How can platelet proteomics best be used to interrogate disease?

Various modifications of proteins and the resulting proteoforms of a protein can associate with many diseases and are also significantly involved in the rapid regulation of hemostasis and thrombosis. For example, the release of prostacyclin from the intact endothelium and the consequent following phosphorylation of VASP in platelets is a post-translational regulation to keep them in a quiescent state. In Alzheimer's disease, proteoforms arise from the altered cleavage of the amyloid precursor protein, which finally causes amyloid plaques in the brain. This changed processing of the amyloid precursor protein can also be detected in platelets, making them an attractive source of biomarkers for this neurodegenerative disease. Age-related or prothrombotic disorders can have multiple origins, including genomic, transcriptional, and translational factors, which together can be mapped at the proteome level. Hence, recording these dynamic protein changes under physiological and pathophysiological conditions is paramount in platelet proteomics. To effectively study diseases through platelet proteomics, it is crucial to consider platelets' primary regulatory mechanism and thoroughly evaluate the disparities between the two leading proteomics technologies, top-down and bottom-up approaches. This commentary provides insights into the differences between these two technologies, which are particularly noticeable in detecting the different proteoforms of a protein.

What is the context?The repertoire of all proteins in a biological sample is the proteome. Proteomics refers to different biochemical technologies that detect and quantify the proteins in a biological sample, such as platelets. If proteome analyses are carried out on a representative number of samples from a specific patient group and compared to a matched control group, disease-dependent changes in proteins can be found that indicate unknown causes of the disease or also be used as biomarkers for diagnosis and prognosis. It is also essential to consider that the proteins in biological samples can occur in various variations, the proteoforms. These proteoforms of a protein can arise, for example, through genetically-based variations or regulatory post-translational protein modifications.What's new?There are two fundamentally different methods in proteomics technology: top-down and bottom-up. For bottom-up proteomics, the proteins must be digested into peptides for technical reasons, whereas top-down proteomics analyzes intact proteins. These different sample processing steps significantly impact the resulting data set. This is particularly crucial for platelet proteomics studies, as primary hemostasis is mainly carried out through post-translational modifications of proteins, resulting in various proteoforms that regulate platelet reactivity and thrombus formation.What's the impact?Bottom-up proteomics can quickly and automatically identify an extensive repertoire of proteins from a platelet sample. This is much more cumbersome with top-down proteomics. In contrast, here, various intact proteoforms of intact proteins can be unbiasedly detected and directly quantified, which is particularly important for examining the global proteome of platelets in clinical samples. This qualitative and quantitative relative assignment to the respective proteoforms of a protein is not possible in bottom-up proteomics.

Development of a new biochip array for APOE4 classification from plasma samples using immunoassay-based methods.

BACKGROUND: Apolipoprotein E (APOE) is a key player in lipid transport and metabolism and exists in three common isoforms: APOE2, APOE3 and APOE4. The presence of the E4 allelic variant is recognized as a major genetic risk factor for dementia and other chronic (neuro)degenerative diseases. The availability of a validated assay for rapid and reliable APOE4 classification is therefore advantageous. METHODS: Biochip array technology (BAT) was successfully applied to identify directly the APOE4 status from plasma within 3 h, through simultaneous immunoassay-based detection of both specific APOE4 and total APOE levels. RESULTS: Samples (n=432) were first genotyped by polymerase chain reaction (PCR), and thereafter, using BAT, the corresponding plasma was identified as null, heterozygous or homozygous for the E4 allele by calculating the ratio of APOE4 to total APOE protein. Two centers based in Austria and Ireland correctly classified 170 and 262 samples, respectively, and achieved 100% sensitivity and specificity. CONCLUSIONS: This chemiluminescent biochip-based sandwich immunoarray provides a novel platform to detect rapidly and accurately an individual's APOE4 status directly from plasma. The E4 genotype of individuals has been shown previously to affect presymptomatic risk, prognosis and treatment response for a variety of diseases, including Alzheimer's disease. The biochip's potential for being incorporated in quantitative protein biomarker arrays capable of analyzing disease stages makes it a superior alternative to PCR-based APOE genotyping and may deliver additional protein-specific information on a variety of diseases in the future.

Proteome analysis of testis from infertile protein C inhibitor-deficient mice reveals novel changes in serpin processing and prostaglandin metabolism.

Serine protease inhibitors (serpin) have therapeutic potential in a variety of pathogenic processes, ranging from thrombosis and altered immune response to liver cirrhosis. To investigate the physiological effects of protein C inhibitor (PCI, serpinA5), its gene was inactivated in a mouse model, resulting in male infertility. In the present report, 2D differential gel electrophoresis was utilized to investigate the molecular mechanisms for PCI involvement in male reproduction. Comparing the testes proteomes of three PCI-knockout mice with three wild types demonstrated similar patterns with the exception of a massive upregulation of prostaglandin reductase 1 (tenfold; p < 0.002) and the complete shifts in the molecular weights of serpinA1C and serpinA3K. All these PCI-dependent proteome changes were immunologically verified. Unbiased proteome analysis indicated that inactivation of serpinA5 strongly influenced both the protein species pattern of other A-clade serpins as well as prostaglandin metabolism in the testes.

