The drebrin/EB3 pathway regulates cytoskeletal dynamics to drive neuritogenesis in embryonic cortical neurons.

Co-ordinating the dynamic behaviour of actin filaments (F-actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill-defined. The drebrin/end-binding protein 3 (EB3) pathway is a candidate pathway for linking F-actin to microtubules in filopodia. Drebrin binds F-actin and, simultaneously, the microtubule-binding protein EB3 when bound to microtubule plus-ends. We assessed the effect on neuritogenesis of gain- or loss-of-function of proteins in the drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss-of-function of drebrin by gene editing or pharmacological inhibition of drebrin binding to F-actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over-expression of drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end-binding protein 1 (EB1), a related protein that does not bind to drebrin, did not affect neuritogenesis. Over-expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus-ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F-actin zippering in filopodia orchestrated by the drebrin/EB3 pathway.

Comparison of polyspecific versus IgG specific ELISA in predominately cardiac patients with suspected heparin induced thrombocytopenia.

A diagnosis of heparin induced thrombocytopenia (HIT) must often be made based on clinical and laboratory evidence. This was a quasi-experimental study of patients admitted from June 2016 to October 2017. The primary endpoint was the incidence of false positive results in polyspecific and IgG specific enzyme-linked immunosorbent assay (ELISA); then we compared the sensitivity and specificity of each assays in predominately cardiac patients with suspected HIT. A sensitivity/specificity analysis was conducted using serotonin release assay (SRA) as the 'gold standard'. The secondary outcome measures included length of hospital stay. We identified a total of 155 patients who met the inclusion criteria. Confirmatory tests with SRA on both groups were completed; false positive result was higher in the polyspecific group when compared to the IgG group [60% vs. 5%]. The IgG specific ELISA test yielded a sensitivity of 100% and a specificity of 95% however, the polyspecific ELISA had a low yield for specificity of 24% but maintained 100% sensitivity. In the IgG specific group with HIT-, their median length of stay was halved compared to those who were HIT + ; hospital LOS in days, IQR [30 (27-81) vs. 15 (7-33) p = 0.023] and a shorter median LOS in the ICU, IQR [24 (5-47) vs. 6 (2-14); p = 0.079]. Hospital or ICU LOS was the same in both (HIT+ and HIT-) groups managed with polyspecific ELISA. The IgG specific test had few false positive results and a high sensitivity score. Ensuring appropriate testing can bring a substantial decrease in drug expenditure, reduced length of stay and prevent unnecessary anticoagulation.

Risk of pump thrombosis and stroke in patients with continuous-flow left ventricular assist devices and gastrointestinal bleeding.

Gastrointestinal (GI) bleeding is a common complication following the placement of continuous-flow left ventricular assist devices (LVADs) in patients with advanced heart failure. Secondary events arising as a result of GI bleeding have not been well-described. Furthermore, attribution of these events to bleeding is complicated by the interruption or de-intensification of antithrombotic therapy, while bleeding is controlled. The purpose of this study was to assess the incidence of pump thrombosis and ischemic stroke in patients with LVADs who experience GI bleeding, while on support. This was a single-center, retrospective, observational cohort study of consecutive patients with LVADs implanted from January 2012 to June 2018. Patients were assigned to comparator groups based on whether they experienced GI bleeding while on LVAD support. The primary endpoint assessed was the composite of pump thrombosis or ischemic stroke. Secondary endpoints assessed included incidence of pump thrombosis or ischemic stroke. A total of 250 patients were included after screening for exclusion criteria, 101 (40.4%) in the GI bleeding group, and 149 (59.6%) in the non-bleeding group. The incidence of pump thrombosis or ischemic stroke was not significantly greater in patients experiencing GI bleeding [23 (22.8%) vs. 21 (14.1%); P = .09]; however, the incidence of ischemic stroke alone was significantly greater [17 (16.8%) vs. 10 (6.7%); P = .01]. We conclude that GI bleeding in LVAD patients may be associated with a greater risk of ischemic stroke.

