C. Van Geet

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.

Polymorphisms and mutations of human TMPRSS6 in iron deficiency anemia.

Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.

Congenital disorders of glycosylation type Ia and IIa are associated with different primary haemostatic complications

Congenital disorders of glycosylation (CDG) type I are mostly due to a deficient phosphomannomutase activity, called CDG Ia. CDG IIa (mutations in the MGAT2 gene) results from a deficient activity of the Golgi enzyme N-acetylglucosaminyltransferase II. CDG Ia patients predominantly have a thrombotic tendency, whereas our CDG IIa patient has an increased bleeding tendency, despite similar coagulation factor abnormalities in both types. We have investigated whether abnormally glycosylated platelet membrane glycoproteins are involved in the haemostatic complications of both CDG groups. In flow cytometry, the binding of Ricinus communis lectin (reactive with β-galactose primarily) to control platelets increased after neuraminidase treatment: this increase was smaller (p < 0.01) in CDG Ia patients (3.1 ± 0.08 times) than in control platelets (8.5 ± 1.8 times) and did not occur in the CDG IIa patient. Platelet-rich plasma from CDG Ia patients, but not a CDG IIa patient, aggregated spontaneously and gel-filtered platelets from CDG Ia patients agglutinated at very low concentrations of ristocetin, independently of von Willebrand factor (vWF). Accordingly, in stirred whole blood, the rate of single platelet disappearance of CDG Ia patients was twice that of control platelets. In contrast, perfusion of whole anticoagulated blood of the CDG IIa patient over collagen yielded markedly decreased platelet adherence to collagen at shear rates involving glycoprotein (GP) Ib–vWF interactions. Thus, abnormal glycosylation of platelet glycoproteins in CDG Ia enhances nonspecific platelet interactions, in agreement with a thrombotic tendency. The reduced GP Ib-mediated platelet reactivity with vessel wall components in the CDG IIa patient under flow conditions provides a basis for his bleeding tendency.

Carbohydrate-deficient glycoprotein syndrome type II

The carbohydrate-deficient glycoprotein syndromes (CDGS) are a group of autosomal recessive multisystemic diseases characterized by defective glycosylation of N-glycans. This review describes recent findings on two patients with CDGS type II. In contrast to CDGS type I, the type II patients show a more severe psychomotor retardation, no peripheral neuropathy and a normal cerebellum. The CDGS type II serum transferrin isoelectric focusing pattern shows a large amount (95%) of disialotransferrin in which each of the two glycosylation sites is occupied by a truncated monosialo-monoantennary N-glycan. Fine structure analysis of this glycan suggested a defect in the Golgi enzyme UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) which catalyzes an essential step in the biosynthetic pathway leading from hybrid to complex N-glycans. GnT II activity is reduced by over 98% in fibroblast and mononuclear cell extracts from the CDGS type II patients. Direct sequencing of the GnT II coding region from the two patients identified two point mutations in the catalytic domain of GnT II, S290F (TCC to TTC) and H262R (CAC to CGC). Either of these mutations inactivates the enzyme and probably also causes reduced expression. The CDG syndromes and other congenital defects in glycan synthesis as well as studies of null mutations in the mouse provide strong evidence that the glycan moieties of glycoproteins play essential roles in the normal development and physiology of mammals and probably of all multicellular organisms.

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.

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

Summary.  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.

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

Summary.  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.

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.

Video reveals self‐stimulation in infancy

The medical literature on early childhood masturbation is sparse. Only 12 patients who presented with infantile self‐stimulation under the age of 1 y are described. During the last 2 y, five girls under 1 y of age presented at our department with self‐stimulating behaviour. The diagnosis of this behaviour was difficult, but could be made by watching a video of the attacks. Infantile self‐stimulation is often misdiagnosed and unnecessary investigations and useless treatments are often prescribed. Video recording can be of great help to put forward the correct diagnosis. Masturbation is not so uncommon and treatment consists mostly in reassuring the parents. It can, however, be associated with behavioural problems. Few data are available on the clinical outcome of childhood masturbation, but most children seem to develop normally.

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 Albrights 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.