Megakaryocyte apoptosis in immune thrombocytopenia.

The mechanisms of platelet underproduction in immune thrombocytopenia (ITP) remain unknown. While the number of megakaryocytes is normal or increased in ITP bone marrow, further studies of megakaryocyte integrity are needed. Megakaryocytes are responsible for the production of platelets in the bone marrow, and they are possible targets of immune-mediated injury in ITP. Since the biological process of megakaryocyte apoptosis impacts platelet production, we investigated megakaryocyte DNA fragmentation as a marker of apoptosis from ITP bone marrow biopsies. Archived bone marrow biopsy specimens from ITP patients, bone marrow specimens from controls with normal platelet counts, and bone marrow specimens from thrombocytopenic controls with myelodysplastic syndrome (MDS) were evaluated. Sections were stained with anti-CD61 for megakaryocyte enumeration, and terminal deoxynucleotidyl transferase dUTP nick-end labeling was used as an apoptotic indicator. In ITP patients, megakaryocyte apoptosis was reduced compared to nonthrombocytopenic controls. Megakaryocyte apoptosis was similarly reduced in thrombocytopenic patients with MDS. These results suggest a link between megakaryocyte apoptosis and platelet production.

Effect of a thrombopoietin receptor agonist on use of intravenous immune globulin in patients with immune thrombocytopenia.

BACKGROUND: Thrombopoietin receptor agonists are new treatments for patients with chronic immune thrombocytopenia (ITP). How one of these agent, romiplostim, has impacted practice patterns, especially the use of intravenous immune globulin (IVIG), has not been evaluated outside of clinical trials. STUDY DESIGN AND METHODS: This was a retrospective cohort study of adult ITP patients treated with romiplostim in four Canadian centers. Patients had primary or secondary ITP and were followed for 1 year before starting weekly romiplostim treatment. We compared IVIG use, clinical outcomes, and cost before and after romiplostim. RESULTS: Twenty-nine patients with ITP received romiplostim. Median age was 54 years (interquartile range [IQR], 45-63 years) and patients had a median of two prior ITP treatments (IQR, 1-4) including splenectomy (n = 7). Median platelet (PLT) count was 23 × 10(9) before and 124 × 10(9) after romiplostim. Median duration of romiplostim treatment was 3.7 months. Patients used a median of two IVIG infusions per year before and 0.7 per year after starting romiplostim (p = 0.16). For patients who received weekly romiplostim for at least 1 month (n = 19), IVIG infusions were three (IQR, 1-5) per year before and 0.7 (IQR, 0.4-1.6) per year after romiplostim. Results were squewed by two high IVIG users. Nineteen (66%) patients discontinued romiplostim treatment during follow-up because of lack of response (n = 8), sustained response (n = 5), toxicities (n = 4), or response to splenectomy (n = 2). Overall health care costs were similar before and after romiplostim when concomitant treatments, nursing resources, and hospitalizations were considered. CONCLUSIONS: Romiplostim was associated with improved PLT counts and fewer IVIG infusions for most ITP patients. In practice, romiplostim was generally not continued long term and was cost neutral for overall ITP management.

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.

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.

Vascular endothelial growth factor is an important determinant of sepsis morbidity and mortality.

Sepsis, the systemic inflammatory response to infection, is a leading cause of morbidity and mortality. The mechanisms of sepsis pathophysiology remain obscure but are likely to involve a complex interplay between mediators of the inflammatory and coagulation pathways. An improved understanding of these mechanisms should provide an important foundation for developing novel therapies. In this study, we show that sepsis is associated with a time-dependent increase in circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) in animal and human models of sepsis. Adenovirus-mediated overexpression of soluble Flt-1 (sFlt-1) in a mouse model of endotoxemia attenuated the rise in VEGF and PlGF levels and blocked the effect of endotoxemia on cardiac function, vascular permeability, and mortality. Similarly, in a cecal ligation puncture (CLP) model, adenovirus-sFlt-1 protected against cardiac dysfunction and mortality. When administered in a therapeutic regimen beginning 1 h after the onset of endotoxemia or CLP, sFlt peptide resulted in marked improvement in cardiac physiology and survival. Systemic administration of antibodies against the transmembrane receptor Flk-1 but not Flt-1 protected against sepsis mortality. Adenovirus-mediated overexpression of VEGF but not PlGF exacerbated the lipopolysaccharide-mediated toxic effects. Together, these data support a pathophysiological role for VEGF in mediating the sepsis phenotype.

