Determination of Antibody Activity by Platelet Aggregation.

Platelets play an important role in hemostasis by forming clots and stopping bleeding. In immune thrombotic conditions, platelets and leukocytes are aberrantly activated by pathogenic antibodies resulting in platelet aggregates and NETosis, leading to thrombosis and thrombocytopenia. A simple assay that assesses platelet function and antibody activity is light transmission aggregometry. This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). Briefly, for detection of pathogenic antibody, platelet-rich plasma (PRP) is treated with a specific agent (e.g., patient sera or purified patient antibodies) with constant stirring. Upon activation, platelets undergo a shape change and adhere to each other forming aggregates. This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with 14C-serotonin release assay, another common assay for detecting antibody activity. Key features • This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT. • Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies. • Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist. • For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results.

NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. Inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast, in vivo blockage of FcγRIIa abrogates both thrombosis and thrombocytopenia suggesting these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via FcγRIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and FcγRIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively, leading to better patient outcomes.

Circulating platelet-neutrophil aggregates characterize the development of type 1 diabetes in humans and NOD mice.

Platelet-neutrophil aggregates (PNAs) facilitate neutrophil activation and migration and could underpin the recruitment of neutrophils to the pancreas during type 1 diabetes (T1D) pathogenesis. PNAs, measured by flow cytometry, were significantly elevated in the circulation of autoantibody-positive (Aab+) children and new-onset T1D children, as well as in pre-T1D (at 4 weeks and 10-12 weeks) and T1D-onset NOD mice, compared with relevant controls, and PNAs were characterized by activated P-selectin+ platelets. PNAs were similarly increased in pre-T1D and T1D-onset NOD isolated islets/insulitis, and immunofluorescence staining revealed increased islet-associated neutrophil extracellular trap (NET) products (myeloperoxidase [MPO] and citrullinated histones [CitH3]) in NOD pancreata. In vitro, cell-free histones and NETs induced islet cell damage, which was prevented by the small polyanionic drug methyl cellobiose sulfate (mCBS) that binds to histones and neutralizes their pathological effects. Elevated circulating PNAs could, therefore, act as an innate immune and pathogenic biomarker of T1D autoimmunity. Platelet hyperreactivity within PNAs appears to represent a previously unrecognized hematological abnormality that precedes T1D onset. In summary, PNAs could contribute to the pathogenesis of T1D and potentially function as a pre-T1D diagnostic.

Erythrocyte interaction with neutrophil extracellular traps in coronary artery thrombosis following myocardial infarction.

Cardiovascular disease, including myocardial infarction (MI), is the leading cause of death globally. Current antithrombotic medications used during MI treatment are predominantly directed towards platelet inhibition and, to a lesser extent, anticoagulation. Bleeding is a major risk of such treatment and could be circumvented by targeting other causative factors essential for arterial thrombus formation. We sought to re-evaluate the cellular composition of arterial thrombus in order to better understand mechanisms that lead to coronary artery thrombosis in acute MI. We performed detailed histological and immunohistochemical analysis of coronary artery thrombi aspirated from 26 patients undergoing emergency percutaneous coronary intervention for acute ST elevated myocardial infarction (STEMI). Coronary arterial thrombi had an unanticipated cellular heterogeneity. Neutrophil extracellular traps (NETs) were observed in thrombi as identified by anti-citrullinated histone 3 and anti-myeloperoxidase staining. Increased abundance of NETs was seen directly surrounding erythrocytes. Extracellular iron and erythrocyte fragments were also associated with areas of NETs suggesting a possible link. Our results shed light on potential involvement of erythrocytes in coronary arterial thrombosis through activation of platelets and induction of NETs. If supported by further in vitro and in vivo studies, novel therapies to inhibit NET formation or coagulation activation by erythrocyte release products, could bolster current myocardial infarction treatment.

