Unravelling the disease mechanism for TSPYL1 deficiency.

We describe a lethal combined nervous and reproductive systems disease in three affected siblings of a consanguineous family. The phenotype was characterized by visceroautonomic dysfunction (neonatal bradycardia/apnea, feeding problems, hyperactive startle reflex), severe postnatal progressive neurological abnormalities (including abnormal neonatal cry, hypotonia, epilepsy, polyneuropathy, cerebral gray matter atrophy), visual impairment, testicular dysgenesis in males and sudden death at infant age by brainstem-mediated cardiorespiratory arrest. Whole-exome sequencing revealed a novel homozygous frameshift variant p.Val242GlufsTer52 in the TSPY-like 1 gene (TSPYL1). The truncated TSPYL1 protein that lacks the nucleosome assembly protein domain was retained in the Golgi of fibroblasts from the three patients, whereas control fibroblasts express full-length TSPYL1 in the nucleus. Proteomic analysis of nuclear extracts from fibroblasts identified 24 upregulated and 20 downregulated proteins in the patients compared with 5 controls with 'regulation of cell cycle' as the highest scored biological pathway affected. TSPYL1-deficient cells had prolonged S and G2 phases with reduced cellular proliferation rates. Tspyl1 depletion in zebrafish mimicked the patients' phenotype with early lethality, defects in neurogenesis and cardiac dilation. In conclusion, this study reports the third pedigree with recessive TSPYL1 variants, confirming that TSPYL1 deficiency leads to a combined nervous and reproductive systems disease, and provides for the first time insights into the disease mechanism.

The transcription factor GATA1 regulates NBEAL2 expression through a long-distance enhancer.

Gray platelet syndrome is named after the gray appearance of platelets due to the absence of α-granules. It is caused by recessive mutations in NBEAL2, resulting in macrothrombocytopenia and myelofibrosis. Though using the term gray platelets for GATA1 deficiency has been debated, a reduced number of α-granules has been described for macrothrombocytopenia due to GATA1 mutations. We compared platelet size and number of α-granules for two NBEAL2 and two GATA1-deficient patients and found reduced numbers of α-granules for all, with the defect being more pronounced for NBEAL2 deficiency. We further hypothesized that the granule defect for GATA1 is due to a defective control of NBEAL2 expression. Remarkably, platelets from two patients, and Gata1-deficient mice, expressed almost no NBEAL2. The differentiation of GATA1 patient-derived CD34+ stem cells to megakaryocytes showed defective proplatelet and α-granule formation with strongly reduced NBEAL2 protein and ribonucleic acid expression. Chromatin immunoprecipitation sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream of NBEAL2 covered by a H3K4Me1 mark indicative of an enhancer locus. Luciferase reporter constructs containing this region confirmed its enhancer activity in K562 cells, and mutagenesis of the GATA1 binding sites resulted in significantly reduced enhancer activity. Moreover, DNA binding studies showed that GATA1 and GATA2 physically interact with this enhancer region. GATA1 depletion using small interfering ribonucleic acid in K562 cells also resulted in reduced NBEAL2 expression. In conclusion, we herein show a long-distance regulatory region with GATA1 binding sites as being a strong enhancer for NBEAL2 expression.

Methylome analysis for spina bifida shows SOX18 hypomethylation as a risk factor with evidence for a complex (epi)genetic interplay to affect neural tube development.

