Genetic loci associated with platelet traits and platelet disorders.

Genetic investigations have led to important advances in our knowledge of genes, proteins, and microRNA that influence circulating platelet counts, platelet size, and function. The application of genome-wide association studies (GWAS) to platelet traits has identified multiple loci with a significant association to platelet number, size, and function in aggregation and granule secretion assays. Moreover, the genes altered by disease-causing mutations have now been identified for several platelet disorders, including X-linked recessive, autosomal dominant, and autosomal recessive platelet disorders. Some mutations that cause inherited platelet disorders involve genes that GWAS have associated to platelet traits. Although disease-causing mutations in many rare and syndromic causes of platelet disorders have now been characterized, the genetic mutations that cause common inherited platelet disorders, and impair platelet aggregation and granule secretion, are largely unknown. This review summarizes current knowledge on the genetic loci that influence platelet traits, including the genes with well-characterized mutations in certain inherited platelet disorders.

Quebec platelet disorder: update on pathogenesis, diagnosis, and treatment.

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder associated with reduced platelet counts and a unique gain-of-function defect in fibrinolysis due to increased expression and storage of urokinase plasminogen activator (uPA) by megakaryocytes. QPD increases risks for bleeding and its key clinical feature is delayed-onset bleeding, following surgery, dental procedures or trauma, which responds only to treatment with fibrinolytic inhibitors. The genetic cause of the disorder is a tandem duplication mutation of the uPA gene, PLAU, which upregulates uPA expression in megakaryocytes by an unknown mechanism. The increased platelet stores of uPA trigger plasmin-mediated degradation of QPD α-granule proteins. The gain-of-function defect in fibrinolysis is thought to be central to the pathogenesis of QPD bleeding as the activation of QPD platelets leads to release of uPA from α-granules and accelerated clot lysis. The purpose of this review is to summarize current knowledge on QPD pathogenesis and the recommended approaches to QPD diagnosis and treatment.

Characterization of proteins associating with 5' terminus of PGHS-1 mRNA.

Induction of Prostaglandin Endoperoxide H Synthase-1 (PGHS-1) gene has been previously documented in a few studies during events such as development and cellular differentiation. However, molecular mechanisms governing the regulation of PGHS-1 gene expression and contributing to changes in protein levels are poorly understood. Using the MEG-01 cell model of PGHS-1 gene induction, our laboratory has previously demonstrated that the 5'UTR and the first two exons of PGHS-1 mRNA had a significant impact on decreasing the translational efficiency of a reporter gene and suggested that the presence of a secondary structure is required for conservation of this activity. This 5'end of PGHS-1 mRNA sequence has also been shown to associate with nucleolin protein. In the current study, we set to investigate the protein composition of the mRNP (messenger ribonucleoprotein) associating with the 5'end of PGHS-1 mRNA and to identify its protein members. RNA/protein binding assays coupled with LC-MS analysis identified serpin B1 and NF45 (nuclear factor 45) proteins as potential members of PGHS-1 mRNP complex. Immunoprecipitation experiments using MEG-01 protein extracts validated mass spectrometry data and confirmed binding of nucleolin, serpin B1, NF45 and NF90. The RNA fraction was extracted from immunoprecipitated mRNP complexes and association of RNA binding proteins, serpin B1, NF45 and NF90, to PGHS-1 mRNA target sequence was confirmed by RT-PCR. Together these data suggest that serpin B1, NF45 and NF90 associate with PGHS-1 mRNA and can potentially participate in the formation a single or a number of PGHS-1 ribonucleoprotein complexes, through nucleolin that possibly serves as a docking base for other protein complex members.

Translational regulation of PGHS-1 mRNA: 5' untranslated region and first two exons conferring negative regulation.

