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.

Overall haemostatic potential assay for prediction of outcomes in venous and arterial thrombosis and thrombo-inflammatory diseases.

Thromboembolic diseases including arterial and venous thrombosis are common causes of morbidity and mortality globally. Thrombosis frequently recurs and can also complicate many inflammatory conditions through the process of 'thrombo-inflammation,' as evidenced during the COVID-19 pandemic. Current candidate biomarkers for thrombosis prediction, such as D-dimer, have poor predictive efficacy. This limits our capacity to tailor anticoagulation duration individually and may expose lower risk individuals to undue bleeding risk. Global coagulation assays, such as the Overall Haemostatic Potential (OHP) assay, that investigate fibrin generation and fibrinolysis, may provide a more accurate and functional assessment of hypercoagulability. We present a review of fibrin's critical role as a central modulator of thrombotic risk. The results of our studies demonstrating the OHP assay as a predictive biomarker in venous thromboembolism, chronic renal disease, diabetes mellitus, post-thrombotic syndrome, and COVID-19 are discussed. As a comprehensive and global measurement of fibrin generation and fibrinolytic capacity, the OHP assay may be a valuable addition to future multi-modal predictive tools in thrombosis.

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.

Overall haemostatic potential (OHP) assay can risk stratify for venous thromboembolism recurrence in anticoagulated patients.

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 < 0.001) and OHP (9.3 vs 6.4 units, p < 0.001) as well as lower overall fibrinolytic potential (75.6 v s81.1%, p < 0.001). All 16 VTE recurrences, including 11 unprovoked, occurred above an OCP cut-off of 40th 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.

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.

Global coagulation assays in hypercoagulable 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.

Endothelial Dysfunction in the Brain: Setting the Stage for Stroke and Other Cerebrovascular Complications of COVID-19.

The Coronavirus disease 2019 (COVID)-19 pandemic has already affected millions worldwide, with a current mortality rate of 2.2%. While it is well-established that severe acute respiratory syndrome-coronavirus-2 causes upper and lower respiratory tract infections, a number of neurological sequelae have now been reported in a large proportion of cases. Additionally, the disease causes arterial and venous thromboses including pulmonary embolism, myocardial infarction, and a significant number of cerebrovascular complications. The increasing incidence of large vessel ischemic strokes as well as intracranial hemorrhages, frequently in younger individuals, and associated with increased morbidity and mortality, has raised questions as to why the brain is a major target of the disease. COVID-19 is characterized by hypercoagulability with alterations in hemostatic markers including high D-dimer levels, which are a prognosticator of poor outcome. Together with findings of fibrin-rich microthrombi, widespread extracellular fibrin deposition in affected various organs and hypercytokinemia, this suggests that COVID-19 is more than a pulmonary viral infection. Evidently, COVID-19 is a thrombo-inflammatory disease. Endothelial cells that constitute the lining of blood vessels are the primary targets of a thrombo-inflammatory response, and severe acute respiratory syndrome coronavirus 2 also directly infects endothelial cells through the ACE2 (angiotensin-converting enzyme 2) receptor. Being highly heterogeneous in their structure and function, differences in the endothelial cells may govern the susceptibility of organs to COVID-19. Here, we have explored how the unique characteristics of the cerebral endothelium may be the underlying reason for the increased rates of cerebrovascular pathology associated with COVID-19.

Global coagulation assays in healthy controls: are there compensatory mechanisms within the coagulation system?

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.

Active Micropump-Mixer for Rapid Antiplatelet Drug Screening in Whole Blood.

There is a need for scalable automated lab-on-chip systems incorporating precise hemodynamic control that can be applied to high-content screening of new more efficacious antiplatelet therapies. This paper reports on the development and characterization of a novel active micropump-mixer microfluidic to address this need. Using a novel reciprocating elastomeric micropump design, we take advantage of the flexible structural and actuation properties of this framework to manage the hemodynamics for on-chip platelet thrombosis assay on type 1 fibrillar collagen, using whole blood. By characterizing and harnessing the complex three-dimensional hemodynamics of the micropump operation in conjunction with a microvalve controlled reagent injection system we demonstrate that this prototype can act as a real-time assay of antiplatelet drug pharmacokinetics. In a proof-of-concept preclinical application, we utilize this system to investigate the way in which rapid dosing of human whole blood with isoform selective inhibitors of phosphatidylinositol 3-kinase dose dependently modulate platelet thrombus dynamics. This modular system exhibits utility as an automated multiplexable assay system with applications to high-content chemical library screening of new antiplatelet therapies.

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.

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.

