Gian Marco Podda

ANKRD26 -related thrombocytopenia and myeloid malignancies

To the editor: Since the discovery that mutations in the 5′ untranslated region (UTR) of ANKRD26 are responsible for an autosomal-dominant form of thrombocytopenia ( ANKRD26 -RT),[1][1] 21 affected families were reported.[2][2] A study analyzing this series of patients suggested that ANKRD26 -RT

Clearance of circulating activated platelets in polycythemia vera and essential thrombocythemia.

Essential thrombocythemia (ET) and polycythemia vera (PV) are characterized by persistent platelet activation. The mechanisms involved in their clearance are poorly characterized. In the present study, we report that leukocytes were actively involved in platelet disposal in 51 patients with ET and 30 with PV, but not in 70 age- and sex-matched controls. The fraction of circulating neutrophils and monocytes that had phagocytosed platelets, as assessed by flow cytometry, was significantly higher in patients with PV or ET, independently of hydroxyurea treatment, than in controls. Platelet phagocytosis by circulating leukocytes was confirmed by confocal and electron microscopy. The lack of effect of hydroxyurea, which disrupts the P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) interaction, suggests a P-selectin-independent mechanism. This hypothesis was confirmed in an ad hoc animal model based on the in vivo injection of activated platelets from P-selectin(+/+) and P-selectin(-/-) mice. P-selectin expression was associated with an earlier and effective clearance of platelets by neutrophils. A second delayed, P-selectin-independent phase actively involved monocytes. Our results suggest that phagocytic clearance of platelets by leukocytes occurs in PV and ET, possibly involving P-selectin-dependent and -independent pathways, thus representing a novel mechanism to remove activated platelets from the circulation.

Correlation between platelet phenotype and NBEAL2 genotype in patients with congenital thrombocytopenia and α-granule deficiency

The gray platelet syndrome is a rare inherited bleeding disorder characterized by macrothrombocytopenia and deficiency of alpha (α)-granules in platelets. The genetic defect responsible for gray platelet syndrome was recently identified in biallelic mutations in the NBEAL2 gene. We studied 11 consecutive families with inherited macrothrombocytopenia of unknown origin and α-granule deficiency. All of them underwent NBEAL2 DNA sequencing and evaluation of the platelet phenotype, including a systematic assessment of the α-granule content by immunofluorescence analysis for α-granule secretory proteins. We identified 9 novel mutations hitting the two alleles of NBEAL2 in 4 probands. They included missense, nonsense and frameshift mutations, as well as nucleotide substitutions that altered the splicing mechanisms as determined at the RNA level. All the individuals with NBEAL2 biallelic mutations showed almost complete absence of platelet α-granules. Interestingly, the 13 individuals assumed to be asymptomatic because carriers of a mutated allele had platelet macrocytosis and significant reduction of the α-granule content. However, they were not thrombocytopenic. In the remaining 7 probands, we did not identify any NBEAL2 alterations, suggesting that other genetic defect(s) are responsible for their platelet phenotype. Of note, these patients were characterized by a lower severity of the α-granule deficiency than individuals with two NBEAL2 mutated alleles. Our data extend the spectrum of mutations responsible for gray platelet syndrome and demonstrate that macrothrombocytopenia with α-granule deficiency is a genetic heterogeneous trait. In terms of practical applications, the screening of NBEAL2 is worthwhile only in patients with macrothrombocytopenia and severe reduction of the α-granules. Finally, individuals carrying one NBEAL2 mutated allele have mild laboratory abnormalities, suggesting that even haploinsufficiency has an effect on platelet phenotype.

