Development of endothelial-targeted CD39 as a therapy for ischemic stroke.

BACKGROUND: Ischemic stroke is characterized by a necrotic lesion in the brain surrounded by an area of dying cells termed the penumbra. Salvaging the penumbra either with thrombolysis or mechanical retrieval is the cornerstone of stroke management. At-risk neuronal cells release extracellular adenosine triphosphate, triggering microglial activation and causing a thromboinflammatory response, culminating in endothelial activation and vascular disruption. This is further aggravated by ischemia-reperfusion injury that follows all reperfusion therapies. The ecto-enzyme CD39 regulates extracellular adenosine triphosphate by hydrolyzing it to adenosine, which has antithrombotic and anti-inflammatory properties and reverses ischemia-reperfusion injury. OBJECTIVES: The objective off the study was to determine the efficacy of our therapeutic, anti-VCAM-CD39 in ischaemic stroke. METHODS: We developed anti-VCAM-CD39 that targets the antithrombotic and anti-inflammatory properties of recombinant CD39 to the activated endothelium of the penumbra by binding to vascular cell adhesion molecule (VCAM)-1. Mice were subjected to 30 minutes of middle cerebral artery occlusion and analyzed at 24 hours. Anti-VCAM-CD39 or control agents (saline, nontargeted CD39, or anti-VCAM-inactive CD39) were given at 3 hours after middle cerebral artery occlusion. RESULTS: Anti-VCAM-CD39 treatment reduced neurologic deficit; magnetic resonance imaging confirmed significantly smaller infarcts together with an increase in cerebrovascular perfusion. Anti-VCAM-CD39 also restored blood-brain barrier integrity and reduced microglial activation. Coadministration of anti-VCAM-CD39 with thrombolytics (tissue plasminogen activator [tPA]) further reduced infarct volumes and attenuated blood-brain barrier permeability with no associated increase in intracranial hemorrhage. CONCLUSION: Anti-VCAM-CD39, uniquely targeted to endothelial cells, could be a new stroke therapy even when administered 3 hours postischemia and may further synergize with thrombolytic therapy to improve stroke outcomes.

Rotational ThromboElastometry-guided blood component administration versus standard of care in patients with Cirrhosis and coagulopathy undergoing Invasive ProcEdures (RECIPE): study protocol for a randomised controlled trial.

BACKGROUND: Patients with cirrhosis often undergo invasive procedures both for management of complications of their advanced liver disease, including treatment for hepatocellular carcinoma, as well as underlying comorbidities. Despite a current understanding that most patients with cirrhosis are in a rebalanced haemostatic state (despite abnormalities in conventional coagulation tests, namely INR and platelet count), patients with cirrhosis are still often given prophylactic blood components based on these conventional parameters, in an effort to reduce procedure-related bleeding. Viscoelastic tests such as Rotational Thromboelastometry (ROTEM) provide a global measurement of haemostasis and have been shown to predict bleeding risk more accurately than conventional coagulation tests, and better guide blood product transfusion in a number of surgical and trauma-related settings. The aim of this study is to assess the utility of a ROTEM-based algorithm to guide prophylactic blood component delivery in patients with cirrhosis undergoing invasive procedures. We hypothesise that ROTEM-based decision-making will lead to a reduction in pre-procedural blood component usage, particularly fresh frozen plasma (FFP), compared with standard of care, whilst maintaining optimal clinical outcomes. METHODS: This is a multi-centre randomised controlled trial comparing ROTEM-guided prophylactic blood component administration to standard of care in patients with cirrhosis and coagulopathy undergoing invasive procedures. The primary efficacy outcome of the trial is the proportion of procedures requiring prophylactic transfusion, with the primary safety outcome being procedure-related bleeding complications. Secondary outcomes include the amount of blood products (FFP, platelets, cryoprecipitate) transfused, transfusion-related side effects, procedure-related complications other than bleeding, hospital length of stay and survival. DISCUSSION: We anticipate that this project will lead to improved prognostication of patients with cirrhosis, in terms of their peri-procedural bleeding risk. We hope to show that a significant proportion of cirrhotic patients, deemed coagulopathic on the basis of standard coagulation tests such as INR and platelet count, are actually in a haemostatic balance and thus do not require prophylactic blood product, leading to decreased and more efficient blood component use. TRIAL REGISTRATION: RECIPE has been prospectively registered with the Australia and New Zealand Clinical Trials Registry on the 30th April 2019 ( ACTRN12619000644167 ).

