bluuebunny/crossref_metadata_2025 huggingface dataset:
CLL Tumours with a Deletion of 11q Form Two Functional Subgroups Based on the Mutation Status of the Remaining ATM Allele.
Authors: Austen Belinda , Podinovskaia Maria , Almond Claire , Fews Graham , Gardiner Anne , Oscier David , Moss Paul , Stankovic Tanja
Published: October 2019

Deletions in chromosome 11q are an established prognostic marker in CLL. One copy of the ATM gene is deleted in these tumours, as detected by FISH analysis. However, it remains unclear whether the ATM gene is the main tumour suppressor gene that is accounting for the poor outcome in tumours with 11q deletions.

We have recently reported that patients whose tumours have mutations in the ATM gene have an impaired overall and treatment free survival. In our large cohort of 155 patients, tumours with an ATM mutation only partly correlated with tumours with an 11q deletion. We have therefore investigated the relationship between 11q deletions and mutations in the ATM gene. Using the highly sensitive DHPLC method, we have screened the 60 ATM coding exons for mutations in a cohort of 46 tumours, all with a deletion of chromosome 11q. We have found ATM mutations in 19 tumours, indicating a prevalence of 41%.

The ATM protein is vital in the cell’s response to DNA damage including that induced by chemotherapy. ATM acts upstream from p53 and defects in ATM function, like p53, lead to impaired DNA damage induced apoptosis. Furthermore, we have previously shown that loss of ATM function is associated with both in vitro and in vivo chemo-resistance.

Therefore, we next assessed whether the status of the remaining ATM allele in the 11q deleted tumours affected the response to DNA damage. Firstly we induced DNA damage with irradiation and measured both the phosphorylation of ATM protein targets and the induction of p53 dependent transcription responses in representative samples. We found that 11q deleted tumours with a remaining wild type ATM allele had responses that were similar to those seen in tumours with two wild type ATM alleles. In contrast, 11q deleted tumours with a mutation in the remaining ATM allele had defective DNA damage induced responses. We then analysed the effects of in vitro treatment with Fludarabine. First we demonstrated that fludarabine induces ATM dependent phophosphorylation responses in CLL tumours. Then we analysed its effect in the two 11q deleted CLL subgroups. We showed defective phosphorylation responses to fludarabine in the tumours with a mutation in the second ATM allele, but normal responses in those with a second wild type ATM allele.

In summary, we have shown that approximately 40% of CLL tumours with an 11q deletion have a mutation in their remaining ATM allele. Furthermore, we have demonstrated that the 11q deleted tumours appear to form two functional subgroups based on the presence of a mutation in the remaining ATM allele. In contrast to the subgroup with a wild type ATM allele, CLL tumours with a mutant ATM allele have defective in vitro responses to DNA damage with both irradiation and fludarabine. We expect that the functional differences between the two 11q deleted subsets will translate into differences in clinical outcome.
Outpatient Management of Pulmonary Embolism: Results from the National Multicentre PE Study.
Authors: Rhodes Susan , Bond Sarah , Green Sarah , Wimperis Jennie Z. , Pendry Kate , Davies Christopher W.H. , Medhi Ijaz , Killen Jeremy
Published: October 2019

Pulmonary embolism (PE) is a common complication of deep vein thrombosis(DVT). It has been shown that low molecular weight heparin (LMWH) may be used to treat PE and DVT and that home treatment of DVT with LMWH is safe and effective. A study published in 2000 suggested that outpatient management of PE is feasible and safe for the majority of patients. Phase 1 of this study showed that 44% of patients fulfilled criteria for theoretical outpatient management and that there were no bleeding or theomboembolic complications in the LMWH treatment phase in this group suggesting that these patients could safely have been discharged early.

In Phase 2 of the study all patients with a confirmed PE (high probability VQ scan, positive CT pulmonary angiogarm or clincal features of PE plus radiologically confirmed DVT) were entered into the early discharge scheme if they fulfilled criteria validated in phase 1, patients consented and they were discharged within 72 hours of presentation. Patients were treated with 175 Anti-Xa IU/kg tinzaparin (Innohep®) daily (for a minimum of 6 days) and oral anticoagulant therapy. Patients were excluded if: age < 18, admission for additional monitoring or treatment, active bleeding or bleeding disorder, poor compliance or morbidity, pregnant, previous PE, co-existing major DVT and patient preference. Outcome data were collected at 3 and 6 months including mortality and thromboembolic or bleeding events (early, whilst receiving LMWH, and late, after cessation of LMWH). Patients were asked to complete a satisfaction score using 10-point visual analogue score at the end of LMWH treatment phase and to indicate their preference for inpatient or outpatient management for any future episodes of PE.

107 patients with confirmed PE across 6 centres were discharged early. All patients completed the acute treatment phase with Innohep® and outcome data for this period were available for 100% patients. 3 month outcome data were available in 93 (86%) patients. There were no bleeding, thromboemblic complications, deaths or related readmissions in the early treatment phase. 1 patient experienced an anxiety episode in the early treatment phase and required reassessment but not readmission. Patients received a mean of 7.6 days of Innohep® and this was continued for a mean of 5.5 days after discharge, indicating that outpatient management of PE saved up to 5.5 inpatient bed days. 79.5% of patients completed the satisfaction score - the mean score was 9.3 indicating that patients were highly satisfied with outpatient treatment. 98% of patients expressed a preference for outpatient management. There were no significant adverse events in the acute LMWH treatment phase indicating that selected patients with a confirmed PE can safely be managed as outpatients in schemes similar to outpatient management of DVT and that this is highly acceptable to patients.
Sequential Treatment with the Anti-CD 20 Antibody Rituximab and CHOP+GCSF Chemotherapy in Patients with Post-Transplant Lymphoproliferative Disorder (PTLD): First Interim Analysis of a Multicenter Phase II Study.
Authors: Trappe Ralf , Oertel Stephan , Choquet Sylvain , LeBlond Vernoique , Papp-Vary Matthias , Riess Hanno
Published: October 2019

