High-throughput sequencing for diagnosing platelet disorders: lessons learned from exploring the causes of bleeding disorders.

Inherited platelet disorders (IPDs) are a heterogeneous group of disorders caused by multiple genetic defects. Obtaining a molecular diagnosis for IPD patients using a phenotype- and laboratory-based approach is complex, expensive, time-consuming, and not always successful. High-throughput sequencing (HTS) methods offer a genotype-based approach to facilitate molecular diagnostics. Such approaches are expected to decrease time to diagnosis, increase the diagnostic rate, and they have provided novel insights into the genotype-phenotype correlation of IPDs. Some of these approaches have also focused on the discovery of novel genes and unexpected molecular pathways which modulate megakaryocyte and platelet biology were discovered. A growing number of genetic defects underlying IPDs have been identified and we will here provide an overview of the diverse molecular players. Screening of these genes will deliver a genetic diagnosis for about 40%-50% of the IPDs patients and we will compare different HTS applications that have been developed. A brief focus on gene variant interpretation and classification in a diagnostic setting will be given. Although it is true that successes in diagnostics and gene discovery have been reached, a large fraction of patients still remains without a conclusive diagnosis. In these patients, the sum of non-diagnostic variants in known genes or in potential novel genes might only be proven informative in future studies with larger patient cohorts and by data sharing among the diverse genome medicine initiatives. Finally, we still do not understand the role of the non-coding genome space for IPDs.

High-throughput sequencing approaches for diagnosing hereditary bleeding and platelet disorders.

Hereditary bleeding and platelet disorders (BPDs) are characterized by marked genetic heterogeneity, far greater than previously appreciated. The list of genes involved in the regulation of megakaryopoiesis, platelet formation, platelet function and bleeding has been growing rapidly since the introduction of high-throughput sequencing (HTS) approaches in research. Thanks to the gradual adoption of HTS in diagnostic practice, these discoveries are improving the diagnostic yield for BPD patients, who may or may not present with bleeding problems and often have other clinical symptoms unrelated to the blood system. However, it was previously found that screening for all known etiologies gives a diagnostic yield of over 90% when the phenotype closely matches a known BPD but drops to 10% when the phenotype is indicative of a novel disorder. Thus, further research is needed to identify currently unknown etiologies for BPDs. Novel genes are likely to be found to be implicated in BPDs. New modes of inheritance, including digenic inheritance, are likely to play a role in some cases. Additionally, identifying and interpreting pathogenic variants outside exons is a looming challenge that can only be tackled with an improved understanding of the regulatory landscape of relevant cell types and with the transition from targeted sequencing to whole-genome sequencing in the clinic.

The effect of paternal methyl-group donor intake on offspring DNA methylation and birth weight.

Most nutritional studies on the development of children focus on mother-infant interactions. Maternal nutrition is critically involved in the growth and development of the fetus, but what about the father? The aim is to investigate the effects of paternal methyl-group donor intake (methionine, folate, betaine, choline) on paternal and offspring global DNA (hydroxy)methylation, offspring IGF2 DMR DNA methylation, and birth weight. Questionnaires, 7-day estimated dietary records, whole blood samples, and anthropometric measurements from 74 fathers were obtained. A total of 51 cord blood samples were collected and birth weight was obtained. DNA methylation status was measured using liquid chromatography-tandem mass spectrometry (global DNA (hydroxy)methylation) and pyrosequencing (IGF2 DMR methylation). Paternal betaine intake was positively associated with paternal global DNA hydroxymethylation (0.028% per 100 mg betaine increase, 95% CI: 0.003, 0.053, P=0.03) and cord blood global DNA methylation (0.679% per 100 mg betaine increase, 95% CI: 0.057, 1.302, P=0.03). Paternal methionine intake was positively associated with CpG1 (0.336% per 100 mg methionine increase, 95% CI: 0.103, 0.569, P=0.006), and mean CpG (0.201% per 100 mg methionine increase, 95% CI: 0.001, 0.402, P=0.049) methylation of the IGF2 DMR in cord blood. Further, a negative association between birth weight/birth weight-for-gestational age z-score and paternal betaine/methionine intake was found. In addition, a positive association between choline and birth weight/birth weight-for-gestational age z-score was also observed. Our data indicate a potential impact of paternal methyl-group donor intake on paternal global DNA hydroxymethylation, offspring global and IGF2 DMR DNA methylation, and prenatal growth.