The role of pyruvate-induced enhancement of oxygen metabolism in extracellular purinergic signaling in the post-cardiac arrest rat model.

Purine nucleotide adenosine triphosphate (ATP) is a source of intracellular energy maintained by mitochondrial oxidative phosphorylation. However, when released from ischemic cells into the extracellular space, they act as death-signaling molecules (eATP). Despite there being potential benefit in using pyruvate to enhance mitochondria by inducing a highly oxidative metabolic state, its association with eATP levels is still poorly understood. Therefore, while we hypothesized that pyruvate could beneficially increase intracellular ATP with the enhancement of mitochondrial function after cardiac arrest (CA), our main focus was whether a proportion of the raised intracellular ATP would detrimentally leak out into the extracellular space. As indicated by the increased levels in systemic oxygen consumption, intravenous administrations of bolus (500 mg/kg) and continuous infusion (1000 mg/kg/h) of pyruvate successfully increased oxygen metabolism in post 10-min CA rats. Plasma ATP levels increased significantly from 67 ± 11 nM before CA to 227 ± 103 nM 2 h after the resuscitation; however, pyruvate administration did not affect post-CA ATP levels. Notably, pyruvate improved post-CA cardiac contraction and acidemia (low pH). We also found that pyruvate increased systemic CO2 production post-CA. These data support that pyruvate has therapeutic potential for improving CA outcomes by enhancing oxygen and energy metabolism in the brain and heart and attenuating intracellular hydrogen ion disorders, but does not exacerbate the death-signaling of eATP in the blood.

Global coagulation assays in patients with chronic kidney disease and their role in predicting thrombotic risk.

BACKGROUND: Despite cardiovascular diseases and thrombosis being major causes of death in patients with chronic kidney disease (CKD), there remains no effective biomarker to predict thrombotic risk in this population. OBJECTIVE: To evaluate global coagulation assays in patients with CKD and correlate the biomarkers to clinical outcomes. MATERIAL AND METHODS: Patients with eGFR<30 mL/min/1.73m2 were recruited (n = 90) in this prospective observational study. Blood samples were collected for global coagulation assays, including thromboelastography, calibrated automated thrombogram (CAT), overall hemostatic potential (OHP) and tissue factor pathway inhibitor (TFPI). RESULTS: Following adjustment for age and gender, CKD subjects (mean age 66 years, 36 % female) had increased maximum amplitude on thromboelastography (70.1 vs 60.2 mm, p < 0.001), higher peak thrombin (233.2 vs 219.7 mm, p = 0.030) and increased OHP (16.1 vs 6.4 units, p < 0.001) compared to healthy controls (n = 153). TFPI was also increased in CKD patients (36.4 vs 14.5 ng/mL, p < 0.001). Compared to hemodialysis patients (n = 43), peritoneal-dialysis patients (n = 25) had more hypercoagulable parameters. Thirty-five CKD patients reported thrombotic complications - key predictors included dialysis, higher fibrinogen, reduced endogenous thrombin potential, elevated D-dimer and increased TFPI. Using the dialysis cohort, the predictive risk model based on the key predictors performed better than Framingham heart score and number of cardiovascular risk factors (Harrell's C-stat 0.862 vs 0.585 vs 0.565). CONCLUSION: CKD appears to confer a hypercoagulable state compared to healthy controls. Interestingly, reduced thrombin generation and raised TFPI was paradoxically associated with increased thrombotic risks, highlighting possible complex compensatory mechanisms within the coagulation system, which may be important in predicting clinical outcomes.