Post-transplant lymphoproliferative disorders (PTLD) are a main causative factor for mortality after organ transplantation. In January 2003 we started a multicenter phase II trial investigating a sequential treatment of the anti-CD 20 antibody rituximab and CHOP + GCSF chemotherapy. 29 patients have been enrolled so far. 14 patients were renal transplant recipients, 5 were heart transplant recipients, 7 were liver transplant recipients, 1 was a combined heart/lung transplant recipient, one was a combined kidney/pancreas transplant recipient and one patient was after bone marrow transplantation. Patients were treated with four weekly doses of 375mg/m2 of rituximab as a single therapeutic agent at days 1, 8, 15 and 22. CHOP chemotherapy had been administered at days 50, 72, 94 and 116 and was followed by GCSF starting at day 3 of each chemotherapy cycle. In case of disease progression during administration of rituximab or during the 4-week interval between antibody therapy and CHOP administration the patients directly entered chemotherapy. Histology divided in 21 diffuse large cell-, 2 marginal zone-, 1 Burkitt lymphoma and 3 polymorphic lymphoproliferations. 18 patients had stage III or stage IV disease. Patient age ranged from 16 to 80 years with a median age of 55 years and a mean age of 50.9 years. Time from transplantation to diagnosis divided the patients into two groups. The first group is defined by a median time from transplantation to diagnosis of 6 months (range 1 to 11 months, 11 patients, early PTLD), and a second one is defined by a median time from transplantation to diagnosis of 90 months (range 19 to 204 months, 16 patients, late PTLD). EBV-status was available for 19 patients with 10 EBV-associated PTLD and 9 non-EBV associated PTLD. In August 2005 24 patients were evaluable for treatment response, 25 were evaluable for treatment toxicity. In 84 applied chemotherapy cycles WHO 3°–4° leucopenia occurred in 29 cycles. 9 patients suffered from WHO 3°–4° infections. There were 3 early deaths due to therapy-associated complications. 15 patients (62.5%) achieved complete remission. Partial remission was observed in 5 patients (20.8%). 5 patients experienced progressive disease. With a mean follow up of 10.7 months four patients relapsed. There was no difference in response to rituximab therapy and in response to the sequential treatment of rituximab followed by CHOP chemotherapy for EBV-positive and EBV-negative PTLD. In summary sequential treatment of PTLD with rituximab and CHOP+GCSF is associated with an acceptable toxicity profile and is highly effective with a overall response rate of 83.3%.
PRTX-100 Inhibits Platelet Phagocytosis In Vitro.
Authors: Yatko Chris , Herrem Christopher , Siddiqui Samia , Sloan Victor S.
Published: October 2019

Background: In idiopathic thrombocytopenic purpura (ITP), autoantibodies bind to platelets which are then phagocytosed by monocytes/macrophages and removed by the reticuloendothelial system. PRTX-100 (Staphylococcal protein A) is being investigated for the treatment of ITP.

Objective: To assess the effect of PRTX-100 on phagocytosis of platelets in an in vitro assay.

Methods: Human monocytes were isolated from whole blood peripheral blood mononuclear cells (PBMCs) by adherence and cultured for 6 days in RPMI + 5% human serum. 48 hours prior to phagocytosis assay, PRTX-100 was added at 250, 25, and 2.5ng/ml. Human platelets were labeled with a fluorescent (PerCP) lipophilic dye and opsonized with an antibody to MHC Class I (W632). 2×10−5 monocytes were co-cultured with 2×10−7 labeled platelets for 1 hour at 37 ° C. All conditions were performed in triplicate. After an hour, phycoerythrin (PE) labeled anti-CD61 antibody was added to assess surface bound platelets versus ingested platelets. Phagocytosis was determined by flow cytometric analysis. The monocyte population was gated upon by forward and side scatter properties, then verified by staining with CD14-FITC. Percent phagocytosis was calculated as the fraction of ingested platelets (PerCP +/CD61−) to the total PerCP population (PerCP +/CD61−) + ( PerCP+/CD61+) within the gated monocyte population.

Results: PRTX-100 inhibits the phagocytosis of W632 opsonized platelets by human monocytes. Phagocytosis of W632 opsonized platelets was 40%, while phagocytosis in the presence of PRTX-100 at concentrations of 250, 25, and 2.5ng/ml was 18.3%, 23%, and 24.3%, respectively. Phagocytosis at 250ng/ml and 25ng/ml was significantly different from control phagocytosis with p values of 0.014 and 0.001 respectively by Student’s t test.

Conclusions: PRTX-100 inhibits the phagocytosis of platelets by monocytes, the effector limb of ITP. Prevention of platelet phagocytosis is an important treatment goal in ITP. PRTX-100 has been shown to be generally safe and well-tolerated in a phase I study in healthy volunteers (J Clin Pharmacol, in press). PRTX -100 is a promising therapeutic option for ITP and deserves further study.

Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets Effect of PRTX-100 on In Vitro Phagocytosis of Opsonized Human Platelets
Prevalence and Clinical Significance of Elevated Antiphospholipid Antibodies in Adults with Immune Thrombocytopenic Purpura.
Authors: Pierrot-Deseilligny Carole , Khellaf Mehdi , Gouault Michele , Intrator Liliane , Michel Marc , Bierling Philippe , Godeau Bertrand
Published: October 2019

Background: The clinical and pathogenic significances of the presence of antiphospholipid antibodies (APA) [anticardiolipin antibodies (aCL) and lupus-like anticoagulant (LA)] in patients with immune thrombocytopenic purpura (ITP) remain controversial.