BACKGROUND: Neural tube defects (NTDs) are severe congenital malformations that arise from failure of neurulation during early embryonic development. The molecular basis underlying most human NTDs still remains largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, DNA methylation changes could play a role in NTDs. We performed a methylome analysis for patients with myelomeningocele (MMC). Using a candidate CpG analysis for HOX genes, a significant association between HOXB7 hypomethylation and MMC was found. METHODS: In the current study, we analyzed leukocyte methylome data of ten patients with MMC and six controls using Illumina Methylation Analyzer and WateRmelon R-packages and performed validation studies using larger MMC and control cohorts with Sequenom EpiTYPER. RESULTS: The methylome analysis showed 75 CpGs in 45 genes that are significantly differentially methylated in MMC patients. CpG-specific methylation differences were next replicated for the top six candidate genes ABAT, CNTNAP1, SLC1A6, SNED1, SOX18, and TEPP but only for the SOX18 locus a significant overall hypomethylation was observed (P value = 0.0003). Chemically induced DNA demethylation in HEK cells resulted in SOX18 hypomethylation and increased expression. Injection of sox18 mRNA in zebrafish resulted in abnormal neural tube formation. Quantification of DNA methylation for the SOX18 locus was also determined for five families where parents had normal methylation values compared to significant lower values for both the MMC as their non-affected child. SOX18 methylation studies were performed for a MMC patient with a paternally inherited chromosomal deletion that includes BMP4. The patient showed extreme SOX18 hypomethylation similar to his healthy mother while his father had normal methylation values. CONCLUSIONS: This is the first genome-wide methylation study in leukocytes for patients with NTDs. We report SOX18 as a novel MMC risk gene but our findings also suggest that SOX18 hypomethylation must interplay with environmental and (epi)genetic factors to cause NTDs. Further studies are needed that combine methylome data with next-generation sequencing approaches to unravel NTD etiology.

A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.

The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.

Platelet studies in autism spectrum disorder patients and first-degree relatives.

BACKGROUND: Platelets have been proven to be a useful cellular model to study some neuropathologies, due to the overlapping biological features between neurons and platelets as granule secreting cells. Altered platelet dense granule morphology was previously reported in three autism spectrum disorder (ASD) patients with chromosomal translocations that disrupted ASD candidate genes NBEA, SCAMP5, and AMYSIN, but a systematic analysis of platelet function in ASD is lacking in contrast to numerous reports of elevated serotonin levels in platelets and blood as potential biomarker for ASD. METHODS: We explored platelet count, size, epinephrine-induced activation, and dense granule ATP secretion in a cohort of 159 ASD patients, their 289 first-degree relatives (103 unaffected siblings, 99 mothers, and 87 fathers), 45 adult controls, and 65 pediatric controls. For each of the responses separately, a linear mixed model with gender as a covariate was used to compare the level between groups. We next investigated the correlation between platelet function outcomes and severity of impairments in social behavior (social responsiveness score (SRS)). RESULTS: The average platelet count was increased in ASD patients and siblings vs. controls (ASD 320.3 × 10(9)/L, p = 0.003; siblings 332.0 × 10(9)/L, p < 0.001; controls 283.0 × 10(9)/L). The maximal platelet secretion-dependent aggregation response to epinephrine was not significantly lower for ASD patients. However, secondary wave responses following stimulation with epinephrine were more frequently delayed or absent compared to controls (ASD 52 %, siblings 45 %, parents 53 %, controls 22 %, p = 0.002). In addition, stimulated release of ATP from dense granules was reduced in ASD patients, siblings, and parents vs. controls following activation of platelets with either collagen (ASD 1.54 µM, p = 0.001; siblings 1.51 µM, p < 0.001; parents 1.67 µM, p = 0.021; controls 2.03 µM) or ADP (ASD 0.96 µM, p = 0.003; siblings 1.00 µM, p = 0.012; parents 1.17 µM, p = 0.21; controls 1.40 µM). Plasma serotonin levels were increased for ASD patients (n = 20, p = 0.005) and siblings (n = 20, p = 0.0001) vs. controls (n = 16). No significant correlations were found in the different groups between SRS scores and count, size, epinephrine aggregation, or ATP release. CONCLUSIONS: We report increased platelet counts, decreased platelet ATP dense granule secretion, and increased serotonin plasma levels not only in ASD patients but also in their first-degree relatives. This suggests that potential genetic factors associated with platelet counts and granule secretion can be associated with, but are not fully penetrant for ASD.

DNA methylation analysis of Homeobox genes implicates HOXB7 hypomethylation as risk factor for neural tube defects.

