Comprehensive investigation of platelet function in patients with cirrhosis.

Cirrhosis presents with thrombocytopenia and possibly thrombocytopathy. Previous studies exploring platelet function gave conflicting results and most controversies are explained by the variety of methods employed for investigation. We sought to assess in-vitro the overall platelet function in cirrhosis. We investigated 34 patients by using the following tests. (i)Aggregometry. (ii)Measurement of the content of platelet granules. (iii)Cytometric platelet activation. (iv)Plasmatic markers of in-vivo platelet activation. (v)Platelet procoagulant activity by thrombin generation (TG) in platelet-rich plasma (PRP). TG measured in PRP for patients and controls was similar. Platelets from patients with cirrhosis showed reduction of aggregation and secretion of ATP. Similar results were observed for platelet activation parameters such as P-selectin expression and PAC-1 platelet binding. Plasma levels of ßeta-thromboglobulin and soluble P-selectin, were increased in patients-vs-controls. In contrast, there were no patients-vs-controls differences for plasmatic platelet-factor-4. Results are consistent with a state of in-vivo platelet activation and decreased in-vitro aggregation. Since bleeding events following invasive procedures are uncommon in cirrhosis, we speculate that in-vitro aggregometry testing does not reflect the situation occurring in-vivo. Results of the study and pathophysiological considerations support the conclusion that platelet function in cirrhosis as determined by aggregometry, although somewhat impaired, may support the overall hemostatic potential, which is needed for most invasive interventions. These conclusions are in line with the recommendations of international guidelines, warning against indiscriminate use of prophylactic preprocedural administration of platelets before invasive procedures. Decision on platelet support should not be made based on in-vitro laboratory testing for platelet function.

Patients with Essential Thrombocythemia may be Poor Responders to Enteric-Coated Aspirin, but not to Plain Aspirin.

Essential thrombocythemia (ET) patients are treated with aspirin (acetylsalicylic acid [ASA]) to prevent thrombosis. Previous studies showed that serum thromboxane (Tx) B2 was high 24 hours after enteric-coated (EC)-ASA in ET patients, due to increased number of noninhibited reticulated platelets (RPs), consequent to high platelet turnover, and that ASA should be given twice a day to ET patients. We studied ET patients (n = 17) and healthy subjects (n = 10) on 100 mg EC-ASA once daily; experiments were repeated after 14-day treatment with 100 mg plain-ASA once daily. Serum TxB2, plasma ASA, and salicylic acid (SA) were measured before the morning dose and up to 8 hours thereafter. Blood activity of ASA-deacethylating esterases, in vitro inhibition of collagen-induced TxB2 production by ASA (10-1,000 µM), and number of RP were measured. TxB2 inhibition by ASA in vitro and esterases activities were normal in all subjects. EC-ASA elicited highly variable responses; 6 ET patients were poor responders, as their serum TxB2 was high after EC-ASA; their plasma levels of ASA and SA were low/undetectable. In contrast to EC-ASA, plain ASA decreased serum TxB2 and increased plasma ASA and SA in all subjects. Serum TxB2 was high in ET patients at 24 hours and significantly correlated with RP count (but not RP percentage) and platelet count. Plain ASA should be used in ET patients to inhibit platelets efficiently. The identification of ET patients who might benefit from twice a day ASA could simply be based on their platelet count: since their platelet turnover is not increased, ET patients with normalized platelet count should not need twice a day ASA treatment.

Novel variant in HPS3 gene in a patient with Hermansky Pudlak syndrome (HPS) type 3.

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder caused by defects in 10 human HPS genes, characterized by oculocutaneous albinism (OCA) and bleeding diathesis associated to platelet δ-storage pool defect (SPD). We report a case of 4-year-old boy from non-consanguineous parents with OCA and negative personal and familiar hemorrhagic history, referred to us for severe bleeding after mild trauma. His platelet function, studied by lumi-aggregometry, showed normal first wave of aggregation in response to exogenous agonists and impaired second wave with defective ATP release. This, in combination with impaired platelet δ-granules content (serotonin, ATP, ADP) and the OCA phenotype suggested the HPS diagnosis. HPS3: sequencing revealed a novel pathogenic homozygous variant (NM_032383.4:c.7>T, p.Gln3*) resulting in a premature stop codon at the amino acid 3. Moreover, our report highlights the importance of evaluating platelet function in children with OCA without bleeding diathesis to identify HPS early and prevent bleeding complications.

