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Hereditary thrombocytopenias (HTPs) constitute a heterogeneous group of diseases characterized by a reduction in platelet count and a potential bleeding risk. As a result of advances in diagnostic methods, HTPs are increasingly being identified, and appear to be less rare than previously thought. Most HTPs do not have effective treatments, except for platelet transfusion when bleeding occurs and in preparation for procedures associated with a risk of bleeding. Preliminary clinical evidence suggests that thrombopoietin receptor agonists (TPO-RAs) with an established use in the treatment of certain acquired thrombocytopenias are well tolerated and provide clinical benefits in patients with some forms of HTP. These drugs may therefore be considered for the treatment of HTPs in clinical practice. However, caution and close monitoring are recommended, owing to the absence of long-term safety data and the potential risks posed by prolonged bone marrow stimulation in certain HTPs. In this review, we summarize the available clinical data on TPO-RAs in the treatment of HTPs, and discuss their use in patients with these disorders. We believe that TPO-RAs will play a major role in the treatment of HTPs, particularly myosin heavy chain 9-related disease, Wiskott-Aldrich syndrome, X-linked thrombocytopenia, and thrombocytopenia caused by THPO mutations., (© 2018 International Society on Thrombosis and Haemostasis.)

Essentials Extramedullary hematopoiesis (EMH) represents a pathologic finding in adult life. We report a mass-like EMH in the presacral space in a patient with ANKRD26-related thrombocytopenia. We found possible correlation between EMH and conditions causing lifelong thrombocytopenia. EMH can cause masses of unknown origin in patients with inherited thrombocytopenias., Summary: Most commonly located in the liver and spleen, extramedullary hematopoiesis (EMH) is the presence of hematopoietic tissue outside the bone marrow. MYH9-related thrombocytopenia (MYH9-RD) and ANKRD26-related thrombocytopenia (ANKRD26-RT) are two of the most frequent forms of inherited thrombocytopenia (IT). Until recently, EMH has been associated with neoplastic and non-neoplastic hematologic conditions in which ITs were not included. We describe a case of mass-like EMH in the presacral space in a patient affected with ANKRD26-RT, comparing it with another case of paravertebral EMH we recently described in a subject with MYH9-RD. The surprisingly similitude of such a finding in the context of a group of rare disorders induces us to speculate about the possible pathogenic relationship between EMH and conditions causing lifelong thrombocytopenia, particularly the entity of ITs. Finally, we suggest that EMH has to be taken into consideration in the diagnostic work-up of masses of unknown origin in subjects affected with ITs., (© 2017 International Society on Thrombosis and Haemostasis.)

Bleeding diathesis has been considered for a long time the main clinical issue impacting the lives of patients affected by inherited thrombocytopaenias. However, the number of known inherited thrombocytopaenias greatly increased in recent years, and careful evaluation of hundreds of patients affected by these 'new' disorders revealed that most of them are at risk of developing additional life-threatening disorders during childhood or adult life. These additional disorders are usually more serious and dangerous than low platelet count. For instance, it is known that mutations in RUNX1, ANKRD26 and ETV6 cause congenital thrombocytopaenia, but we now know that they also predispose to haematological malignancies. Similarly, MYH9 mutations result in congenital thrombocytopaenia and increase the risk of developing kidney failure, cataracts and hearing loss at a later stage, while MPL mutations cause a congenital thrombocytopaenia that almost always evolves into deadly bone marrow failure. Thus, identification of patients with these disorders is essential for evaluation of their prognosis, enabling effective genetic counselling, personalizing follow-up and giving appropriate treatments in case of development of additional diseases. Careful clinical evaluation and peripheral blood film examination are extremely useful tools in guiding the diagnostic process and identifying the candidate genes to be sequenced., (© 2017 John Wiley & Sons Ltd.)

Essentials There are many hereditary platelet disorders (HPD) but diagnosing these is challenging. We provide a method to diagnose several HPDs using standard blood smears requiring < 100 µL blood. By this approach, the underlying cause of HPD was characterized in ~25-30% of referred individuals. The method facilitates diagnosis of HPD for patients of all ages around the world., Summary: Background Many hereditary thrombocytopenias and/or platelet function disorders have been identified, but diagnosis of these conditions remains challenging. Diagnostic laboratory techniques are available only in a few specialized centers and, using fresh blood, often require the patient to travel long distances. For the same reasons, patients living in developing countries usually have limited access to diagnosis. Further, the required amount of blood is often prohibitive for pediatric patients. Objectives By a collaborative international approach of four centers, we aimed to overcome these limitations by developing a method using blood smears prepared from less than 100 μL blood, for a systematic diagnostic approach to characterize the platelet phenotype. Methods We applied immunofluorescence labelling (performed centrally) to standard air-dried peripheral blood smears (prepared locally, shipped by regular mail), using antibodies specific for proteins known to be affected in specific hereditary platelet disorders. Results By immunofluorescence labelling of blood smears we characterized the underlying cause in 877/3217 (27%) patients with suspected hereditary platelet disorders (HPD). Currently about 50 genetic causes for HPD are identified. Among those, the blood smear method was especially helpful to identify MYH9 disorders/MYH9-related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia and gray platelet syndrome. Diagnosis could be established for GATA1 macrothrombocytopenia, GFI1B macrothrombocytopenia, ß1-tubulin macrothrombocytopenia, filamin A-related thrombocytopenia and Wiskott-Aldrich syndrome. Conclusion Combining basic and widely available preanalytical methods with the immunomorphological techniques presented here, allows detailed characterization of the platelet phenotype. This supports genetic testing and facilitates diagnosis of hereditary platelet disorders for patients of all ages around the world., (© 2017 International Society on Thrombosis and Haemostasis.)