Inhibition of NADPH oxidase blocks NETosis and reduces thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is associated with severe and potentially lethal thrombotic complications. NETosis was recently shown to be an important driver of thrombosis in HIT. We investigated the role of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and their contributions to thrombus development in HIT. We showed that neutrophil activation by HIT immune complexes induced ROS-dependent NETosis. Analysis of thrombi formed in a microfluidics system showed ROS production in both platelets and neutrophils, and abundant neutrophil extracellular traps (NETs) and ROS distributed throughout the clot. Neutrophil-targeted ROS inhibition was sufficient to block HIT-induced NETosis and thrombosis using human blood. Inhibition of NOX2 with diphenyleneiodonium chloride or GSK2795039 abrogated HIT-induced thrombi in vivo using FcγRIIa+/hPF4+-transgenic mice. Thrombocytopenia in mice remained unaffected by ROS inhibition. Increased ROS production in activated neutrophils was also confirmed using fresh blood from patients with active HIT. Our findings show that ROS and NOX2 play a crucial role in NETosis and thrombosis in HIT. This enhances our understanding of the processes driving thrombosis in HIT and identifies NOX2 as a potential new therapeutic target for antithrombotic treatment of HIT.

Management of heparin-induced thrombocytopenia: systematic reviews and meta-analyses.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic adverse drug reaction occurring in <0.1% to 7% of patients receiving heparin products depending on the patient population and type of heparin. Management of HIT is highly dependent on a sequence of tests for which clinicians may or may not have the results when care decisions need to be made. We conducted systematic reviews of the effects of management strategies in persons with acute HIT, subacute HIT A or B, and remote HIT. We searched Medline, EMBASE, and the Cochrane Database through July 2019 for previously published systematic reviews and primary studies. Two investigators independently screened and extracted data and assessed the certainty of the evidence using the Grading of Recommendations Assessment, Development and Evaluation approach. We found primarily noncomparative studies and case series assessing effects of treatments, which led to low to very low certainty evidence. There may be little to no difference in the effects between nonheparin parenteral anticoagulants and direct oral anticoagulants in acute HIT. The benefits of therapeutic-intensity may be greater than prophylactic-intensity anticoagulation. Using inferior vena cava filters or platelet transfusion may result in greater harm than not using these approaches. Evidence for management in special situations, such as for patients undergoing cardiovascular interventions or renal replacement therapy, was also low to very low certainty. Additional research to evaluate nonheparin anticoagulants is urgently needed, and the development of novel treatments that reduce thrombosis without increasing hemorrhage should be a priority.

Evolving concepts of pathogenesis of heparin-induced thrombocytopenia: Diagnostic and therapeutic implications.

Heparin-induced thrombocytopenia (HIT) is an immune reaction to heparin. It often causes severe thrombosis which may lead to limb gangrene and thrombosis-associated death. The concept of its pathogenesis has been evolving during the past five decades. Initially, HIT was thought to be caused by disseminated intravascular coagulation. Later it became clear that HIT was mediated by an immune mechanism whereby an IgG antibody induced platelet aggregation, release of procoagulant materials and consequently thrombus formation. The antigen comprises Platelet Factor 4 (PF4) and heparin which have a tendency to form ultralarge complexes. The HIT immune response has atypical features. IgG antibody appears early without IgM precedence and lasts transiently. One explanation is that there is prior priming by bacterial infection. Another unique characteristic is that it is processed as if it is a particulate antigen involving complement activation and B cells. Antigen-presenting cells/monocytes are also involved but the role of T cells is controversial. Recent advances have provided new insights into the underlying mechanisms of HIT-related thrombosis. Previously, platelets were believed to play a central role; their activation and consequently the induction of blood coagulation was the basis of the hypercoagulability in HIT. More recently, several studies have provided clear evidence that neutrophil and NETosis, monocytes and endothelial cells contribute significantly to the thrombosis in HIT. These new insights may result in development of better diagnostic laboratory assays and more effective treatments for HIT.

c-Mpl and TPO expression in the human central nervous system neurons inhibits neuronal apoptosis.

Thrombopoietin (TPO) is a growth factor for the megakaryocytic/platelet lineage. In this study, we investigated the expression of TPO and its receptor, c-Mpl, in the human central nervous system (CNS) and their roles after a neural insult. Our results demonstrate that both TPO and c-Mpl are expressed in the neurons of the human CNS. TPO was also detected in human cerebrospinal fluid. TPO was found to be neuroprotective in hypoxic-ischemic neonatal rat brain models. In these rat models, treatment with TPO reduced brain damage and improved sensorimotor functions. In addition, TPO promoted C17.2 cell proliferation through activation of the PI3K/Akt signaling pathway. Via the Bcl-2/BAX signaling pathway, TPO exerted an antiapoptotic effect by suppressing mitochondrial membrane potentials. Taken together, our results indicate that TPO is neuroprotective in the CNS.