Acquired platelet dysfunction and overproduction of platelet cyclic AMP in two patients with myeloid malignancies.

The pathophysiology of impaired platelet function in acquired disorders is often poorly understood. We report two unrelated patients with hematologic malignancies associated with acquired severe bleeding diathesis, and complex platelet function abnormalities, including overproduction of the physiological inhibitor cyclic-AMP (cAMP). Patient 1, with mild macrocytic anemia and thrombocytopenia (100 x 109/L), was diagnosed with chronic myelomonocytic leukemia a few months after the onset of her bleeding diathesis and our analysis of platelet function. Patient 2, with bleeding diathesis of recent onset, was studied when his myelodysplastic syndrome with excess blasts had already progressed to acute myeloid leukemia. In both patients, platelet aggregation/ATP secretion, serum thromboxane B2, intraplatelet content of ADP, ATP, serotonin, and fibrinogen were severely impaired. Baseline platelet cAMP levels were mildly elevated and markedly increased after stimulation by prostaglandin E1. In conclusion, these are the first patients with myeloid malignancies associated with acquired severe platelet dysfunction and overproduction of cAMP.

Complications of whole-exome sequencing for causal gene discovery in primary platelet secretion defects.

Primary platelet secretion defects constitute a heterogeneous group of functional defects characterized by reduced platelet granule secretion upon stimulation by different agonists. The clinical and laboratory heterogeneity of primary platelet secretion defects warrants a tailored approach. We performed a pilot study in order to develop DNA sequence analysis pipelines for gene discovery and to create a list of candidate causal genes for platelet secretion defects. Whole-exome sequencing analysis of 14 unrelated Italian patients with primary secretion defects and 16 controls was performed on Illumina HiSeq. Variant prioritization was carried out using two filtering approaches: identification of rare, potentially damaging variants in platelet candidate genes or by selecting singletons. To corroborate the results, exome sequencing was applied in a family in which platelet secretion defects and a bleeding diathesis were present. Platelet candidate gene analysis revealed gene defects in 10/14 patients, which included ADRA2A, ARHGAP1, DIAPH1, EXOC1, FCGR2A, ITPR1, LTBP1, PTPN7, PTPN12, PRKACG, PRKCD, RAP1GAP, STXBP5L, and VWF The analysis of singletons identified additional gene defects in PLG and PHACTR2 in two other patients. The family analysis confirmed a missense variant p.D1144N in the STXBP5L gene and p.P83H in the KCNMB3 gene as potentially causal. In summary, exome sequencing revealed potential causal variants in 12 of 14 patients with primary platelet secretion defects, highlighting the limitations of the genomic approaches for causal gene identification in this heterogeneous clinical and laboratory phenotype.

Studies of the interaction of ticagrelor with the P2Y13 receptor and with P2Y13-dependent pro-platelet formation by human megakaryocytes.

Ticagrelor is an antagonist of the platelet P2Y12 receptor for ADP, approved for the prevention of thromboembolic events in patients with acute coronary syndrome. Previous studies showed that ticagrelor has no significant activity versus P1 receptors for adenosine and other known P2Y receptors, with the exception of P2Y13, which was not tested. The P2Y12 antagonist cangrelor has been shown to also inhibit P2Y13 and to decrease the P2Y13-regulated capacity of megakaryocytes to produce pro-platelets. We tested whether or not ticagrelor inhibits P2Y13 signalling and function. The in vitro effects of ticagrelor, its active (TAM) and inactive (TIM) metabolites, cangrelor and the P2Y13 antagonist MRS2211 were tested in two experimental models: 1) a label-free cellular response assay in P2Y13-transfected HEK293 T-REx cells; and 2) pro-platelet formation by human megakaryocytes in culture. Ticagrelor, TAM, cangrelor and MRS2211, but not TIM, inhibited the cellular responses in P2Y13-transfected cells. In contrast, only MRS2211 and cangrelor, confirming previous results, inhibited pro-platelet formation by megakaryocytes in vitro. The platelet count of patients randomised to treatment with ticagrelor in the PLATO trial did not change during treatment and was comparable to those of patients randomised to clopidogrel. In conclusion, ticagrelor and TAM act as P2Y13 antagonists in a transfected cell system in vitro but this does not translate into any impact on pro-platelet formation in vitro or altered platelet count in patients.