Abscisic acid (ABA) is a phytohormone involved in pivotal physiological functions in higher plants. Recently, ABA has been proven to be also secreted and active in mammals, where it stimulates the activity of innate immune cells, mesenchymal and hematopoietic stem cells, and insulin-releasing pancreatic β cells through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). In addition to behaving like an animal hormone, ABA also holds promise as a nutraceutical plant-derived compound in humans. Many biological functions of ABA in mammals are mediated by its binding to the LANCL-2 receptor protein. A putative binding of ABA to GRP78, a key regulator of endoplasmic reticulum stress, has also been proposed. Here we investigated the role of exogenous ABA in modulating thrombopoiesis, the process of platelet generation. Our results demonstrate that expression of both LANCL-2 and GRP78 is up-regulated during hematopoietic stem cell differentiation into mature megakaryocytes (Mks). Functional ABA receptors exist in mature Mks because ABA induces an intracellular Ca 2+ increase ([Ca 2+ ] i ) through PKA activation and subsequent cADPR generation. In vitro exposure of human or murine hematopoietic progenitor cells to 10 μm ABA does not increase recombinant thrombopoietin (rTpo)-dependent Mk differentiation or platelet release. However, under conditions of cell stress induced by rTpo and serum deprivation, ABA stimulates, in a PKA- and cADPR-dependent fashion, the mitogen-activated kinase ERK 1/2, resulting in the modulation of lymphoma 2 (Bcl-2) family members, increased Mk survival, and higher rates of platelet production. In conclusion, we demonstrate that ABA is a prosurvival factor for Mks in a Tpo-independent manner., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The European Hematology Association (EHA) Roadmap for European Hematology Research highlights major achievements in diagnosis and treatment of blood disorders and identifies the greatest unmet clinical and scientific needs in those areas to enable better funded, more focused European hematology research. Initiated by the EHA, around 300 experts contributed to the consensus document, which will help European policy makers, research funders, research organizations, researchers, and patient groups make better informed decisions on hematology research. It also aims to raise public awareness of the burden of blood disorders on European society, which purely in economic terms is estimated at €23 billion per year, a level of cost that is not matched in current European hematology research funding. In recent decades, hematology research has improved our fundamental understanding of the biology of blood disorders, and has improved diagnostics and treatments, sometimes in revolutionary ways. This progress highlights the potential of focused basic research programs such as this EHA Roadmap.The EHA Roadmap identifies nine 'sections' in hematology: normal hematopoiesis, malignant lymphoid and myeloid diseases, anemias and related diseases, platelet disorders, blood coagulation and hemostatic disorders, transfusion medicine, infections in hematology, and hematopoietic stem cell transplantation. These sections span 60 smaller groups of diseases or disorders.The EHA Roadmap identifies priorities and needs across the field of hematology, including those to develop targeted therapies based on genomic profiling and chemical biology, to eradicate minimal residual malignant disease, and to develop cellular immunotherapies, combination treatments, gene therapies, hematopoietic stem cell treatments, and treatments that are better tolerated by elderly patients., (Copyright© Ferrata Storti Foundation.)

Some familial platelet disorders are associated with predisposition to leukemia, myelodysplastic syndrome (MDS) or dyserythropoietic anemia. We identified a family with autosomal dominant thrombocytopenia, high erythrocyte mean corpuscular volume (MCV) and two occurrences of B cell-precursor acute lymphoblastic leukemia (ALL). Whole-exome sequencing identified a heterozygous single-nucleotide change in ETV6 (ets variant 6), c.641C>T, encoding a p.Pro214Leu substitution in the central domain, segregating with thrombocytopenia and elevated MCV. A screen of 23 families with similar phenotypes identified 2 with ETV6 mutations. One family also had a mutation encoding p.Pro214Leu and one individual with ALL. The other family had a c.1252A>G transition producing a p.Arg418Gly substitution in the DNA-binding domain, with alternative splicing and exon skipping. Functional characterization of these mutations showed aberrant cellular localization of mutant and endogenous ETV6, decreased transcriptional repression and altered megakaryocyte maturation. Our findings underscore a key role for ETV6 in platelet formation and leukemia predisposition.

Background: Single nucleotide polymorphisms (SNPs) in platelet-associated genes partly explain inherent variability in platelet counts. Patients with monoallelic Bernard Soulier syndrome due to the Bolzano mutation (GPIBA A156V) have variable platelet counts despite a common mutation for unknown reasons., Objectives: We investigated the effect of the most common SNP (R307H) in the hematopoietic-specific tubulin isotype β-1 in these Bernard Soulier patients and potential microtubule-based mechanisms of worsened thrombocytopenia., Patients/methods: Ninety-four monoallelic Bolzano mutation patients were evaluated for the R307H β-1 SNP and had platelet counts measured by three methods; the Q43P SNP was also evaluated. To investigate possible mechanisms underlying this association, we used molecular modeling of β-1 tubulin with and without the R307H SNP. We transfected SNP or non-SNP β-1 tubulin into MCF-7 and CMK cell lines and measured microtubule regrowth after nocodazole-induced depolymerization., Results: We found that patients with at least one R307H SNP allele had significantly worse thrombocytopenia; manual platelet counting revealed a median platelet count of 124 in non-SNP patients and 76 in SNP patients (both ×10(9)  L(-1) ; P < 0.01). The Q43P SNP had no significant association with platelet count. Molecular modeling suggested a structural relationship between the R307H SNP and microtubule stability via alterations in the M-loop of β tubulin; in vitro microtubule recovery assays revealed that cells transfected with R307H SNP β-1 had significantly impaired microtubule recovery., Conclusions: Our data show that the R307H SNP is significantly associated with the degree of thrombocytopenia in congenital and acquired platelet disorders, and may affect platelets by altering microtubule behavior., (© 2014 International Society on Thrombosis and Haemostasis.)

Background: The performance of pocket mobile ultrasound devices (PUDs) is comparable with that of standard ultrasonography, whereas the accuracy of a physical examination is often poor requiring further tests to assess diagnostic hypotheses. Adding the use of PUD to physical examination could lead to an incremental benefit., Aim: We assessed whether the use of PUD in the context of physical examination can reduce the prescription of additional tests when used by physicians in different clinical settings., Methods: We conducted a cohort impact study in four hospital medical wards, one gastroenterological outpatient clinic, and 90 general practices in the same geographical area. The study involved 135 physicians who used PUD, after a short predefined training course, to examine 1962 consecutive patients with one of 10 diagnostic hypotheses: ascites, pleural effusion, pericardial effusion, urinary retention, urinary stones, gallstones, biliary-duct dilation, splenomegaly, abdominal mass, abdominal aortic aneurysm. According to the physicians' judgment, PUD examination could rule out or in the diagnostic hypothesis or require further testing; the concordance with the final diagnosis was assessed. The main outcome was the proportion of cases in which additional tests were required after PUD. The PUD diagnostic accuracy was assessed in patients submitted to further testing., Findings: The 1962 patients included 37% in-patients, 26% gastroenterology outpatients, 37% from general practices. Further testing after PUD examination was deemed unnecessary in 63%. Only 5% of patients with negative PUD not referred for further testing were classified false negatives with respect to the final diagnosis. In patients undergoing further tests, the sensitivity was 91%, and the specificity 83%., Conclusions: After a simple and short training course, a PUD examination can be used in addition to a physical examination to improve the answer to ten common clinical questions concerning in- and outpatients, and can reduce the need for further testing.