Updated international consensus report on the investigation and management of primary immune thrombocytopenia.

Over the last decade, there have been numerous developments and changes in treatment practices for the management of patients with immune thrombocytopenia (ITP). This article is an update of the International Consensus Report published in 2010. A critical review was performed to identify all relevant articles published between 2009 and 2018. An expert panel screened, reviewed, and graded the studies and formulated the updated consensus recommendations based on the new data. The final document provides consensus recommendations on the diagnosis and management of ITP in adults, during pregnancy, and in children, as well as quality-of-life considerations.

CRISTAL: protocol for a cluster randomised, crossover, non-inferiority trial of aspirin compared to low molecular weight heparin for venous thromboembolism prophylaxis in hip or knee arthroplasty, a registry nested study.

INTRODUCTION: Venous thromboembolism (VTE) is a serious complication following hip arthroplasty (HA) and knee arthroplasty (KA). This study aims to determine whether aspirin is non-inferior to low molecular weight heparin (LMWH) in preventing symptomatic VTE following HA and KA. METHODS AND ANALYSIS: This is a cluster randomised, crossover, non-inferiority, trial nested within the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR). The clusters will consist of Australian hospitals performing at least 250 HA and/or KA procedures per annum. All adult patients undergoing HA or KA will be included. The intervention will be aspirin, orally, 85-150 mg daily. The comparator will be LMWH (enoxaparin) 40 mg, subcutaneously, daily. Both drugs will commence within 24 hours postoperatively and continue for 35 days after HA and 14 days after KA. Each hospital will be randomised to commence with aspirin or LMWH and then crossover to the alternative treatment after meeting the recruitment target. Data will be collected through the AOANJRR via patient-reported surveys. The primary outcome is symptomatic VTE within 90 days post surgery, verified by AOANJRR staff. The primary analysis will include only patients undergoing elective primary total hip arthroplasty and total knee arthroplasty for osteoarthritis. Secondary outcomes will include symptomatic VTE for all HA and KA (including partial and revision) within 90 days, readmission, reoperation, major bleeding and death within 90 days and reoperation, death and patient-reported pain, function and health status at 6 months. If aspirin is found to be inferior, a cost-effectiveness analysis will be conducted. The study will aim to recruit 15 562 patients from 31 hospitals. ETHICS AND DISSEMINATION: Ethics approval has been granted. Trial results will be submitted for publication. The trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12618001879257, pre-results) and is endorsed by the Australia and New Zealand Musculoskeletal Clinical Trials Network.

Diagnosis and management of heparin-induced thrombocytopenia: a consensus statement from the Thrombosis and Haemostasis Society of Australia and New Zealand HIT Writing Group.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder that occurs following the administration of heparin and is caused by antibodies to platelet factor 4 and heparin. Diagnosis of HIT is essential to guide treatment strategies using non-heparin anticoagulants and to avoid unwanted and potential fatal thromboembolic complications. This consensus statement, formulated by members of the Thrombosis and Haemostasis Society of Australia and New Zealand, provides an update on HIT pathogenesis and guidance on the diagnosis and management of patients with suspected or confirmed HIT. MAIN RECOMMENDATIONS: A 4Ts score is recommended for all patients with suspected HIT prior to laboratory testing. Further laboratory testing with a screening immunoassay or confirmatory functional assay is not recommended in individuals with a low 4Ts score. However, if there are missing or unreliable clinical data, then laboratory testing should be performed. A positive functional assay result confirms the diagnosis of HIT and should be performed to confirm a positive immunoassay result. Heparin exposure must be ceased in patients with suspected or confirmed HIT and initial treatment with a non-heparin alternative instituted. Non-heparin anticoagulants (danaparoid, argatroban, fondaparinux and bivalirudin) used to treat HIT should be given in therapeutic rather than prophylactic doses. Direct oral anticoagulants may be used in place of warfarin after patients with HIT have responded to alternative parenteral anticoagulants with platelet count recovery. CHANGES IN MANAGEMENT AS A RESULT OF THIS STATEMENT: These are the first Australasian recommendations for diagnosis and management of HIT, with a focus on locally available diagnostic assays and therapeutic options. The importance of examining both clinical and laboratory data in considering a diagnosis of HIT cannot be overstated.

Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.

Eltrombopag treatment during induction chemotherapy for acute myeloid leukaemia: a randomised, double-blind, phase 2 study.

BACKGROUND: Patients with acute myeloid leukaemia frequently have thrombocytopenia during induction chemotherapy. Eltrombopag, an oral thrombopoietin receptor agonist, stimulates platelet production by a similar mechanism to endogenous thrombopoietin. This study investigated safety and efficacy of eltrombopag versus placebo during anthracycline-based induction treatment of patients with acute myeloid leukaemia. METHODS: In this randomised, double-blind, phase 2 study, treatment-naive patients were recruited from clinical centres across 10 countries (Australia, Belgium, Canada, Greece, Hungary, Israel, South Korea, Poland, Russia, and the USA). Patients with acute myeloid leukaemia of any subtype except M3 and M7 were stratified by antecedent malignant haematological disorder (yes or no) and age (18-60 years or >60 years) and were then randomly assigned (1:1) using an automated interactive voice-response system randomisation schedule. Investigators and patients were blinded to study treatment. Starting on day 4, patients received standard induction chemotherapy (daunorubicin bolus intravenous infusion on days 1-3 [90 mg/m2 for patients aged 18-60 years or 60 mg/m2 for patients aged >60 years], plus cytarabine continuous intravenous infusion on days 1-7 [100 mg/m2]), with eltrombopag 200 mg (100 mg for east Asians) or placebo once daily, until platelet counts were 200 × 109/L or higher, until remission, or after 42 days from the start of induction chemotherapy. The primary objective of the study was safety and tolerability assessed by adverse events, changes in left ventricular ejection fraction (LVEF), and clinical laboratory parameters in all treated patients. This study has been completed and is registered with ClinicalTrials.gov, number NCT01890746. FINDINGS: Between Sept 7, 2013, and Jan 30, 2015, 149 patients were assessed for eligibility and 148 were then randomly assigned to receive eltrombopag (n=74) and placebo (n=74). Groups were matched in mean (SD) age (56·7 years [12·3] in the eltrombopag group vs 56·6 years [11·6] in the placebo group), mean (SD) initial platelet count (59·5 × 109/L [43·3] vs 63·7 × 109/L [48·0]), and poor-risk karyotype (16 [22%] of 74 patients in both groups). The most common grade 3-4 adverse events (≥10% in either group) were febrile neutropenia (31 [42%] vs 28 [39%]), decreased white blood cell count (8 [11%] vs 5 [7%]), and hypophosphataemia (3 [4%] vs 9 [13%]). Serious adverse events occurred in 24 (32%) patients in the eltrombopag group compared with 14 (20%) patients in the placebo group. 39 (53%) patients in the eltrombopag group died versus 29 (41%) patients in the placebo group. Thromboembolic events (5 [7%] vs 4 [6%]) and mean (SD) change in LVEF (-2·5% [7·8] vs -4·3% [8·5]) were similar. INTERPRETATION: Data from this trial do not support combining eltrombopag with induction chemotherapy in patients with acute myeloid leukaemia. FUNDING: Novartis Pharma AG.

Romiplostim in adult patients with newly diagnosed or persistent immune thrombocytopenia (ITP) for up to 1 year and in those with chronic ITP for more than 1 year: a subgroup analysis of integrated data from completed romiplostim studies.

The thrombopoietin receptor agonist romiplostim is approved for second-line use in chronic immune thrombocytopenia (ITP), but its effects in patients with ITP for ≤1 year are not well characterized. This analysis of pooled data from 9 studies included patients with ITP for ≤1 year (n = 311) or >1 year (n = 726) who failed first-line treatments and received romiplostim, placebo or standard of care. In subgroup analysis by ITP duration, patient incidences for platelet response at ≥75% of measurements were higher for romiplostim [ITP ≤1 year: 74% (204/277); ITP >1 year: 71% (450/634)] than for placebo/standard of care [ITP ≤1 year: 18% (6/34); ITP >1 year: 9% (8/92)]. Of patients with ≥9 months on study, 16% with ITP ≤1 year and 6% with ITP >1 year discontinued romiplostim and maintained platelet counts ≥50 × 109 /l for ≥6 months without ITP treatment (treatment-free remission). Independent of ITP duration, rates of serious adverse events and bleeding were lower with romiplostim than placebo/standard of care and thrombotic events occurred at similar rates. In this analysis, romiplostim and placebo/standard of care had similar safety profiles and romiplostim increased platelet counts in patients with either ITP ≤1 year or ITP >1 year, with more treatment-free remission in those with ITP ≤1 year.