N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

Obesity and the Risk of Venous Thromboembolism after Major Lower Limb Orthopaedic Surgery: A Literature Review.

The risk of venous thromboembolism following total joint arthroplasty is significantly greater than those of other types of elective orthopaedic procedures. This risk is increased in obesity due to the associated prothrombotic physiological and hematological changes that predispose to embolic events. The prevalence of obesity is increasing in the aging population, which contributes to a further increase in the risk of postoperative thrombosis in the older patients. There is a lack of clear evidence regarding dosing information for thromboprophylaxis medications in patients with obesity. As a result, the currently available thromboprophylaxis guidelines do not provide specific recommendations for this group. Suboptimal dosing regimens for these medications can place these patients at a risk of bleeding or clotting complications postsurgery. Hence any increase in dosage may require intensive surveillance for the residual anticoagulant effects and careful balancing of risks and benefits on an individual basis. Our review discusses the basis for increased thrombotic risk in obesity, the evidence supporting dosage recommendations, and the implications of the current guidelines for pharmacological thromboprophylaxis in patients with obesity undergoing lower limb arthroplasty.

CD39 in the development and progression of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating progressive disease characterised by pulmonary arterial vasoconstriction and vascular remodelling. Endothelial dysfunction has emerged as a contributing factor in the development of PAH. However, despite progress in the understanding of the pathophysiology of this disease, current therapies fail to impact upon long-term outcomes which remain poor in most patients. Recent observations have suggested the disturbances in the balance between ATP and adenosine may be integral to the vascular remodelling seen in PAH. CD39 is an enzyme important in regulating these nucleos(t)ides which may also provide a novel pathway to target for future therapies. This review summarises the role of adenosine signalling in the development and progression of PAH and highlights the therapeutic potential of CD39 for treatment of PAH.

The potential role of protease systems in hemophilic arthropathy.

Hemophilic arthropathy (HA) is characterized by joint damage following recurrent joint bleeds frequently observed in patients affected by the clotting disorder hemophilia. Joint bleeds or hemarthroses trigger inflammation in the synovial tissue, which promotes damage to the articular cartilage. The plasminogen activation system is integral to fibrinolysis, and the urokinase plasminogen activator, or uPA in particular, is strongly upregulated following hemarthroses. uPA is a serine protease that catalyzes the production of plasmin, a broad-spectrum protease that can degrade fibrin as well as proteins of the joint extracellular matrix and cartilage. Both uPA and plasmin are able to proteolytically generate active forms of matrix metalloproteinases (MMPs). The MMPs are a family of >20 proteases that are secreted as inactive proenzymes and are activated extracellularly. MMPs are involved in the degradation of all types of collagen and proteoglycans that constitute the extracellular matrix, which provides structural support to articular cartilage. The MMPs have an established role in joint destruction following rheumatoid arthritis (RA). They degrade cartilage and bone, indirectly promoting angiogenesis. MMPs are also implicated in the pathology of osteoarthritis (OA), characterized by degradation of the cartilage matrix that precipitates joint damage and deformity. HA shares a number of overlapping pathological characteristics with RA and OA. Here we discuss how the plasminogen activation system and MMPs might exacerbate joint damage in HA, lending insight into novel possible therapeutic targets to reduce the comorbidity of hemophilia.

Age, sex and racial differences in fibrin formation and fibrinolysis within the healthy population.

