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The homeobox gene,Hoxa1, has two different isoforms generated by alternative splicing: a full-length homeodomain-containingHoxa1(Hoxa1-FL), and a truncatedHoxa1(Hoxa1-T), that lacks the homeodomain. Oncoretroviral overexpression of wildtypeHoxa1cDNA (WT-Hoxa1), which generates bothHoxa1isoforms, in murine hematopoietic stem and progenitor cells (HSPCs) perturbed hematopoiesis, resulting in myelodysplastic syndromes (MDS) in mice. Overexpression of a mutatedHoxa1cDNA (MUT-Hoxa1) that generatesHoxa1-FLbut notHoxa1-Tled to a more severe MDS capable of transforming to secondary acute myeloid leukemia (sAML). Similar to human MDS, DNA damage repair pathways were downregulated inHoxa1-overexpressing hematopoietic progenitor cells. Conditional knock-in mouse models revealed aHoxa1-FLdosage-dependent effect on MDS disease severity. Our data reveal that increased expression ofHoxa1-FLin HSPCs is sufficient to initiate MDS in mice. CD34+ cells from up to 50% of patients with MDS had elevatedHOXA1-FLexpression, highlighting the clinical relevance of our mouse models.Statement of SignificanceOur study demonstrates thatHoxa1is a key regulator of HSPCs and that increased expression of the transcriptionally activeHoxa1-FLcan initiate MDS in mice. Furthermore,HOXA1-FLexpression is upregulated in a significant proportion of human MDS patients and likely contributes to the disease in these patients.

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. Indicated by the increased levels in systemic oxygen consumption and brain ATP levels, intravenous administrations of bolus (500 mg/kg) and continuous infusion (1000 mg/kg/hr) of pyruvate successfully increased oxygen and energy metabolism in post 10-min CA rats. The plasma ATP levels increased significantly from 67 ± 11 nM before CA to 227 ± 100 nM 2 hours after the resuscitation, while the pyruvate injection did not affect post-CA ATP levels. Notably, the pyruvate injection 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 iron disorders, but does not exacerbate the death-signaling of eATP in the blood.

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 IIβ (CaMKIIβ). 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 CaMKIIβ, 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.

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.

Assessing the risk of recurrent venous thromboembolism (VTE), particularly when patients are anticoagulated, remains a major challenge largely due to the lack of biomarkers. Blood was sampled from adult VTE patients recruited between January 2018 and September 2020, while receiving therapeutic anticoagulation. Results were compared to 144 healthy subjects (34.7% male, median age 42 years). Overall haemostatic potential (OHP) assay, a spectrophotometric assay, was performed on platelet-poor plasma, in which fibrin formation (triggered by small amounts of thrombin (overall coagulation potential, OCP)) and fibrinolysis (by the addition of thrombin and tissue plasminogen activator (OHP)) are simultaneously measured. Results were obtained from 196 patients (52.6% male, mean age 57.1 years). Compared to healthy subjects, VTE patients displayed significantly higher OCP (39.6 vs 34.5 units, p th percentile (recurrence rate 4.32/100 patient-years (100PY), 95% confidence interval (CI) 2.39–7.80, p = 0.002). Of 97 patients who subsequently discontinued anticoagulation, all unprovoked VTE recurrences (n = 9) occurred above the 40th OCP percentile (recurrence rate 9.10/100PY, 95% CI 4.74–17.49, p = 0.005) and the 40th OHP percentile (recurrence rate 8.46/100PY, 95% CI 4.40–16.25, p = 0.009). Our pilot study demonstrates that the OHP assay can detect a hypercoagulable and hypofibrinolytic state in anticoagulated VTE patients and may be able to risk stratify VTE recurrence, allowing for more individualised decision on long-term anticoagulation. Further larger prospective studies are required.

Excitotoxicity is a neuronal death process initiated by over-stimulation 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 IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbation of 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 CaMKIIβ undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, our findings inform excitotoxic neuronal death mechanism and suggest potential therapeutic strategies for neuroprotection.In BriefAmeen, et al. used a proteomic method called N-terminomics to identify proteolytic events occurring in neurons during excitotoxicity. They found that most proteolytic processing is mediated by calpains, resulting in the generation of stable truncated fragments with the potential to induce synaptic dysfunction and loss, eventually leading to neuronal death. They further showed that some of these proteolytic processed proteins, such as the protein kinases Src and CaMKII, are potential targets for neuroprotection.HighlightsIdentification of over 300 neuronal proteins cleaved by calpains to form stable truncated fragments during excitotoxicity.The calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons.These pathological proteolytic events potentially induce synaptic dysfunction and loss, which likely contribute to excitotoxic neuronal death.Some of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection.Graphical abstract: Pathological proteolytic events in neurons during excitotoxicity unveiled by N-terminomic analyses(A) N-terminomic and global proteomic analyses identified neo-N-terminal sites and neuronal proteins undergoing significant abundance changes during excitotoxicity. (B) Informatic analysis of the proteomic results predicted (i) the preferred sequences of proteolytic processing of neuronal proteins catalyzed by calpains during excitotoxicity and (ii) perturbation of synaptic organization and functions as the major consequence of calpain-mediated proteolytic events. (C) Validation of these predictions and further experimentations unveiled: (i) calpain-mediated cleavage of proteins associated with synaptic damage in excitotoxic neurons, (ii) a new mechanism of dysregulation of CaMKIIα and CaMKIIβ, which are key protein kinases governing synaptic dysfunctions and excitotoxic neuronal death and (iii) potential therapeutic targets such as the protein kinases Src and CaMKII for neuroprotectionOne Sentence SummaryProteolytic events in neurons during excitotoxicity inform neuronal death mechanism and potential therapeutic strategies for neuroprotection.

