Srikanth Nagalla

The complex transcriptional landscape of the anucleate human platelet

BackgroundHuman blood platelets are essential to maintaining normal hemostasis, and platelet dysfunction often causes bleeding or thrombosis. Estimates of genome-wide platelet RNA expression using microarrays have provided insights to the platelet transcriptome but were limited by the number of known transcripts. The goal of this effort was to deep-sequence RNA from leukocyte-depleted platelets to capture the complex profile of all expressed transcripts.ResultsFrom each of four healthy individuals we generated long RNA (≥40 nucleotides) profiles from total and ribosomal-RNA depleted RNA preparations, as well as short RNA (<40 nucleotides) profiles. Analysis of ~1 billion reads revealed that coding and non-coding platelet transcripts span a very wide dynamic range (≥16 PCR cycles beyond β-actin), a result we validated through qRT-PCR on many dozens of platelet messenger RNAs. Surprisingly, ribosomal-RNA depletion significantly and adversely affected estimates of the relative abundance of transcripts. Of the known protein-coding loci, ~9,500 are present in human platelets. We observed a strong correlation between mRNAs identified by RNA-seq and microarray for well-expressed mRNAs, but RNASeq identified many more transcripts of lower abundance and permitted discovery of novel transcripts.ConclusionsOur analyses revealed diverse classes of non-coding RNAs, including: pervasive antisense transcripts to protein-coding loci; numerous, previously unreported and abundant microRNAs; retrotransposons; and thousands of novel un-annotated long and short intronic transcripts, an intriguing finding considering the anucleate nature of platelets. The data are available through a local mirror of the UCSC genome browser and can be accessed at:http://cm.jefferson.edu/platelets_2012/.

VAMP8/endobrevin is overexpressed in hyperreactive human platelets: suggested role for platelet microRNA

Summary.  Background: Variation in platelet reactivity contributes to disorders of hemostasis and thrombosis, but the molecular mechanisms are not well understood. Objectives: To discover associations between interindividual platelet variability and the responsible platelet genes, and to begin to define the molecular mechanisms altering platelet gene expression. Subjects/methods: Two hundred and eighty‐eight healthy subjects were phenotyped for platelet responsiveness. Platelet RNA from subjects demonstrating hyperreactivity (n = 18) and hyporeactivity (n = 11) was used to screen the human transcriptome. Results: Distinctly different mRNA profiles were observed between subjects with differing platelet reactivity. Increased levels of mRNA for VAMP8/endobrevin, a critical v‐SNARE involved in platelet granule secretion, were associated with platelet hyperreactivity (Q = 0.0275). Validation studies of microarray results showed 4.8‐fold higher mean VAMP8 mRNA levels in hyperreactive than hyporeactive platelets (P = 0.0023). VAMP8 protein levels varied 13‐fold among platelets from these normal subjects, and were 2.5‐fold higher in hyperreactive platelets (P = 0.05). Among our cohort of 288 subjects, a VAMP8 single‐nucleotide polymorphism (rs1010) was associated with platelet reactivity in an age‐dependent manner (P < 0.003). MicroRNA‐96 was predicted to bind to the 3′‐untranslated regionof VAMP8 mRNA and was detected in platelets. Overexpression of microRNA‐96 in VAMP8‐expressing cell lines caused a dose‐dependent decrease in VAMP8 protein and mRNA, suggesting a role in VAMP8 mRNA degradation. Conclusions: These findings support a role for VAMP8/endobrevin in the heterogeneity of platelet reactivity, and suggest a role for microRNA‐96 in the regulation of VAMP8 expression.

Racial differences in human platelet PAR4 reactivity reflect expression of PCTP and miR-376c

Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. Platelet aggregation and calcium mobilization induced by the PAR4 thrombin receptor were significantly greater in black subjects. Numerous differentially expressed RNAs were associated with both race and PAR4 reactivity, including PCTP (encoding phosphatidylcholine transfer protein), and platelets from black subjects expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked PAR4- but not PAR1-mediated activation of platelets and megakaryocytic cell lines. miR-376c levels were differentially expressed by race and PAR4 reactivity and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. miR-376c regulated the expression of PC-TP in human megakaryocytes. A disproportionately high number of microRNAs that were differentially expressed by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus and were expressed at lower levels in platelets from black subjects. These results suggest that PC-TP contributes to the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race and emphasize a need to consider the effects of race when developing anti-thrombotic drugs.