Increased fibrin generation and reduced fibrinolytic potential have been detected using global coagulation assays in several hypercoagulable states including cardiovascular disease and venous thromboembolism. We aimed in this study to define the impact of age, sex and race on fibrin generation and lysis using the Overall Haemostatic Potential (OHP) assay in a group of stringently defined healthy adults. Healthy adult patients not receiving anticoagulation and without a history of thrombotic disease were prospectively recruited. Iindividuals with cardiovascular risk factors (e.g. hypertension, diabetes, smoking), receiving hormonal therapy, antiplatelet agents or with abnormal routine blood tests were also excluded. Platelet-poor plasma was obtained and the OHP assay, which evaluates fibrin formation with and without tissue plasminogen activator, was performed on all plasma samples. 144 healthy subjects (34.7% male) with median age 42 years (interquartile range 20, 77) were recruited. After multivariate analysis, age at least 50 years and female sex were associated with significantly increased fibrin generation parameters (overall coagulation potential, OHP, maximum optical density, fibrin) as well as reduced markers of fibrinolysis (overall fibrinolytic potential and time-to-50% lysis). There were no significant differences in OHP parameters between whites, East Asians and South Asians after accounting for age and sex. This study defines age, sex and racial differences of fibrin generation and fibrinolysis as measured by the OHP assay in a sample of healthy subjects. Further studies are warranted in diseased populations, where there is growing awareness of the role of global coagulation assay in defining prothrombotic and hypofibrinolytic states.

Global coagulation assays in patients with diabetes mellitus.

BACKGROUND: There is significant heterogeneity in the incidence and severity of diabetes-associated vascular complications and there is no routine biomarker that accurately predicts these outcomes. This pilot study investigates the role of global coagulation assays in patients with diabetes mellitus. METHODS: In this cross-sectional study, patients with diabetes not on anticoagulation or dialysis and without active malignancy were recruited from endocrinology clinics. Blood samples were collected for global coagulation assays including thromboelastography (TEG), thrombin generation using calibrated automated thrombogram (CAT), and fibrin generation and fibrinolysis using the overall hemostatic potential (OHP) assay. The results were compared with healthy controls. RESULTS: A total of 147 adult patients including 19 with type 1 diabetes (T1DM), 120 with type 2 diabetes (T2DM), and eight with latent autoimmune diabetes were recruited. Compared with 153 healthy controls, patients with diabetes demonstrated higher maximum amplitude (68.6 vs 60.2 mm, p < 0.001) on TEG, and higher OHP (9.3 vs 6.4, p < 0.001) with comparable CAT parameters. Patients with T2DM were more hypercoagulable than those with T1DM on most biomarkers. Higher maximum amplitude, velocity index, and OHP were associated with increased risk of complications (C-stat 0.82). Patients with history of microvascular complications appear to have more hypercoagulable thrombin and fibrin generation than those without. CONCLUSION: Patients with diabetes have more hypercoagulable profiles on global coagulation assays, particularly patients with T2DM and those with microvascular complications. Further studies with longitudinal follow-up are ongoing to evaluate the utility of global coagulation assays in predicting long-term patient outcomes.

Role of Purinergic Signalling in Endothelial Dysfunction and Thrombo-Inflammation in Ischaemic Stroke and Cerebral Small Vessel Disease.

The cerebral endothelium is an active interface between blood and the central nervous system. In addition to being a physical barrier between the blood and the brain, the endothelium also actively regulates metabolic homeostasis, vascular tone and permeability, coagulation, and movement of immune cells. Being part of the blood-brain barrier, endothelial cells of the brain have specialized morphology, physiology, and phenotypes due to their unique microenvironment. Known cardiovascular risk factors facilitate cerebral endothelial dysfunction, leading to impaired vasodilation, an aggravated inflammatory response, as well as increased oxidative stress and vascular proliferation. This culminates in the thrombo-inflammatory response, an underlying cause of ischemic stroke and cerebral small vessel disease (CSVD). These events are further exacerbated when blood flow is returned to the brain after a period of ischemia, a phenomenon termed ischemia-reperfusion injury. Purinergic signaling is an endogenous molecular pathway in which the enzymes CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia and CSVD, eATP is released from dying neurons as a damage molecule, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response. Evidently, therapies that promote adenosine generation or boost CD39 activity at the site of endothelial injury have promising benefits in the context of atherothrombotic stroke and can be extended to current CSVD known pathomechanisms. Here, we have reviewed the rationale and benefits of CD39 and CD39 therapies to treat endothelial dysfunction in the brain.