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.

Thrombosis is one of the major global causes of morbidity and mortality, and predicting the risk of thrombotic and cardiovascular complications remains one of the key challenges in modern medicine. Conventional coagulation testing does not provide sufficient information, primarily because they measure the time to start of blood clotting and do not evaluate total thrombin generation. Possible adjunctive tools that may be helpful are global coagulation assays, which includes the assessment of the final products of the coagulation cascade, namely thrombin and fibrin. Whilst these assays have been more widely investigated in bleeding states, their role in thrombotic disorders is less established. We have previously investigated the use of assays such as thromboelastography, calibrated automated thrombogram and overall haemostatic potential assay in several hypercoagulable states including cardiovascular disease, haematological disorders and influence of hormone status as well as healthy controls. We provide a review of the use and limitations of global coagulation assays in healthy controls as well as hypercoagulable conditions.

Global coagulation assays (GCAs) may provide a more comprehensive individual hemostatic profiling. We aim to evaluate GCAs (thromboelastography, thrombin generation) in healthy controls, and correlate results with age, gender, lipid status, tissue factor pathway inhibitor (TFPI) and P-selectin. Blood samples were collected from healthy controls ( 18 years of age) not taking anticoagulation or antiplatelet agents and without known cardiovascular disease. Thromboelastography (TEG) was performed on citrated whole blood while calibrated automated thrombogram (CAT), P-selectin (endothelial marker) and TFPI (principle inhibitor of tissue factor-initiated coagulation) were performed on platelet-poor plasma. 153 healthy controls (mean age 42 years, 98 females (64%)) were recruited. Female controls demonstrated more hypercoagulable TEG and CAT parameters while those over 50 years of age demonstrated more hypercoagulable TEG parameters despite comparable thrombin generation. Paradoxically, individuals with "flattened" thrombin curves (lower velocity index (rate of thrombin generation) despite preserved endogenous thrombin potential (amount of thrombin)) were more likely to be male (49% vs 20%, p = 0.003) with increased low-density lipoprotein cholesterol (3.3 vs 2.6 mmol/L, p = 0.003), P-selectin (54.2 vs 47.3 ng/mL, p = 0.038) and TFPI (18.7 vs 8.6 ng/ml, p = 0.001). In addition to reduced velocity index and thrombin peak, controls in the highest TFPI tertile also demonstrated a poorer lipid profile. GCAs can detect subtle changes of the hemostatic profile. Interestingly, reduced thrombin generation was paradoxically associated with increased cardiovascular risk factors, possibly attributable to increased TFPI. This finding may suggest compensation by the coagulation system in response to endothelial activation and represent a biomarker for early cardiovascular disease. A larger prospective study evaluating these assays in the cardiovascular disease population is ongoing.

Venous thromboembolism (VTE) is a major cause of morbidity and mortality with significant heterogeneity in its management, both within our local practice and in international guidelines.To provide a holistic evaluation of 'real-world' Australian experience in the warfarin era, including how we compare to international guidelines.Retrospective evaluation of VTE from July 2011 to December 2012 at two major hospitals in Melbourne, Australia. These results were compared to recommendations in the international guidelines.A total of 752 episodes involving 742 patients was identified. Contrary to international guidelines, an unwarranted heritable thrombophilia screen was performed in 22.0% of patients, amounting to a cost of AU$29 000. The duration of anticoagulation was longer compared to international recommendations, although the overall recurrence (3.2/100 person-years) and clinically significant bleeding rates (2.4/100 person-years) were comparable to 'real-world' data. Unprovoked VTE (hazard ratio 2.06; P = 0.01) was a risk factor for recurrence, and there was no difference in recurrence between major VTE (proximal deep vein thrombosis (DVT) and/or pulmonary embolism) and isolated distal DVT (3.02 vs 3.94/100 person-years; P = 0.25). Fourteen patients were subsequently diagnosed with malignancy, and patients with recurrent VTE had increased risk of prospective cancer diagnosis (relative risk 6.68; P 0.001).While our 'real-world' VTE experience during the warfarin era largely correlates with international guidelines, there remains heterogeneity in the management strategies, including excessive thrombophilia screening and longer duration of anticoagulation. This audit highlights the need for national VTE guidelines, as well as prospective auditing of VTE management, in the direct oral anticoagulant era for future comparison.

Patients undergoing surgery for colorectal cancer are at high risk of post-operative venous thromboembolism (VTE). Thromboprophylaxis has been shown to have significant risk reduction, although there remains some controversy surrounding the optimal duration of pharmacological prophylaxis. Our institution does not routinely practise extended prophylaxis. The aim of this study was to retrospectively review the rate of post-operative thromboprophylaxis in colorectal cancer patients, and incidence of symptomatic VTE.We conducted a retrospective audit of 200 consecutive patients who underwent colorectal surgery for cancer. Data to 90 days post-operatively were collected from medical records and imaging and phone calls to patients and family practitioners.Of the patients, 98% received pharmacological prophylaxis, with a median duration of eight days. Eight (4%) symptomatic VTEs were diagnosed within the 90-day follow-up period: two deep vein thrombosis (DVTs), five pulmonary emboli (PE) and one patient with both PE and DVT. A higher proportion of patients developed DVT/PE if they received prophylaxis other than low molecular weight heparin and similarly there was a trend in increased risk of DVT in the presence of metastatic disease. However, using univariate analysis, these results were not statistically significant (P = 0.18 and 0.11, respectively).The use of thromboprophylaxis was high in our centre, and the incidence of VTE was low when patients received a median of 8 days pharmacological prophylaxis combined with mechanical prophylaxis. The VTE incidence of 4% is similar to previous studies using extended prophylaxis. Our study findings do not support changing local protocol to extended prophylaxis.