Platelet-related Diseases Research Articles

Platelet Releasate Proteome Profiling Reveals a Core Set of Proteins with Low Variance between Healthy Adults.

Upon activation, platelets release a powerful cocktail of soluble and vesicular signals, collectively termed the "platelet releasate" (PR). Although several studies have used qualitative/quantitative proteomic approaches to characterize PR; with debated content and significant inter-individual variability reported, confident, and reliable insights have been hindered. Using label-free quantitative (LFQ)-proteomics analysis, a reproducible, quantifiable investigation of the 1U mL-1 thrombin-induced PR from 32 healthy adults was conducted. MS proteomics data are available via ProteomeXchange, identifier PXD009310. Of the 894 proteins identified, 277 proteins were quantified across all donors and form a "core" PR. Bioinformatics and further LFQ-proteomic analysis revealed that the majority (84%) of "core" PR proteins overlapped with the protein composition of human platelet-derived exosomes. Vesicles in the exosomal-size range were confirmed in healthy-human PR and reduced numbers of similar-sized vesicles were observed in the PR of a mouse model of gray platelet syndrome, known to be deficient in platelet alpha-granules. Lastly, the variability of proteins in the PR was assessed, and reproducible secretion levels were found across all 32 healthy donors. Taken together, the PR contains valuable soluble and vesicular cargo and has low-population variance among healthy adults, rendering it a potentially useful platform for diagnostic fingerprinting of platelet-related disease.

Mitochondrial PIP3-binding protein FUNDC2 supports platelet survival via AKT signaling pathway.

Platelets undergo apoptosis in response to a variety of stimuli in the circulation. Mitochondria in platelets are essential for their apoptosis. Specifically, pro-survival protein BCL-xL on mitochondria is the key regulator of platelet lifespan. Here we identify an outer mitochondrial membrane protein FUNDC2 for platelet survival. FUNDC2 knockout mice carrying excessively apoptotic platelets exhibit thrombocytopenia in response to hypoxia. Mechanistically, FUNDC2 binds the lipid PIP3 via its unique, highly conserved N-terminal motif. FUNDC2 deficiency abrogates the phosphorylation of AKT and its substrate BAD in a PIP3/PI3K-dependent manner, which suppresses BCL-xL. Indeed, FUNDC2 deficiency shortens the platelet lifespan under stress. Thus, this FUNDC2/AKT/BCL-xL axis signifies a balance between platelet survival and apoptosis at the single organelle level and provides new insight for platelet-related diseases as well.

Association of human platelet antigen polymorphisms with platelet count and mean platelet volume

ABSTRACTObjectives: Although recent genome-wide association studies have identified a number of single nucleotide polymorphisms associated with platelet count and mean platelet volume (MPV), it is unclear whether polymorphisms in the human platelet antigens (HPA) genes are associated with platelet count and MPV. The aim of this study was to determine the association of the HPA-2, -3, -5 and -15 polymorphisms with platelet count and MPV.Methods: The HPA were genotyped by 5ˊ-nuclease assay in 139 healthy Chinese Han individuals, while platelet count and MPV from the same samples were measured using an hematology cell analyzer.Results: The platelet count was significantly lower in the individuals with the HPA-2aa genotype compared to those with HPA-2ab (P = 0.020), and significantly higher in individuals with HPA-5aa and HPA-15aa genotypes compared to those with HPA-5ab (P = 0.045) and HPA-15ab/bb (P = 0.032), respectively. On the other hand, platelet count of individuals with the HPA-3aa and HPA-3ab/bb genotypes did not differ significantly (P = 0.084). The MPV was significantly lower in individuals with HPA-5aa genotype compared to those with HPA-5ab (P = 0.001) but did not differ among the HPA-2, -3 and -15 genotypes. Furthermore, HPA-2, -5 and -15 polymorphisms were identified as independent factors for the platelet count and HPA-5 polymorphism was shown as an independent factor for MPV.Conclusions: This study demonstrates that HPA-2, -5 and -15 polymorphisms are associated with the platelet count while HPA-5 polymorphism is associated with MPV. This finding will further our understanding of the association of HPA polymorphisms with platelet-related diseases.

Application of Next Generation Sequencing in Genetic Diagnosis of Hereditary Platelet Disorders - Review

Hereditary platelet disorders are a heterogeneous group of disorders characterized by abnormal number or function of platelets, sometimes even involving other systems apart from blood abnormalities. The great clinical and genetic heterogeneity makes the diagnosis and treatment of hereditary platelet disorders as a huge challenge for clinicians. At present, only a small number of patients have received a clear molecular diagnosis of hereditary platelet diseases, and a lot of pathogenic genetic variations still remain unknown. The popularity of next generation sequencing (NGS) promotes the development of individualized gene sequencing. Researchers have made great progress in the field of hemostasis and thrombosis using whole genome sequencing (WGS), whole exone sequencing (WES) and target gene sequencing (TGS). The development of NGS has not only promoted the individualized molecular diagnosis of hereditary platelet diseases, but also laid a solid foundation for gene therapy in the future. In this review, the new progress of the diagnosis of platelet-related diseases by using next generation sequencing techniques, is summarized.