Prognostic utility and characterization of cell-free DNA in patients with severe sepsis.

INTRODUCTION: Although sepsis is the leading cause of death in noncoronary critically ill patients, identification of patients at high risk of death remains a challenge. In this study, we examined the incremental usefulness of adding multiple biomarkers to clinical scoring systems for predicting intensive care unit (ICU) mortality in patients with severe sepsis. METHODS: This retrospective observational study used stored plasma samples obtained from 80 severe sepsis patients recruited at three tertiary hospital ICUs in Hamilton, Ontario, Canada. Clinical data and plasma samples were obtained at study inclusion for all 80 patients, and then daily for 1 week, and weekly thereafter for a subset of 50 patients. Plasma levels of cell-free DNA (cfDNA), interleukin 6 (IL-6), thrombin, and protein C were measured and compared with clinical characteristics, including the primary outcome of ICU mortality and morbidity measured with the Multiple Organ Dysfunction (MODS) score and Acute Physiology and Chronic Health Evaluation (APACHE) II scores. RESULTS: The level of cfDNA in plasma at study inclusion had better prognostic utility than did MODS or APACHE II scores, or the biomarkers measured. The area under the receiver operating characteristic (ROC) curves for cfDNA to predict ICU mortality is 0.97 (95% CI, 0.93 to 1.00) and to predict hospital mortality is 0.84 (95% CI, 0.75 to 0.94). We found that a cfDNA cutoff value of 2.35 ng/µl had a sensitivity of 87.9% and specificity of 93.5% for predicting ICU mortality. Sequential measurements of cfDNA suggested that ICU mortality may be predicted within 24 hours of study inclusion, and that the predictive power of cfDNA may be enhanced by combining it with protein C levels or MODS scores. DNA-sequence analyses and studies with Toll-like receptor 9 (TLR9) reporter cells suggests that the cfDNA from sepsis patients is host derived. CONCLUSIONS: These studies suggest that cfDNA provides high prognostic accuracy in patients with severe sepsis. The serial data suggest that the combination of cfDNA with protein C and MODS scores may yield even stronger predictive power. Incorporation of cfDNA in sepsis risk-stratification systems may be valuable for clinical decision making or for inclusion into sepsis trials.

Pathophysiology and management of chronic immune thrombocytopenia: focusing on what matters.

Immune thrombocytopenia (ITP) is a common autoimmune disease characterized by low platelet counts and an increased risk of bleeding. Antibody-mediated platelet destruction has been the prevailing hypothesis to explain ITP pathogenesis, supported by the efficacy of B-cell depletion therapy; however, the recent success of thrombopoietin receptor agonists lends support to the notion that platelet production is also insufficient. Best practice for the management of chronic ITP has not yet been established because data from comparative trials are lacking. Despite renewed interest in novel drugs capable of increasing platelet counts, ultimate treatment goals for ITP patients must be kept in mind: to improve patients' health and well-being. In this article, the pathophysiology of ITP is reviewed and key remaining questions about mechanism are explored. A rational approach to the management of ITP in adults is outlined, acknowledging evidence and evidence gaps, and highlighting the need for clinically important endpoints in future clinical trials.

Angiopoietin-1 and angiopoietin-2 as clinically informative prognostic biomarkers of morbidity and mortality in severe sepsis.