The amplitude of coagulation curves from thrombin time tests allows dysfibrinogenemia caused by the common mutation FGG-Arg301 to be distinguished from hypofibrinogenemia.

INTRODUCTION: Thrombin time (TT) tests are useful for diagnosing coagulation disorders involving abnormal fibrinogen but do not allow us to distinguish between qualitative and quantitative defects. However, with the widening availability of optical coagulation automates, more information about the coagulation process is becoming increasingly accessible. METHODS: In this study, we compared the coagulation curves of TT tests carried out with plasma from healthy donors with those from patients with acquired low Clauss fibrinogen levels or with dysfibrinogenemia caused by a heterozygous point mutation in the fibrinogen γ-chain that results in a p.Arg301(275)Cys substitution. The functional fibrinogen levels of these three groups of samples were also measured with the Clauss method, and their fibrinogen protein levels were determined by ELISA. RESULTS: Our data indicate that the amplitude and maximal velocity of coagulation curves from plasma samples from FGG p.Arg301(275)Cys dysfibrinogenemic patients were comparable to those from plasma samples with fibrinogen in the normal range, whereas the amplitude of coagulation curves from patients with acquired low fibrinogen levels was lower. CONCLUSIONS: Examination of the amplitude of coagulation curves generated during TT tests may provide additional information to enable the differential diagnoses of diseases following a low fibrinogen measurement by the Clauss method.

DNA methylation in imprinted genes IGF2 and GNASXL is associated with prenatal maternal stress.

Epigenetic regulation of imprinted genes during embryonic development is influenced by the prenatal environment. Our aim was to examine the effect of maternal emotional stress and cortisol levels during pregnancy on methylation of imprinted genes, insulin-like growth factor 2 (IGF2) and guanine nucleotide-binding protein, alpha stimulating extra-large (GNASXL), using umbilical cord blood DNA. Maternal depressed mood (Edinburgh Depression Scale; EDS), pregnancy-related anxiety questionnaire (PRAQ) and cortisol day profiles were assessed throughout pregnancy. At birth, a cord blood sample (n = 80) was taken to study DNA methylation of IGF2 DMR0 (differentially methylated region), IGF2 anti-sense (IGF2AS) and GNASXL using Sequenom EpiTYPER. Linear mixed models were used to examine the relationship between DNA methylation and maternal stress, while correcting for confounders. We also studied the association of DNA methylation with the child ponderal index at birth. We found a cytosine-guanine dinucleotide (CpG)-specific association of PRAQ subscales with IGF2 DMR0 (CpG5, P < 0.0001) and GNASXL (CpG11, P = 0.0003), while IGF2AS was associated with maternal EDS scores (CpG33, P = 0.0003) and cortisol levels (CpG33, P = 0.0006; CpG37-38, P = 0.0005). However, there was no association of methylation with ponderal index at birth. In conclusion, maternal stress during pregnancy, as defined by cortisol measurements, EDS and PRAQ scores, is associated with DNA methylation of imprinted genes IGF2 and GNASXL. Our results provide further evidence that prenatal adversity can influence imprinted gene methylation, although future studies are needed to unravel the exact mechanisms.

