Platelet Protein Content Research Articles

Platelet proteomic profiling in sitosterolemia suggests thrombocytopenia is driven by lipid disorder and not platelet aberrations

AbstractSitosterolemia is a rare autosomal recessive genetic disorder in which patients develop hypercholesterolemia and may exhibit abnormal hematologic and/or liver test results. In this disease, dysfunction of either ABCG5 or ABCG8 results in the intestinal hyperabsorption of all sterols, including cholesterol and, more specifically, plant sterols or xenosterols, as well as in the impaired ability to excrete xenosterols into the bile. It remains unknown how and why some patients develop hematologic abnormalities. Only a few unrelated patients with hematologic abnormalities at the time of diagnosis have been reported. Here, we report on 2 unrelated pedigrees who were believed to have chronic immune thrombocytopenia as their most prominent feature. Both consanguineous families showed recessive gene variants in ABCG5, which were associated with the disease by in silico protein structure analysis and clinical segregation. Hepatosplenomegaly was absent. Thrombopoietin levels and megakaryocyte numbers in the bone marrow were normal. Metabolic analysis confirmed the presence of strongly elevated plasma levels of xenosterols. Potential platelet proteomic aberrations were longitudinally assessed following dietary restrictions combined with administration of the sterol absorption inhibitor ezetimibe. No significant effects on platelet protein content before and after the onset of treatment were demonstrated. Although we cannot exclude that lipotoxicity has a direct and platelet-specific impact in patients with sitosterolemia, our data suggest that thrombocytopenia is neither caused by a lack of megakaryocytes nor driven by proteomic aberrations in the platelets themselves.

Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

Enrichment of Complement, Immunoglobulins, and Autoantibody Targets in the Proteome of Platelets from Patients with Systemic Lupus Erythematosus.

Background Systemic lupus erythematosus (SLE) is a complex disease characterized by autoimmunity toward apoptotic cells, excessive amounts of circulating immune complexes, and complement activation. A decreased platelet size has been observed in SLE and their nonhemostatic functions may play an active role in the disease. The main objective of this study was to find clues that could explain their decreased size and functional role, analyzing the entire platelet proteome. Methods Platelets were isolated from 23 patients with SLE. The five individuals with the highest and lowest average platelet forward scatter were selected for further analysis. Platelet protein content was analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and compared with platelets from five healthy controls. Data are available via ProteomeXchange with identifier PXD031202. Results Out of 2,572 proteins identified, 396 had significantly different levels (ANOVA q -value ≤ 0.01). Forty proteins, including immunoglobulin-, complement- and phosphatidylserine-binding proteins had higher abundance in platelets from SLE patients, largely independent of size (fold difference of ≥1.5 and a t -test p -value of ≤0.05 as cut-off). Functional characterization revealed increased degranulation and skewed hemostatic balance in platelets from SLE patients. In the SLE proteome, immunoglobulin proteins were negatively correlated to serum complement C3 and C4 and the highest relative levels were detected in platelets of normal size. Conclusion Platelets from SLE patients shared a specific protein profile, including immunoglobulins, complement proteins, and autoantigens, largely independent of the platelet size and in agreement with an integrated role for platelets in SLE.

Platelets in Healthy and Disease States: From Biomarkers Discovery to Drug Targets Identification by Proteomics.

Platelets are a heterogeneous small anucleate blood cell population with a central role both in physiological haemostasis and in pathological states, spanning from thrombosis to inflammation, and cancer. Recent advances in proteomic studies provided additional important information concerning the platelet biology and the response of platelets to several pathophysiological pathways. Platelets circulate systemically and can be easily isolated from human samples, making proteomic application very interesting for characterizing the complexity of platelet functions in health and disease as well as for identifying and quantifying potential platelet proteins as biomarkers and novel antiplatelet therapeutic targets. To date, the highly dynamic protein content of platelets has been studied in resting and activated platelets, and several subproteomes have been characterized including platelet-derived microparticles, platelet granules, platelet releasates, platelet membrane proteins, and specific platelet post-translational modifications. In this review, a critical overview is provided on principal platelet proteomic studies focused on platelet biology from signaling to granules content, platelet proteome changes in several diseases, and the impact of drugs on platelet functions. Moreover, recent advances in quantitative platelet proteomics are discussed, emphasizing the importance of targeted quantification methods for more precise, robust and accurate quantification of selected proteins, which might be used as biomarkers for disease diagnosis, prognosis and therapy, and their strong clinical impact in the near future.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.