Objective: To determine the prevalence and clinical significance of positive APA in a large monocentric cohort of adults with newly diagnosed ITP.

Methods: aCL (IgG and IgM) and LA were searched at time of diagnosis in 260 adults with ITP and a platelet count < 50×109/L over a 15 years period. Thirty-five patients with definite systemic lupus, 5 with primary antiphospholipid syndrome and 4 with HIV infection were excluded and the analysis was performed on 216 patients.

Results: APA (aCL and LA) were detected at ITP diagnosis in 55 patients (25%) including: IgG-aCL in 43 patients (20%) with a level between 15-39 UI/L in 31 and ≥40UI/l in 12; IgM-aCL in 11 patients (5%); and LA in 16 patients (7%). LA was associated with IgG-aCL in 14 patients, IgM-aCL in 1 patient, and was isolated in 1 patient. Positive test for LA was correlated with the presence of highly positive IgG-aCL (≥40UI/L) (p=0.001). Age, gender, initial platelet count, acute or chronic outcome of ITP were not associated with the presence of APA (aCL and LA), aCL and LA status except for age which was lower in LA positive group (mean age 22 years vs 42 years in LA negative group, p=0.002). After a median follow-up of 31 months (range: 1 to 185 months), 15/216 (7%) patients experienced a thrombosis (arterial = 3, deep venous thrombosis and/or pulmonary embolism = 12); 5 of whom (33%) had positive aCL (with a level ≥ 40 UI/L in 4). In 4 of these 5 patients, aCL were associated with positive LA. In multivariate analysis, thrombosis events were associated with age [Hazard ratio = 1.5 (IC95% : 1.1–2.0], presence of LA [Hazard ratio = 6.5 (IC95% : 1.7–25.5] or presence of IgG-aCL at high level (≥40UI/L) [Hazard ratio = 6.1 (IC95% : 1.6–23.7]. In contrast, there was no association of thrombosis events with sex, platelet count at diagnosis, acute or chronic evolution (≥ 6 months), antinuclear antibodies or treatments used.

Conclusions: Prevalence of APA (25%) in adults with newly diagnosed ITP is lower in our cohort than previously observed in other studies. Though the incidence of thrombosis is significantly increased in patients with LA and/or highly positive aCL, this risk seems too low to recommend a systematic testing in patients with ITP and no previous history of thrombosis.
Mice Overexpressing Histone Deacetylase 9 Display Abnormal B Cell Development and Proliferation.
Authors: Gil Veronica S. , Petrie Kevin , Chornet Manuel Boix , Howell Louise , Sebire Neil J. , Zelent Arthur
Published: October 2019

Histone acetylation plays a key role in regulating chromatin structure and gene expression. The activities of histone deacetylases (HDAC), enzymes that remove acetyl groups from lysines located at the amino-terminal tails of core histones, have been implicated in the pathogenesis of hematological malignancies. Consequently, inhibitors of these enzymes (HDACi) are considered an important new class of drugs for use in anti-cancer therapy and are now in various stages of development and clinical trials. For example, depsipeptide has now been granted a fast track designation by the FDA for development as a treatment for CTCL. To date, eleven HDACs have been identified and grouped on the basis of sequence homology to class I (HDACs 1–3 and 8), IIa (4, 5, 7 and 9) and IIb (6, 10 and 11). Depsipeptide and other currently used HDACi lack class and enzyme specificities. Additionally, the contribution of each HDAC to the pathogenesis of a given malignant disease is poorly understood. Improvements in HDACi specificities, together with advances in the understanding of the roles that individual HDACs play in normal hematopoiesis and distinct hematological neoplasms, are required for an effective use of these agents in anti-cancer therapies. We have previously identified and characterized HDAC9 as a class IIa enzyme that within the hematopoietic system is preferentially expressed in the lymphoid lineage. HDAC9 appears to be overexpressed in B lymphoid malignancies and associates at physiological levels with the BCL6 oncoprotein (Petrie et al, JBC278:16059–72, 2003), a transcriptional repressor implicated in the pathogenesis of non-Hodgkin’s lymphoma, suggesting a role for this chromatin modifying enzyme in B-cell transformation. In order to examine the function of HDAC9 in lymphoid development and cancer, we have generated transgenic mice that express the protein throughout the B lymphoid compartment under the control of the immunoglobulin heavy chain enhancer (Eμ). Initial data show that at an average of seven months these mice display splenomegaly and alterations to the splenic and bone marrow lymphoid populations. The presence of immature lymphocytes was also detected in peripheral blood. Consistent with these results and the above hypothesis, knockdown of HDAC9 results in strong inhibition of cell growth. These data indicate that aberrant expression of HDAC9 confers a proliferative advantage and leads to a phenotype resembling a pre-malignant condition.
Leukemogenic Cascade Induced by a Constitutively Active Neurotrophin Receptor, ΔTrkA.
Authors: Li Zhixiong , Meyer Johann , Rhein Mathias , Kustikova Olga , Rudolph Cornelia , Kamino Kenji , Neumann Thomas , Yang Min , Wahlers Anke , Fehse Boris , Reuther Gary W. , Schlegelberger Brigitte , Ganser Arnold , Baum Christopher
Published: October 2019

