Zinc (Zn 2+ ) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn 2+ storage and release. To visualize Zn 2+ storage in human and mouse platelets, the Zn 2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d −/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn 2+ release upon activation. Platelets from Nbeal2 −/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn 2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn 2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2 −/− and Unc13d −/− mice, and the impairment could be partially restored by extracellular Zn 2+ . Altogether, we conclude that the release of ionic Zn 2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn 2+ in platelet-dependent fibrin formation.

The investigators explore the role of PDK1 (phosphoinositide-dependent protein kinase 1) in the cytoskeletal regulation of platelet production and furnish new insights into megakaryocyte maturation and proplatelet formation.

Thrombocytopenia absent radii (TAR) syndrome is clearly defined by the combination of radial aplasia and reduced platelet counts. The genetics of TAR syndrome has recently been resolved and comprises a microdeletion on Chromosome 1 including the RBM8A gene and a single nucleotide polymorphism (SNP) either at the 5' untranslated region (5'UTR) or within the first intron of RBM8A. Although phenotypically readily diagnosed after birth, the genetic determination of particular SNPs in TAR syndrome harbours valuable information to evaluate disease severity and treatment decisions. Here, we present clinical data in a cohort of 38 patients and observed that platelet counts in individuals with 5'UTR SNP are significantly lower compared to patients bearing the SNP in intron 1. Moreover, elevated haemoglobin values could only be assessed in patients with 5'UTR SNP whereas white blood cell count is unaffected, indicating that frequently observed anaemia in TAR patients could also be SNP-dependent whereas leucocytosis does not correlate with genetic background. However, this report on a large cohort provides an overview of important haematological characteristics in TAR patients, facilitating evaluation of the various traits in this disease and indicating the importance of genetic validation for TAR syndrome.

Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.

BackgroundAcute myeloid leukemia with complex karyotype (CK-AML) is a distinct biological entity associated with a very poor outcome. Since complex karyotypes frequently contain deletions of the chromosomal region 12p13 encompassing the tumor suppressor genes ETV6 and CDKN1B, we aimed to unravel their modes of inactivation in CK-AML.ResultsTo decipher deletions, mutations and methylation of ETV6 and CDKN1B, arrayCGH, SNP arrays, direct sequencing of all coding exons and pyrosequencing of the 5′UTR CpG islands of ETV6 and CDKN1B were performed. In total, 39 of 79 patients (49%) showed monoallelic deletions of 12p13 according to karyotypic data and 20 of 43 patients (47%) according to genomic profiling. Genomic profiling led to the minimal deleted region covering the 3′-UTR of ETV6 and CDKN1B. Direct sequencing revealed one novel monoallelic frameshift mutation in ETV6 while no mutations in CDKN1B were identified. Furthermore, methylation levels of ETV6 and CDKN1B did not indicate transcriptional silencing of any of these genes. ETV6 and CDKN1B had reduced expression levels in CK-AML patients with deletion in 12p13 as compared to CK-AML without deletion in 12p13, while the other genes (BCL2L14, LRP6, DUSP16 and GPRC5D) located within the minimal deleted region in 12p13 had very low or missing expression in CK-AML irrespective of their copy number status.ConclusionsETV6 and CDKN1B are mainly affected by small monoallelic deletions, whereas mutations and hypermethylation play a minor role in CK-AML. Reduced gene dosage led to reduced gene expression levels, pointing to haploinsufficiency as the relevant mechanism of inactivation of ETV6 and CDKN1B in CK-AML.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-784) contains supplementary material, which is available to authorized users.