A single-chain antibody-CD39 fusion protein targeting activated platelets protects from cardiac ischaemia/reperfusion injury.

Aims: CD39 is a cell membrane NTPase with anti-inflammatory and anti-platelet effects. However, its clinical use is limited by its bleeding side effect. With the goal of harnessing its therapeutic potential while avoiding haemostatic problems, we designed a fusion protein consisting of the extracellular domain of CD39 and a single-chain antibody (Targ-CD39) that specifically binds to activated glycoprotein (GP)IIb/IIIa and thus to activated platelets. Through this enrichment at activated platelets, the required systemic dose is below the dose impairing haemostasis. Methods and results: Using an ischaemia/reperfusion mouse model (left anterior descending artery ligated for 1 h) we achieved remarkable protection of the reperfused tissue with Targ-CD39 compared with Non-targ-CD39 (mutated, non-binding version of Targ-CD39) and PBS control. Targ-CD39 restored ejection fraction and fractional shortening to a level indistinguishable from pre-injury status, while controls showed functional deterioration. Employing advanced clinically relevant methods of ultrasound analysis, we observed that both radial and longitudinal strain and strain rate showed infarct-typical changes of myocardial deformation in controls, but not in Targ-CD39 treated mice. Histological assessment confirmed strong reduction of infarct size and increase in neovascularization. Furthermore, attenuation of post-ischaemic inflammation was seen in cytokine profiling. Conclusion: Overall, we demonstrate that Targ-CD39 holds promise for treatment of myocardial infarction.

Direct Oral Anticoagulants and the Paradigm Shift in the Management of Venous Thromboembolism.

The advent of direct oral anticoagulants (DOACs) has revolutionized anticoagulation management in both stroke prevention and venous thromboembolism (VTE) treatment/prevention. Clinical trials and secondary real-world data have shown that DOACs have similar efficacy and, in some cases, improved bleeding safety profiles compared with vitamin K antagonists. Together with benefits of patient convenience, this has shifted the risk-benefit ratio toward long-term anticoagulation. However, current VTE risk assessment models are based on vitamin K antagonists and do not take into account the new paradigm of DOACs. Therefore, challenges to the thrombosis community remain to determine patients who would benefit from long-term anticoagulation in the DOAC era. Here, the authors review the current literature on risks and benefits of DOACs and their potential role in long-term VTE thromboprophylaxis as well as in current risk assessment models. The increasing use of DOACs, led by their convenience of use and generally lower bleeding rates, calls for a reevaluation of the current models as the benefits of long-term anticoagulation may begin to outweigh risks and inconvenience associated with their predecessors.

CD39 and CD73 activity are protective in a mouse model of antiphospholipid antibody-induced miscarriages.

OBJECTIVE: Antiphospholipid syndrome (APS) is a systemic autoimmune disorder of young adults associated with devastating pregnancy complications (recurrent miscarriages, preeclampsia and low birth weight) and vascular complications including thrombosis. The key components implicated in pathogenesis of APS are the complement cascade and tissue factor (TF) activity causing inflammation and coagulation. Purinergic signalling involving catabolism of ATP to adenosine by cell-surface enzymes CD39 and CD73 has anti-inflammatory and anti-thrombotic effects. We studied whether activities of CD39 and CD73 are important in preventing the development of miscarriages in APS. METHODS: We studied frequency of miscarriages and decidual pathology following passive transfer of human aPL-ab to pregnant wildtype mice, and mice deficient in CD39 and CD73, and also transgenic mice exhibiting 2-3X higher CD39 activity. RESULTS: aPL-ab infusion in pregnant CD39-or CD73-knockout mice triggers an increase in miscarriages, associated with increased TF expression and complement deposition as well as elevated oxidative stress and pro-inflammatory TNF-α and IL-10 expression within the placental decidua. In contrast, aPL-ab induced miscarriages are prevented in mice over-expressing CD39, with reduced decidual TF expression and C3d deposition, diminished lipid peroxidation (4-hydroxynonenal or 4-HNE positive lipid adducts), and reduced TNF-α expression. CONCLUSION: We demonstrate a protective role for CD39 in APS and provide rationale for both the development of endothelial cell-targeted soluble CD39 as a novel therapeutic for APS and analysis of perturbations in the purinergic pathway to explain human disease.

Retrospective evaluation of venous thromboembolism: Are all transient provoking events the same?