Platelet-protein-profiling of healthy controls and patients with early- and late-stage colorectal cancers

Aims: Early tumor detection and intervention are important determinants of survival in patients with cancer. During early tumor growth, platelets actively and selectively sequester proteins and, as such, the platelet protein content can be used as a marker of early tumor growth. We therefore aimed at identifying differenzially expressed proteins of platelets that distinguish healthy controls from patients with early- and late-stage colorectal cancer. Such markers might serve for innovative early detection, prognosis and therapeutic intervention.

Variations in platelet protein associated with arterial thrombosis

Introduction Hyperactivity of platelets has been associated with thrombotic episodes by molecular mechanisms not yet elucidated. The present work aimed at identifying whether the platelet protein content from patients who had suffered an arterial thrombosis episode differed from that of platelets obtained from normal healthy donors. Methods Differential platelet protein profiles were determined by 2-dimensional (2-D) gel electrophoresis and Western blot analysis of total platelet lysates. Identification of differentially expressed proteins was carried out by mass spectrometry (MALDI–TOF). Results We found a decreased platelet content of three protein spots in patients of arterial thrombosis: integrin linked kinase (ILK), fructose bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) whereas the content of four other protein spots was increased: actin binding protein, coronine like (p57), non-muscle myosin heavy chain (NMMHC-A), pyruvate kinase M2 isoenzyme (PK) and phosphoglycerate kinase (PGK). The variations in ILK, GAPDH and PK were validated by Western blot analysis. The proteins showing a decreased platelet content in arterial thrombosis patients are associated with the cytoskeletal insoluble fraction and the detected increase in some proteins seems to be due to the generation of peptides caused by a limited proteolysis. Differences in the protein profiles of circulating platelets from arterial thrombosis were maintained months after the acute thrombotic event and disappear in the long term. Conclusions The observed variations in some platelet proteins suggest the existence of a perturbation in the cytoskeletal organization and increased proteolysis, both indicative of a platelet pro-active state, persistent after the thrombotic event.

Funktionelle Proteomanalyse humaner Thrombozyten

Platelets are anucleated cells and therefore ideal research objects for modern proteome analyses. Despite their importance in thrombosis and hemostasis the protein content of platelets is still poorly characterized in major parts. In preparation for bioinformatic and functional studies a series of proteomic analyses was conducted for platelet subproteomes as well as for posttranslational modifications. Thereby, the identification of 489 proteins, over 550 phosphorylations and 326 N-glycosylation sites was possible, which were not identified in previous proteome studies of platelets. Those results represent new research possibilities for functional characterization of platelet proteins as well as their modifications.

Proteomic Analysis of Gray Platelet Syndrome by iTRAQ Labelling and Mass Spectroscopy: A Potential New Diagnostic Strategy for Platelet Disorders.

The recent introduction of novel proteomic techniques to evaluate normal human platelet proteins and their modifications following activation may also help to elucidate the causes and improve diagnoses of platelet-related bleeding disorders. Previous studies from this and other laboratories have demonstrated the sensitivity and reliability of this new technique to detect minor changes (+/−20%) in the protein content of platelets. More than 600 proteins shared by normal platelets were identified, and >200 in platelet-derived microparticles (MPs) generated by ionophore or Thrombin Receptor Activating Peptide (TRAP) activation. As a prelude to our ultimate goal of establishing this new technology, we applied the iTRAQ method (multiplexed relative protein quantitation by mass spectrometry) in the study of two unrelated patients with the Gray Platelet Syndrome (GPS).Megakaryocytes of patients with GPS can synthesize alpha-granule proteins and enclose them within membranes. However, the membranes lack structure linked latency, and enclosed proteins leak out of the organelles before platelets are delivered to circulating blood. Thus, GPS platelets contain empty alpha-granule vacuoles instead of alpha-granules. Due to large changes in platelet properties, it was surprising to find that cytoskeletal proteins of GPS were present in similar abundance to normal platelets (i.e. in the range of 0.80–1.20). Receptor proteins showed a similar distribution. As expected, the content of most alpha-granule proteins in the platelets from the 2 GPS patients was markedly reduced, with individual protein abundance ratios of 0.10 to 0.52 compared to normal platelets. However, certain trans-membrane proteins of alpha-granules (such as vesicle-associated proteins and P-selectin) were better preserved in GPS platelets, with ratios in the range of 0.73 to 1.16 compared to normal platelets. These results showed that many structural components (such as cytoskeleton and receptor proteins) are unchanged in GPS platelets. The results support our previous data indicating that detection of a change of as little as 20% may indicate a pathological change in the platelet proteome that may impact normal function. Our results therefore demonstrate that highly sensitive methods, capable of detecting small protein changes, may be needed to fully appreciate pathological disorders. The iTRAQ technique is able to detect low level changes and may be a leading tool that can generate new insights into the molecular pathophysiology and genetic variations of platelet disorders.