Pituitary adenylate cyclase-activating polypeptide deficiency associated with increased platelet count and aggregability in nephrotic syndrome.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) was recently identified as an inhibitor of megakaryopoiesis and platelet aggregability. OBJECTIVE: We studied PACAP levels in children with nephrotic syndrome (NS), which is associated with thrombocytosis, platelet hyperaggregability, and an increased risk of thrombosis. PATIENTS/METHODS: In four children with congenital NS (CNS) and 24 children with idiopathic NS (INS), plasma and urine levels of PACAP and ceruloplasmin were measured, as were platelet counts and platelet aggregation responses to collagen. In CNS patients, in vitro megakaryopoiesis and nuclear factor-κB expression in platelet lysates were also measured. All tests were performed during the nephrotic state and the non-nephrotic state. RESULTS: Urinary losses of PACAP and ceruloplasmin were observed during the nephrotic state, and disappeared during the non-nephrotic state. Plasma PACAP deficiency was more pronounced in CNS patients than in INS patients. Thrombocytosis was observed in all CNS patients and in 11 of 29 INS patients during the nephrotic state. During the PACAP-deficient state, in vitro megakaryopoiesis was increased for CNS patients, and this effect could be reversed by the addition of recombinant PACAP. Platelet hyperaggregability was observed during the nephrotic state in both CNS and INS patients. In INS patients, the addition of recombinant PACAP to patients' platelets was studied, and resulted in decreased aggregation during the nephrotic state. Platelet aggregation correlated inversely with plasma PACAP levels, but not with serum albumin levels. CONCLUSIONS: We demonstrate urinary losses of PACAP and plasma PACAP deficiency in children with NS, associated with thrombocytosis and platelet hyperaggregability.

Update on the causes of platelet disorders and functional consequences.

Platelets are derived from megakaryocytes in the bone marrow that create the cellular machinery the platelet needs to participate in the different processes of primary hemostasis including adhesion, activation and clot formation at the site of injury. Defects related to megakaryocyte differentiation, platelet formation, and/or platelet function can result in bleeding. Patients with thrombopathies can present with mucous membrane bleeding but may also present with bleeding following trauma or surgery. In this review, we have classified inherited platelet bleeding disorders (IPD) according to their underlying defective pathway: transcription regulation, TPO signaling, cytoskeletal organization, apoptosis, granule trafficking, and receptor signaling. Platelet function testing has provided insights into the underlying molecular defects that can result in bleeding. A major step forward was made during the last 3 years using new-generation genetic approaches that resulted in the discovery of novel genes such as NBEAL2, RBM8A, ACTN1, and GFI1B for the well-known IPD that cause gray platelet syndrome, thrombocytopenia-absent radius syndrome, and autosomal dominant thrombocytopenias, respectively. In the near future, it is expected that a similar approach will identify many novel genes that cause IPD of unknown etiology, which are common. The future challenge will be to use a functional, systems biology approach to study the genes mutated in IPD and determine their roles in megakaryocyte and platelet biology and pathology.

Proteomic analysis of platelet N-glycoproteins in PMM2-CDG patients.

UNLABELLED: PMM2-CDG, the most frequent congenital disorder of N-glycosylation, is an autosomal recessive disease with a multisystem presentation. PMM2-CDG patients show an increased risk for thrombosis, which might be in part due to spontaneous platelet aggregations as previously described. A potential hypoglycosylation of platelet proteins in these patients might explain this increased reactivity, as removal of sialic acid from platelets, particularly of GPIbα, leads to enhance platelet aggregation and clearance from the circulation. This study is the first one that has evaluated the glycosylation status of platelet proteins in 6 PMM2-CDG patients using different approaches including immunoblot, RCA120 lectin binding to platelets and expression of different membrane platelet N-glycoproteins by flow cytometry, as well as by platelet N-glycoproteome analysis. RCA120 lectin binding to the platelet membrane of PMM2-CDG patients showed evidence for decreased sialic acid content. However, immunoblot and flow cytometric analysis of different platelet N-glycoproteins, together with the more sensitive 2D-DIGE analysis, suggest that platelet N-glycoproteins, including GPIbα, seem to be neither quantitatively nor qualitatively significantly affected. The increased binding of RCA120 lectin could be explained by the abnormal glycosylation of hepatic proteins being attached to the platelets. CONCLUSIONS: This is the first study that has evaluated the platelet N-glycoproteome. Our findings suggest that platelet proteins are not significantly affected in PMM2-CDG patients. Further studies are still warranted to unravel the mechanism(s) that increase(s) the risk of thrombosis in these patients.