A platelet protein biochip rapidly detects an Alzheimer's disease-specific phenotype.

Alzheimer's disease (AD), a multifactorial neurodegenerative condition caused by genetic and environmental factors, is diagnosed using neuropsychological tests and brain imaging; molecular diagnostics are not routinely applied. Studies have identified AD-specific cerebrospinal fluid (CSF) biomarkers but sample collection requires invasive lumbar puncture. To identify AD-modulated proteins in easily accessible blood platelets, which share biochemical signatures with neurons, we compared platelet lysates from 62 AD, 24 amnestic mild cognitive impairment (aMCI), 13 vascular dementia (VaD), and 12 Parkinson's disease (PD) patients with those of 112 matched controls by fluorescence two-dimensional differential gel electrophoresis in independent discovery and verification sets. The optimal sum score of four mass spectrometry (MS)-identified proteins yielded a sensitivity of 94 % and a specificity of 89 % (AUC = 0.969, 95 % CI = 0.944-0.994) to differentiate AD patients from healthy controls. To bridge the gap between bench and bedside, we developed a high-throughput multiplex protein biochip with great potential for routine AD screening. For convenience and speed of application, this array combines loading control-assisted protein quantification of monoamine oxidase B and tropomyosin 1 with protein-based genotyping for single nucleotide polymorphisms (SNPs) in the apolipoprotein E and glutathione S-transferase omega 1 genes. Based on minimally invasive blood drawing, this innovative protein biochip enables identification of AD patients with an accuracy of 92 % in a single analytical step in less than 4 h.

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

A proteomics study reveals a predominant change in MaoB expression in platelets of healthy volunteers after high protein meat diet: relationship to the methylation cycle.

Studies investigating the impact of high meat intake on cognition have yielded contradictory results as some show improved cognitive performance, whereas others report an increase of risk factors for dementia. However, few studies were designed to directly assess the effect of a high protein (HP) diet on both cognitive performance and corresponding biochemical parameters. A randomised intervention study was conducted with 23 healthy males (aged 19-31 years) to investigate the effects of a usual (UP) versus a HP diet on cognitive function and on the platelet proteome a well-established model for neurons. The study individuals were assigned to either a UP diet (15% energy) or a HP diet (30% energy) for 3 weeks with controlled intake of food and beverages. Blood samples were taken along with measurements of cognitive functions at the beginning and at the end of the intervention period. Among 908 reproducibly studied platelet proteins only the level of monoamine oxidase B (MaoB), a neurotransmitter degrading enzyme, decreased by 26% significantly (adjusted P value < 0.05) due to the HP diet. In addition, we found a correlation (r = 0.477; P < 0.02) between the decrease of MaoB expression and the shortened reaction time (cognitive function) which is in accordance with reports that dementia patients show increased MaoB activity. Plasma vitamin B(12) concentration was increased by the HP diet and correlates inversely with platelet MaoB expression (r = -0.35; P < 0.02). Healthy young males on a HP diet showed improved cognitive function and counteract well-known dementia biomarkers such as platelet MaoB and components of the methylation cycle such as vitamin B(12) and homocysteine.

Alterations of the Platelet Proteome in Lung Cancer: Accelerated F13A1 and ER Processing as New Actors in Hypercoagulability.

In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins calreticulin (CALR), endoplasmic reticulum chaperone BiP (HSPA5) and protein disulfide-isomerase (P4HB) were significantly elevated. Accelerated conversion of the fibrin stabilising factor XIII was detected in platelets of patients with lung cancer by elevated levels of a coagulation factor XIII (F13A1) 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma levels of the plasmin-α-2-antiplasmin complex and D-dimer suggests its enhanced degradation by the fibrinolytic system. Protein association network analysis showed that lung cancer-related proteins were involved in platelet degranulation and upregulated ER protein processing. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Platelet Phenotype Analysis of COVID-19 Patients Reveals Progressive Changes in the Activation of Integrin αIIbß3, F13A1, the SARS-CoV-2 Target EIF4A1 and Annexin A5.

