Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis-like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin-elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca(2+) activity as well as Ca(2+) -dependent proteolytic processing of µ-calpain. Pyocyanin further up-regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin-induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl-ester labelling, pyocyanin-treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis-inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection.

Producing megakaryocytes from a human peripheral blood source.

BACKGROUND: Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood-derived human hematopoietic stem and progenitor cells (HSPCs). STUDY DESIGN AND METHODS: HSPCs (CD34+) were isolated from peripheral blood by positive selection and expanded using an optimal CD34+ expansion supplement. We evaluated megakaryocyte growth, maturation, and morphology in response to thrombopoietin (TPO) stimulation using flow cytometry and electron microscopy. TPO demonstrated a dose-dependent stimulatory effect on both megakaryocyte number and maturation. RESULTS: From 90 to 120 mL of unmanipulated peripheral blood, we isolated a mean of 1.5 × 10(5) HSPCs (1.5 × 10(3) cells/mL of whole blood). HSPCs expanded nine-fold after a 4-day culture using an expansion supplement. Expanded cells were cultured for an additional 8 days with TPO (20 ng/mL), which resulted in a 2.9-fold increase in megakaryocytic cells where 83% of live cells expressed CD41a+, a marker of megakaryocyte commitment, and 50% expressed CD42b+, a marker for megakaryocyte maturation. The expanded HSPCs responded to TPO stimulation to yield more than 1.0 × 10(6) megakaryocytes. This cell number was sufficient for morphologic studies that demonstrated these expanded HSPCs produced mature polyploid megakaryocytes capable of forming proplatelet extensions. CONCLUSIONS: Peripheral blood HSPCs can be expanded and differentiated into functional, mature megakaryocytes, a finding that supports the use of this process to study inherent platelet (PLT) production disorders as well as study factors that impair normal PLT production.

Pitfalls in the diagnosis of heparin-Induced thrombocytopenia: A 6-year experience from a reference laboratory.

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies against complexes of platelet factor 4 (PF4) and heparin. The diagnosis of HIT is contingent on accurate and timely laboratory testing. Recently, alternative anticoagulants for the treatment of HIT have been introduced along with algorithms for better HIT diagnosis. However, the increased reliance on immunoassays for the diagnosis of HIT may have harmful consequences due to the high rate of false positive results. To compare trends and implications of current HIT testing approaches, we analyzed results over a six-year period from the McMaster University Platelet Immunology Reference Laboratory. From 2008 to 2013, 8,546 samples were investigated for HIT using both an in-house IgG-specific anti-PF4/heparin enzyme immunoassay (EIA) and the serotonin-release assay (SRA). Of 8,546 samples tested, 13.4% were true-positives (positive in both assays); 65.6% were true-negatives (negative in both assays); 20.9% were presumed false positive for HIT (EIA-positive/SRA-negative); and 0.2% were EIA-negative/SRA-positive. The frequency of EIA-positive/SRA-negative results increased over time (from 12.9% in 2008 to 22.9% in 2013). We found that the number of SRA-negative samples was reduced from referring centers that used an immunoassay as an initial screen; however, 41% of those samples tested negative in the immunoassay and in the SRA at the reference laboratory. The suspicion of HIT continues at a high rate and the agreement between the EIA and SRA test results remains problematic.

The platelet serotonin-release assay.

Few laboratory tests are as clinically useful as The platelet serotonin-release assay (SRA): a positive SRA in the appropriate clinical context is virtually diagnostic of heparin-induced thrombocytopenia (HIT), a life- and limb-threatening prothrombotic disorder caused by anti-platelet factor 4 (PF4)/heparin antibodies that activate platelets, thereby triggering serotonin-release. The SRA's performance characteristics include high sensitivity and specificity, although caveats include indeterminate reaction profiles (observed in ∼4% of test sera) and potential for false-positive reactions. As only a subset of anti-PF4/heparin antibodies detectable by enzyme-immunoassay (EIA) are additionally platelet-activating, the SRA has far greater diagnostic specificity than the EIA. However, requiring a positive EIA, either as an initial screening test or as an SRA adjunct, will reduce risk of a false-positive SRA (since a negative EIA in a patient with a "positive" SRA should prompt critical evaluation of the SRA reaction profile). The SRA also provides useful information on whether a HIT serum produces strong platelet activation even in the absence of heparin: such heparin-"independent" platelet activation is a marker of unusually severe HIT, including delayed-onset HIT and severe HIT complicated by consumptive coagulopathy with risk for microvascular thrombosis.