Various neurotrophins (NT) are expressed in the hematopoietic microenvironment to deliver their signals through TRK receptor tyrosine kinases. Although previous reports suggested a transforming potential of activated TRK signaling in hematopoiesis, the target cells and underlying mechanisms are largely unknown. In this study, we investigated in vivo leukemogenesis of ΔTrkA, a mutant of TRKA isolated from a patient with acute myeloid leukemia (AML). Retroviral expression of ΔTrkA in myeloid 32D cells induced AML at ~4 weeks after transplantation into syngeneic C3H/Hej mice (n=11). C57BL/6J mice (n=15) transplanted with ΔTrkA-transduced primary bone marrow cells developed myeloid and/or lymphoid leukemia with infiltration in multiple organs including bone marrow, spleen, liver, lung, and central nervous system. While activation of a tyrosine kinase generally is not sufficient to cause AML, surprisingly, 7 out of 15 C57BL/6J mice developed a polyclonal AML with a latency of <12 days. This strongly suggests that ΔTrkA is sufficient to block differentiation and promote proliferation of short-term repopulating myeloid progenitor cells. This simultaneous interference with two complementary leukemogenic pathways has not been reported for other oncogenic tyrosine kinases. However, as half of the mice survived this disorder, ΔTrkA does not seem to confer self-renewal properties to transformed blasts. Supporting this hypothesis, transplantation into lethally irradiated secondary recipients did not re-induce polyclonal AML, but rather gave rise to clonal acute lymphoblastic leukemia (ALL) after a latency of >78 days. This suggests that ΔTrkA can also transform long-term repopulating cells with a lymphoid potential. Retroviral insertion site analyses and spectral karyotyping revealed that induction of ALL by ΔTrkA required additional genetic lesions: Leukemic cells showed retroviral insertions in proto-oncogenes Bcl11a and Bcl11b, among others. Characterization of signal transduction demonstrated that PI3K and mTOR-raptor were crucial components of the transforming pathway induced by ΔTrkA. Phospholipase D was an important contributing factor, whereas STAT and MAP kinase pathways were not involved. In summary, our findings reveal potent transforming properties of altered NT receptor signaling in leukemia induction, which are in many respects distinct from other oncogenic tyrosine kinases. We would suggest to further evaluate the role of NT receptor signaling in leukemia pathogenesis, prognosis and treatment.
Egr-1 Functions as a Tumor Suppressor for Leukemias Caused by Different Oncogenes.
Authors: Gibbs John D. , Liebermann Dan A. , Hoffman Barbara
Published: October 2019

Deregulated expression of the proto-oncogenes c-myc, E2F-1 and c-myb in M1 myeloid leukemic cells rendered these cells incapable of undergoing myeloid terminal differentiation and its associated growth arrest, and prevented the loss in leukemogenicity normally observed when M1 cells are induced to differentiate. We recently showed that expressing Egr-1 in M1myc cells abrogated the c-myc-mediated block in terminal myeloid differentiation, with the cells undergoing macrophage maturation in the absence of growth arrest. Importantly, Egr-1 also abrogated c-myc-driven leukemogenicity. In this study, we asked if Egr-1 functions as a tumor suppressor by abrogating the block in differentiation caused by other oncogenes. It was shown that expressing Egr-1 in M1E2F-1 cells completely overrode the block in terminal myeloid cell differentiation, with the cells undergoing macrophage maturation, and unlike in myc/Egr-1 expressing cells, also becoming growth arrested. In contrast, expression of Egr-1 in M1myb cells promoted progression of the differentiation program to an intermediate stage of differentiation, but the cells became neither fully matured nor growth arrested, continuing to proliferate indefinitely. As seen for expression of Egr-1 in M1myc cells, Egr-1 expression in M1E2F-1 cells also abrogated E2F-1 driven leukemogenicity. In contrast, Egr-1 co-expression with c-myb in M1myb cells did not abrogate myb driven leukemia. Taken together, it can be concluded that Egr-1 is dominant to both E2F-1 and c-myc with regard to terminal morphological differentiation and the ability to suppress leukemias, whereas the proto-oncogene c-myb is dominant to Egr-1. These findings indicate that the tumor suppressor Egr-1 provides important tools for differentiation therapy of multiple leukemic phenotypes, dependent on the molecular nature of the activated oncogene.
Microparticles Are a Major Determinant of Thrombin Generation Measured by Technothrombin®TGA.
Authors: Kaufmann Viktoria , Mager Jutta , Eichinger Sabine , Binder Bernd R.
Published: October 2019

We have recently shown that a thrombin generation assay using low concentrations of phospholipids (3,2 mM; 20% PS) can discriminate patients with an increased risk for recurrent venous thromboembolism after cessation of anticoagulant therapy from those with a lower risk (Hron et al JAMA 2006). Patients with peak thrombin of ≥400nM had a ~3-fold higher relative risk of recurrence than those with peak thrombin <400nM. This difference was not explained by a larger proportion of patients with clotting factor abnormalities among those with high peak thrombin. We therefore investigated which other factors might influence peak thrombin generation. For this purpose 35 healthy male plasma donors were analyzed. Thrombin generation was measured in standard platelet poor plasma and in plasma made microparticle (MP) free by centrifugation for 120 minutes at 35,250 x g using low (3,2μM) and high (32μM ) phospholipids concentrations at the same tissue factor concentration (71,6 pM). For control purposes, peak thrombin was measured under similar conditions in 12 individuals each having either heterozygous FVLeiden mutation, being on vitamin K-antagonist therapy (VKA; mean INR=3.2) or having a lupus anticoagulant. In healthy plasma donors peak thrombin as generated by tissue factor and low phospholipids (RCL) in platelet poor plasma was 110±40 nM and increased to 306±99 nM in the presence of high phospholipids (RCH). When MP free plasma was used, however, peak thrombin dropped to 23±13 nM (RCL) and 109±43 nM thrombin (RCH), respectively. When MPs were re-added to MP-free plasma samples, a dose dependent increase in peak thrombin was found both for RCL and RCH reagents. At a MP concentration of 25,000 added per 1 ml MP free plasma, peak thrombin was 381±24 nM for RCL and 399±63 nM for RCH. The effects of MPs on peak thrombin using RCL were much more pronounced than the differences in peak thrombin seen between plasmas from individuals with FVLeiden, during anticoagulant therapy or with lupus anticoagulant as compared to controls (FVLeiden : 161±50 nM; VKA: 50±12 nM; lupus: 37±23 nM; controls: 110±40 nM). These results indicate that MPs contained in platelet poor plasma are a major determinant of peak thrombin in thrombin generation assays when low phospholipids are used and might cause increased peak thrombin levels found in patients with recurrent venous thromboembolism.
High Affinity Autoantibodies to ADAMTS13 in the Antiphospholipid Syndrome.
Authors: Austin Steven K. , Starke Richard D. , Purdy Gordon , Lawrie Andrew S. , Machin Samuel J. , Mackie Ian J.
Published: October 2019