Neural tube defects (NTDs) are common birth defects of complex etiology. Though family- and population-based studies have confirmed a genetic component, the responsible genes for NTDs are still largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, epigenetic factors, such as DNA methylation, are predicted to be involved in NTDs. Homeobox (HOX) genes play a role in spinal cord development and are tightly regulated in a spatiotemporal and collinear manner, partly by epigenetic modifications. We have quantified DNA methylation for the different HOX genes by subtracting values from a genome-wide methylation analysis using leukocyte DNA from 10 myelomeningocele (MMC) patients and 6 healthy controls. From the 1575 CpGs profiled for the 4 HOX clusters, 26 CpGs were differentially methylated (P-value < 0.05; ß-difference > 0.05) between MMC patients and controls. Seventy-seven percent of these CpGs were located in the HOXA and HOXB clusters, with the most profound difference for 3 CpGs within the HOXB7 gene body. A validation case-control study including 83 MMC patients and 30 unrelated healthy controls confirmed a significant association between MMC and HOXB7 hypomethylation (-14.4%; 95% CI: 11.9-16.9%; P-value < 0.0001) independent of the MTHFR 667C>T genotype. Significant HOXB7 hypomethylation was also present in 12 unaffected siblings, each related to a MMC patient, suggestive of an epigenetic change induced by the mother. The inclusion of a neural tube formation model using zebrafish showed that Hoxb7a overexpression but not depletion resulted in deformed body axes with dysmorphic neural tube formation. Our results implicate HOXB7 hypomethylation as risk factor for NTDs and highlight the importance for future genome-wide DNA methylation analyses without preselecting candidate pathways.

NPC1 defect results in abnormal platelet formation and function: studies in Niemann-Pick disease type C1 patients and zebrafish.

Niemann-Pick type C is a lysosomal storage disease associated with mutations in NPC1 or NPC2, resulting in an accumulation of cholesterol in the endosomal-lysosomal system. Niemann-Pick type C has a clinical spectrum that ranges from a neonatal rapidly fatal disorder to an adult-onset chronic neurodegenerative disease combined with remarkably, in some cases, hematological defects such as thrombocytopenia, anemia and petechial rash. A role of NPC1 in hematopoiesis was never shown. Here, we describe platelet function abnormalities in three unrelated patients with a proven genetic and biochemical NPC1 defect. Their platelets have reduced aggregations, P-selectin expression and ATP secretions that are compatible with the observed abnormal alpha and reduced dense granules as studied by electron microscopy and CD63 staining after platelet spreading. Their blood counts were normal. NPC1 expression was shown in platelets and megakaryocytes (MKs). In vitro differentiated MKs from NPC1 patients exhibit hyperproliferation of immature MKs with different CD63(+) granules and abnormal cellular accumulation of cholesterol as shown by filipin stainings. The role of NPC1 in megakaryopoiesis was further studied using zebrafish with GFP-labeled thrombocytes or DsRed-labeled erythrocytes. NPC1 depletion in zebrafish resulted in increased cell death in the brain and abnormal cellular accumulation of filipin. NPC1-depleted embryos presented with thrombocytopenia and mild anemia as studied by flow cytometry and real-time QPCR for specific blood cell markers. In conclusion, this is the first report, showing a role of NPC1 in platelet function and formation but further studies are needed to define how cholesterol storage interferes with these processes.

Methylation defect in imprinted genes detected in patients with an Albright's hereditary osteodystrophy like phenotype and platelet Gs hypofunction.

BACKGROUND: Pseudohypoparathyroidism (PHP) indicates a group of heterogeneous disorders whose common feature is represented by impaired signaling of hormones that activate Gsalpha, encoded by the imprinted GNAS gene. PHP-Ib patients have isolated Parathormone (PTH) resistance and GNAS epigenetic defects while PHP-Ia cases present with hormone resistance and characteristic features jointly termed as Albright's Hereditary Osteodystrophy (AHO) due to maternally inherited GNAS mutations or similar epigenetic defects as found for PHP-Ib. Pseudopseudohypoparathyroidism (PPHP) patients with an AHO phenotype and no hormone resistance and progressive osseous heteroplasia (POH) cases have inactivating paternally inherited GNAS mutations. METHODOLOGY/PRINCIPAL FINDINGS: We here describe 17 subjects with an AHO-like phenotype that could be compatible with having PPHP but none of them carried Gsalpha mutations. Functional platelet studies however showed an obvious Gs hypofunction in the 13 patients that were available for testing. Methylation for the three differentially methylated GNAS regions was quantified via the Sequenom EpiTYPER. Patients showed significant hypermethylation of the XL amplicon compared to controls (36 ± 3 vs. 29 ± 3%; p<0.001); a pattern that is reversed to XL hypomethylation found in PHPIb. Interestingly, XL hypermethylation was associated with reduced XLalphaS protein levels in the patients' platelets. Methylation for NESP and ExonA/B was significantly different for some but not all patients, though most patients have site-specific CpG methylation abnormalities in these amplicons. Since some AHO features are present in other imprinting disorders, the methylation of IGF2, H19, SNURF and GRB10 was quantified. Surprisingly, significant IGF2 hypermethylation (20 ± 10 vs. 14 ± 7%; p<0.05) and SNURF hypomethylation (23 ± 6 vs. 32 6%; p<0.001) was found in patients vs. controls, while H19 and GRB10 methylation was normal. CONCLUSION/SIGNIFICANCE: In conclusion, this is the first report of methylation defects including GNAS in patients with an AHO-like phenotype without endocrinological abnormalities. Additional studies are still needed to correlate the methylation defect with the clinical phenotype.