Antibody binding to megakaryocytes in vivo in patients with immune thrombocytopenia.

OBJECTIVES: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder caused by increased platelet destruction and impaired platelet production. Antibody binding to megakaryocytes may occur in ITP, but in vivo evidence of this phenomenon is lacking. METHODS: We determined the proportion of megakaryocytes bound with immunoglobulin G (IgG) in bone marrow samples from primary patients with ITP (n = 17), normal controls (n = 13) and thrombocytopenic patients with myelodysplastic syndrome (MDS; n = 10). Serial histological sections from archived bone marrow biopsies were stained for CD61 and IgG. IgG binding and the number of bone marrow megakaryocytes were determined morphologically by a hematopathologist with four assessors after a calibration exercise to ensure consistency. RESULTS: The proportion of ITP patients with high IgG binding (>50% of bone marrow megakaryocytes) was increased compared with normal controls [12/17 (71%) vs. 3/13 (23%), P = 0.03]. However, the proportion of ITP patients with high IgG binding was no different than thrombocytopenic patients with MDS [12/17 (71%) vs. 7/10 (70%), P = 1.00]. IgG binding was associated with increased megakaryocyte numbers. Like platelet-associated IgG, megakaryocyte-associated IgG is related to thrombocytopenia but may not be specific for ITP. CONCLUSION: Mechanistic studies in ITP should focus on antibody specificity and include thrombocytopenic control patients.

Novel treatments for immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is caused by platelet autoantibodies and T-cell dysregulation. Both platelets and their precursor megakaryocytes may be targeted leading to platelet destruction and underproduction. Current treatments for ITP are inadequate since they do not reverse the disease process and generally do not result in durable remissions. In addition, many treatments are limited by side effects including infection and potentially thrombosis. Novel agents that are currently in development target certain key steps in the disease process, including: (1) the interaction between T-cell and antigen presenting cells (CD40-CD154 interaction); (2) the binding of the Fc portion of platelet autoantibodies to Fc-receptors on macrophages (soluble Fc-RIIb); and (3) the signaling pathways leading to platelet phagocytosis by macrophages (Syk inhibition). Other strategies have been to augment platelet production by simulating thrombopoiesis or by neutralizing physiological inhibitors of megakaryopoiesis. Targeted therapies in ITP have the potential to improve disease morbidity and mortality while limiting systemic side effects. Before these agents can be used in practice, additional clinical studies are needed with rational study outcomes including platelet count, bleeding and quality of life. An individualized treatment strategy is needed for patients since ITP is a distinctly heterogeneous disease.

Approach to the diagnosis and management of drug-induced immune thrombocytopenia.

Drug-induced immune thrombocytopenia (DITP) is a challenging clinical problem that is under-recognized, difficult to diagnose and associated with severe bleeding complications. DITP may be caused by classic drug-dependent platelet antibodies (eg, quinine); haptens (eg, penicillin); fiban-dependent antibodies (eg, tirofiban); monoclonal antibodies (eg, abciximab); autoantibody formation (eg, gold); and immune complex formation (eg, heparin). A thorough clinical history is essential in establishing the diagnosis of DITP and should include exposures to prescription medications, herbal preparations and even certain foods and beverages. Clinical and laboratory criteria have been established to determine the likelihood of a drug being the cause of thrombocytopenia, but these criteria can only be applied retrospectively. The most commonly implicated drugs include quinine, quinidine, trimethoprim/sulfamethoxazole and vancomycin. We propose a practical approach to the diagnosis of the patient with suspected DITP. Key features are: the presence of severe thrombocytopenia (platelet nadir <20×10(9)/L); bleeding complications; onset 5 to 10days after first drug exposure, or within hours of subsequent exposures or after first exposure to fibans or abciximab; and exposure to drugs that have been previously implicated in DITP reactions. Treatment involves stopping the drug(s), administering platelet transfusions or other therapies if bleeding is present and counselling on future drug avoidance. The diagnosis can be confirmed by a positive drug re-challenge, which is often impractical, or by demonstrating drug-dependent platelet reactive antibodies in vitro. Current test methods, which are mostly flow cytometry-based, must show drug-dependence, immunoglobulin binding, platelet specificity and ideally should be reproducible across laboratories. Improved standardization and accessibility of laboratory testing should be a focus of future research.