Regulators of G protein signaling: role in hematopoiesis, megakaryopoiesis and platelet function.

Regulators of G protein signaling (RGS) are intracellular signaling regulators that bind activated G protein α subunits (Gα) and increase their intrinsic GTPase activity via their common RGS homology domain. In addition to their GTPase accelerating activity (GAP), RGS proteins also contain other domains that regulate their receptor selectivity, their interaction with other proteins such as adenylyl cyclase or their subcellular localization via interaction with scaffold proteins such as tubulin, 14-3-3 or spinophilin. There are at least 37 different RGS family members in humans and numerous physiological functions have been assigned to these proteins, which have rather a tissue-specific expression pattern. The role of some RGS proteins was shown to be important for hematopoiesis. More recent studies also focused on their expression in platelets, and for R4 RGS subfamily members RGS2, RGS16 and RGS18, it could be demonstrated that they regulate megakaryopoiesis and/or platelet function. These functional studies mostly comprised in vitro experiments and in vivo studies using small animal models. Their role in human pathology related to platelet dysfunction remains still largely unknown, except for a case report with a RGS2 gain of function mutation. In addition to an introduction on RGS signaling and different effectors with a special focus on the R4 subfamily members, we here will give an overview of the studies related to the role of RGS proteins in hematopoiesis, megakaryopoiesis and platelet function.

Proteomics to unravel platelet-related diseases and identify novel anti-platelet drugs.

Blood platelets play a fundamental role in primary haemostasis and wound repair, but are also involved in several thrombotic and bleeding disorders for which the underlying mechanisms are still largely unknown. Elucidating platelet biology would help in finding novel disease biomarkers and drug targets in complex and/or genetically unknown platelet-related disorders. Proteomics, which allows studying thousands of gene products at once, represents an efficient tool to quali-quantitatively analyze and compare the platelet protein patterns of different samples (i.e. control/patient, treated/untreated, drug sensitive/resistant), to investigate post-translation modifications, protein-protein interactions and the underlying molecular pathways. This review gives an overview of the applications of proteomic strategies to study platelet biology and function, as well as to unravel differences in protein expression according to specific platelet conditions (i.e. basic versus activated), compartments (i.e. membrane or granules) and fractions (i.e. phosphoproteins and glycoproteins). The use of innovative powerful proteomic technologies can lead to the identification of proteins whose expression is altered in pathological conditions, allowing the identification of candidate biomarkers for: i) understanding the molecular defects underlying platelet disorders, ii) obtaining novel insights in more complex diseases that involve platelets, iii) unraveling the drug mode of action or identifying the mechanisms of drug resistance and iv) detecting novel therapeutic antiplatelet targets based on fundamental platelet research studies. Several studies on how proteomics proved to be useful in our understanding of platelet function and its diseases are discussed. Eventually, this could result in the discovery of novel drug targets for antiplatelet therapy.

Inherited platelet disorders.

Inherited diseases of the megakaryocyte lineage give rise to bleeding when platelets fail to fulfill their hemostatic function upon vessel injury. Platelet defects extend from the absence or malfunctioning of adhesion (GPIb-IX-V, Bernard-Soulier syndrome) or aggregation receptors (integrin αIIbß3, Glanzmann thrombasthenia) to defects of primary receptors for soluble agonists, secretion from storage organelles, activation pathways and the generation of procoagulant activity. In disorders such as the Chediak-Higashi, Hermansky-Pudlak, Wiskott-Aldrich and Scott syndromes the molecular lesion extends to other cells. In familial thrombocytopenia (FT), platelets are produced in insufficient numbers to assure hemostasis. Some FT affect platelet morphology and give rise to the 'giant platelet' syndromes (e.g. MYH9-related diseases) with changes in megakaryocyte maturation within the bone marrow and premature release of platelets. Diseases of platelet production may also affect other cells and in some cases interfere with development and/or functioning of major organs. Diagnosis of platelet disorders requires platelet function testing, studies often aided by the quantitative analysis of receptors by flow cytometry and fluorescence and electron microscopy. New generation DNA-based procedures including whole exome sequencing offer an exciting new perspective. Transfusion of platelets remains the most common treatment of severe bleeding, management with desmopressin is often used for mild disorders. Substitute therapies are available including rFVIIa and the potential use of thrombopoietin analogues for FT. Stem cell or bone marrow transplantation has been successful for several diseases while gene therapy shows promise in the Wiskott-Aldrich syndrome.