Antiplatelet Therapy Bridging With Cangrelor in Patients With Coronary Stents: A Case Series.

BACKGROUND: Cangrelor is an intravenous P2Y12 receptor antagonist approved for use during percutaneous coronary intervention (PCI) to reduce ischemic events associated with new stent placement and has been used off-label at reduced doses guided by platelet function testing as a "bridge" from discontinuation of oral P2Y12 receptor antagonists to surgical procedures when the long-term effects of oral agents are undesirable. OBJECTIVE: To describe the dosing, laboratory monitoring, and clinical outcomes of a series of patients who received cangrelor as a "bridging" antiplatelet agent. METHODS: This study is a retrospective analysis of all patients within the study center with coronary stents who received cangrelor as a bridge to surgical procedure and had VerifyNow monitoring during treatment. RESULTS: A total of 11 patients were identified for inclusion. The median cangrelor dose was 0.5 µg/kg/min (interquartile range = 0.5-0.5) and was maintained in 7 of 11 patients. Doses ranged from 0.25 to 2 µg/kg/min during therapy, and 81.6% of VerifyNow results assessed were within goal range (⩽208 P2Y12 reaction units). Bleeding complications during therapy occurred in 3 patients, all of whom were receiving concomitant heparin infusions, and no stent thrombosis was reported. Conclusion and Relevance: Low-dose cangrelor may represent an effective option for bridging antiplatelet therapy in patients with coronary stents. This study demonstrated that the majority of patients received adequate platelet inhibition without any incidence of stent thrombosis on 0.5 µg/kg/min using the VerifyNow assay to monitor platelet inhibition, which represents a lower dose than previously reported in the literature.

Bivalirudin for left ventricular assist device thrombosis.

Pump thrombosis remains a serious complication of implantable ventricular assist device therapy and is associated with increased risk of morbidity and mortality. Optimal management strategies remain controversial and are guided largely by limited literature and expert opinion. Medical management of pump thrombosis, including the use of direct thrombin inhibitors, has been associated with mixed results. The purpose of this study is to report the outcomes associated with bivalirudin therapy in LVAD patients with suspected pump thrombosis. A single-center, retrospective observational study of 15 patients with suspected pump thrombosis that were all treated with bivalirudin therapy was conducted. The majority of subjects' initial treatment courses were unsuccessful [9/15 (60%)]; however, 6/15 (40%) achieved an initial improvement in serum lactate dehydrogenase (LDH) levels and were stabilized to be successfully discharged from the hospital. Of the subjects discharged, there was a high rate of recurrence of pump thrombosis within 6 months [5/6 (83.3%)]. Bivalirudin therapy was not associated with a consistent reduction in LDH among all subjects studied, and clinical responses to therapy appear to be associated with high rates of thrombosis recurrence. This study analyzes the largest cohort to date of LVAD patients with pump thrombosis treated with bivalirudin therapy, and suggests that alternative therapies should be considered in management.

The Actin-Binding Protein Drebrin Inhibits Neointimal Hyperplasia.

OBJECTIVE: Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple subfamily C TRP (TRPC) channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton. APPROACH AND RESULTS: Wild-type (WT) and congenic Dbn(-/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4±0.3-fold greater in Dbn(-/+) than in WT mice. Levels of globular actin were equivalent in Dbn(-/+) and WT SMCs, but there was a 2.4±0.5-fold decrease in filamentous actin in Dbn(-/+) SMCs compared with WT. Filamentous actin was restored to WT levels in Dbn(-/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(-/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(-/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer. CONCLUSIONS: Drebrin reduces SMC activation through its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds.

Phosphorylation of Drebrin and Its Role in Neuritogenesis.