A retrospective analysis of donor deferral characteristics for plateletpheresis in a tertiary care hospital, South India

Background: There is an increasing demand for platelets in the recent years due to increase in cancer cases, dengue fever, and other platelet-related diseases. Blood bank plays a vital role in supplying platelet units which have better yield and fewer complications. Single donor platelet (SDP) unit is a useful platelet product in many diseases for raising the platelet count. For better yield, a healthy and young donor is required which is a great challenge for the blood bank staff. Materials and Methods: This retrospective study was carried out for a period of 4 years from January 2014 to December 2017. Data were collected from the records maintained by the Department of Transfusion Medicine and Immunohematology in a Tertiary Care Hospital, Hyderabad, India. Results: Out of 860, a total of 705 Plateletpheresis donors were selected for SDP donation, and the remaining 155 (18.02%) donors were deferred for various reasons. The predominant age range of the deferred donors was 25–34 years (50.32%). Among the deferred donors, males were 90.32% and females were 9.68%. Temporarily deferred donors account for 98.07% and permanently deferred donors were 1.93%. The most common causes for deferral were low platelet count (31.61%), low hemoglobin (27.09%), and poor venous access (20.64%) and the least common cause was dermatitis at the venipuncture site (1.29%). The most common reason for permanent deferral was seropositivity for HBsAg. Conclusion: Selection of plateletpheresis donor with utmost stringency will give good yield of platelets. Temporary deferred donor should be counseled properly so as to encourage them for the future donations when they become eligible, and this bridges the gap between the demand and supply of apheresis platelets.

The novel compound MP407 inhibits platelet aggregation through cyclic AMP-dependent processes

Platelet hyperactivity plays a critical role for initiating several vascular diseases such as atherothrombosis. Therefore, development of effective antiplatelet agents is necessary for ameliorating platelet-related diseases. In this study, we investigated the effects of the new synthesized compound, MP407 on platelet aggregation and further elucidated the underlying mechanisms. Our results demonstrated that MP407 dose-dependently inhibited collagen-induced platelet aggregation, thromboxane B2 (TXB2) production, intracellular Ca2+ mobilization, platelet membrane GPIIb/IIIa expression, and the phosphorylation of Akt, GSK3β, p38MAPK, and phospho (Ser) PKC substrate (p47). Moreover, MP407 is able to increase the cyclic AMP formation both in resting and activated platelets. However, blocking cyclic AMP formation with 2′5′-ddAdo, an inhibitor of adenylate cyclase, greatly reversed the antiplatelet activity of MP407 and related platelet-activating pathways. MP407 also enhanced VASP phosphorylation at Ser157 in collagen-stimulated platelets, which was attenuated by addition of 2′5′-ddAdo. Therefore, the antiplatelet activity of MP407 may be modulated by cyclic AMP-dependent regulation of Akt, GSK3β, p38MAPK and VASP phosphorylation. Notably, treatment with MP407 markedly reduced the pulmonary thrombosis and the numbers of paralysis and death in mice induced by ADP injection, but did not affect the bleeding time. Taken together, MP407 may be a potential candidate or lead compound for developing novel antiplatelet or antithrombotic agents for platelet hyperactivity-triggered disease therapy.

Analysis of the proteins associated with platelet detergent resistant membranes.

Proteomic studies have facilitated the identification of proteins associated with the detergent-resistant membrane (DRM) fraction in a variety of cell types. Here, we have undertaken label-free quantitative (LFQ) proteomic profiling of the proteins associated with detergent-resistant plasma and internal membranes from resting and activated platelets. One hundred forty-one proteins were identified and raw data is available via ProteomeXchange with identifier PXD002554. The proteins identified include a myriad of important platelet signaling and trafficking proteins including Rap1b, Src, SNAP-23, syntaxin-11, and members of the previously unattributed Ragulator complex. Mean LFQ intensities calculated across three technical replicates for the three biological donors revealed that several important platelet signaling proteins altered their detergent solubility upon activation, including GPIbα, GPIbβ, Src, and 14-3-3ζ. Altered detergent solubility for GPIbα, following activation using a variety of platelet agonists, was confirmed by immunoblotting and further coimmunoprecipitation experiments revealed that GPIbα forms a complex with 14-3-3ζ that shifts into DRMs following activation. Taken together, proteomic profiling of platelet DRMs allowed greater insight in the complex biology of both DRMs and platelets and will be a useful subproteome to study platelet-related disease. All MS data have been deposited in the ProteomeXchange with identifier PXD002554 (http://proteomecentral.proteomexchange.org/dataset/PXD002554).

Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer

Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.

Inhibition of Protein Kinase a Induces Platelet Apoptosis

Background:Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelet, or in the development of some platelet-related diseases. However, up to now, little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase A (PKA) is highly expressed and plays central roles in signal transduction in human platelets. The certain level of PKA activity is indispensability for maintaining circulating platelets in resting state. In nucleated cells, PKA stimulated proliferation or promoted cell death via apoptosis in different cell types. However, it is still unclear whether PKA plays a role in platelets apoptosis. The aim of the current study is to investigate the role of PKA in platelets apoptosis.Methods and Results:Apoptotic events were assessed in platelets by flow cytometry, western blotting. PKA inhibitor H89 dose-dependently elucidated depolarization of mitochondrial inner membrane potential (ΔΨm), activation of caspase-3, -9, cleavage of gelsolin, phosphatidylserine (PS) exposure and membrane shrinkage in platelets. Incubation of platelets with PKA catalytic subunit-α (PKACα)-siRNA specific reduced the production PKACα and obviously elucidated platelet apoptosis. Platelet aggregation induced by ADP, botrocetin, or α-thrombin was significantly reduced in platelets treated with H89. However, H89 does not incur P-selectin surface expression and PAC-1 binding in platelets. After intraperitoneal injection of PKA inhibitor (Rp-cAMPS), the circulating platelets were reduced at 8 hour time point and recovered within 24 hours. H89 down-regulated the expression of Bad in platelets, and phosphorylation of Bad at Ser 112 was reduced in platelets. Cyclosporin A and ROS inhibitor (NAC) significantly reduced H89-induced platelet ΔΨm depolarization. However, z-DEVE-fmk, GM6001, MDL28170, forskolin, SB203580, BAPTA had no obvious effect on depolarization of ΔΨm.Conclusions:These data indicate that inhibition of PKA results in ROS-mediated platelet apoptosis. The findings suggest a novel mechanism for PKA in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis. DisclosuresNo relevant conflicts of interest to declare.

Protein kinase C activation induces platelet apoptosis

Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

The production of soluble C-type lectin-like receptor 2 is a regulated process

C-type lectin-like receptor 2 (CLEC-2) is a newly identified type II transmembrane protein belonging to the C-type lectin family molecules, which acts as a cell-surface receptor for snake venom toxin rhodocytin and tumor antigen podoplanin. We previously demonstrated that the full-length mouse CLEC-2 (mCLEC-2) can be cleaved into soluble form. Elevated levels of soluble forms of membrane proteins in circulating blood may reflect increased expression of membrane proteins and disease activities. In the present study, we clarified the domain and sites contributing to the production of soluble mCLEC-2. The shedding process can be positively regulated by protein kinase C (PKC). Moreover, we explored the possibility that human CLEC-2 (hCLEC-2) may also be proteolyticly cleaved and released as a soluble form. We have observed that the production of soluble hCLEC-2 could be induced by phorbol ester (PMA) in cells stably transfected with hCLEC-2 cDNA. Further studies may explore therapeutic and diagnostic applications of soluble hCLEC-2 in platelet-related diseases.

Proteomics applied to the study of platelet-related diseases: Aiding the discovery of novel platelet biomarkers and drug targets

Platelets play a fundamental role in hemostasis. Because they do not have a nucleus, proteomics is an ideal way to approach their biochemistry. Platelet proteomics is still a young field that emerged a decade ago. Initial platelet proteomic research focused on general proteome mapping followed by the exploration of sub-cellular compartments, the membrane proteome, and signaling pathways. The initial studies were later completed with the analysis of the platelet releasate and microparticle proteome. The success of these studies led to the application of platelet proteomics to the study of several pathologies where platelets play a fundamental role. Those include platelet-related disorders, such as storage pool disease, gray platelet syndrome, and Quebec platelet disorder; diseases where unwanted platelet activation is highly relevant, such as thrombosis and cardiovascular disease; and other diseases, such as cystic fibrosis, uremia, or Alzheimer's disease. In the present review article, we revise the most relevant proteomic studies on platelet-related diseases carried out to date, paying special attention to sample preparation requirements for platelet clinical proteomic studies. This article is part of a Special Issue entitled: Integrated omics.