Prostaglandin endoperoxide H synthase-1 gene expression is described as inducible in a few contexts such as differentiation of megakaryoblastic MEG-01 cells into platelet-like structures. In the MEG-01 cells model of PGHS-1 gene induction, we previously reported a delay in protein synthesis and identified the translational step of gene expression as being regulated. In the current study, we mapped PGHS-1 mRNA sequences regulating translational efficiency and identified an RNA binding protein. The 5'UTR and first two exons of the PGHS-1 5' mRNA decreased the synthesis of Luciferase protein by approximately 80% without significant changes in mRNA levels when compared to controls. Both the PGHS-1 5'-UTR and the first two exons were required for activity. Sucrose density gradient fractionations of cytoplasmic extracts from MEG-01 cells infected with reporter constructs, either controls or containing PGHS-1 sequence, presented a similar profile of distribution of reporter transcripts between polysomal and non-polysomal fractions. RNA/protein interaction studies revealed nucleolin binding to the 135 nt PGHS-1 sequence. Mutation of the two NRE elements located in the 5'end of PGHS-1 mRNA sequence partially reduced the negative activity of the 135 nt sequence. Stable secondary structures predicted at the 5' end of the transcript are potentially involved in translational regulation. We propose that the 5'end of PGHS-1 mRNA represses translation and could delay the synthesis of PGHS-1 enzyme.

Antiplatelet therapies: aspirin at the heart of new directions.

When introduced over 100 years ago, aspirin was prescribed as an analgesic drug to arthritic patients for pain relief. The prevalence of users grew quite rapidly and to this day, aspirin remains widely used in clinical practice. The popularity of aspirin resulted not only from its analgesic properties but also from a second benefit recognized later as an anti-platelet effect. It was this important activity of aspirin that made it one of the most recommended drugs for the treatment and prevention of cardiovascular diseases. The anti-platelet effect of aspirin emerged from the first few case reports published in the early 1900s and was described as a mild bleeding. The molecular mechanisms involved were described in 1971 and constituted the irreversible inhibition of cyclooxygenase-1 enzyme and prevention of platelet aggregation. Today, the contribution of aspirin to our understanding of cardiovascular health persists and remains considerable. Observations from large cohorts of aspirin users generate massive amount of valuable information used in the identification of factors influencing the potential risk for cardiovascular diseases, including sex, age and genetic predisposition. Aspirin and the path of discovery leading to its anti-platelet activity has taken a hundred years was based on manifestations of effects observed in its users, and it remains a successful strategy for the identification of new avenues to treat cardiovascular diseases associated with hyper-platelet activity. The contribution of aspirin to the understanding of cardiovascular diseases and to the design of effective treatment and prevention strategies, remains of high importance in our society.

Cyclooxygenase inhibitors: instrumental drugs to understand cardiovascular homeostasis and arterial thrombosis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and Aspirin target cyclooxygenase (cox) enzymes and inhibit the synthesis of prostanoids. These drugs were originally developed to reduce the cardinal signs of inflammation, primarily pain. Prior to understanding their mechanism of action, investigations of Aspirin response in humans have revealed a protective effect on the cardiovascular system. Daily low-dose Aspirin is a well-established and prevailing treatment for the prevention of arterial thrombosis. Platelet inhibition by Aspirin results from the irreversible inhibition of cyclooxygenase-1 enzyme and prevention of thromboxane A2, a potent aggregatory agent, formation. In an effort to develop drugs with a safer profile for the stomach, a new form of cyclooxygenase was discovered. Subsequently and with the development of cloning strategies, cyclooxygenase-2 was cloned and characterized to have a profile of induction associated with the inflammatory reaction. This provided the rationale to target cox-2 enzyme and development of cox-2 selective drugs such as Vioxx and Celebrex. Coxibs were initially a successful treatment for arthritic patients also providing a reduction in gastric ulceration compared to traditional NSAIDs. Further investigations on the drug response to coxibs revealed a detrimental effect; the increase of myocardial infarctions, and the withdrawal of Vioxx from the market. The current theory to explain the harmful effect of coxib suggests the disruption of the platelet-endothelium interaction and selective inhibition of endothelium cox-2 activity depriving the cardiovascular system of vascular prostacyclin with anti-aggregatory activity. The balancing prostanoid theory to explain coxib cardiovascular complications was recently opposed. Recent investigations of Aspirin drug response have unraveled genetic variations in the cox-1 gene that are associated with the occurrence of Aspirin sensitivity or lack of protections against cardiovascular accidents. Screening for cox-1 gene variants will identify susceptible patients and reduce undesirable side-effects associated with Aspirin. Here we review recent findings in the cyclooxygenase-1 pathway and potential impact for the development of therapeutics that would segregate antithrombotic benefit from bleeding risk. Over 100 years following the initial use of Aspirin, cyclooxygenase inhibitors continue to be instrumental in our understanding of cardiovascular homeostasis and how the cyclooxygenase pathways are disrupted in disease.