OBJECTIVE: To determine the utility of angiopoietin-1 and angiopoietin-2 as potentially novel biomarkers of morbidity and mortality in patients with severe sepsis. DESIGN: Multicenter longitudinal cohort study. SETTING: Three tertiary hospital intensive care units in Hamilton, Ontario, Canada. PATIENTS: A total of 70 patients with severe sepsis were enrolled within 24 hrs of meeting the inclusion criteria for severe sepsis and followed until day 28, hospital discharge, or death. INTERVENTIONS: Clinical data and plasma samples were obtained at intensive care unit admission for all 70 patients and then daily for 1 wk and weekly thereafter for a subset of 43 patients. Levels of angiopoietin-1 and angiopoietin-2 in stored plasma samples were measured and compared with clinical characteristics, including the primary outcomes of 28-day mortality and morbidity measured by the Multiple Organ Dysfunction score. MEASUREMENTS AND MAIN RESULTS: Lower angiopoietin-1 plasma levels (≤ 5.5 ng/mL) at admission were associated with increased likelihood of death (relative risk 0.49 [95% confidence interval of 0.25-0.98], p = .046). Lower angiopoietin-1 levels remained a significant predictor of 28-day mortality in a multiple logistic regression model (adjusted odds ratio of 0.282 [95% confidence interval of 0.086-0.93], p = .037). Analysis of serial data using linear mixed models confirmed that sepsis survivors had higher levels of angiopoietin-1 (p = .012) and lower daily levels of angiopoietin-2 (p = .022) than nonsurvivors. Furthermore, survivors had higher peak angiopoietin-1 levels (median 13 vs. 10 ng/mL, p = .019) and lower nadir angiopoietin-2 levels (median 2.8 vs. 6.2 ng/mL, p = .013) than nonsurvivors. A score incorporating angiopoietin-1 and angiopoietin-2 and three other markers of endothelial activation discriminated with high accuracy between fatal and nonfatal cases (c-index of 0.80 [95% confidence interval of 0.69-0.90], p < .001). Plasma levels of angiopoietin-2 correlated with clinical markers of organ dysfunction and molecular markers of endothelial cell activation. CONCLUSIONS: Angiopoietin-1 levels at admission and both angiopoietin-1 and angiopoietin-2 levels measured serially correlated with 28-day mortality in severe sepsis. Angiopoietin-2 levels also correlated with organ dysfunction/injury and a validated clinical sepsis score. These results suggest the use of angiopoietins as clinically informative biomarkers of disease severity and patient outcome in severe sepsis.

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.

Activated protein C up-regulates IL-10 and inhibits tissue factor in blood monocytes.

The protective effect of recombinant activated protein C therapy in patients with severe sepsis likely reflects the ability of recombinant activated protein C to modulate multiple pathways implicated in sepsis pathophysiology. In this study, we examined the effects of recombinant activated protein C on the anti-inflammatory cytokine IL-10 and on the procoagulant molecule tissue factor (TF) in LPS-challenged blood monocytes. Treatment of LPS-stimulated monocytes with recombinant activated protein C resulted in an up-regulation of IL-10 protein production and mRNA synthesis. The up-regulation of IL-10 required the serine protease activity of recombinant activated protein C and was dependent on protease-activated receptor-1, but was independent of the endothelial protein C receptor. At the intracellular level, p38 MAPK activation was required for recombinant activated protein C-mediated up-regulation of IL-10. We further observed that incubation of LPS-stimulated monocytes with recombinant activated protein C down-regulated TF Ag and activity levels. This anticoagulant effect of recombinant activated protein C was dependent on IL-10 since neutralization of endogenously produced IL-10 abrogated the effect. In patients with severe sepsis, plasma IL-10 levels were markedly higher in those treated with recombinant activated protein C than in those who did not receive recombinant activated protein C. This study reveals novel regulatory functions of recombinant activated protein C, specifically the up-regulation of IL-10 and the inhibition of TF activity in monocytes. Our data further suggest that these activities of recombinant activated protein C are directly linked: the recombinant activated protein C-mediated up-regulation of IL-10 reduces TF in circulating monocytes.

Protective effects of activated protein C in sepsis.

Sepsis remains a complex syndrome associated with significant morbidity and mortality. It is now widely accepted that the pathways of inflammation, coagulation, apoptosis, and endothelial permeability are intimately linked in sepsis pathophysiology. The clinical success of activated protein C (APC), a natural anticoagulant, in reducing mortality in patients with severe sepsis has fuelled basic and preclinical research on the protective effects of this molecule. Over the past 15 years, impressive research advances have provided novel insights into the multifunctional activities of APC. APC is now viewed not only as an anticoagulant, but also as a cell signaling molecule that dampens the excessive or insufficiently controlled host response during sepsis. This review attempts to summarize the pleiotropic activities of APC with focus on its ability to inhibit coagulation, inflammation, apoptosis, and endothelial barrier breakdown. A comprehensive PUBMED literature review up to May 2008 was conducted.