Inherited dysfunctional platelet P2Y12 receptor mutations associated with bleeding disorders.

The platelet adenosine 5'-diphosphate (ADP) receptor P2Y12 (P2Y12R) plays a critical role in platelet aggregation. The present report illustrates an update of dysfunctional platelet P2Y12R mutations diagnosed with congenital lifelong bleeding problems. Described patients with heterozygous or homozygous substitution in the P2Y12R gene and qualitative abnormalities of the platelet P2Y12R are summarized. Recently, a further dysfunctional variant of P2Y12R has been identified in two brothers who presented with a lifelong severe bleeding disorder. During in vitro aggregation studies, the patient´s platelets show a markedly reduced and rapid reversible ADP-promoted aggregation. A homozygous c.561T>A substitution that changes the codon for His187 to Gln (p.His187Gln) in the P2Y12R gene has been identified. This mutation causes no change in receptor expression but decreases the affinity of the ligand for the receptor, even at high concentrations. Structure modelling studies indicated that the p.His187Gln mutation, located in the fifth transmembrane spanning domain (TM5), impairs conformational changes of the receptor. Structural integrity of the TM5 region is necessary for agonist and antagonist binding and for correct receptor function.

Zonisamide in the management of epilepsy in the elderly.

Zonisamide (ZNS), a second-generation antiepileptic drug, indicated as add-on treatment of focal epilepsy, has been recently approved as monotherapy for the treatment of partial seizures in adults affected by newly diagnosed epilepsy in Europe. Evidence on the efficacy and tolerability of antiepileptic drugs in the elderly is still lacking as these patients are frequently excluded from clinical trials. Here, a comprehensive overview of available data regarding the use of ZNS in the treatment of epilepsy in elderly people is provided. In a pooled analysis conducted in patients aged ≥65 years, no new/unexpected safety findings have emerged. Few data from uncontrolled investigations suggest that ZNS may be effective and well tolerated when administered as monotherapy or adjunctive antiepileptic treatment in the elderly. However, evidence from these observational studies is less than satisfactory, and randomized controlled trials focused on these patients are still needed.

Usefulness of the INNOVANCE PFA P2Y test cartridge for the detection of patients with congenital defects of the platelet P2Y12 receptor for adenosine diphosphate.

INTRODUCTION: The platelet function analyzer (PFA)-100 is used in clinical practice to screen patients with bleeding diathesis and suspected defects of primary hemostasis. A new cartridge, INNOVANCE PFA P2Y, has been specifically developed to monitor patients' response to drugs inhibiting the platelet P2Y12 receptor for ADP. In this study, we compared the ability of INNOVANCE PFA P2Y to detect congenital defects of the platelet P2Y12 receptor to that of standard cartridge formulations currently in clinical use. MATERIALS AND METHODS: We studied two patients with severe P2Y12 deficiency, one patient with heterozygous P2Y12 deficiency and one with dysfunctional P2Y12 receptor. Closure times were measured using 3 cartridges: collagen/ADP, collagen/epinephrine, and INNOVANCE PFA P2Y. The results obtained in the four patients with P2Y12 defects were compared to those obtained for 20 healthy controls. RESULTS: In 2 patients with severe P2Y12 deficiency, closure times of INNOVANCE PFA P2Y and collagen/ADP cartridges were >300 s, while those of collagen/epinephrine cartridge were variable (186s and >300 s). In the patient with dysfunctional P2Y12, closure time of INNOVANCE PFA P2Y was >300 s, while closure times of collagen/ADP and collagen/epinephrine were normal. Closure times of all cartridges were normal in the patient with heterozygous P2Y12 deficiency. CONCLUSION: Our study provides the first evidence that INNOVANCE PFA P2Y cartridge is sensitive to congenital severe and moderate defects of the platelet P2Y12 receptors.

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.

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.