The effect of rituximab on vaccine responses in patients with immune thrombocytopenia.

B-cell depletion may impair vaccine responses and increase infection risk in patients with immune thrombocytopenia (ITP). We investigated the effects of rituximab on antibody and cellular responses to Streptococcus pneumoniae polysaccharide and Haemophilus influenzae type b (Hib) vaccines in ITP patients. Of 60 patients in the main trial, 24 patients received both vaccines 6 months after rituximab (n = 17) or placebo (n = 7). Among 20 evaluable patients, 3 of 14 (21%) in the rituximab group and 4 of 6 (67%) in the placebo group achieved a fourfold increase in anti-pneumococcal antibodies (P = .12). For anti-Hib antibodies, 4 of 14 (29%) and 5 of 6 (83%), respectively, achieved a fourfold increase (P < .05). Fewer patients in the rituximab group demonstrated Hib killing (2 of 14 [14%], 5 of 6 [83%], P < .05). Three of 14 rituximab-treated patients failed to respond to vaccines by any criteria. After vaccinations, preplasma cell blasts and interferon-γ-secreting T cells were reduced in rituximab-treated patients. Antibody responses were impaired for at least 6 months after rituximab. Cellular immunity was reduced in parallel with depleted B-cell pools. These findings have implications for the timing of vaccinations and the mechanism of infection after rituximab in ITP patients.

Sustained remissions of immune thrombocytopenia associated with the use of thrombopoietin receptor agonists.

BACKGROUND: Thrombopoietin receptor agonists (TRAs) are effective treatments for immune thrombocytopenia (ITP). However, continuous therapy is generally required to maintain platelet (PLT) count responses. STUDY DESIGN AND METHODS: In this case series, we describe ITP patients from our practice who achieved durable responses to the TRAs romiplostim and eltrombopag. Patients were classified as having a definite TRA-induced remission if PLT counts increased above 100 × 10(9) /L after TRA treatment and remained above 100 × 10(9) /L even after the medication was discontinued; or a possible TRA-induced remission if PLT counts increased above 100 × 10(9) /L, remained elevated for at least 3 months after the medication was discontinued, but a subsequent relapse occurred or the effect of other disease-modifying therapies could not be excluded. RESULTS: Of 31 patients with chronic ITP treated with TRAs in our practice, nine patients achieved a PLT count response with either romiplostim (n = 6) or eltrombopag (n = 3) that was maintained even after the medications were discontinued. Three patients met criteria for a definite TRA-induced remission, each after exposure to romiplostim. Patients had ITP for a median of 7.8 years and had failed a median of four prior therapies including eight patients who had a splenectomy. We documented a progressive decline in anti-glycoprotein IIbIIIa PLT autoantibodies in one patient while on treatment. CONCLUSION: Some patients with ITP can achieve sustained PLT count responses after the use of TRAs. This observation raises the possibility that these agents may restore immune tolerance to PLT antigens in some patients and supports the practice of down titrating the dose.

A blinded study of bone marrow examinations in patients with primary immune thrombocytopenia.

OBJECTIVE: The role of bone marrow examinations in patients with primary immune thrombocytopenia (ITP) is uncertain. The objectives of this study were to determine the inter-rater reliability of bone marrow examinations and to identify distinguishing morphological features of ITP bone marrows under controlled conditions. METHODS: Histological slides of bone marrow biopsy specimens and aspirates from 32 adult patients with severe primary ITP who had failed a median of two treatments, and 51 non-thrombocytopenic controls were retrieved from hospital archives. Slides were arranged in random order in a slide box and coded. Blinded to the diagnosis and platelet counts, three independent hematopathologists were asked to identify the ITP bone marrows and to evaluate megakaryocyte number, morphology, and distribution. RESULTS: Overall chance-corrected agreement on ITP classification among the three raters was poor [kappa (κ) = 0.30; 95% confidence interval 0.22-0.38]. Raters were generally unable to correctly identify the ITP bone marrows from controls. Increased number of megakaryocytes, while an uncommon finding, was more frequent among ITP patients compared with controls (6/32, 18.8%; vs. 2/51, 3.9%; P = 0.05), and abnormal megakaryocyte morphology often led individual raters to reach a diagnosis of ITP. Overall sensitivity and specificity of bone marrow examinations were 24% and 90%, respectively. CONCLUSIONS: This study confirms methodologically that bone marrow examinations are unreliable and frequently non-diagnostic in ITP. Thus, they are not useful for patients with typical disease. Rare subsets of patients with severe ITP demonstrated unique features such as increased number of megakaryocytes.