Early Endothelial Activation in a Mouse Model of Graft vs Host Disease Following Chemotherapy.

Allogenic hematopoietic stem cell transplant (allo-HSCT) can lead to sinusoidal obstruction syndrome (SOS) and graft-versus-host disease (GvHD) in some individuals. GvHD is characterised by an immune triggered response that arises due to donor T cells recognizing the recipient tissue as "foreign". SOS results in impaired liver function due to microvascular thrombosis and consequent obstruction of liver sinusoids. Endothelial damage occurs following chemotherapy and allo-HSCT and is strongly associated with GvHD onset as well as hepatic SOS. Animal models of GvHD are rarely clinically relevant, and endothelial dysfunction remains uncharacterised. Here we established and characterised a clinically relevant model of GvHD wherein Balb/C mice were subjected to myeloablative chemotherapy followed by transplantation of bone marrow (BM) cells± splenic T-cells from C57Bl6 mice, resulting in a mismatch of major histocompatibility complexes (MHC). Onset of disease indicated by weight loss and apoptosis in the liver and intestine was discovered at day 6 post-transplant in mice receiving BM+T-cells, with established GvHD detectable by histology of the liver within 3 weeks. Together with significant increases in pro-inflammatory cytokine gene expression in the liver and intestine, histopathological signs of GvHD and a significant increase in CD4+ and CD8+ effector and memory T-cells were seen. Endothelial activation including upregulation of vascular cell adhesion molecule (VCAM)- 1 and downregulation of endothelial nitric oxide synthase (eNOS) as well as thrombosis in the liver indicated concomitant hepatic SOS. Our findings confirm that endothelial activation is an early sign of acute GvHD and SOS in a clinically relevant mouse model of GvHD based on myeloablative chemotherapy. Preventing endothelial activation may be a viable therapeutic strategy to prevent GvHD.

CSK-homologous kinase (CHK/MATK) is a potential colorectal cancer tumour suppressor gene epigenetically silenced by promoter methylation.

Hyperactivation of SRC-family protein kinases (SFKs) contributes to the initiation and progression of human colorectal cancer (CRC). Since oncogenic mutations of SFK genes are rare in human CRC, we investigated if SFK hyperactivation is linked to dysregulation of their upstream inhibitors, C-terminal SRC kinase (CSK) and its homolog CSK-homologous kinase (CHK/MATK). We demonstrate that expression of CHK/MATK but not CSK was significantly downregulated in CRC cell lines and primary tumours compared to normal colonic tissue. Investigation of the mechanism by which CHK/MATK expression is down-regulated in CRC cells uncovered hypermethylation of the CHK/MATK promoter in CRC cell lines and primary tumours. Promoter methylation of CHK/MATK was also observed in several other tumour types. Consistent with epigenetic silencing of CHK/MATK, genetic deletion or pharmacological inhibition of DNA methyltransferases increased CHK/MATK mRNA expression in CHK/MATK-methylated colon cancer cell lines. SFKs were hyperactivated in CHK/MATK-methylated CRC cells despite expressing enzymatically active CSK, suggesting loss of CHK/MATK contributes to SFK hyperactivation. Re-expression of CHK/MATK in CRC cell lines led to reduction in SFK activity via a non-catalytic mechanism, a reduction in anchorage-independent growth, cell proliferation and migration in vitro, and a reduction in tumour growth and metastasis in a zebrafish embryo xenotransplantation model in vivo, collectively identifying CHK/MATK as a novel putative tumour suppressor gene in CRC. Furthermore, our discovery that CHK/MATK hypermethylation occurs in the majority of tumours warrants its further investigation as a diagnostic marker of CRC.