Analysis of 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia

Pregnancy in women with inherited thrombocytopenias is a major matter of concern as both the mothers and the newborns are potentially at risk of bleeding. However, medical management of this condition cannot be based on evidence because of the lack of consistent information in the literature. To advance knowledge on this matter, we performed a multicentric, retrospective study evaluating 339 pregnancies in 181 women with 13 different forms of inherited thrombocytopenia. Neither the degree of thrombocytopenia nor the severity of bleeding tendency worsened during pregnancy and the course of pregnancy did not differ from that of healthy subjects in terms of miscarriages, fetal bleeding and pre-term births. The degree of thrombocytopenia in the babies was similar to that in the mother. Only 7 of 156 affected newborns had delivery-related bleeding, but 2 of them died of cerebral hemorrhage. The frequency of delivery-related maternal bleeding ranged from 6.8% to 14.2% depending on the definition of abnormal blood loss, suggesting that the risk of abnormal blood loss was increased with respect to the general population. However, no mother died or had to undergo hysterectomy to arrest bleeding. The search for parameters predicting delivery-related bleeding in the mother suggested that hemorrhages requiring blood transfusion were more frequent in women with history of severe bleedings before pregnancy and with platelet count at delivery below 50 × 109/L.

State of the art of new P2Y12 antagonists

The interaction of ADP with its platelet receptor P2Y12 plays a crucial role in platelet activation and thrombogenesis. This article reviews the pharmacology and clinical trials of specific antagonists of P2Y12. Clopidogrel is a thienopyridine with proven antithrombotic efficacy, but it has some important drawbacks: (a) it is a pro-drug that needs to be metabolized to its active metabolite; (b) it has a delayed onset and offset of action and (c) there is high inter-individual variability in pharmacological response. Prasugrel is also a thienopyridine, with faster onset of action and a more uniform inhibition of platelet function compared to clopidogrel, accounting for lower incidence of ischemic events in patients with acute coronary syndromes (ACS) undergoing percutaneous coronary intervention (PCI) and higher incidence of both non-CABG-related bleeding complications. Two direct and reversible P2Y12 antagonists, Cangrelor and ticagrelor, are characterized by rapid onset and reversal of platelet inhibition. Cangrelor is not superior to clopidogrel in preventing thrombotic events in patients undergoing PCI. Ticagrelor is superior to clopidogrel in preventing major adverse cardiac events in ACS patients, but, like prasugrel, is associated with a higher frequency of non-CABG-related bleeding complications. A shorter period of drug discontinuation before surgery is necessary in ticagrelor-treated patients compared to clopiodgrel-treated patients to limit the severity of post-surgical bleeding.

Prediction of optimal warfarin maintenance dose using advanced artificial neural networks

BACKGROUND In recent years, pharmacogenetic algorithms were developed for estimating the appropriate dose of vitamin K antagonists. AIM To evaluate the performance of new generation artificial neural networks (ANNs) to predict the warfarin maintenance dose. METHODS Demographic, clinical and genetic data (CYP2C9 and VKORC1 polymorphisms) from 377 patients treated with warfarin were used. The final prediction model was based on 23 variables selected by TWIST® system within a bipartite division of the data set (training and testing) protocol. RESULTS The ANN algorithm reached high accuracy, with an average absolute error of 5.7 mg of the warfarin maintenance dose. In the subset of patients requiring ≤21 mg and 21-49 mg (45 and 51% of the cohort, respectively) the absolute error was 3.86 mg and 5.45 with a high percentage of subjects being correctly identified (71 and 73%, respectively). CONCLUSION ANN appears to be a promising tool for vitamin K antagonist maintenance dose prediction.

EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin

INTRODUCTION This study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE(2)-mediated TP receptor contraction of human arteries was also investigated. MATERIALS AND METHODS Fifty-two type-2 diabetics with CAD on chronic aspirin (100 mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100 nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE(2). RESULTS Arachidonic acid (1 mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63 ± 4 AU), which was significantly reduced by in vitro exposure to EV-077 (38 ± 3 AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE(2) (P<0.01). CONCLUSIONS Aspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE(2) was effectively inhibited by EV-077.