Recent advances in GNAS epigenetic research of pseudohypoparathyroidism.

Endocrinopathies in patients with hypocalcemia and hyperphosphatemia that share resistance to parathyroid hormone (PTH) are grouped under the term pseudohypoparathyroidism (PHP). Patients with PHP type Ia (PHP-Ia) often present with additional hormonal resistance and show characteristic physical features that are jointly termed as having an Albright's hereditary osteodystrophy (AHO) phenotype. Alternatively, PHPIb patients predominantly have PTH and sometimes TSH resistance but do not present with AHO features. Most of these PHP forms are caused by defects in GNAS, an imprinted gene locus consisting of maternal, paternal and biallelic transcripts. PHP-Ia is caused by heterozygous inactivating mutations in those exons of GNAS encoding the alpha subunit of the stimulatory guanine nucleotide-binding protein (Gsalpha) while PHPIb results from epigenetic GNAS defects. Familial and sporadic forms of PHP-Ib have distinct GNAS imprinting patterns: familial PHP-Ib patients have an exon A/B-only imprinting defect whereas sporadic PHP-Ib cases have abnormal imprinting of the three differentially methylated regions (DMRs) in GNAS. This classification of PHP was made years ago but was recently questioned since different studies showed GNAS epigenetic defects in PHP-Ia patients. In this review, we focus on the epigenetic description and screening methods of GNAS, the associated pathology and the recent need for a PHP reclassification.

Regulators of platelet cAMP levels: clinical and therapeutic implications.

Platelets are indispensable for primary haemostasis, but their function needs to be tightly regulated to prevent excessive platelet activity, possibly leading to atherothrombotic events. An important mediator of the platelet activity is cyclic AMP (cAMP), which inhibits platelet aggregation. Intracellular cAMP levels are regulated via the Gs and Gi alpha subunits of heterotrimeric G proteins, which couple to adenylyl cyclase to respectively stimulate or inhibit cAMP production. Binding of a ligand to its G protein-coupled seven-transmembrane receptor activates these G proteins. In this review, we discuss a Gs-coupled receptor on platelets, VPAC1, and 2 important Gi-coupled receptors, the ADP receptor P2Y(12) and the prostaglandin E(2) receptor EP3. The regulation of platelet cAMP levels at the level of the receptors themselves or the G proteins coupled to them is analyzed. Alterations in Gsα and Giα function are associated with altered platelet reactivity. An increase in Gs function, or alternatively a defective Gi signaling, can be a risk factor for bleeding, while a loss of Gs function can result in a prothrombotic state. Regulator of G protein signaling (RGS) proteins accelerate the rate of inactivation of G protein-mediated signaling. One of the RGS proteins, RGS2, inhibits Gs signaling by interacting directly with adenylyl cyclase. The thienopyridine class of antiplatelet agents is based on cAMP-mediated regulation of platelet function through modification of the P2Y(12) receptor. Clopidogrel and some other novel cAMP regulators are discussed. Secondly, we review the use of prostacyclin derivatives to treat pulmonary arterial hypertension.

Platelet Gs hypofunction and abnormal morphology resulting from a heterozygous RGS2 mutation.

SUMMARY BACKGROUND: Regulator of G-protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. OBJECTIVE: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. METHODS: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild-type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. RESULTS: The mutation leads to reduced cAMP production after stimulation of Gs-coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild-type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal alpha-granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. CONCLUSIONS: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.