Piecing together the humoral and cellular mechanisms of immune thrombocytopenia.

The precise mechanisms leading to platelet-targeted autoimmunity in immune thrombocytopenia (ITP) are not known. Cellular checkpoints normally regulate immunological self-reactivity during the development of B and T cells through cell deletion, receptor editing, induction of anergy, and extrinsic cellular suppression. When these checkpoints fail, tolerance to self-antigens may be lost. In this review, we summarize the various immune mechanisms contributing to the development of ITP and relate them back to the checkpoint model of autoimmunity. These mechanisms, including increased levels of lymphocyte growth factors, resistance to death signals, and loss of T-regulatory function, result in an environment permissive to the development of platelet-reactive B and T cells. The mechanisms that lead to thrombocytopenia once tolerance for platelet antigens is lost are examined, including complement-dependent and apoptotic pathways. An improved understanding of ITP pathogenesis will ultimately guide the development of better therapies.

ß-Lactone natural products and derivatives inactivate homoserine transacetylase, a target for antimicrobial agents.

Homoserine transacetylase (HTA) catalyzes the transfer of an acetyl group from acetyl-CoA to the hydroxyl group of homoserine. This is the first committed step in the biosynthesis of methionine (Met) from aspartic acid in many fungi, Gram-positive and some Gram-negative bacteria. The enzyme is absent in higher eukaryotes and is important for microorganism growth in Met-poor environments, such as blood serum, making HTA an attractive target for new antimicrobial agents. HTA catalyzes acetyl transfer via a double displacement mechanism facilitated by a classic Ser-His-Asp catalytic triad located at the bottom of a narrow actives site tunnel. We explored the inhibitory activity of several ß-lactones to block the activity of HTA. In particular, the natural product ebelactone A, a ß-lactone with a hydrophobic tail was found to be a potent inactivator of HTA from Haemophilus influenzae. Synthetic analogs of ebelactone A demonstrated improved inactivation characteristics. Covalent modification of HTA was confirmed by mass spectrometry, and peptide mapping identified Ser143 as the modified residue, consistent with the known structure and mechanism of the enzyme. These results demonstrate that ß-lactone inhibitors are excellent biochemical probes of HTA and potential leads for new antimicrobial agents.

A vancomycin photoprobe identifies the histidine kinase VanSsc as a vancomycin receptor.

Inducible resistance to the glycopeptide antibiotic vancomycin requires expression of vanH, vanA and vanX, controlled by a two-component regulatory system consisting of a receptor histidine kinase, VanS, and a response regulator, VanR. The identity of the VanS receptor ligand has been debated. Using a synthesized vancomycin photoaffinity probe, we show that vancomycin directly binds Streptomyces coelicolor VanS (VanSsc) and this binding is correlated with resistance and required for vanH, vanA and vanX gene expression.

Combination immunosuppressant therapy for patients with chronic refractory immune thrombocytopenic purpura.

Treatment options for patients with chronic refractory immune thrombocytopenic purpura (ITP) are limited. Because combination immunosuppressant therapy appeared to be effective in ITP and other disorders, we used this approach in patients with particularly severe and refractory ITP. In this retrospective, observational study, we determined the response (platelet count above 30 x 10(9)/L and doubling of baseline) among 19 refractory ITP patients. Treatment consisted of azathioprine, mycophenolate mofetil, and cyclosporine. The patients had failed a median of 6 prior treatments, including splenectomy (in all except 1). Of 19 patients, 14 (73.7%) achieved a response lasting a median of 24 months, after which time 8 (57.1%) relapsed. Of the 8 relapsing patients, 6 responded to additional treatments. Of the 14 patients who achieved an initial response, 2 (14.3%) remained in remission after eventually stopping all medications. Severe adverse events did not occur. Combination immunosuppressant therapy can produce a rise in the platelet count that is sometimes sustained in refractory ITP patients.

Platelet count response to H. pylori treatment in patients with immune thrombocytopenic purpura with and without H. pylori infection: a systematic review.