Global Coagulation Assays in Transgender Women on Oral and Transdermal Estradiol Therapy.

CONTEXT: The thrombotic effects of estradiol therapy in transgender women are unclear. Global coagulation assays (GCA) may be better measures of hemostatic function compared with standard coagulation tests. OBJECTIVE: To assess the GCA profiles of transgender women in comparison to cisgender controls and to compare how GCA differ between routes of estradiol therapy in transgender women. DESIGN: Cross-sectional case-control study. SETTING: General community. PARTICIPANTS: Transgender women, cisgender male and cisgender female controls. MAIN OUTCOME MEASURES: Citrated blood samples were analyzed for (i) whole blood thromboelastography (TEG®5000), (ii) platelet-poor plasma thrombin generation (calibrated automated thrombogram); and (iii) platelet-poor plasma fibrin generation (overall hemostatic potential assay). Mean difference (95% confidence intervals) between groups are presented. RESULTS: Twenty-six transgender women (16 oral estradiol, 10 transdermal estradiol) were compared with 98 cisgender women and 55 cisgender men. There were no differences in serum estradiol concentration (P = 0.929) and duration of therapy (P = 0.496) between formulations. Transgender women demonstrated hypercoagulable parameters on both thromboelastography (maximum amplitude + 6.94 mm (3.55, 10.33); P < 0.001) and thrombin generation (endogenous thrombin potential + 192.62 nM.min (38.33, 326.91); P = 0.009; peak thrombin + 38.10 nM (2.27, 73.94); P = 0.034) but had increased overall fibrinolytic potential (+4.89% (0.52, 9.25); P = 0.024) compared with cisgender men. No significant changes were observed relative to cisgender women. Route of estradiol delivery or duration of use did not influence the GCA parameters. CONCLUSION: Transgender women on estradiol therapy demonstrated hypercoagulable GCA parameters compared with cisgender men with a shift towards cisgender female parameters. Route of estradiol delivery did not influence the GCA parameters.

Hepatitis B management during immunosuppression for haematological and solid organ malignancies: an Australian consensus statement.

INTRODUCTION: Individuals with chronic hepatitis B virus (HBV) infection or past exposure to HBV infection have a substantial risk of reactivation during immunosuppressive cancer therapy. HBV reactivation can lead to liver failure, cancer treatment interruption or death. Clinical concordance with screening and treatment guidelines is inconsistent in practice, and existing international guidelines are not specific to the Australian context. We developed an Australian consensus statement with infectious diseases, hepatology, haematology and oncology specialists to inform hepatitis B screening and antiviral management for immunocompromised patients with haematological and solid organ malignancies in Australia. MAIN RECOMMENDATIONS: Recommendations address four key areas of HBV infection management for immunocompromised patients with haematological and solid organ malignancies: who to test for HBV infection, when to start antiviral agents, when to stop antiviral agents, and how to monitor patients during cancer therapy. We recommend testing all patients undergoing cancer treatment for hepatitis B (including HBV surface antigen [HBsAg], HBV core antibody [anti-HBc], and HBV surface antibody) before cancer treatment. Individuals with chronic HBV infection (HBsAg positive) or past exposure (HBsAg negative and anti-HBc positive) receiving higher risk chemotherapy require antiviral prophylaxis using entecavir or tenofovir. CHANGES IN MANAGEMENT AS A RESULT OF THIS STATEMENT: This consensus statement will simplify the approach to testing and prophylaxis for HBV infection during cancer therapy, and harmonise approaches to discontinuing and monitoring individuals which have been highly variable in practice. We advocate for broader Medicare Benefits Schedule and Pharmaceutical Benefits Scheme access to HBV testing and treatment for patients undergoing cancer therapy.

Hemopoietic Cell Kinase amplification with Protein Tyrosine Phosphatase Receptor T depletion leads to polycythemia, aberrant marrow erythoid maturation, and splenomegaly.

