The Genotype-Tissue Expression (GTEx) project was established to characterize genetic effects on the transcriptome across human tissues and to link these regulatory mechanisms to trait and disease associations. Here, we present analyses of the version 8 data, examining 15,201 RNA-sequencing samples from 49 tissues of 838 postmortem donors. We comprehensively characterize genetic associations for gene expression and splicing in cis and trans, showing that regulatory associations are found for almost all genes, and describe the underlying molecular mechanisms and their contribution to allelic heterogeneity and pleiotropy of complex traits. Leveraging the large diversity of tissues, we provide insights into the tissue specificity of genetic effects and show that cell type composition is a key factor in understanding gene regulatory mechanisms in human tissues.

Platelet-derived microparticles (PMPs) are associated with enhancement of metastasis and poor cancer outcomes. Circulating PMPs transfer platelet microRNAs (miRNAs) to vascular cells. Solid tumor vasculature is highly permeable, allowing the possibility of PMP-tumor cell interaction. Here, we show that PMPs infiltrate solid tumors in humans and mice and transfer platelet-derived RNA, including miRNAs, to tumor cells in vivo and in vitro, resulting in tumor cell apoptosis. MiR-24 was a major species in this transfer. PMP transfusion inhibited growth of both lung and colon carcinoma ectopic tumors, whereas blockade of miR-24 in tumor cells accelerated tumor growth in vivo, and prevented tumor growth inhibition by PMPs. Conversely, -deleted mice, which had reduced circulating microparticles (MPs), supported accelerated tumor growth which was halted by PMP transfusion. PMP targeting was associated with tumor cell apoptosis in vivo. We identified direct RNA targets of platelet-derived miR-24 in tumor cells, which included mitochondrial, and , a noncoding small nucleolar RNA. These RNAs were suppressed in PMP-treated tumor cells, resulting in mitochondrial dysfunction and growth inhibition, in an miR-24-dependent manner. Thus, platelet-derived miRNAs transfer in vivo to tumor cells in solid tumors via infiltrating MPs, regulate tumor cell gene expression, and modulate tumor progression. These findings provide novel insight into mechanisms of horizontal RNA transfer and add multiple layers to the regulatory roles of miRNAs and PMPs in tumor progression. Plasma MP-mediated transfer of regulatory RNAs and modulation of gene expression may be a common feature with important outcomes in contexts of enhanced vascular permeability.

Summary. The bleeding problems associated with common and rare inherited platelet disorders illustrate the importance of platelets to normal haemostasis. At sites of injury, platelets normally adhere, undergo activation, secretion and aggregate formation, and they provide the membrane surface for the assembly of coagulation to generate thrombin. The causes of inherited disorders that alter platelet haemostatic functions are quite diverse, ranging from defects in receptors critical to platelet adhesion and aggregation, to defects in signalling molecules or in transcription factors important for production of functional platelets. The mechanisms of impaired platelet function are largely unknown for the more common disorders that alter platelet activation, secretion and the secondary wave of platelet aggregation. The diagnostic evaluation of congenital platelet disorders has been challenging as some Ôplatelet-typeÕ bleeding symptoms, such as bruising, are quite common in the general population. Moreover, the diagnostic tests used by clinical laboratories to evaluate disorders of platelet function have not been standardized. In individuals recognized to have an inherited defect in platelet function, therapy is important for controlling and preventing bleeding episodes. Presently, there are a number of choices to consider for the management of bleeding symptoms, including menorrhagia. This paper reviews the causes, diagnostic evaluation and therapies for common and rare congenital platelet disorders.

The mechanisms by which agonists activate glycoprotein (GP) IIb-IIIa function remain unclear. We have reported data on a patient with thrombocytopenia and impaired receptor-mediated aggregation, phosphorylation of pleckstrin (a protein kinase C [PKC] substrate), and activation of the GPIIb-IIIa complex. Abnormalities in hematopoietic transcription factors have been associated with thrombocytopenia and platelet dysfunction. To define the molecular mechanisms, we amplified from patient platelet RNA exons 3 to 6 of core-binding factor A2 (CBFA2) cDNA, which encompasses the DNA-binding Runt domain; a 13-nucleotide (nt) deletion was found (796-808 nt). The gDNA revealed a heterozygous mutation (G>T) in intron 3 at the splice acceptor site for exon 4, leading to a frameshift with premature termination in the Runt domain. On immunoblotting, platelet CBFA2, PKC-, albumin, and IgG were decreased, but pleckstrin, PKC-␣, -␤I, -␤II, -, -⑀, -␦, and -, and fibrinogen were normal. Our conclusions are that (1) CBFA2 mutation is associated with not only thrombocytopenia, but also impaired platelet protein phosphorylation and GPIIb-IIIa activation; (2) proteins regulated by CBFA2 are required for inside-out signal transduction-dependent activation of GPIIbIIIa; and (3) we have documented the first deficiency of a human PKC isozyme (PKC-), suggesting a major role of this isozyme in platelet production and function. IntroductionSignal transduction-dependent activation of integrin glycoprotein (GP) IIb-IIIa complex and subsequent fibrinogen binding is a prerequisite for platelet aggregation on exposure to receptormediated agonists. However, the molecular mechanisms by which GPIIb-IIIa function is regulated on cell activation are not fully understood. We have previously reported 1 detailed studies in a patient with lifelong mucocutaneous bleeding manifestations, mild thrombocytopenia, and markedly abnormal platelet aggregation (including in primary wave) and dense granule secretion in response to multiple agonists. The patient's father and grandfather also had a history of easy bruising, and the father (who had thrombocytopenia) had died of acute leukemia. Phosphorylation of pleckstrin (a protein kinase C [PKC] substrate) and myosin light chain (MLC) was diminished in the patient's platelets on activation with platelet-activating factor (PAF) and thrombin. PKC activation has been linked to insideout signal transduction-dependent activation of GPIIb-IIIa on platelets. 2,3 In detailed studies in this patient, we demonstrated by flow cytometry 1 that the platelets had a full complement of GPIIb-IIIa complexes but receptor-mediated signal transductiondependent activation of GPIIb-IIIa was impaired on stimulation with PAF, adenosine diphosphate (ADP), and protease-activated receptor 1 (PAR-1) agonist SFLLRN. The expression of ligandinduced binding sites (LIBSs) induced by a number of agonists was normal. We concluded 1 that the patient's platelets had a defect in GPIIb-IIIa activation due to an upstream defect in the signaling mechanisms...