Autoantibodies to ADAMTS13 are well known to play an important role in the development of microthrombosis and organ ischaemia in thrombotic thrombocytopenic purpura (TTP). Antiphospholipid syndrome (APS) is an autoimmune disorder associated with thrombosis and pregnancy failure, but in severe cases of APS, microvascular thrombosis can occur similar to that seen in TTP - suggesting possible mutual pathogenic factors. The role of ADAMTS13 in APS patients is unknown and we hypothesize that ADAMTS13 may play a role in the development of thrombosis in the APS population. We evaluated ADAMTS13 in 68 patients: 52 with APS (as defined by the revised Sapporo Criteria), and 16 with anti-phospholipid antibodies (aPL) only. Of the 68 patients, 38 (56%) had IgG antibody to ADAMTS13 (median 13.2 ug/ml, range 9.8 – 28.3 ug/ml, NR< 9.6 ug/ml) by ELISA (Imubind ELISA, American Diagnostica Inc). 25/68 patients (37%) demonstrated low ADAMTS13 activity by our in-house collagen binding assay (median 33.2%, range 0–64%, NR 66–126%), with 12 of the 25 patients demonstrating detectable antibody to ADAMTS13. Low activity was not associated with elevated VWF:Ag levels suggesting that high VWF turnover did not account for the low ADAMTS Activity. In addition, ADAMTS13 Antigen levels (Imubind ELISA, American Diagnostica Inc) were elevated in 45/68 (66%) (median 850 ng/ml, range 890–1379 ng/ml, NR350-730ng/ml) indicating discordance with the observed low ADAMTS13 activity levels. The increased ADAMTS13 antigen may reflect a compensatory response to the presence of autoantibodies to ADAMTS13 in APS. The abnormal ADAMTS13 findings were not associated with the presence of anticardiolipin antibody, anti-β2glycoprotein1 or lupus anticoagulant. Similarly, no association was seen in analysis of clinical subgroups (arterial or venous thrombosis, recurrent miscarriage). We subsequently purified IgG from plasma of 3 of the APS patients with detectable anti-ADAMTS13 autoantibody and observed strong binding to ADAMTS13 via ELISA. Furthermore using the purified IgG, increasing NaCl concentrations did not prevent antibody binding to ADAMTS suggesting that these antibodies are high affinity. These findings suggest that ADAMTS13 autoantibodies may have a role in other autoimmune thrombotic conditions aside from TTP. In APS, the prevalence of these high affinity antibodies may well be a pathogenic feature integral to shifting the haemostatic balance in favour of thrombosis.
Myeloproliferative Disease in the Pathogenesis and Survival of Budd-Chiari Syndrome.
Authors: Smalberg Jasper H. , Murad Sarwa D. , Braakman Eric , Valk Peter , Kappers-Klunne Mies C. , Janssen Harry L. , Leebeek Frank W.G.
Published: October 2019

Budd-Chiari syndrome (BCS) is a rare disorder caused by obstruction of the hepatic veins or the suprahepatic inferior vena cava. Myeloproliferative diseases (MPD) are the most important aetiological factor in BCS. The aim of this study was to evaluate multifactorial aetiology in BCS patients with MPD, to assess potential added value of the recently discovered JAK2 mutation in the diagnostics of MPD in BCS, and to determine the survival of MPD patients in BCS. All patients referred to our university hospital with primary, non-malignant BCS between January 1980 and January 2006 were included in this study (n=40). Median age was 28.4 years (18.4–53.3), 26 patients (65%) were female and in nine patients (23%) additional portal vein thrombosis (PVT) was present. Overall mean follow-up was 7.1 ± 6.9 years. Only two patients were lost to follow-up, of which one MPD patient. In addition to standard MPD work-up according to WHO criteria, JAK2 mutation analysis was performed in 17 patients. MPD was present in 33% of the patients: Polycythemia Vera (n=6), Essential Thrombocythemia (ET) (n=6) and unclassifiable MPD (n=1). JAK2 mutation was identified in seven out of the 17 tested BCS patients (41%). In two patients suspect for ET, but who failed to meet WHO criteria, JAK2 mutation analysis led to the diagnosis of MPD. In 38% of patients with MPD additional pro-thrombotic factors were present. Survival rates in patients with MPD at 1, 5, and 10 years remained constant at 92% (95% CI, 78%–100%). Survival rates in patients without MPD at 1, 5, and 10 years were 89% (95% CI, 77%–100%), 64% (95% CI, 44%–85%) and 53% (95% CI, 28%–79%), respectively. There was no significant difference in survival between the two (P=0.18). Additional PVT was only present in patients without MPD in our population, which may account for the slightly lowered survival of these patients, since more extensive thrombosis in the splanchnic area is associated with poorer survival. In addition, three patients with and one patients without MPD underwent liver transplantation, which may have affected survival rate in favour of MPD patients. In conclusion, we report MPD in 33% of our BCS patients. In 38% of the patients with MPD, additional pro-thrombotic factors were present. These results enforce the necessity of extensive screening for additional pro-thrombotic conditions. Our study clearly confirms the importance of JAK2 mutation analysis in patients with BCS. Long term follow-up did not show a significant difference in survival between patients with and without MPD.
Methionine-Loading and Random Homocysteine Tests Have No Added Value in Risk Assessment for Venous and Arterial Thrombosis.
Authors: Lijfering Willem M. , Veeger Nic J.G.M. , Brouwer Jan-Leendert P. , van de Poel Marlene H.W. , van der Meer Jan
Published: October 2019