Boron neutron capture therapy (BNCT) is an anticancer treatment based on the accumulation in the tumor cells of (10) B-containing molecules and subsequent irradiation with low-energy neutrons, which bring about the decay of (10) B to very toxic (7) Li(3+) and (4) He(2+) ions. The effectiveness of BNCT is limited by the low delivery and accumulation of the used (10) B-containing compounds. Here, we report the development of folic acid-conjugated 4-amino-phenylboronate as a novel possible compound for the selective delivery of (10) B in BNCT. An extensive analysis about its biocompatibility to mature blood cells and platelet progenitors revealed that the compound markedly supports platelet aggregation, neutrophil oxidative burst, and inhibition of megakaryocyte development, while it does not have any manifest effect on red blood cells., (© 2013 John Wiley & Sons A/S.)

Boron neutron capture therapy (BNCT) is a radiotherapy treatment based on the accumulation in the tumor of a (10)B-containing drug and subsequent irradiation with low energy neutrons, which bring about the decay of (10)B to (7)Li and an α particle, causing the death of the neoplastic cell. The effectiveness of BNCT is limited by the low delivery and accumulation of the used boron-containing compounds. Here we report the development and the characterization of BPO4 nanoparticles (NPs) as a novel possible alternative drug for BNCT. An extensive analysis of BPO4 NP biocompatibility was performed using both mature blood cells (erythrocytes, neutrophils and platelets) and a model of hematopoietic progenitor cells. A time- and concentration-dependent cytotoxicity study was performed on neoplastic coloncarcinoma and osteosarcoma cell lines. BPO4 functionalization with folic acid, introduced to improve the uptake by tumor cells, appeared to effectively limit the unwanted effects of NPs on the analyzed blood components., From the Clinical Editor: Boron neutron capture therapy (BNCT) is a radiotherapy treatment modality based on the accumulation of a (10)B-containing drug and subsequent irradiation with low energy neutrons, inducing the decay of (10)B to (7)Li and an α particle, causing neoplastic cell death. This team of authors reports on a folic acid functionalized BPO4 nanoparticle with improved characteristics compared with conventional BNCT approaches, as demonstrated in tumor cell lines, and hopefully to be followed by translational human studies., (© 2014.)

In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin αIIbβ3 inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In the present study we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI (calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I), is a novel substrate for the cAMP-activated PKA (protein kinase A). CalDAG-GEFI phosphorylation occurred in intact platelets treated with the cAMP-increasing agent forskolin and was inhibited by the PKA inhibitor H89. Purified recombinant CalDAG-GEFI was also phosphorylated in vitro by the PKA catalytic subunit. By screening a panel of specific serine to alanine residue mutants, we identified Ser116 and Ser586 as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK (human embryonic kidney)-293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented the activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets this effect was associated with the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK-293 cells transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. The results of the present study demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.

The diagnosis of inherited thrombocytopenias is difficult, for many reasons. First, as they are all rare diseases, they are little known by clinicians, who therefore tend to suspect the most common forms. Second, making a definite diagnosis often requires complex laboratory techniques that are available in only a few centers. Finally, half of the patients have forms that have not yet been described. As a consequence, many patients with inherited thrombocytopenias are misdiagnosed with immune thrombocytopenia, and are at risk of receiving futile treatments. Misdiagnosis is particularly frequent in patients whose low platelet count is discovered in adult life, because, in these cases, even the inherited origin of thrombocytopenia may be missed. Making the correct diagnosis promptly is important, as we recently learned that some forms of inherited thrombocytopenia predispose to other illnesses, such as leukemia or kidney failure, and affected subjects therefore require close surveillance and, if necessary, prompt treatments. Moreover, medical treatment can increase platelet counts in specific disorders, and affected subjects can therefore receive drugs instead of platelet transfusions when selective surgery is required. In this review, we will discuss how to suspect, diagnose and manage inherited thrombocytopenias, with particular attention to the forms that frequently present in adults. Moreover, we describe four recently identified disorders that belong to this group of disorders that are often diagnosed in adults: MYH9-related disease, monoallelic Bernard-Soulier syndrome, ANKRD26-related thrombocytopenia, and familial platelet disorder with predisposition to acute leukemia., (© 2013 International Society on Thrombosis and Haemostasis.)

Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183-190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a "click chemistry" reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The proline-rich tyrosine kinase Pyk2 is a focal adhesion kinase expressed in blood platelets, and is activated downstream of G-protein coupled receptors as well as integrin α2β1., Objective: In this study we have investigated the involvement of Pyk2 in integrin αIIbβ3 outside-in signaling in human and murine platelets., Methods: We analyzed the stimulation of intracellular signaling pathways in platelets from Pyk2 knockout mice adherent to immobilized fibrinogen., Results: Pyk2 was rapidly phosphorylated and activated in human and murine platelets adherent to fibrinogen through integrin αIIbβ3. Activation of Pyk2 was Src-dependent, but did not require phospholipase Cγ2 activity. Platelets from Pyk2 knockout mice showed a defective ability to adhere and spread on fibrinogen, in association with a dramatic reduction of phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation. Pharmacological and genetic analysis demonstrated that integrin αIIbβ3 engagement selectively stimulated the β-isoform of PI3K (PI3Kβ), and that, as for Pyk2, PI3Kβ activation required Src family kinases activity, but not phospholipase Cγ2. In fibrinogen-adherent platelets, both Pyk2 and PI3Kβ were necessary for stimulation of the small GTPase Rap1b, a regulator of cell adhesion and spreading. Integrin αIIbβ3 engagement triggered the association of the PI3Kβ regulatory subunit p85 with the adaptor protein c-Cbl, which was mediated by the p85 SH3 domain, and was independent of c-Cbl tyrosine phosphorylation. However, p85-associated c-Cbl was tyrosine phosphorylated by activated Pyk2 in fibrinogen adherent platelets., Conclusions: These results identify a novel pathway of integrin αIIbβ3 outside-in signaling and recognize the tyrosine kinase Pyk2 as a major regulator of platelet adhesion and spreading on fibrinogen., (© 2012 International Society on Thrombosis and Haemostasis.)