Drug-induced immune thrombocytopenia: Mapping of the drug binding site to the membrane-proximal region of platelet GPIX.

Drug-induced Immune thrombocytopenia (DIT) is a common complication of several medications, including commonly used antibiotics. The most widely studied DIT is caused by quinine. In DIT, antibodies predominantly bind to the platelet membrane glycoprotein (GP) IX in a drug-dependent fashion resulting in increased platelet clearance. Binding of the sensitizing drug, such as quinine, to GPIX has been proposed but is yet to be established. This work demonstrates that quinine is retained specifically by human GPIX. Quinine binding was first analyzed in wild-type mouse platelets and in transgenic mouse platelet expressing human GPIX using high performance liquid chromatography. Binding of quinine to GPIX was then measured in Chinese hamster ovary (CHO) cells expressing a combination of wild type, human or mouse, three human/mouse chimeric constructs and six mutant GPIX proteins. Quinine was retained by human GPIX. No detectable absorption was observed with mouse GPIX or human GPIbα. The quinine binding site was mapped to residues 110-115 of human GPIX suggesting that quinine interacts with specific residues of the GP. These findings provide further insights into the molecular mechanisms of DIT.

American Society of Hematology 2018 guidelines for management of venous thromboembolism: heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction mediated by platelet-activating antibodies that target complexes of platelet factor 4 and heparin. Patients are at markedly increased risk of thromboembolism. OBJECTIVE: These evidence-based guidelines of the American Society of Hematology (ASH) are intended to support patients, clinicians, and other health care professionals in their decisions about diagnosis and management of HIT. METHODS: ASH formed a multidisciplinary guideline panel balanced to minimize potential bias from conflicts of interest. The McMaster University GRADE Centre supported the guideline development process, including updating or performing systematic evidence reviews. The panel prioritized clinical questions and outcomes according to their importance for clinicians and patients. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to assess evidence and make recommendations, which were subject to public comment. RESULTS: The panel agreed on 33 recommendations. The recommendations address screening of asymptomatic patients for HIT, diagnosis and initial management of patients with suspected HIT, treatment of acute HIT, and special situations in patients with acute HIT or a history of HIT, including cardiovascular surgery, percutaneous cardiovascular intervention, renal replacement therapy, and venous thromboembolism prophylaxis. CONCLUSIONS: Strong recommendations include use of the 4Ts score rather than a gestalt approach for estimating the pretest probability of HIT and avoidance of HIT laboratory testing and empiric treatment of HIT in patients with a low-probability 4Ts score. Conditional recommendations include the choice among non-heparin anticoagulants (argatroban, bivalirudin, danaparoid, fondaparinux, direct oral anticoagulants) for treatment of acute HIT.

Melanoma protective antitumor immunity activated by catalytic DNA.

Melanoma incidence is increasing worldwide, and although drugs such as BRAF/MEK small-molecule inhibitors and immune checkpoint antibodies improve patient outcomes, most patients ultimately fail these therapies and alternative treatment strategies are urgently needed. DNAzymes have recently undergone clinical trials with signs of efficacy and no serious adverse events attributable to the DNAzyme. Here we investigated c-Jun expression in human primary and metastatic melanoma. We also explored the role of T cell immunity in DNAzyme inhibition of primary melanoma growth and the prevention of growth in non-treated tumors after the cessation of treatment in a mouse model. c-Jun was expressed in 80% of melanoma cells in human primary melanomas (n = 17) and in 83% of metastatic melanoma cells (n = 38). In contrast, c-Jun was expressed in only 11% of melanocytes in benign nevi (n = 24). Dz13, a DNAzyme targeting c-Jun/AP-1, suppressed both Dz13-injected and untreated B16F10 melanoma growth in the same mice, an abscopal effect relieved in each case by administration of anti-CD4/anti-CD8 antibodies. Dz13 increased levels of cleaved caspase-3 within the tumors. New, untreated melanomas grew poorly in mice previously treated with Dz13. Administration of anti-CD4/anti-CD8 antibodies ablated this inhibitory effect and the tumors grew rapidly. Dz13 inhibited c-Jun expression, reduced intratumoral vascularity (vascular lumina area defined by CD31 staining), and increased CD4+ cells within the tumors. This study provides the first demonstration of an abscopal effect of a DNAzyme on tumor growth and shows that Dz13 treatment prevents growth of subsequent new tumors in the same animal. Dz13 may be useful clinically as a therapeutic antitumor agent by preventing tumor relapse through adaptive immunity.

