Anna Maria Mezzasoma

Eltrombopag for the treatment of the inherited thrombocytopenia deriving from MYH9 mutations

Platelet transfusion is currently the primary medical treatment for reducing thrombocytopenia in patients with inherited thrombocytopenias. To evaluate whether stimulating megakaryopoiesis could increase platelet count in these conditions, we treated patients with a severe thrombocytopenia induced by MYH9 mutations (MYH9-related disease) with a nonpeptide thrombopoietin receptor agonist, eltrombopag. Twelve adult patients with MYH9-RD and platelet counts of less than 50 × 10(9)/L received 50 mg of eltrombopag orally per day for 3 weeks. Patients who achieved a platelet count higher than 150 × 10(9)/L stopped therapy, those with 100 to 150 platelets × 10(9)/L continued treatment at the same eltrombopag dose for 3 additional weeks, while those with less than 100 platelets × 10(9)/L increased the eltrombopag dose to 75 mg for 3 weeks. Major responses (platelet count of at least 100 × 10(9)/L or 3 times the baseline value) were obtained in 8 patients, minor responses (platelet counts at least twice the baseline value) in 3. One patient did not respond. Bleeding tendency disappeared in 8 of 10 patients with bleeding symptoms at baseline. Mild adverse events were reported in 2 patients. The availability of thrombopoietin mimetics opened new prospects in the treatment of inherited thrombocytopenias. This study is registered at www.clinicaltrials.gov as NCT01133860 (European Union Drug Regulating Authorities Clinical Trials number 2008-001903-42).

Prevention of pulmonary thromboembolism by NCX 4016, a nitric oxide-releasing aspirin.

We studied the antithrombotic activity of 2-acetoxybenzoate 2-[1-nitroxy-methyl]-phenyl ester (NCX 4016), a novel nitric oxide (NO)-releasing aspirin derivative, in vivo in different animal models of platelet-dependent and independent pulmonary thromboembolism and compared it with that of aspirin. NCX 4016 protected mice from death induced by the intravenous (i.v.) injection of collagen plus epinephrine, of 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F(2alpha) (U46619) and of thrombin while aspirin was only active against collagen plus epinephrine. The drop in platelet count and number of lung emboli were reduced by NCX 4016 more effectively than aspirin. NCX 4016 protected mice also from mechanical pulmonary embolism (i.v. injection of hardened rat red blood cells) while aspirin was ineffective. In rabbits, NCX 4016 significantly reduced the accumulation of [111In]oxine-labeled platelets in the pulmonary vasculature induced by collagen and by thrombin while aspirin produced reductions which were significant only versus collagen. In conclusion, NCX 4016 exerts a more pronounced antithrombotic activity than aspirin in vivo in two different animal species, largely due to a deeper inhibitory effect on platelets. NCX 4016 may represent a better antithrombotic agent than aspirin.

Dominant inheritance of a novel integrin β3 mutation associated with a hereditary macrothrombocytopenia and platelet dysfunction in two Italian families