In the present study, we used a knockout murine model to analyze the contribution of the Ca(2+)-dependent focal adhesion kinase Pyk2 in platelet activation and thrombus formation in vivo. We found that Pyk2-knockout mice had a tail bleeding time that was slightly increased compared with their wild-type littermates. Moreover, in an in vivo model of femoral artery thrombosis, the time to arterial occlusion was significantly prolonged in mice lacking Pyk2. Pyk2-deficient mice were also significantly protected from collagen plus epinephrine-induced pulmonary thromboembolism. Ex vivo aggregation of Pyk2-deficient platelets was normal on stimulation of glycoprotein VI, but was significantly reduced in response to PAR4-activating peptide, low doses of thrombin, or U46619. Defective platelet aggregation was accompanied by impaired inside-out activation of integrin α(IIb)β(3) and fibrinogen binding. Granule secretion was only slightly reduced in the absence of Pyk2, whereas a marked inhibition of thrombin-induced thromboxane A(2) production was observed, which was found to be responsible for the defective aggregation. Moreover, we have demonstrated that Pyk2 is implicated in the signaling pathway for cPLA(2) phosphorylation through p38 MAPK. The results of the present study show the importance of the focal adhesion kinase Pyk2 downstream of G-protein-coupled receptors in supporting platelet aggregation and thrombus formation.

Background: Three meta-analyses conducted in the 1990s concluded that the effect of intermittent flushing with heparin at low concentration (10 U/mL) was equivalent to that of 0.9% sodium chloride flushes in preventing occlusion or superficial phlebitis. No firm conclusion was reached on the safety and efficacy of heparin concentrations of 100 U/mL used as an intermittent flush., Purpose: To determine whether flushing peripheral intravenous catheters with 3 mL of a 100 U heparin/mL solution instead of saline improves the outcome of infusion devices., Methods: Cluster-randomized, controlled, two-arm, open trial, conducted in a research and teaching hospital in Northern Italy, involving 214 medical patients without contraindications to heparin: 107 randomly allocated to heparin and 107 to saline flushes (control group). Main outcome measure was catheter occlusion and catheter-related phlebitis., Results: Patients with either phlebitis or occlusion were 45 (42.1%) in the heparin group and 68 (63.6%) in the saline group (OR 0.41; 95% CI 0.24-0.72; p= 0.002); patients with occlusion alone were 23 (21.5%) and 47 (43.9%), respectively (p= 0.03); patients with phlebitis alone were 28 (26.2%) and 56 (52.6%) respectively (p= <0.001). Similar results were obtained when the analysis was based on catheters. No heparin severe side effects were identified., Limitations: Lack of blinding, patient selection, cluster randomization of periods of treatment., Conclusions: Heparin 100 U/mL in the maintenance of peripheral venous catheters was more effective than saline solution, in that it reduced the number of catheter-related phlebitis/occlusions and the number of catheters per patient, with potential advantages to both patients and the health system. It also appeared safe. However, subjects with platelet or coagulation defects were excluded, and, therefore, caution should be used when prescribing this type of catheter maintenance to patients at risk of bleeding., (© 2012 Sigma Theta Tau International.)

Nanomaterials are attracting growing interest for their potential use in several applications as nanomedicine; therefore, the analysis of their potential toxic effects on various cellular models, including circulating blood cells, is mandatory. This study aimed to investigate the effect of three unrelated nanomaterials, namely nanoscale silica, multiwalled carbon nanotubes, and carbon black, on platelet activation and aggregation. We found that these nanomaterials stimulate some of the typical biochemical pathways involved in canonical platelet activation, such as the stimulation of phospholipase C and Rap1b, resulting in the integrin α(IIb)β3-mediated platelet aggregation, through a mechanism largely dependent on the release of the extracellular second messengers ADP and thromboxane A2. Importantly, we found that doses of nanoparticles unable to trigger appreciable responses can synergize with subthreshold amounts of physiological agonists to mediate platelet aggregation, indicating that even small amounts of nanomaterials in the bloodstream might contribute to the development of thrombosis., From the Clinical Editor: In this study, nanosized particles of three virtually unrelated materials (silica, multi-walled carbon nanotubes and carbon black) were investigated regarding their effects on platelet activation and aggregation. All were found to stimulate some of the typical biochemical pathways involved in canonical platelet activation, and were found to have synergistic effects with physiologic platelet activator agonists., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource-limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina., Methods: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin-9 in granulocytes and platelet thrombospondin-1 and molecular screening of candidate genes., Results: Thirty-one patients from 14 pedigrees were included; their median age was 32 (4-72) years and platelet count 72 (4-147)×10(9) L(-1). Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9-related disease in four, while classic and monoallelic Bernard-Soulier syndrome, gray platelet syndrome, X-linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each., Conclusions: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases., (© 2012 International Society on Thrombosis and Haemostasis.)

Integrin α2β1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3Kβ(KD)), but occurred normally in PI3Kγ(KD) platelets. Integrin α2β1 failed to stimulate PI3Kβ in platelets from phospholipase Cγ2 (PLCγ2)-knockout mice, and we found that intracellular Ca(2+) linked PLCγ2 to PI3Kβ activation. Integrin α2β1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca(2+). Whereas activation of Pyk2 occurred normally in PI3Kβ(KD) platelets, stimulation of PI3Kβ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kβ was required for α2β1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbβ3 were reduced after inhibition of PI3Kβ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kβ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kβ downstream of integrin α2β1, and document a novel role for Pyk2 and PI3Kβ in integrin α2β1 promoted inside-out activation of integrin αIIbβ3 and thrombus formation.

The chapter of inherited thrombocytopenias has expanded greatly over the last decade and many "new" forms deriving from mutations in "new" genes have been identified. Nevertheless, nearly half of patients remain without a definite diagnosis because their illnesses have not yet been described. The diagnostic approach to these diseases can still take advantage of the algorithm proposed by the Italian Platelet Study Group in 2003, although an update is required to include the recently described disorders. So far, transfusions of platelet concentrates have represented the main tool for preventing or treating bleedings, while haematopoietic stem cell transplantation has been reserved for patients with very severe forms. However, recent disclosure that an oral thrombopoietin mimetic is effective in increasing platelet count in patients with MYH9-related thrombocytopenia opened new therapeutic perspectives. This review summarizes the general aspects of inherited thrombocytopenias and describes in more detail MYH9-related diseases (encompassing four thrombocytopenias previously recognized as separate diseases) and the recently described ANKRD26-related thrombocytopenia, which are among the most frequent forms of inherited thrombocytopenia.