Neuritogenesis is an early event in neuronal development in which newborn neurons first form growth cones, as a prerequisite for the formation of axons and dendrites. Growth cones emerge from segmented regions of the lamellipodium of embryonic neurons and grow away from the cell body leaving behind a neurite that will eventually polarise into an axon or dendrite. Growth cones also function to navigate precise routes through the embryo to locate an appropriate synaptic partner. Dynamic interactions between two components of the neuronal cytoskeleton, actin filaments and microtubules, are known to be essential for growth cone formation and hence neuritogenesis. The molecular mechanisms that coordinate interactions between actin filaments and dynamic microtubules during neuritogenesis are beginning to be understood. One candidate pathway coupling actin filaments to microtubules consists of the actin filament-binding protein drebrin and the microtubule-binding +TIP protein EB3. This pathway is regulated proximally by cyclin-dependent kinase 5 phosphorylation of drebrin but the upstream elements in the pathway have yet to be identified.

The Role of Drebrin in Cancer Cell Invasion.

Cancer progression is characterized by the capacity of malignant cells to exploit an innate migratory ability in order to invade adjacent tissues, enter the vasculature and eventually metastasize to secondary organs. It is this spread of cancer cells that is the major cause of death in cancer patients. Understanding the basic biology of how cancer cells generate an invasive phenotype will be crucial to the identification of drug targets with the aim of impeding tumour dissemination. Ten years on from its initial description in neuronal cells, drebrin expression was found in a wide variety of non-neuronal cells that importantly included cancer cell lines. Since then mounting evidence suggests that drebrin may be a key player in the advancement of several diverse cancer types where its expression is frequently upregulated. Cancer cell motility and invasion are crucial elements in the metastatic cascade and involve dramatic changes in cellular morphology that are associated with dynamic remodelling of the cytoskeleton. Interestingly, it now appears that drebrin could deliver this role during cancer development.

Treatment of Multiple Myeloma in a Heart Transplant Recipient.

Malignancy following solid organ transplant remains a significant threat to the survival of cardiac transplant recipients. Plasma cell dyscrasias including multiple myeloma have been encountered in this population, and medication treatments traditionally used to treat these disorders demonstrate immunomodulatory effects that may have implications on the transplanted allograft. Lenalidomide is an immunomodulatory agent that has been used to treat plasma cell disorders, including light-chain amyloidosis (AL) and multiple myeloma, and represents such a class of medications in which the risks and benefits in the solid organ transplant population remain to be fully elucidated. This report highlights a clinical practice issue where the treatment of a patient's multiple myeloma with lenalidomide may have potentiated an episode of severe acute cellular rejection and further demonstrates the need for future investigation of the optimal treatment of plasma cell disorders including AL amyloidosis and multiple myeloma following solid organ transplantation.

The role of pharmacotherapy in the prevention of sudden cardiac death in patients with heart failure.

Sudden cardiac death remains a significant threat to the survival of patients with heart failure. Long-term cardiac remodeling predisposes these patients to develop malignant ventricular arrhythmias. Permanent implantable and temporary external defibrillators remain a mainstay for the prevention of sudden cardiac death in this population. For decades, researchers have attempted to identify reliable drug therapies to avoid such arrhythmias; however, to date, success has been inconsistent. This review aims to explore the evidence defining the role of drug therapies for direct and indirect suppression of arrhythmias that may cause sudden cardiac death in patients with heart failure.

Neuronal cytoskeleton in synaptic plasticity and regeneration.

During development, dynamic changes in the axonal growth cone and dendrite are necessary for exploratory movements underlying initial axo-dendritic contact and ultimately the formation of a functional synapse. In the adult central nervous system, an impressive degree of plasticity is retained through morphological and molecular rearrangements in the pre- and post-synaptic compartments that underlie the strengthening or weakening of synaptic pathways. Plasticity is regulated by the interplay of permissive and inhibitory extracellular cues, which signal through receptors at the synapse to regulate the closure of critical periods of developmental plasticity as well as by acute changes in plasticity in response to experience and activity in the adult. The molecular underpinnings of synaptic plasticity are actively studied and it is clear that the cytoskeleton is a key substrate for many cues that affect plasticity. Many of the cues that restrict synaptic plasticity exhibit residual activity in the injured adult CNS and restrict regenerative growth by targeting the cytoskeleton. Here, we review some of the latest insights into how cytoskeletal remodeling affects neuronal plasticity and discuss how the cytoskeleton is being targeted in an effort to promote plasticity and repair following traumatic injury in the central nervous system.