Glanzmann’s thrombasthenia is a bleeding disorder caused by mutations of the ITGA2B or ITGB3 genes. This paper describes two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel mutation in the ITGB3 gene, encoding the integrin β chain β3. This report adds to previous studies of mutant alleles which illustrate the importance of signaling via this integrin for platelet function as well as for platelet formation. Background Defects of integrin αIIbβ3 are typical of Glanzmann’s thrombasthenia, an inherited autosomal recessive bleeding disorder characterized by the failure of platelets to aggregate in response to all physiological agonists, but with no abnormalities in the number or size of platelets. Although large heterogeneity has been described for Glanzmann’s thrombasthenia, no family has so far been described as having an autosomal dominant form of this disease. Design and Methods We describe two Italian families with moderate thrombocytopenia with large platelets, defective platelet function and moderate/severe mucocutaneous bleeding, transmitted as an autosomal dominant trait and associated with a novel integrin β3-gene (ITGB3) mutation. Results The characteristics of our families are moderate macrothrombocytopenia and defective platelet function associated with a mild reduction of surface αIb β3, impaired platelet aggregation to physiological agonists but not to ristocetin, normal clot retraction, reduced fibrinogen binding and expression of activated αIIbβ3 upon stimulation, normal platelet adhesion to immobilized fibrinogen but reduced platelet spreading and tyrosine phosphorylation, indicating defective αIIbβ3-mediated outside-in signaling. Molecular analysis revealed a novel mutation of ITGB3 that determines an in-frame deletion producing the loss of amino acids 647–686 of the βTD ectodomain of integrin β3. Haplotype analysis indicated that the two families inherited the mutation from a common ancestral chromosome. Conclusions This novel autosomal dominant macrothrombocytopenia associated with platelet dysfunction raises interesting questions about the role of integrin β3, and its βTD domain, in platelet formation and function.

In vivo platelet activation and platelet hyperreactivity in abacavir-treated HIV-infected patients

Abacavir (ABC) has been associated with ischaemic cardiovascular events in HIV-infected patients, but the pathogenic mechanisms are unknown. Aim of our study was to assess whether ABC induces in vivo platelet activation and ex vivo platelet hyper-reactivity. In a retrospective, case-control study, in vivo platelet activation markers were measured in 69 HIV-infected patients, before starting therapy and after 6-12 months of either ABC (n=35) or tenofovir (TDF) (n=34), and compared with those from 20 untreated HIV-infected patients. A subgroup of patients was restudied after 28-34 months for ex vivo platelet reactivity. In vivo platelet activation markers were assessed by ELISA or flow cytometry, ex vivo platelet reactivity by light transmission aggregometry (LTA) and PFA-100®. Thein vitro effects of the ABC metabolite, carbovir triphosphate, on aggregation and intra-platelet cGMP were also studied. sPLA2, sPsel and sGPV increased significantly 6-12 months after the beginning of ABC, but not of TDF or of no treatment. Ex vivo platelet function studies showed enhanced LTA, shorter PFA-100® C/ADP closure time and enhanced platelet expression of P-sel and CD40L in the ABC group. The intake of ABC blunted the increase of intraplatelet cGMP induced by nitric oxide (NO) and acutely enhanced collagen-induced aggregation. Preincubation of control platelets with carbovir triphosphate in vitro enhanced platelet aggregation and blunted NO-induced cGMP elevation. In conclusion, treatment with ABC enhances in vivo platelet activation and induces platelet hyperreactivity by blunting the inhibitory effects of NO on platelets. These effects may lead to an increase of ischaemic cardiovascular events.

Laboratory diagnosis and monitoring of desmopressin treatment of von Willebrand’s disease by flow cytometry

Background and Objectives von Willebrand’s disease (VWD) is a heterogeneous bleeding disorder caused by quantitative or qualitative defects in von Willebrand factor (VWF). The diagnosis of VWD requires several laboratory tests. The aim of our study was to validate a flow cytometric test for the diagnosis of VWD and for monitoring the effects of desmopressin therapy. Design and Methods Flow cytometric analysis of ristocetin-induced VWF binding to platelets was performed in platelet-rich plasma (PRP) samples from patients with VWD and from control subjects and in samples of formalin-fixed platelets in the presence of plasma from patients or controls. In 12 VWD patients the test was conducted before and 1 hour after desmopressin infusion. Results were compared with VWF:Ag, VWF:RCo, VWF:CB, RIPA, PFA-100® and the skin bleeding time. Results Ristocetin-induced VWF binding to platelets, evaluated by both flow cytometry-based assays, was significantly reduced in patients with type1, 2A and 2M VWD as compared with that in healthy subjects. Patients with type 2B VWD showed reduced binding of VWF to formalin-fixed platelets, but increased binding to autologous platelets in PRP, similar to RIPA. VWF binding to platelets assessed by both flow cytometric assays correlated significantly with VWF:Ag, VWF:RCo, VWF:CB, RIPA, PFA100® and bleeding time. VWF binding to platelets increased after desmopressin infusion. Interpretation and Conclusions The measurement of ristocetin-induced binding of VWF to platelets by flow cytometry is a sensitive, simple and rapid test for the diagnosis of VWD and for the monitoring of the effects of desmopressin therapy. The flow cytometric assay performed with autologous platelets is useful in the identification of type 2B VWD patients.