ADP-degrading enzymes inhibit platelet activation in bile duct-ligated rats.

BACKGROUND: The effect of cholestatic liver disease on primary hemostasis function remains ill-defined. OBJECTIVES: To determine platelet function and identify the mechanisms involved in the observed platelet function in cholestatic rats. METHODS: Platelet function was studied in a model of 2-week bile duct ligation and compared to that in sham-operated rats with and without a storage pool defect. RESULTS: ADP-induced and collagen-induced platelet aggregation were clearly impaired following bile duct ligation (P<0.01 for areas under the curve). Crossover experiments, with sham platelets in bile duct-ligated plasma and vice versa, demonstrated that this is due to inhibition by a plasmatic factor, as sham platelets aggregated less in cholestatic plasma (P<0.03) and to an equal extent as platelets from bile duct-ligated rats when they were in the same sham or cholestatic plasma. Moreover, in bile duct-ligated rats, platelet ultrastructure was unaffected and platelet aggregation was similar to that of sham platelets when resuspended in the same plasma (P-value not significant). Additionally, studies in storage pool-deficient rats showed no role of platelet exhaustion. The plasmatic factor causing impaired aggregation was shown to be increased total activity of ADP-degrading enzymes upon bile duct ligation (P<0.01), as there was no decreased aggregation with a stable ADP analog in bile duct-ligated rats (P-value not significant vs. sham-operated rats). Furthermore, preincubation of plasma from bile duct-ligated rats with ADP decreased aggregation more than was seen with sham plasma (P<0.01). CONCLUSIONS: Bile duct ligation does not affect intrinsic platelet function, but impairs platelet activation via release of ADP-degrading enzymes in the circulation.

From patient to mouse to therapy: role of the neuropeptide pacap in platelet function and formation.

Megakaryopoiesis and platelet production are very complex processes, orchestrated by different growth factors, cytokines and transcription factors. It's only recently that a role was assigned for the pituitary adenylyl cyclase-activating peptide (PACAP) and the vasoactive intestinal peptide (VIP) via their common Gs-coupled receptor VPAC1 in this process. The basis for this idea originated from studies in two related patients with a partial trisomy 18p11 and therefore carrying 3 copies of the PACAP gene and elevated PACAP concentrations in their plasma which resulted in a bleeding tendency with thrombopathy and a mild thrombocytopenia. This platelet functional and formation defect could be phenocopied in transgenic megakaryocyte specific PACAP-overexpressing mice. The addition of PACAP or VIP to hematopoietic stem cells resulted in an decreased megakaryocyte maturation and DNA polyploidization. In contrast, mice subcutaneously injected with inhibitory anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies presented with increased platelet numbers. This last concept was the basis for the development of these antibodies for the treatment of different types of thrombocytopenia as the therapeutic value for these antibodies was established in mice with a low platelet count due to chemotherapy, anti-platelet antibodies, a congenital factor of after bone marrow transplantation. For all models, the addition of 23A11 or PP1A4 resulted in an increased platelet recovery compared to the control antibody. Further studies are needed to identify the downstream signal transduction components after VPAC1 stimulation in megakaryocytes and platelets.

Human platelet pathology related to defects in the G-protein signaling cascade.

Platelets are highly responsive to signals from their environment. The sensing and processing of some of these stimuli are mediated by G-protein signal transduction cascades. It is well established that proteins involved in signal transduction may be targets for naturally occurring mutations resulting in human diseases. The best-studied molecules in platelets in relation to disease are the G-protein coupled receptors being the most platelet-specific. Many of the other signal transduction genes are often not only present in platelets but also in other tissues. Therefore, the clinical phenotype of signaling defects in platelets, apart from the membrane receptor defects, is seldom isolated to a hemostatic phenotype. Moreover, as platelets are easily accessible cells, and one of the best-studied models regarding signaling, platelets are easily applicable to investigate defects in ubiquitously expressed genes. Apart from a discussion on classical thrombopathies, this review will also deal with the less commonly known relation between platelet signaling defects and disorders with a broader clinical phenotype.