Interactions between immunity, proliferation and molecular subtype in breast cancer prognosis

BackgroundGene expression signatures indicative of tumor proliferative capacity and tumor-immune cell interactions have emerged as principal biology-driven predictors of breast cancer outcomes. How these signatures relate to one another in biological and prognostic contexts remains to be clarified.ResultsTo investigate the relationship between proliferation and immune gene signatures, we analyzed an integrated dataset of 1,954 clinically annotated breast tumor expression profiles randomized into training and test sets to allow two-way discovery and validation of gene-survival associations. Hierarchical clustering revealed a large cluster of distant metastasis-free survival-associated genes with known immunological functions that further partitioned into three distinct immune metagenes likely reflecting B cells and/or plasma cells; T cells and natural killer cells; and monocytes and/or dendritic cells. A proliferation metagene allowed stratification of cases into proliferation tertiles. The prognostic strength of these metagenes was largely restricted to tumors within the highest proliferation tertile, though intrinsic subtype-specific differences were observed in the intermediate and low proliferation tertiles. In highly proliferative tumors, high tertile immune metagene expression equated with markedly reduced risk of metastasis whereas tumors with low tertile expression of any one of the three immune metagenes were associated with poor outcome despite higher expression of the other two metagenes.ConclusionsThese findings suggest that a productive interplay among multiple immune cell types at the tumor site promotes long-term anti-metastatic immunity in a proliferation-dependent manner. The emergence of a subset of effective immune responders among highly proliferative tumors has novel prognostic ramifications.

Human platelet microRNA-mRNA networks associated with age and gender revealed by integrated plateletomics.

There is little data considering relationships among human RNA, demographic variables, and primary human cell physiology. The platelet RNA and expression-1 study measured platelet aggregation to arachidonic acid, ADP, protease-activated receptor (PAR) 1 activation peptide (PAR1-AP), and PAR4-AP, as well as mRNA and microRNA (miRNA) levels in platelets from 84 white and 70 black healthy subjects. A total of 5911 uniquely mapped mRNAs and 181 miRNAs were commonly expressed and validated in a separate cohort. One hundred twenty-nine mRNAs and 15 miRNAs were differentially expressed (DE) by age, and targets of these miRNAs were over-represented among these mRNAs. Fifty-four mRNAs and 9 miRNAs were DE by gender. Networks of miRNAs targeting mRNAs, both DE by age and gender, were identified. The inverse relationship in these RNA pairs suggests miRNAs regulate mRNA levels on aging and between genders. A simple, interactive public web tool (www.plateletomics.com) was developed that permits queries of RNA levels and associations among RNA, platelet aggregation and demographic variables. Access to these data will facilitate discovery of mechanisms of miRNA regulation of gene expression. These results provide new insights into aging and gender, and future platelet RNA association studies must account for age and gender.

Dual roles for immune metagenes in breast cancer prognosis and therapy prediction

BackgroundNeoadjuvant chemotherapy for breast cancer leads to considerable variability in clinical responses, with only 10 to 20% of cases achieving complete pathologic responses (pCR). Biological and clinical factors that determine the extent of pCR are incompletely understood. Mounting evidence indicates that the patients immune system contributes to tumor regression and can be modulated by therapies. The cell types most frequently observed with this association are effector tumor infiltrating lymphocytes (TILs), such as cytotoxic T cells, natural killer cells and B cells. We and others have shown that the relative abundance of TILs in breast cancer can be quantified by intratumoral transcript levels of coordinately expressed, immune cell-specific genes. Through expression microarray analysis, we recently discovered three immune gene signatures, or metagenes, that appear to reflect the relative abundance of distinct tumor-infiltrating leukocyte populations. The B/P (B cell/plasma cell), T/NK (T cell/natural killer cell) and M/D (monocyte/dendritic cell) immune metagenes were significantly associated with distant metastasis-free survival of patients with highly proliferative cancer of the basal-like, HER2-enriched and luminal B intrinsic subtypes.MethodsGiven the histopathological evidence that TIL abundance is predictive of neoadjuvant treatment efficacy, we evaluated the therapy-predictive potential of the prognostic immune metagenes. We hypothesized that pre-chemotherapy immune gene signatures would be significantly predictive of tumor response. In a multi-institutional, meta-cohort analysis of 701 breast cancer patients receiving neoadjuvant chemotherapy, gene expression profiles of tumor biopsies were investigated by logistic regression to determine the existence of therapy-predictive interactions between the immune metagenes, tumor proliferative capacity, and intrinsic subtypes.ResultsBy univariate analysis, the B/P, T/NK and M/D metagenes were all significantly and positively associated with favorable pathologic responses. In multivariate analyses, proliferative capacity and intrinsic subtype altered the significance of the immune metagenes in different ways, with the M/D and B/P metagenes achieving the greatest overall significance after adjustment for other variables.ConclusionsGene expression signatures of infiltrating immune cells carry both prognostic and therapy-predictive value that is impacted by tumor proliferative capacity and intrinsic subtype. Anti-tumor functions of plasma B cells and myeloid-derived antigen-presenting cells may explain more variability in pathologic response to neoadjuvant chemotherapy than previously recognized.

