalence of signalling molecules. This study contributes to a more thorough and holistic understanding of platelet biology, helping to build the basis for future identification of new drug targets and therapeutic strategies.