Deletion of long arm of chromosome 20 [del(20q)] is the second most frequent recurrent chromosomal abnormality in hematological malignancies. It is detected in 10% of myeloproliferative neoplasms, 4-5% of myelodysplastic syndromes, and 1-2% of acute myeloid leukaemia. Recurrent, non-random occurrence of del(20q) indicates that it is a pathogenic driver in myeloid malignancies. Genetic mapping of patient samples has identified two regions of interest on 20q - the "Common Deleted Region" (CDR) and "Common Retained Region" (CRR), which was often amplified. We proposed that the CDR contained tumor suppressor gene(s) (TSG) and the CRR harbored oncogene(s); loss of a TSG together with over-expression of an oncogene favored development of myeloid malignancies. Protein Tyrosine Phosphatase Receptor T (PTPRT) and Hemopoietic cell kinase (HCK) were identified to be the likely candidate TSG and oncogene respectively. Retroviral transduction of HCK into PTPRT-null murine LKS+ stem and progenitor cells resulted in hyperproliferation in colony forming assays and hyperphosphorylation of intracellular STAT3. Furthermore, over half of the murine recipients of these transduced cells developed erythroid hyperplasia, polycythemia and splenomegaly at 12 months, although no leukemic phenotype was observed. The findings suggested that HCK amplification coupled with PTPRT loss in del(20q) leads to development of a myeloproliferative phenotype.

Targeting CD39 Toward Activated Platelets Reduces Systemic Inflammation and Improves Survival in Sepsis: A Preclinical Pilot Study.

OBJECTIVES: Sepsis is associated with a systemic inflammatory reaction, which can result in a life-endangering organ dysfunction. Pro-inflammatory responses during sepsis are characterized by increased activation of leukocytes and platelets, formation of platelet-neutrophil aggregates, and cytokine production. Sequestration of platelet-neutrophil aggregates in the microvasculature contributes to tissue damage during sepsis. At present no effective therapeutic strategy to ameliorate these events is available. In this preclinical pilot study, a novel anti-inflammatory approach was evaluated, which targets nucleoside triphosphate hydrolase activity toward activated platelets via a recombinant fusion protein combining a single-chain antibody against activated glycoprotein IIb/IIIa and the extracellular domain of CD39 (targ-CD39). DESIGN: Experimental animal study and cell culture study. SETTING: University-based experimental laboratory. SUBJECTS: Human dermal microvascular endothelial cells 1, human platelets and neutrophils, and C57BL/6NCrl mice. INTERVENTIONS: Platelet-leukocyte-endothelium interactions were evaluated under inflammatory conditions in vitro and in a murine lipopolysaccharide-induced sepsis model in vivo. The outcome of polymicrobial sepsis was evaluated in a murine cecal ligation and puncture model. To evaluate the anti-inflammatory potential of activated platelet targeted nucleoside triphosphate hydrolase activity, we employed a potato apyrase in vitro and in vivo, as well as targ-CD39 and as a control, nontarg-CD39 in vivo. MEASUREMENTS AND MAIN RESULTS: Under conditions of sepsis, agents with nucleoside triphosphate hydrolase activity decreased platelet-leukocyte-endothelium interaction, transcription of pro-inflammatory cytokines, microvascular platelet-neutrophil aggregate sequestration, activation marker expression on platelets and neutrophils contained in these aggregates, leukocyte extravasation, and organ damage. Targ-CD39 had the strongest effect on these variables and retained hemostasis in contrast to nontarg-CD39 and potato apyrase. Most importantly, targ-CD39 improved survival in the cecal ligation and puncture model to a stronger extent then nontarg-CD39 and potato apyrase. CONCLUSIONS: Targeting nucleoside triphosphate hydrolase activity (CD39) toward activated platelets is a promising new treatment concept to decrease systemic inflammation and mortality of sepsis. This innovative therapeutic approach warrants further development toward clinical application.