Background: MYH9-related disease (MYH9-RD) is a rare autosomal dominant genetic syndrome characterized by congenital thrombocytopenia associated with the risk of developing progressive nephropathy, sensorineural deafness, and presenile cataract. During the collection of a large case-series of patients with MYH9-RD we noticed several cases with unexplained elevation of liver enzymes. Our aim was to evaluate if the alteration of liver tests is a feature of the MYH9-RD and to define its clinical significance., Methods and Findings: Data concerning liver tests, prospectively recorded in the Italian Registry for MYH9-RD, were collected and compared with those of three control populations: patients with autoimmune thrombocytopenia, patients with inherited thrombocytopenias other than MYH9-RD, and the participants to a large epidemiologic survey in an Italian geographic isolate. Thirty-eight of 75 evaluable MYH9-RD patients (50.7%) showed an elevation of ALT and/or AST, and 17 of 63 (27.0%) an increase of GGT. The increases ranged from 1.9 ± 0.7 to 2.7 ± 1.6 fold the upper normal limit. The prevalence of liver test alterations was significantly higher in MYH9-RD patients than in each of the control populations, with odds ratios ranging from 8.2 (95% CIs 2.2-44.8) to 24.7 (14.8-40.8). Clinical follow-up and more detailed liver studies of a subset of patients, including ultrasound liver scan, liver elastography and liver biopsy in one case, did not show any significant structural damage or evolution towards liver insufficiency., Conclusions: Elevation of liver enzymes is a frequent and previously unrecognized feature of the MYH9-RD syndrome; however, this defect does not appear to have poor prognostic value.

We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin. This effect occurred in the absence of Gq stimulation, as it was not associated to intracellular Ca2+ release. Epinephrine-induced pleckstrin phosphorylation was time- and dose-dependent, and was inhibited by the α2-adrenergic antagonist yohimbin. Phosphorylation of pleckstrin did not occur when platelet stimulation with epinephrine was performed in the presence of the ADP scavenger apyrase, and was suppressed by antagonists of both P2Y1 and P2Y12 ADP receptors. Importantly, no release of dense granules was measured in epinephrine-treated platelets. Addition of epinephrine to platelets was also able to stimulate Rap1b activation. Similarly to pleckstrin phosphorylation, however, this effect was prevented in the presence of apyrase or upon pharmacologic blockade of either P2Y1 or P2Y12 receptors. These results indicate that sub-threshold amounts of ADP in the medium are essential to allow epinephrine stimulation of α2-adrenergic receptor to elicit platelet responses, and reveal a novel synergism among strong stimulation of Gz and sub-threshold stimulation of both Gq and Gi, able to dissociate PKC activation from intracellular Ca2+ mobilisation.

A balance between the proteolytic processing of amyloid precursor protein APP through the amyloidogenic and the non-amyloidogenic pathways controls the production and release of amyloid β-protein, whose accumulation in the brain is associated to the onset of Alzheimer Disease. APP is also expressed on circulating platelets. The regulation of APP processing in these cells is poorly understood. In this work we show that platelets store considerable amounts of APP fragments, including sAPPα, that can be released upon stimulation of platelets. Moreover, platelet stimulation also promotes the proteolysis of intact APP expressed on the cell surface. This process is supported by an ADAM metalloproteinase, and causes the release of sAPPα. Processing of intact platelet APP is promoted also by treatment with calmodulin antagonist W7. W7-induced APP proteolysis occurs through the non-amyloidogenic pathway, is mediated by a metalloproteinase, and causes the release of sAPPα. Co-immunoprecipitation and pull-down experiments revealed a physical association between calmodulin and APP. These results document a novel role of calmodulin in the regulation of non-amyloidogenic processing of APP., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood.

Supposedly "homogeneous" red blood cell (RBC) samples are commonly obtained by "washing" whole blood free of plasma, platelets, and white cells with physiological solutions, a procedure that does not result, however, in sufficient removal of polymorphonuclear neutrophils (PMNs), leading to possible artifactual results. Pure RBC samples can be obtained only by leukodepletion procedures. Proposed here is a version of gelatin zymography adapted to detect matrix metalloproteinase 9 (MMP-9), selectively expressed by PMNs, in heterogeneous mixtures of RBCs and PMNs that can reveal contamination at levels as low as 1 PMN/10⁶ RBCs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane skeleton in human erythrocytes and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils that lead to proteolytic disruption of the supramolecular assembly of the membrane skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions that most likely involve the spectrin molecule., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibalpha of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca(2+) mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbalpha by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbalpha binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbalpha proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbalpha cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbalpha., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C(12)E(8)) at 37 degrees C, and by treatment at 4 degrees C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37 degrees C, or from cholesterol-depleted cells at 4 degrees C, for both detergents. For 5-SASL only, increased S values were measured in 4 degrees C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C(12)E(8) DRMs prepared at 4 degrees C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C(12)E(8) DRMs. However, contrary to the 4 degrees C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C(12)E(8) treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C(12)E(8) to the study of DRMs.

Background: Distinguishing inherited thrombocytopenias from immune thrombocytopenia (ITP) can be difficult, and patients are therefore at risk of misdiagnosis and inappropriate treatments. Although it is known that the most common inherited forms of thrombocytopenia are characterized by increased platelet size, the diagnostic power of this feature has never been investigated., Objectives: The aim of this study was to test the hypothesis that platelet size can be used to differentiate ITP from inherited macrothrombocytopenias., Patients/methods: We measured mean platelet volume (MPV) and mean platelet diameter (MPD), within 2 h of blood sampling, in 35 patients with inherited macrothrombocytopenias [15 MYH9-related disease (MYH9-RD), three biallelic and 17 monoallelic Bernard-Soulier syndrome (BSS)], and 56 with ITP. Using receiving operating characteristic analysis, we searched for the best cut-off values to differentiate between these conditions., Results: As expected, platelets were larger in inherited macrothrombocytopenias than in ITP. An MPD larger than 3.3 mum differentiated MYH9-RD and BSS from ITP with 0.89 sensitivity and 0.88 specificity, and an MPV larger than 12.4 fL had 0.83 sensitivity and 0.89 specificity. Combining MPD with MPV increased sensitivity and specificity to 0.97 and 0.89, respectively., Conclusion: Platelet size evaluation by both an appropriate cell counter and blood film examination is useful for differentiating inherited macrothrombocytopenias from ITP.