Regulator of G-protein signaling 18 controls megakaryopoiesis and the cilia-mediated vertebrate mechanosensory system.

RGS18 was originally identified as a R4 subfamily member of regulators of G-protein signaling (RGS) with specific expression in hematopoietic progenitors, myeloerythroid cells, and megakaryocytes, though its physiological role in hematopoiesis remained unknown. Here, we show that lentiviral RGS18 overexpression during differentiation of mouse Sca1(+) hematopoietic stem cells induced a 50% increase of megakaryocyte proliferation. RGS18 depletion in zebrafish results in thrombocytopenia, as 66 to 88% of the embryos lack thrombocytes after injection of an ATG or splice-blocking morpholino, respectively. These embryos have no defects in early hematopoiesis, erythropoiesis, or leukocyte number and migration. In addition, all RGS18 depleted embryos have curly tails and an almost absent response to acoustic stimuli. In situ hybridization in zebrafish, Xenopus, and mouse embryos shows RGS18 expression in thrombocytes and/or hematological tissues but also in brain and otic vesicles. RGS18 interferes with development of cilia in hair cells of the inner ear and neuromast cells. On the basis of literature evidence that RGS-R4 members interact with the G-protein-modulated Wnt/calcium pathway, Wnt5b- but not Wnt5a-depleted embryos phenocopy all RGS18 knockdown effects. In summary, our study is the first to show that RGS18 regulates megakaryopoiesis but also reveals its unexpected role in ciliogenesis, at least in lower vertebrates, via interference with Wnt signaling.

No evidence for GNAS copy number variants in patients with features of Albright's hereditary osteodystrophy and abnormal platelet Gs activity.

Albright's hereditary osteodystrophy (AHO) is characterized by short stature, round face, calcifications, obesity, brachydactyly and intellectual disability. AHO without hormone resistance is called pseudopseudohypoparathyroidism (PPHP), a rare clinical condition difficult to diagnose with highly variable features. PPHP is caused by paternally inherited loss-of-function mutations in the GNAS. Patients with 2q37 microdeletions or HDAC4 mutations are also defined as having an AHO-like phenotype with normal stimulatory G (Gs) function. We have studied 256 patients with AHO features but no other diagnosis. Their platelet Gs activity was determined via the aggregation-inhibition test showing Gs hypo- or hyperfuncton in 24% and 15% of the patients, respectively. Before initiating with detailed (epi)genetic GNAS studies, we here wanted to excluded copy number variants (CNVs) in GNAS as cause of AHO with a novel large-scale screening technique. Multiplex amplicon quantification (MAQ) for CNVs screening was developed for the 20q13.3 region including GNAS and potential long-range imprinting control elements such as STX16. This is the first large-scale GNAS CNV study in patients with common AHO features but no CNVs were detected. In conclusion, CNVs in the GNAS region are not likely to cause an AHO-like phenotype with or without abnormal platelet Gs activity. Future studies will be undertaken to find out whether these AHO patients with abnormal Gs function are characterized by GNAS coding or methylation defects.

Severe gastrointestinal bleeding and thrombocytopenia in a child with an anti-GATA1 autoantibody.