Safety and efficacy of romiplostim in splenectomized and nonsplenectomized patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia is an autoimmune disorder characterized by increased platelet destruction and insufficient platelet production without another identified underlying disorder. Splenectomy may alter responsiveness to treatment and/or increase the risk of thrombosis, infection, and pulmonary hypertension. The analysis herein evaluated the safety and efficacy of the thrombopoietin receptor agonist romiplostim in splenectomized and nonsplenectomized adults with primary immune thrombocytopenia. Data were pooled across 13 completed clinical studies in adults with immune thrombocytopenia from 2002-2014. Adverse event rates were adjusted for time of exposure. Results were considered different when 95% confidence intervals were non-overlapping. Safety was analyzed for 1111 patients (395 splenectomized; 716 nonsplenectomized) who received romiplostim or control (placebo or standard of care). At baseline, splenectomized patients had a longer median duration of immune thrombocytopenia and a lower median platelet count, as well as a higher proportion with >3 prior immune thrombocytopenia treatments versus nonsplenectomized patients. In each treatment group, splenectomized patients used rescue medications more often than nonsplenectomized patients. Platelet response rates (≥50×109/L) for romiplostim were 82% (310/376) for splenectomized and 91% (592/648) for nonsplenectomized patients (P<0.001 by Cochran-Mantel-Haenszel test). Platelet responses were stable over time in both subgroups. Exposure-adjusted adverse event rates were higher for control versus romiplostim for both splenectomized (1857 versus 1226 per 100 patient-years) and nonsplenectomized patients (1052 versus 852 per 100 patient-years). In conclusion, responses to romiplostim were seen in both splenectomized and nonsplenectomized patients, and romiplostim was not associated with an increase in the risk of adverse events in splenectomized patients. clinicaltrials.gov Identifier: 00111475(A)(B), 00117143, 00305435, 01143038, 00102323, 00102336, 00415532, 00603642, 00508820, 00907478, 00116688, and 00440037.

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells.

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

Changes in bone marrow morphology in adults receiving romiplostim for the treatment of thrombocytopenia associated with primary immune thrombocytopenia.

The effects of romiplostim on bone marrow morphology were evaluated in adults with immune thrombocytopenia (ITP). Patients with platelet counts <50 × 10(9)/L, ≥1 prior ITP therapies, and no collagen at baseline received weekly subcutaneous romiplostim starting at 1 µg/kg, adjusted to maintain platelet counts between 50 and 200 × 10(9)/L. Biopsies were scheduled after 1, 2, or 3 years of romiplostim (cohorts 1, 2, and 3, respectively). Irrespective of scheduled time, biopsies were performed earlier if patients discontinued or failed to achieve/maintain a response to romiplostim. Reticulin (silver stain) and collagen (trichrome stain) were graded by two hematopathologists using the modified Bauermeister scale (0-4). Of 169 patients, 131 had evaluable biopsies; 9/131 (6.9 %) had increases of ≥2 grades on the modified Bauermeister scale (cohort 1: 0/34; cohort 2: 2/39; cohort 3: 7/58), including two with collagen. Three of the nine patients had follow-up biopsies, including one patient with collagen; changes were reversible after romiplostim discontinuation. Of the nine patients, one had neutropenia detected by laboratory test and two had adverse events of anemia, both non-serious and not treatment-related. By actual exposure (as some biopsies did not occur as scheduled), the number of patients with grade increases ≥2 were year 1: 3/41, year 2: 1/38, year 3: 5/52. Twenty-four patients sustained platelet counts ≥50 × 10(9)/L for ≥6 months with no ITP medications after discontinuing romiplostim, i.e., they entered clinical remission of their ITP. In conclusion, in patients with ITP receiving romiplostim, bone marrow changes were observed in a small proportion of patients.ClinicalTrials.gov identifier: NCT#00907478.