The relationship between human megakaryocyte nuclear DNA content and gene expression

Increased platelet reactivity has been implicated in various vascular diseases. Since the protein content of platelets is determined mainly by the megakaryocyte, alterations in megakaryocyte mRNA expression may influence platelet protein content and therefore activity. In order to determine whether DNA content (ploidy) of a megakaryocyte influences its mRNA expression, we have developed a method to investigate the relationship between megakaryocyte ploidy and gene expression. By measuring the ploidy and mRNA expression in individual cells, we have shown that in the physiological state there is an increase in mRNA expression for beta-actin, glycoprotein IIb and neuropeptide Y with increase in ploidy and that this increase levels off at high ploidy values. This may have relevance in the understanding of platelet reactivity in pathological events.

Alteration of human platelet protein kinase C with normal aging

Using a two-site enzyme immunoassay, we determined the content of type II protein kinase C (PKC) in human platelets from 24 donors of various ages without any neurological, hematological or malignant disorders. The content of type II PKC in the membranous fraction was positively correlated with aging. The content of PKC in the cytosolic fraction tended to decline with aging, but the correlation was not significant. The total amount of PKC also had no significant correlation with aging. Age-related changes in platelet protein content were not observed. These results suggested that the subcellular distribution of type II PKC in human platelets is altered with normal aging.

The effect of hypercholesterolemia on guinea pig platelets, erythrocytes and megakaryocytes

This study has examined the effect of diet-induced hypercholesterolemia on guinea pig platelets, erythrocytes, megakaryocytes and plasma. The cholesterol/phospholipid ratios of plasma and erythrocytes began to increase after one day on the diet and increased steadily for two weeks and more slowly thereafter until 30 days. In contrast, the cholesterol/phospholipid ratio of platelets remained constant for 4–5 days, then increased until reaching a maximum of about 0.85 in two weeks. Thus, the time-course for increase of the cholesterol/phospholipid ratio is different for platelets than for erythrocytes and plasma. The increase in the cholesterol/phospholipid ratio of megakaryocytes was small and not dependent on the degree of increase in the plasma cholesterol/phospholipid ratio. The cholesterol esters of both platelets and megakaryocytes increased with time for two weeks. The increase in megakaryocyte cholesterol esters appeared to precede that of platelets. The protein content of platelets and megakaryocytes and average megakaryocyte size were increased. Normal platelets incubated in plasma from hypercholesterolemic guinea pigs did not accumulate excess cholesterol, but erythrocyte cholesterol increased 45% in 6 h under the same conditions. Cholesterol synthesis in megakaryocytes was depressed 50–80% by cholesterol feeding and by in vitro incubation of the cells in hypercholesterolemic plasma. The data suggest that the platelets and erythrocytes may accumulate excess cholesterol by different mechanisms. The effects of cholesterol feeding on megakaryocytes and the lag in accumulation of cholesterol in platelets relative to erythrocytes and plasma suggest that a defect in the megakaryocyte may be a primary determinant of accumulation of cholesterol in platelets.

The Amount of Platelet-Bound Albumin Parallels the Amount of IgG on Washed Platelets From Patients With Immune Thrombocytopenia