How do we manage cardiopulmonary bypass coagulopathy?

BACKGROUND: Patients who undergo cardiopulmonary bypass (CPB) are at risk for coagulopathy. Suboptimal turnaround time (TAT) of laboratory coagulation testing results in empiric administration of blood products to treat massive bleeding. We describe our initiative in establishing the coagulation-based hemotherapy (CBH) service, a clinical pathology consultation service that uses rapid TAT coagulation testing and provides comprehensive assessment of bleeding in patients undergoing CPB. A transfusion algorithm that treats the underlying cause of coagulopathy was developed. STUDY DESIGN AND METHODS: The coagulation testing menu includes all aspects of coagulopathy with close proximity of the laboratory to the operating room to allow for rapid test results. The hemotherapy pathologist monitors laboratory results at several stages in surgery and uses a comprehensive algorithm to monitor a patient's hemostasis. The optimal number and type of blood products are selected when the patient is taken off CPB. RESULTS: The CBH service was consulted for 44 ventricular assist device implants, 30 heart transplants, and 31 other cardiovascular surgeries from May 2012 through November 2013. The TAT for laboratory tests was 15 minutes for complete blood count, antithrombin, and coagulation panel and 30 minutes for VerifyNow and thromboelastography, in comparison to 45 to 60 minutes in normal settings. The transfusion algorithms were used with optimal administration of blood components with preliminary data suggestive of reduced blood product usage and better patient outcomes. CONCLUSION: We described the successful introduction of a novel pathology consultation service that uses a rapid TAT coagulation testing menu with transfusion algorithms for improved management of CPB patients.

Improved ticagrelor antiplatelet effect on discontinuation of phenytoin.

OBJECTIVE: To report the influence of phenytoin on the antiplatelet effects of ticagrelor using a validated platelet aggregation study. CASE SUMMARY: A 71-year-old man with coronary artery disease underwent percutaneous coronary intervention to revascularize several major coronary arteries. The patient was previously on phenytoin and was initiated on ticagrelor for antiplatelet therapy following stent placement. While the patient was receiving both drugs, platelet aggregation studies revealed less platelet inhibition than would be expected in a patient not taking a concomitant inducer of ticagrelor metabolism. On discontinuation of phenytoin, platelet inhibition improved. DISCUSSION: Dual antiplatelet therapy with aspirin and a P2Y12 receptor antagonist following placement of coronary stents is critical to prevent stent thrombosis and subsequent myocardial infarction. Ticagrelor is a recently approved P2Y12 receptor antagonist that is subject to drug-drug interactions involving the hepatic cytochrome P450-3A4 enzyme system because of its metabolic elimination pathway. This case demonstrates ticagrelor's drug-drug interaction with phenytoin through a platelet aggregation study and supports the manufacturer recommendation to avoid the combination of ticagrelor with any known inducers of cytochrome P450-3A4 metabolism. CONCLUSION: The combination of ticagrelor and phenytoin may represent a potentially clinically significant drug-drug interaction because of phenytoin induction of ticagrelor metabolism and reduced P2Y12 receptor inhibition in patients who have recently undergone percutaneous coronary intervention and cardiac stent placement.

An ISHLT consensus statement on strategies to prevent and manage hemocompatibility related adverse events in patients with a durable, continuous-flow ventricular assist device.