Summary.  We have reported on a patient with thrombocytopenia, impaired platelet aggregation, secretion, phosphorylation of pleckstrin and myosin light chain (MLC), and GPIIb–IIIa activation, associated with a heterozygous mutation in transcription factor CBFA2 (core binding factor A2, RUNX1 or AML1). To obtain insights into the abnormal platelet mechanisms and CBFA2‐regulated genes, we performed platelet expression profiling in four control subjects and the patient using the Affymetrix U133 GeneChips. In the patient, 298 probe sets were significantly downregulated at least 2‐fold. MLC regulatory polypeptide (MYL9 gene) was decreased ∼77‐fold; this is an important finding because agonist‐stimulated MLC phosphorylation is decreased in patient platelets. Genes downregulated ≥ 5‐fold include those involving calcium binding proteins (CABP5), ion transport (sodium/potassium/Ca exchanger, SLC24A3), cytoskeletal/microtubule proteins (erythrocyte membrane protein band 4.1‐like 3, EPB41L3; tropomyosin 1, TPM1; tubulin, alpha 1, TUBA1), signaling proteins (RAB GTPase activating protein 1‐like, RABGAP1L; β3‐endonexin, ITGB3 BP) and chemokines (platelet factor 4 variant 1, PF4V1; chemokine CXCL5, CXCL5). These and other downregulated genes are relevant to the patient's platelet defects in function and production. These studies provide the first proof of concept that platelet expression profiling can be applied to obtain insights into the molecular basis of inherited platelet defects.

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca 2؉ mobilization. Present studies demonstrated that platelet phospholipase A 2 (cytosolic and membrane) activity in the patient was normal. Receptormediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca 2؉ release induced by guanosine 5-[␥-thio]triphosphate (GTP[␥S]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of ␣-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A 2 analog U46619. Binding of 35 S-labeled GTP[␥S] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I 2 analog) -induced rise in cAMP (mediated by G␣ s ) and its inhibition (mediated by G␣ i ) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of G␣ subunits in the patient's platelet membranes showed a decrease in G␣ q (<50%) but not G␣ i , G␣ z , G␣ 12 , and G␣ 13 . These studies provide evidence for a hitherto undescribed defect in human platelet G-protein ␣-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of G␣ q in thrombin-induced responses.

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Mutations in transcription factor RUNX1 are associated with familial platelet disorder, thrombocytopenia, and predisposition to leukemia. We have described a patient with thrombocytopenia and impaired agonist-induced platelet aggregation, secretion, and glycoprotein (GP) IIb-IIIa activation, associated with a RUNX1 mutation. Platelet myosin light chain (MLC) phosphorylation and transcript levels of its gene MYL9 were decreased. Myosin IIA and MLC phosphorylation are important in platelet responses to activation and regulate thrombopoiesis by a negative regulatory effect on premature proplatelet formation. We addressed the hypothesis that MYL9 is a transcriptional target of RUNX1. Chromatin immunoprecipitation (ChIP) using megakaryocytic cells revealed RUNX1 binding to MYL9 promoter region -729/-542 basepairs (bp), which contains 4 RUNX1 sites. Electrophoretic mobility shift assay showed RUNX1 binding to each site. In transient ChIP assay, mutation of these sites abolished binding of RUNX1 to MYL9 promoter construct. In reporter gene assays, deletion of each RUNX1 site reduced activity. MYL9 expression was inhibited by RUNX1 short interfering RNA (siRNA) and enhanced by RUNX1 overexpression. RUNX1 siRNA decreased cell spreading on collagen and fibrinogen. Our results constitute the first evidence that the MYL9 gene is a direct target of RUNX1 and provide a mechanism for decreased platelet MYL9 expression, MLC phosphorylation, thrombocytopenia, and platelet dysfunction associated with RUNX1 mutations.