The biologic relevance of the increased platelet-associated IgG (PAIgG) on platelets from patients with idiopathic thrombocytopenic purpura (ITP) is unclear. Platelets from ITP patients are often larger than normal, and it is possible that the increased IgG is not specific but passively related to platelet size. The measurement of platelet-bound albumin could provide information concerning the specificity of the platelet-bound IgG, since albumin, like IgG, is a plasma protein, but unlike IgG, is not an active participant in immunologic reactions. Albumin is also a normal constituent of platelet membrane, and increased platelet albumin could indicate an increased platelet mass. Platelet-bound albumin, IgG, and total platelet protein were measured on both intact and disrupted platelets from healthy individuals (n = 25) and patients with ITP (n = 21). Platelet IgG and albumin were measured in an immunoradiometric assay using intact antisera and F(ab')2 fragments prepared from the same antisera. There was no relationship between platelet-bound IgG or albumin, and platelet size measured by either platelet protein or platelet volume, (r less than 0.3 for all interactions). In contrast, there was a significant correlation between platelet-bound albumin and platelet-bound IgG (r = 0.7, n = 21, p less than 0.001). Those patients with elevated platelet PAIgG also had elevated platelet albumin, and this relationship was irrespective of the total platelet protein content or mean platelet volume. It is possible that the increased platelet-bound IgG in ITP reflects an increase in platelet surface area or contaminating platelet fragments that are not manifested as an increase in platelet volume or total platelet protein. Alternatively, a platelet membrane abnormality may occur in ITP that results in the uptake of significant amounts of plasma proteins. Either possibility implies that not all of the IgG on platelets from patients with ITP is pathologic IgG.

The amount of platelet-bound albumin parallels the amount of IgG on washed platelets from patients with immune thrombocytopenia

The biologic relevance of the increased platelet-associated IgG (PAIgG) on platelets from patients with idiopathic thrombocytopenic purpura (ITP) is unclear. Platelets from ITP patients are often larger than normal, and it is possible that the increased IgG is not specific but passively related to platelet size. The measurement of platelet-bound albumin could provide information concerning the specificity of the platelet-bound IgG, since albumin, like IgG, is a plasma protein, but unlike IgG, is not an active participant in immunologic reactions. Albumin is also a normal constituent of platelet membrane, and increased platelet albumin could indicate an increased platelet mass. Platelet-bound albumin, IgG, and total platelet protein were measured on both intact and disrupted platelets from healthy individuals (n = 25) and patients with ITP (n = 21). Platelet IgG and albumin were measured in an immunoradiometric assay using intact antisera and F(ab')2 fragments prepared from the same antisera. There was no relationship between platelet-bound IgG or albumin, and platelet size measured by either platelet protein or platelet volume, (r less than 0.3 for all interactions). In contrast, there was a significant correlation between platelet-bound albumin and platelet-bound IgG (r = 0.7, n = 21, p less than 0.001). Those patients with elevated platelet PAIgG also had elevated platelet albumin, and this relationship was irrespective of the total platelet protein content or mean platelet volume. It is possible that the increased platelet-bound IgG in ITP reflects an increase in platelet surface area or contaminating platelet fragments that are not manifested as an increase in platelet volume or total platelet protein. Alternatively, a platelet membrane abnormality may occur in ITP that results in the uptake of significant amounts of plasma proteins. Either possibility implies that not all of the IgG on platelets from patients with ITP is pathologic IgG.

Relationship of raised platelet IgG in thrombocytopenia to total platelet protein content.

Platelet-associated IgG (PA IgG) of washed platelets of 20 normal controls and 45 thrombocytopenic patients was measured by radioimmunoassay and related to total platelet protein content. Mean values (+/- 2 SD) for 20 normals were 4.4 +/- 3.5 fg IgG per platelet (fg IgG/pl), 2.1 +/= 1.0 pg protein per platelet (pg protein/pl) and a ratio of IgG per g platelet protein of 2.1 +/- 1.8 mg IgG/g protein. Patients could be divided into two groups: Group I (31) with normal IgG/g protein ratios and either normal (16) or raised (15) fg IgG/pl, and Group II (14) with both fg IgG/pl and IgG/g protein raised. Of the 21 patients with idiopathic thrombocytopenia (ITP), 13 were in Group I (seven with raised and six with normal fg IgG/pl), and eight in Group II. There was significant correlation between PA IgG (fg IgG/pl) and platelet protein for the entire Group I (r = 0.95, P less than 0.001). The ITP patients in this group showed a similar correlation (r = 0.77, P less than 0.001), suggesting that raised PA IgG levels in Group I patients may be a result of protein-rich platelets. 60-90% of PA IgG was found in the soluble fraction of lysates of platelets from both normals and patients. Thus, it is either non-specifically associated with platelets or is antibody directed towards easily solubilized platelet antigens. Nine thrombocytopenic patients without immune disorders had raised PA IgG. These results question the assumption that raised PA IgG always indicates specific antiplatelet antibody.