Background: The fatal consequences of an infection with severe acute respiratory syndrome coronavirus 2 are not only caused by severe pneumonia, but also by thrombosis. Platelets are important regulators of thrombosis, but their involvement in the pathogenesis of COVID-19 is largely unknown. The aim of this study was to determine their functional and biochemical profile in patients with COVID-19 in dependence of mortality within 5-days after hospitalization. Methods: The COVID-19-related platelet phenotype was examined by analyzing their basal activation state via integrin αIIbß3 activation using flow cytometry and the proteome by unbiased two-dimensional differential in-gel fluorescence electrophoresis. In total we monitored 98 surviving and 12 non-surviving COVID-19 patients over 5 days of hospital stay and compared them to healthy controls (n = 12). Results: Over the observation period the level of basal αIIbß3 activation on platelets from non-surviving COVID-19 patients decreased compared to survivors. In line with this finding, proteomic analysis revealed a decrease in the total amount of integrin αIIb (ITGA2B), a subunit of αIIbß3, in COVID-19 patients compared to healthy controls; the decline was even more pronounced for the non-survivors. Consumption of the fibrin-stabilizing factor coagulation factor XIIIA (F13A1) was higher in platelets from COVID-19 patients and tended to be higher in non-survivors; plasma concentrations of the latter also differed significantly. Depending on COVID-19 disease status and mortality, increased amounts of annexin A5 (ANXA5), eukaryotic initiation factor 4A-I (EIF4A1), and transaldolase (TALDO1) were found in the platelet proteome and also correlated with the nasopharyngeal viral load. Dysregulation of these proteins may play a role for virus replication. ANXA5 has also been identified as an autoantigen of the antiphospholipid syndrome, which is common in COVID-19 patients. Finally, the levels of two different protein disulfide isomerases, P4HB and PDIA6, which support thrombosis, were increased in the platelets of COVID-19 patients. Conclusion: Platelets from COVID-19 patients showed significant changes in the activation phenotype, in the processing of the final coagulation factor F13A1 and the phospholipid-binding protein ANXA5 compared to healthy subjects. Additionally, these results demonstrate specific alterations in platelets during COVID-19, which are significantly linked to fatal outcome.

Identification of Non-HLA antigens targeted by alloreactive antibodies in patients undergoing chronic hemodialysis.

The only treatment of end-stage renal disease patients undergoing chronic dialysis is kidney transplantation. However, about half of graft recipients encounter organ loss within ten years after renal transplantation. There is emerging evidence that the presence of alloreactive antibodies against non-HLA antigens in the serum of the recipient prior transplantation is associated with higher incidence of chronic rejection. However, the molecular identity of these antigens is largely unknown. To determine the most common non-HLA antigens, we tested lymphocytic extracts from 20 healthy volunteers with sera of 28 patients on the transplantation waiting list by Western blotting. There was a group of five proteins that was recognized by most sera. Using patient's own lymphocytes revealed that autoimmunity plays a minor role in this recognition. Two-dimensional Western blotting experiments followed by mass spectrometry identified the antigens as tubulin beta chain, vimentin, lamin-B1, and Rho GDP-dissociation inhibitor 2. A detailed analysis of vimentin expression revealed that the antigenic 60 kDa isoform is underrepresented in patient's lymphocytes in comparison to those of healthy volunteers. The study revealed that preformed alloreactive antibodies are directed against a small number of specific protein isoforms. Our findings could provide a basis for future improvement of donor-recipient matching.

Altered platelet proteome in lupus anticoagulant (LA)-positive patients-protein disulfide isomerase and NETosis as new players in LA-related thrombosis.

Patients with antiphospholipid syndrome (APS) are at high risk of developing venous and arterial thromboembolism (TE). The role of platelets in the pathogenesis of these prothrombotic conditions is not yet fully understood. The aim of this study was to gain mechanistic insights into the role of platelets in APS by comparing the platelet proteome between lupus anticoagulant (LA)-positive patients with (LA+ TE+) and without a history of TE (LA+ TE-) and healthy controls. The platelet proteome of 47 patients with LA, 31 with a history of TE and 16 without thrombotic history, and 47 healthy controls was analyzed by two-dimensional differential in-gel electrophoresis and mass spectrometry to identify disease-related proteins. Afterward, selected LA-related platelet proteins were validated by western blot and ELISA. Alterations of 25 proteins were observed between the study groups. STRING pathway analysis showed that LA-related protein profiles were involved in platelet activation, aggregation, and degranulation. For example, protein disulfide isomerase family members, enzymes that promote thrombosis, were upregulated in platelets and plasma of LA+ TE+ patients. Leukocyte elastase inhibitor (SERPINB1), an antagonist of neutrophil extracellular trap (NET) formation, was decreased in platelets of LA+ TE+ patients compared to healthy controls. Additionally, citrullinated histone H3, a NET-specific marker, was increased in plasma of LA+ TE+ patients. These findings suggest that decreased platelet SERPINB1 levels favor prothrombotic NETosis, especially in LA+ TE+ patients. Our findings reveal protein abundance changes connected to altered platelet function in LA-positive patients, thus suggesting a pathogenic role of platelets in thrombotic complications in APS.