Diagnosis of platelet-type von Willebrand disease by flow cytometry

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder which is due to a mutation in the gene encoding for platelet glycoprotein Ibα (GPIbα) resulting in enhanced affinity for von Willebrand factor (VWF). PT-VWD is often mistakenly diagnosed as type 2B VWD for the similarities between these two conditions. We characterized a new case of PT-VWD and evaluated the usefulness of a flow cytometric assay in the differential diagnosis between PT-VWD (n=1) and type 2B VWD (n=4). The flow cytometric assay was able to highlight the increased affinity of VWF for GPIbα as much as did RIPA and to differentiate the two diseases through mixing tests. Genetic analysis revealed a heterozygous point mutation in codon 239 of the GPIbα gene leading to a methionine to valine substitution (M239V). Flow cytometry represents a useful tool for the diagnosis of PT-VWD.

A New Case of Acquired Glanzmann's Thrombasthenia: Diagnostic Value of Flow Cytometry

Acquired Glanzmanns thrombasthenia (aGT) is a rare hemorrhagic disorder caused by autoantibodies, alloantibodies, or paraproteins directed against platelet GPIIb/IIIa. Its diagnosis requires several laboratory assays and mixing tests, which are complex and time consuming. We describe here a new case of aGT and compare different tests for the detection of GPIIb/IIIa‐blocking autoantibodies.

Incomplete inhibition of platelet function as assessed by the platelet function analyzer (PFA-100) identifies a subset of cardiovascular patients with high residual platelet response while on aspirin

Sixty-six patients with a history of ischemic events (myocardial infarction, unstable angina, or stroke) on chronic aspirin therapy were studied by different platelet function tests: 37 patients had suffered a recurrent event while on aspirin and 29 were without recurrences. Based on results from light transmission aggregometry (LTA) induced by arachidonic acid (AA) and serum TxB2 both COX-1-dependent methods, only one patient could be identified as aspirin “resistant”. However, when methods only partially-dependent on platelet COX-1 activity were considered, the prevalence of aspirin non-responders ranged, according to the different tests, from 0 to 52%. No difference was observed between patients with recurrences and those without. Among patients with recurrent events, those with an incomplete inhibition of platelet function, as assessed by the PFA-100, had significantly higher residual serum TxB2 (2.4 ± 2.4 ng/mL vs 0.4 ± 0.1 ng/mL, p = 0.03), residual LTA-AA (9.2 ± 10.6% vs 2.0 ± 1.6%, p = 0.008), LTA-Coll (49.3 ± 14.6% vs 10.2 ± 8.3%, p = 0.007) and LTA-ADP (50.9 ± 16.2% vs 34.3 ± 11.0%, p = 0.04). In conclusion, laboratory tests solely exploring the AA-mediated pathway of platelet function, while being the most appropriate to detect the effect of aspirin on its pharmacologic target (platelet COX-1), may fail to reveal the functional interactions between minimal residual TxA2 and additional stimuli or primers potentially leading to aspirin-insensitive platelet aggregation. High residual platelet response in platelet function tests only partially dependent on COX-1 may reveal a condition of persistent platelet reactivity in a subset of aspirin-treated patients characterizing them as a subgroup at higher vascular risk.