Eradication of H. pylori improves thrombocytopenia in some patients with immune thrombocytopenic purpura by mechanisms that remain obscure. Platelet count responses may occur independently of H. pylori infection as a result of the immune modulating effects of macrolide antimicrobials or the removal of other commensal bacteria. We performed a systematic review of the literature to determine the effect of H. pylori eradication therapy in patients with immune thrombocytopenic purpura by comparing the platelet response in patients who were, and who were not infected with H. pylori. MEDLINE, EMBASE, Cochrane central registry and abstracts from the American Society of Hematology (from 2003) were searched in duplicate and independently without language or age restrictions. Eleven studies, 8 from Japan, were included enrolling 282 patients with immune thrombocytopenic purpura who received eradication therapy; 205 were H. pylori-positive and 77 were H. pylori-negative. The odds of achieving a platelet count response following eradication therapy were 14.5 higher (95% confidence interval 4.2 to 83.0) in patients with H. pylori infection (51.2% vs. 8.8%). No study reported bleeding or quality of life. Adverse events were reported in 12 patients. H. pylori eradication therapy was of little benefit for H. pylori-negative patients. These findings strengthen the causal association between H. pylori infection and immune thrombocytopenia in some patients. Randomized trials are needed to determine the applicability of H. pylori eradication therapy across diverse geographical regions.

New treatments for idiopathic thrombocytopenic purpura: rethinking old hypotheses.

BACKGROUND: The efficacy of thrombopoietin (TPO) mimetics in patients with idiopathic thrombocytopenic purpura (ITP) reaffirms that impaired platelet production is an important mechanism. New strategies to reduce platelet destruction, like rituximab, are also effective. OBJECTIVES: To describe the efficacy and safety of rituximab and the TPO mimetics, romiplostim and eltrombopag, and how they relate to ITP pathogenesis. METHODS: Narrative review summarizing full publications and meeting abstracts. RESULTS/CONCLUSIONS: A 4-week course of rituximab is associated with a platelet count response in 60% of patients with ITP, and durable responses have been observed. Subtle increases in infection have been reported. Romiplostim and eltrombopag are each associated with a 60 - 85% response while on treatment. Transient bone marrow reticulin with romiplostim and elevated liver enzymes with eltrombopag are rare side effects. The application of these agents in non-splenectomized patients requires further study.

Role of homoserine transacetylase as a new target for antifungal agents.

Microbial amino acid biosynthesis is a proven yet underexploited target of antibiotics. The biosynthesis of methionine in particular has been shown to be susceptible to small-molecule inhibition in fungi. The first committed step in Met biosynthesis is the acylation of homoserine (Hse) by the enzyme homoserine transacetylase (HTA). We have identified the MET2 gene of Cryptococcus neoformans H99 that encodes HTA (CnHTA) by complementation of an Escherichia coli metA mutant that lacks the gene encoding homoserine transsuccinylase (HTS). We cloned, expressed, and purified CnHTA and determined its steady-state kinetic parameters for the acetylation of L-Hse by acetyl coenzyme A. We next constructed a MET2 mutant in C. neoformans H99 and tested its growth behavior in Met-deficient media, confirming the expected Met auxotrophy. Furthermore, we used this mutant in a mouse inhalation model of infection and determined that MET2 is required for virulence. This makes fungal HTA a viable target for new antibiotic discovery. We screened a 1,000-compound library of small molecules for HTA inhibitors and report the identification of the first inhibitor of fungal HTA. This work validates HTA as an attractive drug-susceptible target for new antifungal agent design.

Crystal structure of homoserine transacetylase from Haemophilus influenzae reveals a new family of alpha/beta-hydrolases.

Homoserine transacetylase catalyzes one of the required steps in the biosynthesis of methionine in fungi and several bacteria. We have determined the crystal structure of homoserine transacetylase from Haemophilus influenzae to a resolution of 1.65 A. The structure identifies this enzyme to be a member of the alpha/beta-hydrolase structural superfamily. The active site of the enzyme is located near the end of a deep tunnel formed by the juxtaposition of two domains and incorporates a catalytic triad involving Ser143, His337, and Asp304. A structural basis is given for the observed double displacement kinetic mechanism of homoserine transacetylase. Furthermore, the properties of the tunnel provide a rationale for how homoserine transacetylase catalyzes a transferase reaction vs hydrolysis, despite extensive similarity in active site architecture to hydrolytic enzymes.