Human Genome-Wide Association and Mouse Knockout Approaches Identify Platelet Supervillin as an Inhibitor of Thrombus Formation Under Shear Stress

Background— High shear force critically regulates platelet adhesion and thrombus formation during ischemic vascular events. To identify genetic factors that influence platelet thrombus formation under high shear stress, we performed a genome-wide association study and confirmatory experiments in human and animal platelets. Methods and Results— Closure times in the shear-dependent platelet function analyzer (PFA)–100 were measured on healthy, nondiabetic European Americans (n=125) and blacks (n=116). A genome-wide association (P<5×10−8) was identified with 2 single-nucleotide polymorphisms within the SVIL gene (chromosome 10p11.23) in African Americans but not European Americans. Microarray analyses of human platelet RNA demonstrated the presence of SVIL isoform 1 (supervillin) but not muscle-specific isoforms 2 and 3 (archvillin, SmAV). SVIL mRNA levels were associated with SVIL genotypes (P⩽0.02) and were inversely correlated with PFA-100 closure times (P<0.04) and platelet volume (P<0.02). Leukocyte-depleted platelets contained abundant levels of the ≈205-kDa supervillin polypeptide. To assess functionality, mice lacking platelet supervillin were generated and back-crossed onto a C57BL/6 background. Compared with controls, murine platelets lacking supervillin were larger by flow cytometry and confocal microscopy and exhibited enhanced platelet thrombus formation under high-shear but not low-shear conditions. Conclusions— We show for the first time that (1) platelets contain supervillin; (2) platelet thrombus formation in the PFA-100 is associated with human SVIL variants and low SVIL expression; and (3) murine platelets lacking supervillin exhibit enhanced platelet thrombus formation at high shear stress. These data are consistent with an inhibitory role for supervillin in platelet adhesion and arterial thrombosis.

Transfection of Human Platelets with Short Interfering RNA

Platelets contain mRNAs and are capable of translating mRNA into protein, and it has been previously demonstrated that platelets increase their levels of integrin β3 overtime while in blood bank storage conditions. We are unaware of prior attempts to introduce nucleic acids into platelets. Considering the potential clinical and research utility of manipulating platelet gene expression, we tested whether small interfering RNAs (siRNAs) could be transfected into normal human platelets. Multiple conditions were tested, including lipofectamine versus electroporation, different amounts of siRNA, the effect of different buffers and the presence of plasma during transfection, and the time for optimal siRNA incorporation after transfection. Using flow cytometry to assess transfection efficiency, we found that optimal transfection was obtained using lipofectamine, washed platelets, and 400 pmoles siRNA. Cell sorting of transfected platelets suggested that the incorporated siRNA was able to knockdown the level of a targeted mRNA. This is the first ever demonstration that nucleic acids can be introduced directly into platelets, and offers proof of concept for manipulating gene expression in platelets by nonviral methods. Future technical improvements may permit improving the quality and/or lifespan of stored human platelets. Clin Trans Sci 2011; Volume 4: 180–182

Platelet RNA chips dip into thrombocytosis

In this issue of Blood , Gnatenko and colleagues have conducted studies to determine whether platelet RNA profiling can predict causes of thrombocytosis.[1][1] The authors show that expression levels of only 4 platelet transcripts are able to predict JAK2 V617F–negative ET in more than 85% of

Reversibility of Apixaban Anticoagulation with a Four‐Factor Prothrombin Complex Concentrate in Healthy Volunteers

It was hypothesized that the four‐factor prothrombin complex concentrate (4F‐PCC) Kcentra 25 unit/kg would reverse impairment of thrombin generation in healthy volunteers dosed with apixaban to steady state. In this randomized, two‐period crossover, assessor‐blinded trial, 12 healthy subjects received 5 mg apixaban every 12 h. Three h after the fifth dose, four‐factor prothrombin complex concentrate (4F‐PCC) 25 unit/kg or saline were infused. Serial blood samples were assessed for thrombin generation using PPP‐reagent and PPP‐reagent low, anti‐Xa, PT, and PTT assays. Geometric mean ratio was calculated at 30 min postinfusion, and at 24, 48, and 72 h. Peak thrombin generation was 76% higher at 30 min postinfusion with 4F‐PCC (p = 0.025). The difference declined to 24% at 24 h and resolved by 48 h. Other thrombin generation parameters were also partially normalized. There was no difference between 4F‐PCC and saline in anti‐Xa assessment at 30 min or later time points.