Activated protein C in sepsis and beyond: update 2006.

Activated protein C (APC), a plasma serine protease, is best known for its ability to inhibit blood clot formation. APC acts as an anticoagulant by degrading coagulation cofactors Va and VIIIa, thereby attenuating the coagulation cascade. Over the past 15 years, impressive research advances have provided novel insights into the diverse biological activities of this molecule. APC is now viewed not only as an anticoagulant but also as a signaling molecule that provides a pivotal link between the pathways of coagulation, inflammation, apoptosis, and vascular permeability. The protective effect of APC supplementation in patients with severe sepsis likely reflects the ability of APC to modulate multiple pathways implicated in sepsis pathophysiology. This review attempts to summarize key studies that support the therapeutic potential of APC in conditions beyond sepsis such as stroke, ischemia-reperfusion injury, lung injury, asthma, pancreatitis, wound healing, and angiogenesis. A comprehensive PUBMED literature review up to May 2006 was conducted.

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.

Elevated plasma matrix metalloproteinases and their tissue inhibitors in patients with severe sepsis.

PURPOSE: Matrix metalloproteinases (MMPs) are essential for tissue remodeling. Our objectives were to determine (1) the concentrations of MMPs and their tissue inhibitors (TIMPs) in plasma obtained from patients with severe sepsis, (2) to correlate changes in MMP and TIMP levels with disease severity, and (3) to investigate recombinant activated protein C (rAPC) actions on plasma MMP2, 9 activities from severe sepsis patients. MATERIALS AND METHODS: Matrix metalloproteinase and TIMP levels were quantified in plasma from patients with severe sepsis using antibody microarrays and gelatin zymography. RESULTS: Plasma MMPs (3, 7, 8, 9) and TIMPs (1, 2, 4) on microarray were increased in severe sepsis on intensive care unit (ICU) day 1, with more than 3-fold increases in MMP3, MMP7, MMP8, MMP9, and TIMP4. Latent forms of MMP2, 9 on zymography were increased in plasma from patients with severe sepsis, whereas only half of severe sepsis patients showed active MMP9. Elevated MMP7 and MMP9 on ICU days 1 and 3 negatively correlated with multiple organ dysfunctions. The temporal activity patterns of MMP2, 9 during 21 ICU days were not altered in patients treated with rAPC or by the addition of exogenous rAPC to plasma. CONCLUSION: Most plasma MMPs and TIMPS were elevated in patients with severe sepsis, but only a limited subset of MMPs (7, 9) negatively correlated with disease severity. Recombinant activated protein C does not appear to directly alter MMP2, 9 activities.

Modulation of monocyte function by activated protein C, a natural anticoagulant.

Activated protein C is the first effective biological therapy for the treatment of severe sepsis. Although activated protein C is well established as a physiological anticoagulant, emerging data suggest that it also exerts anti-inflammatory and antiapoptotic effects. In this study, we investigated the ability of activated protein C to modulate monocyte apoptosis, inflammation, phagocytosis, and adhesion. Using the immortalized human monocytic cell line THP-1, we demonstrated that activated protein C inhibited camptothecin-induced apoptosis in a dose-dependent manner. The antiapoptotic effect of activated protein C requires its serine protease domain and is dependent on the endothelial cell protein C receptor and protease-activated receptor-1. In primary blood monocytes from healthy individuals, activated protein C inhibited spontaneous apoptosis. With respect to inflammation, activated protein C inhibited the production of TNF, IL-1beta, IL-6, and IL-8 by LPS-stimulated THP-1 cells. Activated protein C did not influence the phagocytic internalization of Gram-negative and Gram-positive bioparticles by THP-1 cells or by primary blood monocytes. Activated protein C also did not affect the expression of adhesion molecules by LPS-stimulated blood monocytes nor the ability of monocytes to adhere to LPS-stimulated endothelial cells. We hypothesize that the protective effect of activated protein C in sepsis reflects, in part, its ability to prolong monocyte survival in a manner that selectively inhibits inflammatory cytokine production while maintaining phagocytosis and adherence capabilities, thereby promoting antimicrobial properties while limiting tissue damage.