A. K. Rao

Decreased platelet expression of myosin regulatory light chain polypeptide (MYL9) and other genes with platelet dysfunction and CBFA2/RUNX1 mutation: insights from platelet expression profiling

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 patients 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.

Mapping the platelet proteome: a report of the ISTH Platelet Physiology Subcommittee

Summary.  Proteomic technology has the potential to transform the way we analyze platelet biology, through the determination of platelet protein composition and its modification upon stimulation and with disease. We are a considerable way from achieving these goals, however, because of significant limitations in current methodology. It is therefore important to consider the extent to which these aims can be met and the way that proteomic data should be presented and used. These issues are discussed in the present paper by the Platelet Physiology Subcommittee of the ISTH Scientific Standardisation Committee (SSC). It is recommended that proteomic information be combined with data from other experimental approaches to establish a database on protein expression and function in platelets.

Mechanism of Platelet Factor 4 (PF4) Deficiency with RUNX1 Haplodeficiency: RUNX1 is a Transcriptional Regulator of PF4

Background: Platelet factor 4 (PF4) is an abundant protein stored in platelet α‐granules. Several patients have been described with platelet PF4 deficiency, including the gray platelet syndrome, characterized by a deficiency of α‐granule proteins. Defective granule formation and protein targeting are considered to be the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in the transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its non‐allelic variant PF4V1. Objectives: To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Methods/results: Chromatin immunoprecipitation and gel‐shift assays with phorbol 12‐myristate 13‐acetate‐treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at −1774/−1769 and −157/−152 on the PF4 promoter. In luciferase reporter studies in HEL cells, mutation of each site markedly reduced activity. PF4 promoter activity and PF4 protein level were decreased by small interfering RNA RUNX1 knockdown and increased by RUNX1 overexpression. Conclusions: Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes) levels, we postulate additional defects in RUNX1‐regulated genes involved in vesicular trafficking. These studies advance our understanding of the mechanisms in α‐granule deficiency.

Early growth response transcription factor EGR-1 regulates Gαq gene in megakaryocytic cells

Summary.  Background: Gαq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. Objectives: We studied Gαq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with phorbol 12‐myristate 13‐acetate (PMA) for 24 h to induce megakaryocytic transformation. Methods and results: PMA‐treated HEL cells showed enhanced Gαq expression. Reporter (luciferase) gene studies on 5′ upstream construct (up to −116 bp from ATG) revealed a negative regulatory site at −238/−202 and two positive sites at −203/−138 and −1116/−731. The positive regulatory region −203/−138 contained overlapping Sp1/AP‐2/EGR‐1 consensus sites. Gel shift studies on Gαq oligonucleotides 1 (−203/−175) and 2 (−174/−152) using HEL cell extracts demonstrated protein binding that was due to early growth response factor EGR‐1 at two sites. Mutations in either EGR‐1 site markedly decreased the gene activity, indicating functional relevance. Mutation of consensus E‐Box motif (−185/−180) had no effect. Reduction in the expression of endogenous EGR‐1 with antisense oligonucleotide to EGR‐1 inhibited PMA‐induced Gαq transcription. Correspondingly, Egr‐1 deficient mouse platelets also showed ∼50% reduction in the Gαq expression relative to wild‐type platelets. Conclusions: These studies suggest that Gαq gene is regulated during PMA‐induced megakaryocytic differentiation by EGR‐1, an early growth response transcription factor that regulates a wide array of genes and plays a major role in diverse activities, including cell proliferation, differentiation and apoptosis, and in vascular response to injury and atherosclerosis.

Dysregulation of PLDN (pallidin) is a mechanism for platelet dense granule deficiency in RUNX1 haplodeficiency.

Essentials Platelet dense granule (DG) deficiency is a major abnormality in RUNX1 haplodeficiency patients. The molecular mechanisms leading to the platelet DG deficiency are unknown. Platelet expression of PLDN (BLOC1S6, pallidin), involved in DG biogenesis, is regulated by RUNX1. Downregulation of PLDN is a mechanism for DG deficiency in RUNX1 haplodeficiency.

Platelet function analyzer (PFA)-100Rclosure time in the evaluation of platelet disorders and platelet function: reply to a rebuttal

C. P . M. HAYWA RD, * P . HARR ISON, M. C ATT ANEO, § T . L . ORT EL – and A . K . RAO** *Chair, Working Group on the PFA-100 , ISTH-SSC Platelet Physiology Subcommittee; McMaster University and the Hamilton Regional Laboratory Medicine Program, Hamilton, ON, Canada; Oxford Haemophilia Centre & Thrombosis Unit, Churchill Hospital, Oxford, UK; §Unità di Ematologia e Trombosi, Ospedale San Paolo, DMCO, Università di Milano, Milan, Italy; –Duke University Medical Center, Durham, NC, USA; **Chairperson, ISTH-SSC Platelet Physiology Subcommittee 2001–2004; and Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA

Hematopoietic transcription factor mutations: important players in inherited platelet defects

Transcription factors (TFs) are proteins that bind to specific DNA sequences and regulate expression of genes. The molecular and genetic mechanisms in most patients with inherited platelet defects are unknown. There is now increasing evidence that mutations in hematopoietic TFs are an important underlying cause for defects in platelet production, morphology, and function. The hematopoietic TFs implicated in patients with impaired platelet function and number include runt-related transcription factor 1, Fli-1 proto-oncogene, E-twenty-six (ETS) transcription factor (friend leukemia integration 1), GATA-binding protein 1, growth factor independent 1B transcriptional repressor, ETS variant 6, ecotropic viral integration site 1, and homeobox A11. These TFs act in a combinatorial manner to bind sequence-specific DNA within promoter regions to regulate lineage-specific gene expression, either as activators or repressors. TF mutations induce rippling downstream effects by simultaneously altering the expression of multiple genes. Mutations involving these TFs affect diverse aspects of megakaryocyte biology, and platelet production and function, culminating in thrombocytopenia and platelet dysfunction. Some are associated with predisposition to hematologic malignancies. These TF variants may occur more frequently in patients with inherited platelet defects than generally appreciated. This review focuses on alterations in hematopoietic TFs in the pathobiology of inherited platelet defects.

Spotlight on FLI1, RUNX1, and platelet dysfunction

In this issue of Blood, Stockley et al describe mutations in FLI1 and RUNX1, identified by next-generation sequencing (NGS) studies, in 6 of 13 patients with excessive bleeding and impaired platelet dense granule secretion, and highlight transcription factor (TF) mutations as an important mechanism for inherited platelet dysfunction.

Inherited thrombocytopenias: the beat goes on

In this issue of Blood, Bottega et al document mutations in ACTN1, which encodes the cytoskeletal protein α-actinin 1, in 10 of 239 consecutive probands with an inherited thrombocytopenia--making ACTN1 an important cause of familial thrombocytopenia.

The tale of two COXs

In this issue of Blood , Dragani and colleagues provide evidence in ET for increased reticulated platelets and for a role of COX-2 in the enhanced thromboxane A2 production. These findings highlight the need to rethink the optimum antithrombotic regimens in ET and other states with accelerated