We describe a patient, who developed during the first week of life petechiae and hematomas caused by severe thrombocytopenia and gastrointestinal bleeding due to multiple small gastric hemangiomata. Bone marrow examination showed hypermegakaryocytosis and dysmegakaryopoiesis. Alloimmune thrombocytopenia was excluded. Only 3 y later, platelet counts normalized and bleedings disappeared but small skin hemangiomata remained. Electron microscopy showed enlarged round platelets with a paucity of alpha granules similar as in GATA1-deficient patients but no GATA1 mutation was found. Immunoblot analysis showed a strong interaction between patient Igs and recombinant GATA1, GATA2, and the N finger (Nf) of GATA1. The lymphocyte transformation test with recombinant GATA1Nf was positive. In vitro culturing of normal CD34 cells with purified patient Igs showed a decreased number of megakaryocyte colonies but an increased overall size of the colonies compared with control Igs. Mice injected with patient Igs showed a reduced platelet count compared with mice injected with control Igs. Thrombopoiesis was also reduced after injection of patient Igs in transgenic zebrafish compared with control Igs. In conclusion, this study is the first report of an anti-GATA1 autoantibody leading to severe thrombocytopenia and gastrointestinal bleeding from multiple pinpoint hemangiomata.

GNAS defects identified by stimulatory G protein alpha-subunit signalling studies in platelets.

CONTEXT: GNAS is an imprinted region that gives rise to several transcripts, antisense transcripts, and noncoding RNAs, including transcription of RNA encoding the alpha-subunit of the stimulatory G protein (Gsalpha). The complexity of the GNAS cluster results in ubiquitous genomic imprints, tissue-specific Gsalpha expression, and multiple genotype-phenotype relationships. Phenotypes resulting from genetic and epigenetic abnormalities of the GNAS region include Albright's hereditary osteodystrophy, pseudohypoparathyroidism types Ia (PHPIa) and Ib (PHPIb), and pseudopseudohypoparathyroidism (PPHP). OBJECTIVE: The aim was to study the complex GNAS pathology by a functional test as an alternative to the generally used but labor-intensive erythrocyte complementation assay. DESIGN AND PATIENTS: We report the first platelet-based diagnostic test for Gsalpha hypofunction, supported by clinical, biochemical, and molecular data for six patients with PHPIa or PPHP and nine patients with PHPIb. The platelet test is based on the inhibition of platelet aggregation by cAMP, produced after Gsalpha stimulation. RESULTS: Platelets are easily accessible, and platelet aggregation responses were found to reflect Gsalpha signaling defects in patients, in concordance with the patient's phenotype and genotype. Gsalpha hypofunction in PHPIa and PPHP patients with GNAS mutations was clearly detected by this method. Mildly decreased or normal Gsalpha function was detected in patients with PHPIb with either an overall or exon 1A-only epigenetic defect, respectively. Platelet Gsalpha expression was reduced in both PHPIb patient groups, whereas XLalphas was up-regulated only in PHPIb patients with the broad epigenetic defect. CONCLUSION: The platelet-based test is a novel tool for establishing the diagnosis of Gsalpha defects, which may otherwise be quite challenging.

Compound heterozygous mutations in the GNAS gene of a boy with morbid obesity, thyroid-stimulating hormone resistance, pseudohypoparathyroidism, and a prothrombotic state.

CONTEXT: Pseudohypoparathyroidism type Ia and pseudopseudohypoparathyroidism are characterized by Albright's hereditary osteodystrophy (AHO), respectively, with and without hormone resistance. Both clinical conditions result from decreased expression or function of the alpha-subunit of the stimulatory G protein (Gsalpha) of adenylyl cyclase due to heterozygous inactivating mutations in GNAS. Homozygous GNAS defects have not been described. OBJECTIVE: A genetic and functional GNAS study was undertaken in a boy with morbid obesity (body mass index Z-score of 5 at the age of 3 yr, with a body fat fraction of 40%, which is more than twice normal), TSH resistance, pseudohypoparathyroidism, and a prothrombotic state. RESULTS: The boy was found to be a first case with a compound heterozygous GNAS defect: a de novo R231C mutation on the paternal allele and on the other allele a maternally inherited unique combination of three C to T nucleotide substitutions in exon 7 (I185I), intron 7 (IVS7 + 31), and exon 13 (N371N) leading to aberrant splicing of GNAS. Platelets of this boy displayed a pronounced Gsalpha hypofunction and were spontaneously hyperreactive resulting in a prothrombotic state due to extremely low cAMP levels. CONCLUSION: This report expands the human GNAS genotype-phenotype spectrum to include compound heterozygosity and a prothrombotic state.

PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications.

Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34(+) cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34(+) cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

Increased Gs signalling in platelets and impaired collagen activation, due to a defect in the dystrophin gene, result in increased blood loss during spinal surgery.

Controversy exists regarding the cause of the significantly increased blood loss during spinal surgery in Duchenne muscular dystrophy (DMD) patients compared with similar surgery in other patients. DMD is caused by a mutation in the cytoskeletal dystrophin, which binds to extracellular matrix laminin and which has been described as a G-protein-coupled receptor. We hypothesized that disturbed cytoskeleton organization in DMD patients would alter Gs protein and collagen signalling in platelets, leading to dysfunctional platelets and a haemorrhagic tendency during surgery. In the present study, we found that platelets and skin fibroblasts, respectively, express the Dp71 and Dp116 dystrophin isoforms. Absent or decreased expression of these isoforms in DMD patients correlates with significant Gs alpha upregulation. Moreover, dysfunctional dystrophin in these cells is accompanied with increased Gs signalling and higher cAMP levels after Gs stimulation. Functional analysis showed that DMD platelets responded slower to collagen with an extensive shape change in the aggregometer and with a significantly reduced platelet adhesion to coated collagen under flow. The decreased collagen activation was shown to result from both Gs activation and cytoskeletal disruption and not from decreased expression of platelet membrane receptors or impaired von Willebrand factor (vWF) activity. In conclusion, DMD platelets have a disorganized cytoskeleton and manifest Gs hyperactivity and reduced platelet collagen reactivity. Their increased bleeding during surgery will, at least partly, result from the increased platelet Gs activity after the release of natural Gs agonists as prostacyclin during surgery and an ineffective reactivity to collagen.

What's new in using platelet research? To unravel thrombopathies and other human disorders.

This review on platelet research focuses on defects of adhesion, cytoskeletal organisation, signal transduction and secretion. Platelet defects can be studied by different laboratory platelet functional assays and morphological studies. Easy bruising or a suspected platelet-based bleeding disorder is of course the most obvious reason to test the platelet function in a patient. However, nowadays platelet research also contributes to our understanding of human pathology in other disciplines such as neurology, nephrology, endocrinology and metabolic diseases. Apart from a discussion on classical thrombopathies, this review will also deal with the less commonly known relation between platelet research and disorders with a broader clinical phenotype. Classical thrombopathies involve disorders of platelet adhesion such as Glanzmann thrombastenia and Bernard-Soulier syndrome, defective G protein signalling diseases with impaired phospholipase C activation, and abnormal platelet granule secretion disorders such as gray platelet disorder and delta-storage pool disease. Other clinical symptoms besides a bleeding tendency have been described in MYH9-related disorders and Duchenne muscular dystrophy due to adhesion defects, and also in disorders of impaired Gs signalling, in Hermansky Pudlack disease and Chediak Higashi disease with abnormal secretion. Finally, platelet research can also be used to unravel novel mechanisms involved in many neurological disorders such as depression and autism with only a subclinical platelet defect.

Mechanisms of action and targets for actual and future antiplatelet drugs.

Platelets are key players in arterial thrombosis and have become important targets in the primary and secondary prevention of atherothrombosis. Antiplatelet drugs are primarily directed against platelets and inhibit platelet activation by a number of different mechanisms. They are used for the prevention and treatment of thrombotic processes, especially in the arterial vascular system. Antiplatelet drugs in clinical use and experimental drugs are discussed.

The TUBB1 Q43P functional polymorphism reduces the risk of cardiovascular disease in men by modulating platelet function and structure.