Hyperhomocysteinemia is a risk factor for venous and arterial thrombosis. Different diagnostic strategies are used to identify subjects at risk of thrombosis, related to hyperhomocysteinemia. Measurements of fasting and methionine-loading levels are usually recommended. Alternatively, random homocysteine measurements may simplify the procedure. Random levels < 10 and > 20 μmol/l are considered to indicate normohomocysteinemia and hyperhomocysteinemia, respectively, while consecutive fasting and methionine-loading tests are required at levels 10–20 μmol/l. We performed a study to assess the most suitable strategy in a large cohort of families with hereditary (index) deficiencies of protein S, protein C or antithrombin. Random, fasting and methionine-loading homocysteine samples were measured in 713 relatives. According to predefined cut-off levels hyperhomocysteinemic and normohomocysteinemic relatives were identified and their absolute risks of thrombosis were compared. Relatives with random homocysteine levels > 20 μmol/l were not at risk of venous or arterial thrombosis compared to relatives with levels < 10 μmol/l (relative risks 0.9 [95% CI, 0.4–2.3] and 1.7 [0.5–5.7], respectively). Fasting hyperhomocysteinemia (homocysteine levels > 18.5 μmol/l) was associated with an increased risk of venous and arterial thrombosis (relative risks 2.6 [1.3–4.8] and 3.7 [1.5–8.4)], respectively) (Table). Relatives with normal fasting homocysteine levels, but methionine-loading hyperhomocysteinemia (homocysteine levels > 58.8 μmol/l) were not at risk; relative risk 0.8 (0.2–1.9) for venous thrombosis and 1.1 (0.2–3.9) for arterial thrombosis. Exclusion of relatives with an index deficiency did not alter the risk estimates, while annual incidences of normohomocysteinemic relatives decreased to 0.19% per year (0.12–0.29), which is comparable with the annual incidence in the normal population. As only fasting homocysteine identified subjects at risk of thrombosis, random homocysteine and methionine-loading tests can be omitted in clinical practice.

Venous Thrombosis Observation Relatives Incidence/year (%) Relative Risk years with event (95% CI) (95% CI) * Methionine-loading performed in relatives with no fasting hyperhomocysteinemia Fasting Homocysteine No hyperhomocysteinemia 10408 55 0.53 (0.40–0.69) Reference Hyperhomocysteinemia 804 11 1.37 (0.68–2.45) 2.6(1.3–4.8) Methionine-loading test* No hyperhomocysteinemia 9341 50 0.54 (0.40–0.71) Reference Hyperhomocysteinemia 986 4 0.41 (0.11–1.04) 0.8(0.2–1.9) Arterial Thrombosis Fasting Homocysteine No hyperhomocysteinemia 11096 21 0.19 (0.12–0.29) Reference Hyperhomocysteinemia 1004 7 0.70 (0.28–1.44) 3.7(1.5–8.4) Methionine-loading test* No hyperhomocysteinemia10008 10008 19 0.19 (0.11–0.30) Reference Hyperhomocysteinemia 1000 2 0.20 (0.02–0.72) 1.1(0.2–3.9)
Fibronectin Is Not the Only Important Molecule Required for Fibrinogen/VWF-Independent Platelet Aggregation: Study of Thrombosis in a New Strain of Triple Deficient Mice.
Authors: Reheman Adili , Hong Yang , Spring Christopher M. , Chen Pingguo , Wagner Denisa D. , Fassler Reinhard , Freedman John , Ni Heyu
Published: October 2019

Fibrinogen (Fg) has been considered essential for platelet aggregation. We demonstrated, however, that thrombi do form in Fg-deficient mice and in mice doubly deficient for both fibrinogen and von Willebrand factor (Fg/VWF−/−). We further reported that β3 integrin and thrombin are critical for this Fg/VWF-independent platelet aggregation. In Fg−/− or Fg/VWF−/− mice, platelet fibronectin (Fn) content is increased 3–5 fold. Furthermore, thrombus growth and stability are impaired in plasma Fn conditional deficient (M×-Cre, Fnflox/flox) mice. These data are consistent with the most recent studies of Fn assembly and suggest that Fn may support platelet thrombus formation. To examine whether Fn is the alternative key molecule which mediates platelet aggregation and thrombus formation in Fg/VWF−/− mice, we developed a novel strain of triple knockout (TKO) mice by breeding Fg/VWF−/− mice with M×-Cre+/− Fnflox/flox conditional knockout mice. Cre- littermates delivered from the same parents were used as a control. Fn depletion was induced by i.p. injections of polyinonic-polycytidylic acid. We found that TKO mice are viable with dramatically decreased levels of Fn in both the plasma (<2% of control) and platelets (<20% of control) as determined by immunoblot. No significant difference was found in peripheral blood cell counts or platelet surface adhesion proteins (GPIbα, P-selectin, β3 and β1 integrins) as compared with control mice. Unexpectedly, TKO platelet aggregation induced by thrombin and thrombin receptor activation peptide (TRAP) was enhanced in both platelet rich plasma and PIPES buffer (P<0.05). This phenomenon was also observed in a parallel-plate flow chamber. Significantly more TKO aggregates formed when fluorescently labelled whole blood was perfused over a collagen surface at 500s−1 (P<0.001). We also studied thrombus formation in TKO and Cre- control mice using intravital microscopy. Our preliminary data showed no obvious decrease in thrombus formation in TKO mice and injured arterioles occluded in one of two experimental mice. Our data suggest that Fn is not the only important molecule required for platelet aggregation and thrombus formation in Fg/VWF−/− mice. Further investigation of how Fn may inhibit Fg/VWF-independent platelet aggregation and the identification of what ligand(s) support this novel aggregation pathway may unveil an alternative haemostatic pathway in the absence of Fg and VWF.
Early Growth Response Factor EGR-1 Regulates Gαq Gene in Megakaryocytic Cells.
Authors: Jalagadugula Gauthami , Danny Dhanasekaran N. , Soochong Kim , Kunapuli Satya P. , Rao A. Koneti
Published: October 2019