Life expectancy of patients with a durable, continuous-flow left ventricular assist device (CF-LVAD) continues to increase. Despite significant improvements in the delivery of care for patients with these devices, hemocompatability-related adverse events (HRAEs) are still a concern and contribute to significant morbility and mortality when they occur. As such, dissemination of current best evidence and practices is of critical importance. This ISHLT Consensus Statement is a summative assessment of the current literature on prevention and management of HRAEs through optimal management of oral anticoagulant and antiplatelet medications, parenteral anticoagulant medications, management of patients at high risk for HRAEs and those experiencing thrombotic or bleeding events, and device management outside of antithrombotic medications. This document is intended to assist clinicians caring for patients with a CF-LVAD provide the best care possible with respect to prevention and management of these events.

MAP1B enhances microtubule assembly rates and axon extension rates in developing neurons.

The microtubule-associated protein MAP1B is known to have important roles in neuronal development, particularly during neuronal migration and axonogenesis, but its precise molecular actions are unknown. We used RNA interference silencing of protein expression to specifically knock down MAP1B in cultured embryonic rat cortical neurons. Reduction of MAP1B in these neurons is associated with several abnormal morphological phenotypes including the production of more highly branched and slower growing axons than normal. MAP1B binds to dynamic microtubules and indirect evidence suggests that MAP1B regulates microtubule dynamics. We used the +TIP protein EB3 to assess the dynamic behaviour and orientation of microtubules in neurons in which MAP1B had been knocked down. This revealed a reduction in the speed of microtubule growth in the proximal and distal axon shaft, but not in growth cone filopodia. These observations suggest that the function of MAP1B is to suppress axon branching and enhance axon growth and that this is achieved by maintaining dynamic microtubule growth. To test this hypothesis we expressed MAP1B in a cell line that does not have endogenous MAP1B, this led to an increase in microtubule elongation rates. These findings show that MAP1B enhances microtubule assembly rates and axon extension rates in developing neurons by binding to dynamic microtubules.

Drebrin controls neuronal migration through the formation and alignment of the leading process.

Formation of a functional nervous system requires neurons to migrate to the correct place within the developing brain. Tangentially migrating neurons are guided by a leading process which extends towards the target and is followed by the cell body. How environmental cues are coupled to specific cytoskeletal changes to produce and guide leading process growth is unknown. One such cytoskeletal modulator is drebrin, an actin-binding protein known to induce protrusions in many cell types and be important for regulating neuronal morphology. Using the migration of oculomotor neurons as a model, we have shown that drebrin is necessary for the generation and guidance of the leading process. In the absence of drebrin, leading processes are not formed and cells fail to migrate although axon growth and pathfinding appear grossly unaffected. Conversely, when levels of drebrin are elevated the leading processes turn away from their target and as a result the motor neuron cell bodies move along abnormal paths within the brain. The aberrant trajectories were highly reproducible suggesting that drebrin is required to interpret specific guidance cues. The axons and growth cones of these neurons display morphological changes, particularly increased branching and filopodial number but despite this they extend along normal developmental pathways. Collectively these results show that drebrin is initially necessary for the formation of a leading process and subsequently for this to respond to navigational signals and grow in the correct direction. Furthermore, we have shown that the actions of drebrin can be segregated within individual motor neurons to direct their migration independently of axon guidance.

Managing new oral anticoagulants in the intensive care unit.

Warfarin has been the mainstay of oral anticoagulation for more than half a century. Within the last several years, 2 new classes of oral anticoagulants have been introduced as potential alternatives to warfarin for certain indications. The oral direct thrombin inhibitor, dabigatran, and 2 factor Xa inhibitors, rivaroxaban and apixaban, are the newest agents approved for use in the United States. These agents have been studied in various areas including stroke prophylaxis in atrial fibrillation, prevention and treatment of venous thromboembolism, and for reduction of ischemic events following acute coronary syndromes. While these agents do not require routine monitoring of international normalized ratio, these agents may be more challenging to reverse than traditional warfarin therapy. The following review will focus on describing the areas where the new oral anticoagulant agents have been studied, the basic pharmacologic characteristics of each agent, and how to appropriately manage the reversal of these agents when indicated.