The discoid form of platelets is maintained by a marginal band of tightly coiled microtubules. beta1-tubulin is the major isoform within platelet and megakaryocyte microtubules. In 24.2% of 33 unrelated inherited macrothrombocytopenia patients and in 10.6% of 272 subjects of a healthy population a P for Q substitution in beta1-tubulin was found in the highly conserved residue 43. Heterozygous carriers of the Q43P variant showed a reduced platelet protein beta1-tubulin expression. Transfection of green fluorescent protein (GFP)-tagged Q43P beta1-tubulin in megakaryocytic MEG01 cells resulted in a disturbed tubulin organization. Electron microscopy revealed enlarged spherocytic platelets with a disturbed marginal band and organelle-free zones. In addition, platelets with the Q43P beta1-tubulin variant had reduced adenosine triphosphate (ATP) secretion, thrombin receptor activating peptide (TRAP)-induced aggregation and collagen adhesion. The prevalence of the Q43P beta1-tubulin variant was also 2 times higher (odds ratio, [OR] = 2.1;95% confidence interval [CI], 1.22-3.59) among control subjects than among patients with cardiovascular disease (10.4% versus 5.2%, P < .001). By analyzing this protective factor in men and women separately, this association was only found in men. This study thus presents the functional consequences of the platelet Q43P beta1-tubulin substitution that is frequent in the healthy population and may protect men against arterial thrombosis.

The pituitary adenylate cyclase-activating polypeptide is a physiological inhibitor of platelet activation.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide of the vasoactive intestinal peptide/secretin/glucagon superfamily. Studies in two related patients with a partial trisomy 18p revealed three copies of the PACAP gene and elevated PACAP concentrations in plasma. The patients suffer from severe mental retardation and have a bleeding tendency with mild thrombocytopenia, and their fibroblasts show increased PACAP mRNA levels. The PACAP receptor (vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 [VPAC1]) in platelets and fibroblasts is coupled to adenylyl cyclase activation. Accordingly, we found increased basal cAMP levels in patients' platelets and fibroblasts, providing a basis for the reduced platelet aggregation in these patients. Megakaryocyte-specific transgenic overexpression of PACAP in mice correspondingly increased PACAP release from platelets, reduced platelet activation, and prolonged the tail bleeding time. In contrast, the PACAP antagonist PACAP(6-38) or a monoclonal PACAP antibody enhanced the collagen-induced aggregation of normal human platelets, and in PACAP knockout mice, an increased platelet sensitivity toward collagen was found. Thus, we found that PACAP modulates platelet function and demonstrated what we believe to be the first hemostatic defect associated with PACAP overexpression; our study suggests the therapeutic potential to manage arterial thrombosis or bleeding by administration of PACAP mimetics or inhibitors, respectively.

Functional polymorphisms in the paternally expressed XLalphas and its cofactor ALEX decrease their mutual interaction and enhance receptor-mediated cAMP formation.

The paternally expressed extra-large stimulatory G protein gene (XLalphas) is a splice variant of the stimulatory G-protein gene (Gsalpha) consisting of XL-exon1 and exons 2-13 of Gsalpha. A second open reading frame (ORF) in XL-exon1, that completely overlaps the XL-domain ORF, encodes ALEX, which is translated from the XLalphas mRNA and binds the XL-domain of XLalphas. We previously demonstrated that a paternally inherited functional polymorphism in XL-exon1, consisting of a 36 bp insertion and two nucleotide substitutions, is associated with Gs hyperfunction in platelets, leading to an increased trauma-related bleeding tendency and is accompanied by neurological problems and brachydactyly in two families. Here, we describe eight additional patients with brachydactyly, who inherited the same XLalphas polymorphism paternally and who show Gs hyperfunction in their platelets and fibroblasts. All carriers also have an elongated ALEX protein, as a consequence of the paternally inherited insertion. The in vitro interaction between the two elongated XLalphas and ALEX proteins is markedly reduced. Moreover, XLalphas or ALEX can be co-immunoprecipitated with an antibody against either ALEX or XLalphas in platelets from a control but hardly from patients with the XLalphas/ALEX insertion. In contrast to the strong interaction between the two wild-type proteins, we suggest that this defective association results in unimpeded receptor-stimulated activation of XLalphas. The paternally inherited double XLalphas/ALEX functional polymorphism is also associated with elevated platelet membrane Gsalpha protein levels. Both phenomena contribute to increased Gs signaling in patients with platelet hypersensitivity towards Gs-agonists and may be accompanied by neurological problems or growth deficiency.