Gαq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation. We studied Gαq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic (MK) transformation. RT-PCR analysis of HEL cell RNA revealed that Gαq mRNA was relatively low in untreated cells and it increased after PMA treatment with a peak at 5 h. Immunoblot analysis of HEL lysates showed enhanced Gαq expression with PMA. Luciferase reporter gene studies on full length construct (upto −1116 bp from ATG) and its serial 5′ truncations revealed a negative regulatory site at −238/−202 and two positive sites at −203/−138 and −1116/−731. In the region −238/−202 consensus sites were noted for two transcription factors PU.1 and GATA-1 that are known to regulate several megakaryocytic genes. Deletions of these sites alone or together revealed no change in the transcriptional activity of the gene in reporter studies. The positive region −203/−138 contained two overlapping Sp1/AP-2/EGR-1 consensus sites at −202/−189 and −164/−150. Gel shift studies were performed on oligonucleotides 1 (−203/−175) and 2 (−174/−152) using HEL cell extracts. Protein binding occured with Gαq oligonucleotides 1 and 2, which was competed with excess unlabeled oligos but not by unlabeled Sp1 or AP-2 consensus oligos. Supershift assay using antibody against Sp1 revealed neither competition nor supershift, suggesting that Sp1 does not bind to these oligonucleotides. No protein binding was noted when Gαq oligos 1 or 2 were incubated with extracts known to contain Sp1 or AP-2. These results indicate that Sp1 and AP-2 do not bind to the Gαq oligonucleotide regions. Protein binding to oligonucleotides 1 and 2 was abolished by excess unlabeled consensus EGR-1 oligo, and by immunodepletion of the EGR-1 protein from the nuclear extract with anti-EGR-1 antibody. These experiments reveal that EGR-1 binds to both Gαq oligonucleotides −203/−175 and −174/−152. In luciferase reporter studies mutations in EGR-1 sites present in both oligonucleotides 1 and 2 markedly decreased gene activity indicating functional relevance. In further studies, reduction in endogenous EGR-1 expression with antisense oligonucleotide to EGR-1 inhibited PMA induced Gαq transcription and protein in HEL cells. Lastly, EGR-1 deficient mouse platelets also showed ~50% reduction in the Gαq protein (immunoblotting) relative to wild type platelets. These studies suggest that Gαq gene is regulated during PMA induced differentiation by EGR-1, a transcription factor that regulates a wide array of genes involved in cellular proliferation, differentiation, and apoptosis, and in vascular response to injury and atherosclerosis.
Shedding of Procoagulant Microparticles from Platelets through Integrin Outside-In Signaling Involving WASP and the Actin Cytoskeleton.
Authors: Cauwenberghs Sandra , Feijge Marion A.H. , Harper Alan G.H. , Sage Stewart O. , van Pampus Elisabeth , Curvers Joyce , Heemskerk Johan W.M.
Published: October 2019

Platelet activation by potent calcium-mobilizing agents, like collagen and thrombin or ionomycin, results in Ca2+- and calpain-dependent shedding of microparticles (MP) that are active in coagulation. Here, we describe and characterize a different mechanism of MP formation by platelets under storage in the absence of agonists. This shedding relies on integrin αIIbβ3 activity, since clinically used integrin blockers, like tirofiban or abciximab, reduce storage-induced MP shedding by 83±5.7% (p=0.006); inhibition of intracellular integrin signaling via Src kinase (PP1) or phosphoinositide 3-kinase (PI-3 kinase, wortmannin) inhibits this process by 52.3±14.3% (p=0.035) and 39±7% (p=0.005), respectively. Calpain inhibition suppresses MP formation by only 35±3.8% (p=0.003), while calpain activity is also not increased during storage. Levels of cytosolic Ca2+ concentration remain at 25–30 nM in the stored platelets. Treatment of platelets with recombinant WASP (Wiskott-Aldrich Syndrome Protein) peptide, activating the Arp2/3 complex and promoting actin nucleation, affects storage-induced MP shedding (p=0.001). Rho and Rac1, belonging to the family of Rho GTP-binding proteins, are involved in actin cytoskeleton regulation and platelet spreading. However, these GTP-binding proteins do not seem to be involved in MP shedding, as treatment of platelets with recombinant Rac1 peptide or with Rho kinase inhibitor is ineffective, as in the absence of these GTP-binding proteins. Furthermore, incubation of platelets with cytochalasin D, latrunculin B or jasplakinolide, which compounds affect actin filament polymerization, increases the amount of procoagulant MP (p<0.032). Interestingly, this effect is greatly reverted in the presence of integrin blockers or wortmannin. We determine that coagulation-active MP are abundantly present in stored platelet preparations that are used for transfusion purposes. The contribution of these MP to the coagulation process was studied after transfusion of these preparations to thrombocytopenic patients. Thrombin generation measurements indicate that the transfused MP initiate up to 25 nM concentrations of thrombin, which potentiate the procoagulant effect of platelets in these patients. Taken together, we conclude that the process of integrin-mediated MP shedding from apparently resting platelets is a uniquely regulated cellular response that operates independently of Ca2+ elevation and calpain activation, and contributes to coagulant activity in a clinically relevant way. It involves αIIbβ3 outside-in signaling, leading to WASP-mediated destabilization of the actin cytoskeleton, but independently of Rho and Rac1.
Integrin Linked Kinase Is Expressed in Acute Myeloid Leukemia Blasts and Its Inhibition Is Cytotoxic to Leukemic Progenitors.
Authors: Muranyi Andrew , Dedhar Shokat , Hogge Donna E.
Published: October 2019