OBJECTIVES: Venous thromboembolism (VTE) provoked by transient risk factors has traditionally been classified as a single entity with lower risk of recurrence. We evaluated the association between different categories of transient provoking factors and the relative risk of recurrence. METHODS: Retrospective evaluation of VTE events in non-cancer patients from July 2011 to December 2012 at two tertiary institutions in Australia with a minimum follow-up of 24 months. RESULTS: A total of 747 VTE cases were identified, and following exclusion of cases with mortality within 30 days of presentation (n=26), unprovoked cases (40.2%) had a higher risk of recurrence (4.6 vs 2.3/100 event-years, P=.01). Provoking factors included surgery (40.4%), injury (16.7%), medical-related factors including non-surgical hospitalisation or active infection (22.0%), travel (13.2%) and oestrogen related (6.5%). Air travel had the highest recurrence rate of 5.9/100 event-years, comparable to unprovoked VTE. VTE provoked by surgery showed lower recurrence rate at 1.8/100 event-years (P=.03). 62.5% of patients with provoked VTE recurred with an unprovoked event. CONCLUSION: Transient provoking factors for VTE are heterogeneous with varying potency and should not be considered a single entity. The high recurrence rate after travel-provoked VTE suggests that it is a "minor," if not negligible provoking factor with higher thrombotic predisposition.

Development of a novel strategy to target CD39 antithrombotic activity to the endothelial-platelet microenvironment in kidney ischemia-reperfusion injury.

Kidney ischemia-reperfusion injury (IRI) is common during transplantation. IRI is characterised by inflammation and thrombosis and associated with acute and chronic graft dysfunction. P-selectin and its ligand PSGL-1 are cell adhesion molecules that control leukocyte-endothelial and leukocyte-platelet interactions under inflammatory conditions. CD39 is the dominant vascular nucleotidase that facilitates adenosine generation via extracellular ATP/ADP-phosphohydrolysis. Adenosine signalling is protective in renal IRI, but CD39 catalytic activity is lost with exposure to oxidant stress. We designed a P-selectin targeted CD39 molecule (rsol.CD39-PSGL-1) consisting of recombinant soluble CD39 that incorporates 20 residues of PSGL-1 that bind P-selectin. We hypothesised that rsol.CD39-PSGL-1 would maintain endothelial integrity by focusing the ectonucleotidase platelet-inhibitory activity and reducing leukocyte adhesion at the injury site. The rsol.CD39-PSGL-1 displayed ADPase activity and inhibited platelet aggregation ex vivo, as well as bound with high specificity to soluble P-selectin and platelet surface P-selectin. Importantly, mice injected with rsol.CD39-PSGL-1 and subjected to renal IRI showed significantly less kidney damage both biochemically and histologically, compared to those injected with solCD39. Furthermore, the equivalent dose of rsol.CD39-PSGL-1 had no effect on tail template bleeding times. Hence, targeting recombinant CD39 to the injured vessel wall via PSGL-1 binding resulted in substantial preservation of renal function and morphology after IRI without toxicity. These studies indicate potential translational importance to clinical transplantation and nephrology.

Venous thromboembolism management in Northeast Melbourne: how does it compare to international guidelines and data?

BACKGROUND: 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. AIMS: To provide a holistic evaluation of 'real-world' Australian experience in the warfarin era, including how we compare to international guidelines. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?

The ecto-nucleoside triphosphate diphosphohydrolase CD39 represents a promising antithrombotic therapeutic. It degrades adenosine 5'-diphosphate (ADP), a main platelet activating/recruiting agent. We hypothesized that delayed enrichment of CD39 on developing thrombi will allow for a low and safe systemic concentration and thus avoid bleeding. We use a single-chain antibody (scFv, specific for activated GPIIb/IIIa) for targeting CD39. This should allow delayed enrichment on growing thrombi but not on the initial sealing layer of platelets, which do not yet express activated GPIIb/IIIa. CD39 was recombinantly fused to an activated GPIIb/IIIa-specific scFv (targ-CD39) and a nonfunctional scFv (non-targ-CD39). Targ-CD39 was more effective at preventing ADP-induced platelet activation than non-targ-CD39. In a mouse carotid artery thrombosis model, non-targ-CD39, although protective against vessel occlusion, was associated with significant bleeding on tail transection. In contrast, targ-CD39 concentrated at the thrombus site; hence, a dose ∼10 times less of CD39 prevented vessel occlusion to a similar extent as high-dose non-targ-CD39, without prolonged bleeding time. An equimolar dose of non-targ-CD39 at this low concentration was ineffective at preventing vessel occlusion. Thus, delayed targeting of CD39 via scFv to activated platelets provides strong antithrombotic potency and yet prevents bleeding and thereby promotes CD39 toward clinical use.

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.