The optimal management of bleeding or its prophylaxis in patients with disorders of platelet count or function is controversial. The bleeding diathesis of these patients is usually mild to moderate: therefore, transfusion of platelet concentrates may be inappropriate, as potential adverse effects might outweigh its benefit. The availability of several anti-hemorrhagic drugs further compounds this problem, mainly because the efficacy/suitability of the various treatment options in different clinical manifestations is not well defined. In these guidelines, promoted by the Italian Society for Studies on Haemostasis and Thrombosis (Società Italiana per lo Studio dell'Emostasi e della Trombosi [SISET]), we aim at offering the best available evidence to help the physicians involved in the management of patients with disorders of platelet count or function. Literature review and appraisal of available evidence are discussed for different clinical settings and for different available treatments, including platelet concentrates (PC), recombinant activated factor VII, desmopressin, antifibrinolytics, aprotinin and local hemostatic agents.

Phosphatidylinositol 3-kinase (PI3K) isoforms PI3Kbeta and PI3Kgamma are implicated in platelet adhesion, activation, and aggregation, but their relative contribution is still unclear or controversial. Here, we report the first comparative functional analysis of platelets from mice expressing a catalytically inactive form of PI3Kbeta or PI3Kgamma. We demonstrate that both isoforms were similarly required for maximal activation of the small GTPase Rap1b and for complete platelet aggregation upon stimulation of G protein-coupled receptors for adenosine 5'-diphosphate (ADP) or U46619. Their contribution to these events, however, was largely redundant and dispensable. However, PI3Kbeta, but not PI3Kgamma, enzymatic activity was absolutely required for Akt phosphorylation, Rap1 activation, and platelet aggregation downstream of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor glycoprotein VI (GPVI). Moreover, PI3Kbeta was a major essential regulator of platelet adhesion to fibrinogen and of integrin alpha(IIb)beta(3)-mediated spreading. These results provide genetic evidence for a crucial and selective role of PI3Kbeta in signaling through GPVI and integrin alpha(IIb)beta(3).

Background: Platelet adhesion promoted by integrin alpha2beta1 induces integrin alpha(IIb)beta3 activation through the phospholipase C (PLC)-dependent stimulation of the small GTPase Rap1b., Objective: To analyze the mechanism of PLC activation downstream of alpha2beta1 that is required for regulation of Rap1b and alpha(IIb)beta3., Methods: Human and murine platelets were allowed to adhere to immobilized type I monomeric collagen through alpha2beta1. Tyrosine phosphorylation of PLCgamma2, PLC activation, accumulation of GTP-bound Rap1b and fibrinogen binding were measured and compared., Results: Integrin alpha2beta1 recruitment induced an evident PLC activation that was concomitant with robust tyrosine phosphorylation of PLCgamma2, and was suppressed in platelets from PLCgamma2-knockout mice. Moreover, PLCgamma2(-/-) platelets were unable to accumulate active Rap1b and to activate alpha(IIb)beta3 upon adhesion through alpha2beta1. Inhibition of Src kinases completely prevented tyrosine phosphorylation of PLCgamma2 in adherent platelets, but did not affect its activation, and both Rap1b and alpha(IIb)beta3 stimulation occurred normally. Importantly, alpha(IIb)beta3-induced phosphorylation and activation of PLCgamma2, as well as accumulation of active Rap1b, were totally suppressed by Src inhibition. Integrin alpha2beta1 recruitment triggered the Src kinase-independent activation of the small GTPase Rac1, and activation of Rac1 was not required for PLCgamma2 phosphorylation. However, when phosphorylation of PLCgamma2 was blocked by the Src kinase inhibitor PP2, prevention of Rac1 activation significantly reduced PLCgamma2 activation, GTP-Rap1b accumulation, and alpha(IIb)beta3 stimulation., Conclusions: Src kinases and the Rac GTPases mediate independent pathways for PLCgamma2 activation downstream of alpha2beta1.

Background: Although mutations of GPIb alpha are among the most frequent causes of inherited platelet disorders, the mechanisms for the onset of thrombocytopenia and platelet macrocytosis are still poorly defined., Objective: In this work we analyzed in vitro megakaryocyte differentiation and proplatelet formation in six subjects heterozygous for the Ala156Val mutation in the GPIb alpha (Bolzano mutation)., Methods: Human megakaryocytes were obtained by differentiation of patient cord blood-derived CD34(+) cells and peripheral blood-derived CD45(+) cells. Proplatelet formation was evaluated by phase contrast and fluorescence microscopy., Results: Megakaryocyte differentiation from both cord blood (one patient) and peripheral blood (five patients) was comparable to controls. However, proplatelet formation was reduced by about 50% with respect to controls. An identical defect of proplatelet formation was observed when megakaryocytes were plated on fibrinogen, von Willebrand factor or grown in suspension. Morphological evaluation of proplatelet formation revealed an increased size of proplatelet tips, which was consistent with the increased diameters of patients' blood platelets. Moreover, alpha-tubulin distribution within proplatelets was severely deranged., Conclusions: Megakaryocytes from patients carrying a Bolzano allele of GPIb alpha display both quantitative and qualitative abnormalities of proplatelet formation in vitro. These results suggest that a defect of platelet formation contributes to macrothrombocytopenia associated to the Bolzano mutation, and indicate a key role for GPIb alpha in proplatelet formation.

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4 degrees C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C12E8. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C12E8, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C12E8 in comparison to intact cells, while the difference in the S values between Triton X-100 and C12E8 DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C12E8 detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.

Background: Heterozygous mutations of MYH9, encoding the Non-Muscular Myosin Heavy Chain-IIA (NMMHC-IIA), cause a complex disorder named MYH9-related disease, characterized by a combination of different phenotypic features. At birth, patients present platelet macrocytosis, thrombocytopenia and leukocyte inclusions containing NMMHC-IIA. Moreover, later in life some of them develop the additional features of sensorineural hearing loss, cataracts and/or glomerulonephritis that sometimes leads to end stage renal failure., Results: To clarify the mechanism by which the mutant NMMHC-IIA could cause phenotypic anomalies at the cellular level, we examined the effect of transfection of the full-length mutated D1424H MYH9 cDNAs. We have observed, by confocal microscopy, abnormal distribution of the protein and formation of rod-like aggregates reminiscent of the leukocyte inclusions found in patients. Co-transfection of differently labeled wild-type and mutant full-length cDNAs showed the simultaneous presence of both forms of the protein in the intracellular aggregates., Conclusion: These findings suggest that the NMMHC-IIA mutated in position 1424 is able to interact with the WT form in living cells, despite part of the mutant protein precipitates in non-functional aggregates. Transfection of the entire WT or mutant MYH9 in cell lines represents a powerful experimental model to investigate consequences of MYH9 mutations.

