MicroRNAs (miRNAs) regulate cell physiology by altering protein expression, but the biology of platelet miRNAs is largely unexplored. We tested whether platelet miRNA levels were associated with platelet reactivity by genome-wide profiling using platelet RNA from 19 healthy subjects. We found that human platelets express 284 miRNAs. Unsupervised hierarchical clustering of miRNA profiles resulted in 2 groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed (DE) between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. Using whole genome mRNA expression data on these same subjects, we computationally generated a highpriority list of miRNA-mRNA pairs in which the DE platelet miRNAs had binding sites in 3-untranslated regions of DE mRNAs, and the levels were negatively correlated. IntroductionOn rupture of atherosclerotic plaques, some persons form occlusive platelet thrombi whereas other persons repair the wound without occluding the vessel. The extreme interindividual variation in platelet reactivity probably contributes to the variation in both risk and clinical outcome of ischemic vascular disease because platelet hyper-reactivity has prospectively been shown to be a risk for recurrent coronary syndromes. 1 Although heritability strongly influences the interindividual variation in platelet reactivity, [2][3][4] there is a lack of understanding of the responsible genetic and molecular mechanisms. To understand better the basis for human platelet function, it is critical to define the genes that are expressed in the tissue of interest. We have previously used platelet RNA expression analyses from platelets of differing reactivity to identify differentially expressed (DE) platelet transcripts and proteins. 5 During the course of our studies, we found that a DE platelet microRNA (miRNA) altered the expression of VAMP8, a critical component of platelet granule exocytosis. miRNAs are small (ϳ 22 nucleotides) noncoding RNAs that function post-transcriptionally in regulating gene expression by inducing mRNA degradation or translation inhibition, generally by targeting the 3Ј-untranslated region (UTR) of mRNAs. 6 miRNAs were initially identified as regulators of genes involved in development but have since been shown to affect a broad range of normal physiologic processes, including hematopoietic lineage commitment, as well as pathologic conditions. 7,8 More than 1000 miRNAs have been identified, which are estimated to regulate most (Ͼ 60%) coding genes. 9 The cellular impact of most miRNA-mRNA interactions is a fine-tuning of protein output, and not a major repression of expression. 10 Importantly, as little as a 20% reduction in miRNA levels can produce a disease phenotype. 11 Recent data demonstrate a role for miRNAs in both normal and diseased human megakaryocytopoiesis. 8,12-17 Although we and others have observed miRNAs in platelets, 15,[18][19][20][21][22][23] th...

Adaptation to hypoxic microenvironment is critical for tumor survival and metastatic spread. Hypoxiainducible factor 1␣ (HIF-1␣) plays a key role in this adaptation by stimulating the production of proangiogenic factors and inducing enzymes necessary for anaerobic metabolism. Histone deacetylase inhibitors (HDACIs) produce a marked inhibition of HIF-1␣ expression and are currently in clinical trials partly based on their potent antiangiogenic effects. Although it has been postulated that HDACIs affect HIF-1␣ expression by enhancing its interactions with VHL (von Hippel Lindau), thus promoting its ubiquitination and degradation, the actual mechanisms by which HDACIs decrease HIF-1␣ levels are not clear. Here, we present data indicating that HDACIs induce the proteasomal degradation of HIF-1␣ by a mechanism that is independent of VHL and p53 and does not require the ubiquitin system. This degradation pathway involves the enhanced interaction of HIF-1␣ with HSP70 and is secondary to a disruption of the HSP70/HSP90 axis function that appears mediated by the activity of HDAC-6.

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. PAR4 thrombin receptor induced platelet aggregation and calcium mobilization were significantly greater in black subjects. Numerous differentially expressed (DE) RNAs were associated with both race and PAR4 reactivity, including phosphatidylcholine transfer protein (PCTP), and platelets from blacks expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked activation of platelets or megakaryocytic cell lines through PAR4 but not PAR1. MiR-376c levels were DE by race and PAR4 reactivity, and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. MiR-376c regulated expression of PC-TP in human megakaryocytes. A disproportionately high number of miRNAs DE by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus, and were lower in platelets from blacks. These results support PC-TP as a regulator of 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 race effects when developing anti-thrombotic drugs.

The up-regulation of glycolysis to enhance the production of energy under reduced pO 2 is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1-4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5 -flanking region. Extensive analysis of the 5 pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from ؊1269 to ؊1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at ؊1279 and ؊1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1␣ null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxiainducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.

• Unique dataset of human platelet mRNA, miRNA, and physiology reveals mRNAs and miRNAs that differ by age and gender.• Interactive public web tool (www.plateletomics.com) provides biologic insights into platelet function and gene expression.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. (Blood. 2014;123(16):e37-e45)

• White individuals have a high frequency of the common PAR4 gene (F2RL3) variant Ala120; blacks have a high frequency of Thr120.• PAR4 Thr120 induces greater signaling and is associated with greater platelet aggregation and reduced inhibition by a PAR4 antagonist.Human platelets express 2 thrombin receptors: protease-activated receptor (PAR)-1 and PAR4. Recently, we reported 3.7-fold increased PAR4-mediated aggregation kinetics in platelets from black subjects compared with white subjects. We now show that platelets from blacks (n 5 70) express 14% more PAR4 protein than those from whites (n 5 84), but this difference is not associated with platelet PAR4 function. Quantitative trait locus analysis identified 3 common single nucleotide polymorphisms in the PAR4 gene (F2RL3) associated with PAR4-induced platelet aggregation. Among these single nucleotide polymorphisms, rs773902 determines whether residue 120 in transmembrane domain 2 is an alanine (Ala) or threonine (Thr). Compared with the Ala120 variant, Thr120 was more common in black subjects than in white subjects (63% vs 19%), was associated with higher PAR4-induced human platelet aggregation and Ca 21 flux, and generated greater inositol 1,4,5-triphosphate in transfected cells. A second, less frequent F2RL3 variant, Phe296Val, was only observed in blacks and abolished the enhanced PAR4-induced platelet aggregation and 1,4,5-triphosphate generation associated with PAR4-Thr120. PAR4 genotype did not affect vorapaxar inhibition of platelet PAR1 function, but a strong pharmacogenetic effect was observed with the PAR4-specific antagonist YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole]. These findings may have an important pharmacogenetic effect on the development of new PAR antagonists. (Blood. 2014;124(23):3450-3458)

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Hypoxia inducible factor-1a (HIF-1a) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1a and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1a and thereby enhances HIF-1a ubiquitination and degradation. Furthermore, ARD1 was proposed to be downregulated in hypoxia thus facilitating the stabilization of HIF-1a. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1a. However, we find that hARD1 specifically binds HIF-1a, suggesting a putative, still unclear, connection between these proteins.