Bleeding risk of surgery and its prevention in patients with inherited platelet disorders. The Surgery in Platelet disorders And Therapeutic Approach (SPATA) study

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders. However, very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a worldwide, multicentric, retrospective study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted, and their efficacy in patients with inherited platelet disorders: the Surgery in Platelet disorders And Therapeutic Approach (SPATA) study. We rated the outcome of 829 surgical procedures carried out in 423 patients with well-defined forms of inherited platelet disorders: 238 inherited platelet function disorders and 185 inherited platelet number disorders. Frequency of surgical bleeding was high in patients with inherited platelet disorders (19.7%), with a significantly higher bleeding incidence in inherited platelet function disorders (24.8%) than in inherited platelet number disorders (13.4%). The frequency of bleeding varied according to the type of inherited platelet disorder, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%). Frequency of bleeding was predicted by a pre-operative World Health Organization bleeding score of 2 or higher. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urological surgery. The use of pre-operative pro-hemostatic treatments was associated with a lower bleeding frequency in patients with inherited platelet function disorders but not in inherited platelet number disorders. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with the lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in inherited platelet disorders is substantial, especially in inherited platelet function disorders, and bleeding history, type of disorder, type of surgery and female sex are associated with higher bleeding frequency. Prophylactic pre-operative pro-hemostatic treatments appear to be required and are associated with a lower bleeding incidence.

Bleeding diathesis and gastro-duodenal ulcers in inherited cytosolic phospholipase-A2 alpha deficiency

Arachidonic acid (AA), when cleaved from phospholipids by cytosolic phospholipase A2 alpha (cPLA2a), generates eicosanoids, with pro-hemostatic, pro-inflammatory, vasoactive and gastro-protective functions. We describe a patient (27-year-old man) and his twin-sister with early-onset bleeding diathesis and recurrent gastro-intestinal (GI) ulcers. Platelet aggregation/δ-granules secretion by collagen was impaired, but normal by AA; serum levels of thromboxane (Tx) B2 and 12-hydroxyeicosatetraenoic acid, and urinary levels of 11-dehydro-TxB2 were extremely low. Patients were homozygous for 1723G>C transition in PLA2G4A gene, which changed the codon for Asp575 to His. GI ulcers affected 5/14 heterozygous (< 40 years) and 1/16 wild-type homozygous (> 60 years) family members; none had bleeding diathesis. The proband, his sister and mother also had mildly reduced factor XI levels. Platelet messenger RNA expression did not differ among subjects with different PLA2G4A genotypes. Conversely, platelet cPLA2a was undetectable by Western Blotting in the proband and his sister, and decreased in 1723G>C heterozygous subjects, suggesting that the variant is transcribed, but not translated or translated into an unstable protein. We described a syndromic form of deficiency of cPLA2a , characterised by recurrent GI ulcers and bleeding diathesis, associated with mild inherited deficiency of factor XI. Unlike other reported patients with cPLA2a deficiency, these patients had extremely low levels of platelet TxA2 biosynthesis.

Italian guidelines on thrombolysis indications in ischemic stroke have been revised after IST-3 trial and Cochrane revision: cons

One of the milestones of evidence-based medicine is the critical appraisal [1]. Critical appraisal shifts clinical practice from eminence-based medicine (medicine based on authority) to evidence-based medicine (medicine based on the proof and the judgment of the proof). Clinical practice guideline recommendations should be based on the best available evidence. Therapeutic recommendations should be developed upon randomized controlled trials (RCTs) with clinically relevant (defined as ‘‘hard’’) primary end points, low risk of bias, rigorous internal validity and good external validity [2]. The Italian guidelines on thrombolysis indications in ischemic stroke [3] have been revised after the IST-3 trial [4]. Does IST-3 satisfy the above-mentioned RCT characteristics providing the best evidence on which a clinical practice guideline should be modified? When the IST-3 trial was published many considered it the end of the struggle between ‘‘pros’’ and ‘‘cons’’ about thrombolysis [5–7]. Indeed IST-3 is the largest trial on this topic. The Lancet editorial accompanying the IST-3 trial claims ‘‘The role of stroke and emergency physicians is now not to identify patients who will be given rt-PA, but to identify the few who will not’’ [5]. An authoritative IAEM editorial [8] commenting on the IST-3 trial concludes ‘‘We have no longer to ask the age, but just the onset time!’’ However, some weaknesses should be considered [9] and we suggest that there are still some reasons to introduce some notes of caution. If a large trial has some biases, it should not be considered as the ‘‘best’’ evidence and used to change the recommendations of clinical practice guidelines. The aim of this paper is not a comprehensive critical appraisal of the IST-3 trial, already published [10]. We would like just to highlight some major criticisms indicating that maybe the IST-3 trial results are not adequate to change clinical practice.