New guidelines from the Thrombosis and Haemostasis Society of Australia and New Zealand for the diagnosis and management of venous thromboembolism.

INTRODUCTION: Venous thromboembolism (VTE), including deep vein thrombosis (DVT) and pulmonary embolism (PE), is the third most common cardiovascular disease and, globally, more than an estimated 10 million people have it yearly. It is a chronic and recurrent disease. The symptoms of VTE are non-specific and the diagnosis should actively be sought once considered. The mainstay of VTE treatment is anticoagulation, with few patients requiring additional intervention. A working group of experts in the area recently completed an evidence-based guideline for the diagnosis and management of DVT and PE on behalf of the Thrombosis and Haemostasis Society of Australia and New Zealand (www.thanz.org.au/resources/thanz-guidelines). MAIN RECOMMENDATIONS: The diagnosis of VTE should be established with imaging; it may be excluded by the use of clinical prediction rules combined with D-dimer testing. Proximal DVT or PE caused by a major surgery or trauma that is no longer present should be treated with anticoagulant therapy for 3 months. Proximal DVT or PE that is unprovoked or associated with a transient risk factor (non-surgical) should be treated with anticoagulant therapy for 3-6 months. Proximal DVT or PE that is recurrent (two or more) and provoked by active cancer or antiphospholipid syndrome should receive extended anticoagulation. Distal DVT caused by a major provoking factor that is no longer present should be treated with anticoagulant therapy for 6 weeks. For patients continuing with extended anticoagulant therapy, either therapeutic or low dose direct oral anticoagulants can be prescribed and is preferred over warfarin in the absence of contraindications. Routine thrombophilia testing is not indicated. Thrombolysis or a suitable alternative is indicated for massive (haemodynamically unstable) PE. CHANGES IN MANAGEMENT AS A RESULT OF THE GUIDELINE: Most patients with acute VTE should be treated with a factor Xa inhibitor and be assessed for extended anticoagulation.

A comparison of global coagulation assays between normal controls and patients with thrombocytopenia.

INTRODUCTION: Some patients with thrombocytopenia may be at risk of bleeding although quantitative platelet count is not always a sufficient predictive factor. Global coagulation assays such as thromboelastography (TEG® ), calibrated automated thrombogram (CAT) and overall haemostatic potential (OHP) may provide a better assessment of an individual's haemostatic profile. METHODS: Blood samples were collected from thrombocytopenic patients. TEG® was performed on citrated whole blood, while CAT and OHP were performed on platelet-poor plasma. Results were compared to our previously collected normal controls. RESULTS: Fifty-eight participants (24 immune thrombocytopenia, 34 chemotherapy/malignancy-related) with mean age of 57.5 years were recruited. Compared to normal controls, thrombocytopenic participants had comparable maximum amplitude but reduced clot lysis (0.0% vs 0.6%; P < 0.001) on TEG® with reduced endogenous thrombin potential on CAT (1252.2 vs 1353.0 nmol/L/min; P = 0.040). No differences were seen in the OHP parameters. TEG® showed significant difference between marked and mild thrombocytopenia groups with minimal differences seen on CAT and OHP. Those with marked thrombocytopenia showed reduced maximum amplitude (47.2 vs 57.8 mm; P = 0.002) as expected while participants with mild thrombocytopenia (platelet count 100-150 × 109 /L) paradoxically demonstrated increased maximum amplitude (66.4 vs 57.8 mm; P < 0.001). CONCLUSION: Global coagulation assays, particularly TEG® , can detect subtle differences in coagulation in thrombocytopenic patients. While patients with marked thrombocytopenia showed reduced maximum amplitude, patients with mild thrombocytopenia appear to paradoxically show increased maximum amplitude, suggesting compensatory activity within the coagulation pathway which may in part explain why not all thrombocytopenic patients have bleeding complications.