Further Studies of Canine von Willebrand's Disease

Abstract Additional characterization of von Willebrand's disease (VWD) in a family of German shepherd dogs is presented. Genetic studies of three generations of affected dogs indicate that about 50% of the progeny are affected if one parent has VWD and about 60% if both parents have the defect. Some of these progeny manifested an incomplete form of VWD, suggesting autosomal dominant inheritance with variable expressivity. The disease become progressively less severe with advancing age and repeated pregnancies. Ristocetin-induced platelet aggregation was significantly reduced in VWD dogs as compared with normal, thrombopathic, and hemophilic carrier dogs. Immunodiffusion and electroimmunodiffusion studies with rabbit anticanine factor VII showed the level of factor VII-related antigen to be low in VWD dogs but present in increased amounts in hemophilic dogs. VWD affected dogs had markedly delayed hemostatic plug formation, but their plugs appeared normal by light and electron microscopy. Their platelet nucleotides, ATP/ADP ration, and platelet protein content were normal. Platelet and fibrinogen survival times with [75Se] selenomethionine were also normal, although platelets from VWD dogs incorporated more radioactivity than did those from normal dogs or from dogs with incomplete VWD.

Further studies of canine von Willebrand's disease

Additional characterization of von Willebrand's disease (VWD) in a family of German shepherd dogs is presented. Genetic studies of three generations of affected dogs indicate that about 50% of the progeny are affected if one parent has VWD and about 60% if both parents have the defect. Some of these progeny manifested an incomplete form of VWD, suggesting autosomal dominant inheritance with variable expressivity. The disease become progressively less severe with advancing age and repeated pregnancies. Ristocetin-induced platelet aggregation was significantly reduced in VWD dogs as compared with normal, thrombopathic, and hemophilic carrier dogs. Immunodiffusion and electroimmunodiffusion studies with rabbit anticanine factor VII showed the level of factor VII-related antigen to be low in VWD dogs but present in increased amounts in hemophilic dogs. VWD affected dogs had markedly delayed hemostatic plug formation, but their plugs appeared normal by light and electron microscopy. Their platelet nucleotides, ATP/ADP ration, and platelet protein content were normal. Platelet and fibrinogen survival times with [75Se] selenomethionine were also normal, although platelets from VWD dogs incorporated more radioactivity than did those from normal dogs or from dogs with incomplete VWD.

Platelet metabolism in acute leukemia.

Abstract Carbohydrate metabolism was studied in platelets from 9 patients with untreated acute leukemia to evaluate one possible mechanism underlying the previously reported defect in function that is characteristic of these cells. Rates of utilization of 14 C-labeled glucose and rates of production of lactate and 14 CO 2 were measured in resting platelets and in platelets stimulated with thrombin. The results were compared to those obtained using normal platelets incubated at similar concentrations and were evaluated on the basis of both platelet number and platelet protein content. The mean protein content of leukemic platelets was 3.44 ± 0.30 (1 S.E.M.) mg. per 10 9 platelets as compared to normal mean of 2.00 ± 0.05 (1 S.E.M.) mg. per 10 9 platelets. The platelet life-span, measured in 7 of the 9 patients, ranged from 2 to 3.5 days (normal 8.5 ± 0.2 (1 S.E.M.) days). When expressed on the basis of platelet number, the rates of 14 C-glucose utilization, lactate production, and 14 CO 2 production averaged 1.8, 1.8, and 2.2 times normal, respectively. When expressed per milligram protein, each was normal. The average net increases in the rates of 14 C-glucose utilization, lactate production, and oxidation of glucose-6- 14 C to 14 CO 2 with thrombin stimulation were 0.6 to 0.9 times normal when expressed on a cellular basis, and were 0.3 to 0.5 times normal when expressed per milligram protein. Induction of remission in 1 patient was associated with a decrease in platelet protein content and a partial improvement in the metabolic response to thrombin. The results indicate that platelets from patients with acute leukemia are larger and younger than normal platelets. Although metabolic activity of resting platelets may be normal, the net increase in energy production with thrombin stimulation, as determined from production rates of CO 2 and lactate, is impaired. The subnormal metabolic response to thrombin stimulation may be 1 factor responsible for the impaired functional activity of platelets from patients with acute leukemia.