Comparison of survival by vasoactive-inotropic score in patients receiving veno-arterial extracorporeal life support.

After the initiation of veno-arterial extracorporeal membrane oxygenation (V-A ECMO) for hemodynamic support, patients often require vasopressor and inotropic medications to support their blood pressure and cardiac contractility. The vasoactive-inotropic score (VIS) is a standardized calculation of vasopressor and inotrope equivalence, which uses coefficients for each medication to calculate a total value. This study evaluated the association between the 30-day survival of patients receiving V-A ECMO support and the VIS calculated 24 h after ECMO cannulation (VIS24). This was a single-center, retrospective, observational cohort study. The median VIS24 of the entire cohort was 6.0, and was determined as a cutoff for comparison. Patients with a VIS24 < 6.0 were assigned to a group, and those with a VIS24 ≥ 6.0 were assigned to a second group. Patients with a VIS24 < 6.0 had higher 30-day survival than those with a VIS24 ≥ 6.0 (54.5% vs 41.4%; p = 0.03). The group with a VIS24 < 6.0 also had significantly improved survival to decannulation of ECMO support; however, there was no difference in the survival to hospital discharge. We conducted a secondary analysis of quartiles and determined that individuals with a VIS24 > 11.4 had the lowest survival in the cohort. This finding may help identify patients with the lowest probability of 30-day survival in those receiving V-A ECMO for hemodynamic support.

Evaluation of Vasoactive-Inotropic Score and Survival to Decannulation in Adult Patients on Venoarterial Extracorporeal Life Support: An Observational Cohort Study.

Extracorporeal life support with venoarterial extracorporeal membrane oxygenation (VA-ECMO) is used to assist circulation in patients with severe cardiogenic shock or cardiac arrest. The vasoactive-inotropic score (VIS) is a standardized calculation of vasoactive medication support which uses coefficients for each medication that converts them to an equivalent value. The purpose of this study was to assess the VIS as an early prognostication tool for survival to decannulation patients on adult VA-ECMO support. This was a single-center, observational cohort study of adult patients who received VA-ECMO support compared based on their survival to decannulation. The primary endpoint was the VIS at hour 24 postcannulation. Among the 265 patients included in this study, 140 patients (52.8%) survived to decannulation of VA-ECMO. At 24 hours postcannulation, a lower VIS was observed in the group that survived decannulation (6.5 ± 7.5 vs. 12.3 ± 16.9; p < 0.001). Multivariate analysis performed also demonstrates an association between 24-hour VIS and survival to decannulation (odds ratio 0.95; 95% confidence interval, 0.91-0.95). This study suggests that the 24-hour VIS may be an early prognostic indicator in patients on VA-ECMO patients. http://links.lww.com/ASAIO/B39.

Cytoskeletal dynamics in growth-cone steering.

Interactions between dynamic microtubules and actin filaments are essential to a wide range of cell biological processes including cell division, motility and morphogenesis. In neuronal growth cones, interactions between microtubules and actin filaments in filopodia are necessary for growth cones to make a turn. Growth-cone turning is a fundamental behaviour during axon guidance, as correct navigation of the growth cone through the embryo is required for it to locate an appropriate synaptic partner. Microtubule-actin filament interactions also occur in the transition zone and central domain of the growth cone, where actin arcs exert compressive forces to corral microtubules into the core of the growth cone and thereby facilitate microtubule bundling, a requirement for axon formation. We now have a fairly comprehensive understanding of the dynamic behaviour of the cytoskeleton in growth cones, and the stage is set for discovering the molecular machinery that enables microtubule-actin filament coupling in growth cones, as well as the intracellular signalling pathways that regulate these interactions. Furthermore, recent experiments suggest that microtubule-actin filament interactions might also be important for the formation of dendritic spines from filopodia in mature neurons. Therefore, the mechanisms coupling microtubules to actin filaments in growth-cone turning and dendritic-spine maturation might be conserved.