Background: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized., Objective: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors., Methods: Human megakaryocytes were obtained by differentiation of cord blood-derived CD34(+) cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy., Results: We found that human megakaryocytes extended proplatelets in a time-dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin alphaIIbbeta3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb-IX-V, or upon cleavage of GPIbalpha by the metalloproteinase mocarhagin. Membrane-associated VWF was detected exclusively on proplatelet-forming megakaryocytes, but not on round mature cells that do not extend proplatelets., Conclusions: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio-temporal regulation orchestrated by adhesive proteins, GPIb-IX-V and myosin IIA.

Stimulation of G(q)-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca(2+) mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the G(q)-coupled P2Y1 receptor but also upon blockade of the G(i)-coupled P2Y12 receptor. The role of G(i) on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon G(q) stimulation by the thromboxane A(2) analogue U46619. Importantly, activation of phospholipase C and intracellular Ca(2+) mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the G(i)-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.

Rap1b and Rap2b are the only members of the Rap family of GTPases expressed in circulating human platelets. Rap1b is involved in the inside-out activation of integrins, while the role of Rap2b is still poorly understood. In this work, we investigated the localization of Rap proteins to specific microdomains of plasma membrane called lipid rafts, implicated in signal transduction. We found that Rap1b was not associated to lipid rafts in resting platelets, and did not translocate to these microdomains in stimulated cells. By contrast, about 20% of Rap2b constitutively associated to lipid rafts, and this percentage did not increase upon platelet stimulation. Rap2b interaction with lipid rafts also occurred in transfected HEK293T cell. Upon metabolic labelling with [(3)H]palmitate, incorporation of the label into Rap2b was observed. Palmitoylation of Rap2b did not occur when Cys176 or Cys177 were mutated to serine, or when the C-terminal CAAX motif was deleted. Contrary to CAAX deletion, Cys176 and Cys177 substitution did not alter the membrane localization of Rap2b, however, relocation of the mutants within lipid rafts was completely prevented. In intact platelets, disruption of Rap2b interaction with lipid rafts obtained by cholesterol depletion caused a significant inhibition of aggregation. Importantly, agonist-induced activation of Rap2b was concomitantly severely impaired. These results demonstrate that Rap2b, but not the more abundant Rap1b, is associated to lipid rafts in human platelets. This interaction is supported by palmitoylation of Rap2b, and is important for a complete agonist-induced activation of this GTPase.

L-Methionine (Met), in its free form or when inserted in proteins, is sensitive to oxidation of its thioether group by reactive oxygen species from exogenous or endogenous sources. Two stable diastereomers of Met sulfoxide [Met-(O)] may be formed [Met-S-(O) and Met-R-(O)], but these can be reduced by two classes of Methionine-sulfoxide-reductase (Msr) enzymes: MsrA, which reduces the S, and MsrB, which reduces the R sulfoxide. In this study, we have examined the levels of expression of Msr in human blood cells by enzymatic activity assay, Western blotting, and RT-PCR of purified populations of polymorphonuclear neutrophils and eosinophils, mononuclear cells, platelets, and erythrocytes. Our data indicate that of the blood cells analyzed, neutrophils expressed the highest activity, which was mainly of MsrB type. During degranulation of activated neutrophils, Msr activity was not released but remained confined within the cell, indicating a non-granular localization. Immunoprecipitation and RT-PCR studies indicated the almost complete lack of mitochondrial forms of Msrs in granulocytes. It is thus likely that Msrs are important as antioxidant/repair systems for neutrophils, cells with enormous capacity for the generation of reactive oxidants and hence, susceptible to oxidative damage.

Activation of human platelets by cross-linking of the low-affinity receptor for immunoglobulin G (FcgammaRIIA) is initiated by Src kinase-mediated phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) within the receptor, but the identity of the enzyme responsible for its dephosphorylation and inactivation is unknown. Here we report that the 18-kDa low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) is expressed in human platelets and undergoes subcellular redistribution upon FcgammaRIIA cross-linking. In vitro, LMW-PTP was found to efficiently dephosphorylate activated FcgammaRIIA and LAT, but not Syk or phospholipase Cgamma2. In the megakaryocytic cell line DAMI, antibody-induced phosphorylation of FcgammaRIIA was rapid and transient. The late dephosphorylation of FcgammaRIIA was dramatically delayed upon reduction of LMW-PTP expression by siRNA. Strikingly, overexpression of LMW-PTP resulted in the inhibition of antibody-induced phosphorylation of FcgammaRIIA, and caused a more rapid dephosphorylation. In addition, overexpression of LMW-PTP inhibited activation of Syk downstream of FcgammaRIIA and reduced intracellular Ca(2+) mobilization. These results demonstrate that LMW-PTP is responsible for FcgammaRIIA dephosphorylation, and is implicated in the down-regulation of cell activation mediated by this ITAM-bearing immunoreceptor.

In this work we have investigated the ability of epinephrine to trigger the release of intracellular Ca2+ in thrombin-desensitized platelets. Addition of thrombin to platelets in the presence of extracellular EGTA caused a rapid and transient release of Ca2+ from intracellular stores and rendered platelets unresponsive to a second addition of the same agonist. Although epinephrine alone had no effect on intracellular Ca2+ mobilization, its addition to thrombin-desensitized platelets was associated to a rapid and evident secondary release of intracellular Ca2+. This effect of epinephrine was not observed when platelets were desensitized with other agonists able to induce phospholipase C activation, including convulxin, U46619, and ADP. Although the platelet receptor for epinephrine is coupled to the Gi family member Gz, no secondary Ca2+ release was seen in thrombin-desensitized platelets upon stimulation of other Gi-coupled receptors, including the P2Y12 receptor and the CXCR4. Addition of hirudin to thrombin-desensitized platelets prevented epinephrine-promoted secondary release of Ca2+, indicating that thrombin, rather than epinephrine itself, is actually responsible for this event as a consequence of thrombin receptors resensitization. Studies with platelets stimulated with specific PAR1- and PAR4- activating peptides proved that neither one of these thrombin receptors were involved in the secondary epinephrine-assisted Ca2+ release. Moreover, we found that thrombin was still able to induce a reduced, but evident release of Ca2+ from internal stores in PAR1- and PAR4-desensitized platelets, which could be followed by a secondary Ca2+ release upon subsequent addition of epinephrine. Importantly, both the primary and the secondary Ca2+ release induced by thrombin and epinephrine in PAR1- and PAR4-desensitized platelets were abrogated upon cleavage of GPIbalpha by the metalloproteinase mocarhagin. These results demonstrate a direct role of thrombin binding to GPIb-IX-V in the mobilization of Ca2+ from intracellular stores, and reveal that epinephrine can restore this process in desensitized platelets, thus prolonging the effect of thrombin stimulation.