Defective platelet β-N-acetyl hexosaminidase content and release in chronic myeloproliferative disorders

Background and objectives: Abnormalities of platelet function or structure are a hallmark of chronic myeloproliferative disorders (MPD). In vivo platelet activation with the release of α- and δ-granules in the circulation is one of the most frequently described alterations in MPD. Platelets contain and release upon activation also lysosomes, and in particular β-N-acetylhexosaminidase (Hex). We have assessed whether the content and in vivo release of Hex of platelets from MPD patients is altered. Design and methods: Twenty-three MPD patients were compared with 19 age- and sex-matched healthy controls. The activity of platelet β-N-acetylhexosaminidase was measured in plasma, serum and in the capillary blood emerging from the skin wound inflicted for the measurement of the bleeding time. Lysosome integral membrane protein (LIMP or CD63), lysosome-associated membrane protein (LAMP-2 or CD107b) and P-selectin were evaluated by flow cytometry. Platelet aggregation in vitro and the release of β-N-acetylhexosaminidase, ATP and β-thromboglobulin were performed to study platelet reactivity. Results: Hex levels in plasma were significantly higher in MPD than in controls while the release of Hex in the bleeding time blood, i.e. at a localized site of in vivo platelet plug formation, was lower in MPD and the platelet content of Hex was reduced. These changes were accompanied by in vivo platelet activation. Finally, the isoenzymatic pattern of Hex was altered in platelets of MPD patients, with a reduced amount of the Hex A isoform as compared with controls. Interpretations and conclusions: MPD patients present an altered platelet Hex content and release; prospective studies to assess whether altered platelet Hex is related to thrombotic/hemorrhagic complications and/or tissue fibrosis in MPD are warranted.

Inhibition of PAF synthesis by stimulated human polymorphonuclear leucocytes with cloricromene, an inhibitor of phospholipase A2 activation.

1 A phospholipase A2 (PLA2) represents the key enzyme in the remodelling pathway of platelet‐activating factor (PAF) synthesis in human polymorphonuclear (PMN) leucocytes. 2 PLA2 activation is also the rate‐limiting step for the release of the arachidonic acid utilized for the synthesis of leukotrienes in stimulated leucocytes; however, it is unknown whether the PLA2s involved in the two biosynthetic pathways are identical. 3 Cloricromene (8‐monochloro‐3‐β‐diethylaminoethyl‐4‐methyl‐7‐ethoxy‐carbonylmethoxy coumarin) is an antithrombotic coumarin derivative which inhibits platelet and leucocyte function and suppresses arachidonic acid liberation by interfering with PLA2 activation. 4 The aim of the present study was to assess whether chloricromene inhibits PAF synthesis by stimulated human polymorphonuclear leucocytes (PMNs). 5 Cloricromene (50–500 μm) inhibited in a concentration‐dependent manner the release of PAF, as measured by h.p.l.c. bioassay, from A23187‐stimulated PMNs. Significant inhibition (45%) of PAF‐release was obtained with 50 μm cloricromene and the IC50 was 85 μm. Mepacrine (500 μm), a non‐specific PLA2 inhibitor, strikingly reduced PAF release. 6 The incorporation of [3H]‐acetate into [3H]‐PAF induced by serum‐treated zymosan in human PMNs was also inhibited concentration‐dependently by cloricromene, with an IC50 of 105 μm. Mepacrine also suppressed [3H]‐acetate incorporation into [3H]‐PAF. 7 Cloricromene did not affect the activities of the enzymes involved in PAF‐synthesis acetyltransferase or phosphocholine transferase. 8 Our data demonstrate that cloricromene, an inhibitor of PLA2‐activation in human leucocytes, reduces the synthesis of PAF by stimulated PMNs. This finding has a twofold implication: the PLA2s (or the mechanisms that regulate their activation) involved in PAF synthesis and arachidonate release in human leucocytes are either identical or else indistinguishable by their sensitivity to cloricromene; the inhibition of PAF release by activated leucocytes may contribute to the antithrombotic and anti‐ischaemic activities exerted by cloricromene.