Inside platelets…

In this issue of Blood, Pleines and colleagues show how deletion of the GTPase family member RhoA in murine megakaryocytes/platelets induces macrothrombocytopenia but also protects against occlusive thrombosis or cerebral infarction,providing new insights into both RhoA function as well as platelet-related diseases.

P-selectin–dependent platelet aggregation and apoptosis may explain the decrease in platelet count during Helicobacter pylori infection

P-selectin expression has been shown in Helicobacter pylori-infected persons, an infection that has been clinically associated with platelet-related diseases, such as idiopathic thrombocytopenic purpura. However, the role of P-selectin expression during H pylori infection remains unclear. In this study, we hypothesized that P-selectin expression was associated with platelet aggregation during H pylori infection. Using flow cytometry, we examined the levels of adhesion between H pylori and platelets as well as the levels of P-selectin expression and platelet phosphatidylserine (PS) expression during H pylori infection. Significantly high levels of adhesion between pro-aggregatory bacteria and platelets were observed. We identified that H pylori IgG is required for bacteria to induce P-selectin expression and that a significant release of P-selectin is essential for H pylori to induce aggregation. In addition, cellular apoptotic signs, such as membrane blebbing, were observed in platelet aggregates. PS expression was also detected in platelets during infection with both pro-aggrogatory and nonaggregatory strains of H pylori. These results suggest that the decrease in platelet counts seen during H pylori infection is the result of P-selection-dependent platelet aggregation and PS expression induced by the bacteria.

Protective effects of d-glucaro-1,4-lactone against oxidative modifications in blood platelets

Background and aim Peroxynitrite (ONOO −), a highly reactive species, modulates platelet activation and function. Modifications of platelet proteins induced by ONOO − may be an important factor in the pathogenesis of platelet-related diseases. The defence mechanisms against ONOO − are therefore crucial for normal cellular function. Recently, there has been an increased interest in the screening of natural products present in the diet and herbals for possible antioxidative agents (ONOO − scavengers). d-glucaro-1,4-lactone (1,4-GL), formed from d-glucaric acid (GA), a natural compound found in fruits and vegetables, possesses detoxifying and anticancerogenic properties. However, the effect of this compound on platelet activation is unknown. Methods and results We investigated the effects of 1,4-GL on nitrative and oxidative alteration of platelet proteins and lipid peroxidation caused by two strong oxidants: ONOO − and hydroperoxide (H 2O 2). The action of 1,4-GL on platelet aggregation induced by adenosine diphosphate (ADP) was also studied. Exposure of platelets to ONOO − or H 2O 2 resulted in an increase in the level of carbonyl groups (approximately three-fold and two-fold, respectively). In the presence of 1,4-GL, a significant decrease (about 50% for the highest concentration of 1,4-GL) in carbonyl group formation was observed; however the level of nitrotyrosine residues in platelets treated with ONOO − remained unchanged. Conclusions We demonstrated an inhibitory effect of 1,4-GL on lipid peroxidation in platelets treated with ONOO − (0.1 mM) or H 2O 2 (2 mM). 1,4-GL inhibited platelet lipid peroxidation by about 40%. In the presence of 1,4-GL, peroxidation of plasma lipids was also reduced by about 40%. These results demonstrate that 1,4-GL possesses antioxidative properties and reduces the activation of blood platelets.

Modèles murins de pathologies plaquettaires

Platelet-related diseases correspond to functional defects or abnormal production (thrombopoiesis) of hereditary and immunological origins. Recent progress in the manipulation of the mouse genome (transgenesis, gene inactivation or insertion) has resulted in the generation of numerous strains exhibiting defective platelet function or production. Some strains reproduce known hereditary diseases affecting haemostasis (Glanzmann thrombasthenia, Bernard-Soulier syndrome (BSS) or thrombopoiesis (Wiscott-Aldrich or May-Hegglin syndrome). More often the mutated strains have no human equivalent and represent useful models to study: (i) the role of adhesive or signalling receptors or of signalling proteins in platelet-dependent haemostasis and thrombosis or; (ii) to study the poorly characterized mechanisms of thrombopoiesis, which implicate transcription factors (GATA, Fli1), growth factors and receptors (TPO, cMPL), and cytoskeletal or contractile proteins (tubulin, myosin). Additional mouse strains result from the selection of spontaneous mutants many of which affect intracellular platelet granules, representing models of storage pool diseases (SPD) such as the Gray platelet syndrome (αSPD) or Hermansky-Pudlack syndrome (δSPD). More recently, a systematic chemical mutagenesis approach has also identified genes involved in thrombopoiesis and platelet survival. Finally, mouse models of auto- or allo-immune thrombocytopenia have been developed to study the mechanisms of platelet destruction or removal.