Background: We report a novel case of gray platelet syndrome (GPS). A 14-year-old boy had bleeding diathesis, mild thrombocytopenia, giant platelets with severe defect of alpha-granule secretory proteins, myelofibrosis and splenomegaly., Methods and Results: Platelet function studies showed a marked reduction of aggregation and Ca(2+) mobilization by thrombin, protease-activated receptor 1 (PAR1)-activating peptide (AP) and PAR4-AP, PAR1 expression at 55% of normal levels, and a more than two hundred fold reduction of in vitro whole-blood thromboxane B(2) (TXB(2)) production. Sequencing of coding regions of the PAR1 gene failed to show abnormalities. This patient was initially classified as a sporadic case of GPS, as electron microscopy failed to identify giant platelets and/or alpha-granule deficiency in his relatives. However, further studies on the father and three other relatives showed a relative lack of platelet alpha-granule proteins by immunofluorescence microscopy, a defective platelet response to PAR4-AP, and severely reduced in vitro whole-blood TXB(2) production. On this basis, we suggest that in this family, GPS was transmitted in a dominant fashion with highly variable penetrance., Conclusions: Our study suggests that current diagnostic criteria fail to identify some patients with a mild GPS phenotype and that such patients might be identified by the methods cited above. It also better characterizes the pathogenesis of defective platelet responses to thrombin, and raises interesting questions on the correlation between abnormal PAR function and the lack of alpha-granule content in GPS.

The low affinity receptor for immunoglobulin G, FcgammaRIIA, is expressed in human platelets, mediates heparin-induced thrombocytopenia and participates to platelet activation induced by von Willebrand factor. In this work, we found that stimulation of platelets with agonists acting on G-protein-coupled receptors resulted in the tyrosine phosphorylation of FcgammaRIIA, through a mechanism involving a Src kinase. Treatment of platelets with the blocking monoclonal antibody IV.3 against FcgammaRIIA, but not with control IgG, inhibited platelet aggregation induced by TRAP1, TRAP4, the thromboxane analogue U46619, and low concentrations of thrombin. By contrast, platelet aggregation induced by high doses of thrombin was unaffected by blockade of FcgammaRIIA. We also found that the anti-FcgammaRIIA monoclonal antibody IV.3 inhibited pleckstrin phosphorylation and calcium mobilization induced by low, but not high, concentrations of thrombin. In addition, thrombin- or U46619-induced tyrosine phosphorylation of several substrates typically involved in FcgammaRIIA-mediated signalling, such as Syk and PLCgamma2, was clearly reduced by incubation with anti-FcgammaRIIA antibody IV.3. Upon stimulation with thrombin, FcgammaRIIA relocated in lipid rafts, and thrombin-induced tyrosine phosphorylation of FcgammaRIIA occurred within these membrane domains. Controlled disruption of lipid rafts by depleting membrane cholesterol prevented tyrosine phosphorylation of FcgammaRIIA and impaired platelet aggregation induced by U46619 or by low, but not high, concentrations of thrombin. These results indicate that FcgammaRIIA can be activated in human platelets downstream G-protein-coupled receptors and suggest a novel general mechanism for the reinforcement of platelet activation induced by low concentrations of agonists.

Myosins of class II constitute part of a superfamily of several classes of proteins expressed in almost all eukaryotic cell types. Differences in the heavy chains produce three isoforms of class II non-muscle myosins (A, B and C), which are widely distributed in most tissues and thought to be components of the cell motor systems, although specific functional roles are largely unknown. In particular, it is still a matter of debate whether they interact and have overlapping or distinct functions. This argument is relevant not only to cell physiology, but also to human pathology since mutations of the MYH9 gene encoding non-muscle myosin heavy chain II A (NMMHC-A) cause MYH9-related disease (MYH9-RD), an autosomal dominant disorder characterized by platelet macrocytosis, thrombocytopenia and leukocyte inclusions, variably associated with sensorineural hearing loss, cataracts and/or glomerulonephritis. In this study, we report the results of yeast two-hybrid screening showing that the C-terminals of NMMHC-A and -B interact. This interaction was confirmed by immunoprecipitation in transfected COS-7 cells and in skin fibroblasts naturally expressing both isoforms. Moreover, our immunomorphological study revealed that isoforms A and B co-localize in fibroblasts, erythroblasts and kidney cells. These results suggest that isoforms A and B are strictly related molecules and support the hypothesis that their interrelationship could be involved both in the variability of clinical phenotype and selectivity of tissue damage of MYH9-RD.

The involvement of the small GTPase Rap1b in platelet integrin alpha2beta1-dependent outside-in signaling was investigated. Platelet adhesion to 4 different specific ligands for integrin alpha2beta1, monomeric collagen, decorin, and collagen-derived peptides CB8(II) and CB11(II), induced a robust and rapid activation of Rap1b. This process did not require secreted ADP or thromboxane A2 production but was critically regulated by phospholipase C (PLC)-derived second messengers. Both Ca2+ and protein kinase C were found to organize independent but additive pathways for Rap1b activation downstream of integrin-alpha2beta1, which were completely blocked by inhibition of PLC with U73122. Moreover, integrin alpha2beta1 engagement failed to trigger Rap1b activation in murine platelets lacking CalDAG-GEFI, a guanine nucleotide exchange factor regulated by Ca2+ and diacylglycerol, despite normal phosphorylation and activation of PLCgamma2. In addition, CalDAG-GEFI-deficient platelets showed defective integrin alpha2beta1-dependent adhesion and spreading. We found that outside-in signaling through integrin alpha2beta1 triggered inside-out activation of integrin alphaIIbbeta3 and promoted fibrinogen binding. Similarly to Rap1b stimulation, this process occurred downstream of PLC activation and was dramatically impaired in murine platelets lacking the Rap1 exchange factor CalDAG-GEFI. These results demonstrate that Rap1b is an important element in integrin-dependent outside-in signaling during platelet adhesion and regulates the cross talk between adhesive receptors.

Background: Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets., Aim: To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out., Methods: Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the dchip and pam algorithms., Results: A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the 'MK-core' isolated by informatics tools., Conclusions: This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.