A compound heterozygous mutation in glycoprotein VI in a patient with a bleeding disorder.

BACKGROUND: The physiological relevance of the collagen glycoprotein VI (GPVI) receptor was known prior to its recognition as a platelet membrane receptor as several patients lacking GPVI as a consequence of autoantibody inhibition presented with a mild bleeding diathesis. Remarkably, patients with a proven GPVI gene mutation have not yet been identified. RESULTS: In the present study, we describe a patient with a lifelong history of bleeding problems, structurally normal platelets but a functional platelet defect. Platelet aggregations are normal except for an absent response to Horm collagen, convulxin and the collagen-related peptide (CRP). ATP dense granule secretion is normal with ADP but absent with Horm collagen. Thrombus formation on a collagen surface in flowing blood is reduced but more single platelets are attached. Remarkably, the platelet function analyzer-100 shows a shortened collagen/ADP closure time. Flow cytometry demonstrates an absent expression of GPVI whereas immunoblot analysis shows strongly reduced levels of GPVI. The patient is compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation S175N in a highly conserved residue of the 2nd Ig-like GPVI domain. The parents without clinical bleeding problems are heterozygous carriers. The mother carries the S175N mutation and presents with a mild functional platelet defect. In vitro studies show a reduced membrane expression and convulxin binding with the mutated S175N compared with the wild-type (WT) GPVI receptor. CONCLUSIONS: This study presents the first patient with a proven genetic GPVI defect.

Review article: blood platelet number and function in chronic liver disease and cirrhosis.

BACKGROUND: The liver plays a central role in coagulation and fibrinolysis but is also closely intertwined with the function and number of blood platelets. AIM: To describe and integrate all literature concerning blood platelets and liver disease by performing a thorough literature research. METHODS: A thorough literature research on 'blood platelets' and 'liver disease' was performed. RESULTS: Thrombocytopenia is a marked feature of chronic liver disease and cirrhosis. Traditionally, this thrombocytopenia was attributed to passive platelet sequestration in the spleen. More recent insights suggest an increased platelet breakdown and to a lesser extent decreased platelet production plays a more important role. Besides the reduction in number, other studies suggest functional platelet defects. This platelet dysfunction is probably both intrinsic to the platelets and secondary to soluble plasma factors. It reflects not only a decrease in aggregability, but also an activation of the intrinsic inhibitory pathways. The net effect, finally, is a decreased platelet function in the various types of chronic liver diseases and cirrhosis. Finally, recent data suggest that platelets are not only affected by but can also contribute to the liver disease process, as for instance, in viral hepatitis and cholestatic liver disease. CONCLUSION: Platelet research in liver disease is a growing area of investigation and could provide new pathophysiological insights.

Genetic variation of the extra-large stimulatory G protein alpha-subunit leads to Gs hyperfunction in platelets and is a risk factor for bleeding.

Alternatively spliced GNAS1 and XL-GNAS1, encoding respectively the stimulatory G-protein alpha-subunit (Gsalpha) and the extra-large stimulatory G-protein alpha-subunit (XLsalpha), are located on the imprinted chromosomal region 20q13.12-13. We presently report a functional polymorphism in the imprinted XL-GNAS1 gene. In three patients, a 36 bp insertion and two basepair substitutions flanking this insertion were found in the paternally inherited XL-GNAS1 exon 1. They clinically manifest an enhanced trauma-related bleeding tendency and a variable degree of mental retardation. A platelet aggregation inhibition test to evaluate Gs function was developed. Their platelets display Gs hyperfunction and an enhanced cAMP generation upon stimulation of Gs-coupled receptors. The prevalence of the XLsalpha insertion in a normal control group was 2.2%. Normal controls, inheriting the insertion maternally, had a normal platelet Gs activity, whereas controls inheriting the insertion paternally had increased inducible platelet Gs activity, defining the insertion as a functional polymorphism. This paternally inherited XLsalpha insertion represents a new genetic cause of an inherited bleeding tendency, although to a variable degree.