Constitutive activation of the phosphatidylinositol-3-kinase (PI-3K) pathway is frequent in acute myeloid leukemia (AML) blasts and down-regulation of this pathway results in apoptotic death of these cells from many patient samples. Integrin linked kinase (ILK) is stimulated by activated PI-3K and ILK, in turn, activates AKT, a key downstream effector of the PI-3K pathway. The expression and activity of ILK are increased in a range of solid tumors. Small-molecule inhibitors of ILK activity have been identified and shown to inhibit tumour growth, invasion and angiogenesis. We investigated the possible role of ILK in AML blast and colony forming cell (AML-CFC) survival. Using Western blotting, ILK protein was detected in 30 of 35 primary AML blast samples although the levels seen were variable. ILK kinase activity correlated strongly with ILK protein expression as quantitated by densitometry (r = 0.91) in 6 samples where both were studied. Activation of the PI-3K pathway, as measured by detection of AKT phosphorylation on serine 473 (ser473), was also found in 27 of 30 samples expressing ILK protein. QLT0267 is a potent second generation small molecule inhibitor (from QLT Inc., Vancouver, Canada) which selectively inhibits the kinase activity of ILK but not a variety of other kinases. Treatment of AML blasts with QLT0267 resulted in a time and dose dependent decrease in p-AKT ser473 expression as well as downstream targets of AKT (p-S6, p-GSK-3β). 27 AML and 5 normal bone marrow (NBM) samples were incubated for 48h with QLT0267 or 10 μM of the PI-3K inhibitor, LY294002, and then plated in CFC assay. There was a direct correlation between the % AML-CFC kill seen with LY204002 and QLT0267 (r= 0.70 and 0.60 comparing % kill with LY294002 and QLT0267 at 3 and 10 μM, respectively). The IC50 of QLT0267 was ≤ 3μM for 6 of 23 AML samples and 0 of 5 NBM samples (range % kill of AML and normal CFC after treatment with 3μM; 0 – 86% and 0 – 23%, respectively). The IC90 for this inhibitor was ≤ 10μM for 9 of 27 AML and 0 of 5 NBMs (range % kill 6 – 99 and 30 – 68, respectively, for AML and normal CFC treated with 10μM QLT0267). Interestingly, AML-CFC from 4 AML samples in which ILK protein could not be detected were resistant to killing with QLT0267. Thus, ILK is expressed in a large proportion of AML samples. Inhibition of ILK kinase is cytotoxic to leukemic progenitors suggesting that this molecule is important for the survival of these cells. Approximately one third of AML samples tested were more susceptible to killing by ILK inhibition than NBM cells suggesting that selective targeting of malignant rather than normal hematopoietic cells may be achieved in some cases. Furthermore, it may be possible to predict which AML samples will be sensitive to inhibitors such as QLT0267 by measuring expression of the target protein.
Protoporphyrin IX Down-Regulates DMT1 Associated Protein (DAP) in K562 Cells.
Authors: Okazaki Yasumasa , Yin Hong , Ma Yuxiang , Yeh Mary , Yeh Kwo-yih , Glass Jonathan
Published: October 2019

The final steps of heme biosynthesis include the transport of coproporphyrin with the transport step probably mediated by the peripheral benzodiazepine receptor (PBR). Within the mitochondria copropoprhyrin is then converted to protoporphyrin IX (PPIX) which in turn is converted to hemin with insertion of iron by ferrochelatase. PBR is ubiquitously expressed and has been implicated in steriodogenesis, apoptosis, erythroid differentiation, and inflammation. Interestingly, PPIX is among several high affinity ligands for PBR. Various cytosolic proteins that interact with PBR have also been defined including PBR associated protein 7 (PAP7). The various PBR ligands including PPIX may affect the binding of these proteins to PBR. We have demonstrated (Blood, Nov 2004; 104: 53) that DAP, a protein highly homologous to PAP7, binds to the C-terminus of DMT1 and may have a role in regulation of intracellular iron transport. We, therefore, examined the effects of PPIX on the functions of DAP and other proteins that affect cellular iron metabolism. DAP is 526 amino acid protein with a nuclear localization signal domain (aa 212–229) and a Golgi localization domain (aa 380–524), and is distributed in the cytoplasm, Golgi apparatus, and nuclei of K562 cells. K562 cells were grown in the presence of 5 μM PPIX for 24 hours and then the expression of DAP, transferrin receptor 1 (TfR1), and ferritin examined by western blot analysis. In addition, cells were grown in medium of either normal iron content (3.5 μM from ferri-transferrin), high iron content (217 μM from the addition of ferric ammonium citrate), or low iron content (by the addition of 50 mM desferroxamine). Under all three iron conditions PPIX induced differentiation but down-regulated ferritin expression and up-regulated TfR1 expression. Additionally, PPIX had a striking effect on DAP expression markedly decreasing DAP levels but only in cells grown either in normal or low iron medium. In addition, PPIX affected the expression of the iron transporter DMT1in parallel with DAP. As PPIX induced erythroid differentiation of K562 cells we examined the effects of hemin which can also induce differentiation of K562 cells. In contrast to PPIX, hemin caused strong down-regulation of TfR and up-regulation of ferritin and DAP. The down-regulation of DAP induced by PPIX was restored by the addition of hemin. These results indicate that PPIX affects DAP expression and other important elements involved in cellular iron metabolism and that these effects are partially modified by the iron status of the cell.