Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

Constituents of platelet membranes regulate the activity of the prothrombinase complex. We demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PE) bind factor Va with high affinity (Kd = ∼10 nm) in the absence of phosphatidylserine (PS). These membranes support formation of a 60–70% functional prothrombinase complex at saturating factor Va concentrations. Although reduced interfacial packing does contribute to factor Va binding in the absence of PS, it does not correlate with the enhanced activity of the Xa-Va complex assembled on PE-containing membranes. Instead, specific protein-PE interactions appear to contribute to the effects of PE. In support of this, soluble C6PE binds to recombinant factor Va2 (Kd = ∼6.5 μm) and to factor Xa (Kd = ∼91 μm). C6PE and C6PS binding sites of factor Xa are specific, distinct, and linked, because binding of one lipid enhances the binding and activity effects of the other. C6PE triggers assembly (Kdapp = ∼40 nm) of a partially active prothrombinase complex between factor Xa and factor Va2, compared with Kdapp for C6PS ∼2 nm. These findings provide new insights into the possible synergistic roles of platelet PE and PS in regulating thrombin formation, particularly when exposed membrane PS may be limiting

Barriers to clinical adoption of next generation sequencing: Perspectives of a policy Delphi panel

AbstractThis research aims to inform policymakers by engaging expert stakeholders to identify, prioritize, and deliberate the most important and tractable policy barriers to the clinical adoption of next generation sequencing (NGS). A 4-round Delphi policy study was done with a multi-stakeholder panel of 48 experts. The first 2 rounds of online questionnaires (reported here) assessed the importance and tractability of 28 potential barriers to clinical adoption of NGS across 3 major policy domains: intellectual property, coverage and reimbursement, and FDA regulation. We found that: 1) proprietary variant databases are seen as a key challenge, and a potentially intractable one; 2) payer policies were seen as a frequent barrier, especially a perceived inconsistency in standards for coverage; 3) relative to other challenges considered, FDA regulation was not strongly perceived as a barrier to clinical use of NGS. Overall the results indicate a perceived need for policies to promote data-sharing, and a desire for consistent payer coverage policies that maintain reasonably high standards of evidence for clinical utility, limit testing to that needed for clinical care decisions, and yet also flexibly allow for clinician discretion to use genomic testing in uncertain circumstances of high medical need

Creating a data resource: What will it take to build a medical information commons?

National and international public-private partnerships, consortia, and government initiatives are underway to collect and share genomic, personal, and healthcare data on a massive scale. Ideally, these efforts will contribute to the creation of a medical information commons (MIC), a comprehensive data resource that is widely available for both research and clinical uses. Stakeholder participation is essential in clarifying goals, deepening understanding of areas of complexity, and addressing long-standing policy concerns such as privacy and security and data ownership. This article describes eight core principles proposed by a diverse group of expert stakeholders to guide the formation of a successful, sustainable MIC. These principles promote formation of an ethically sound, inclusive, participant-centric MIC and provide a framework for advancing the policy response to data-sharing opportunities and challenges

Phosphatidylserine and FVa regulate FXa structure

Human coagulation factor Xa (FXa) plays a key role in blood coagulation by activating prothrombin to thrombin on “stimulated” platelet membranes in the presence of its cofactor factor Va (FVa). Phosphatidylserine (PS) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used fluorescence resonance energy transfer (FRET) to monitor changes in FXa length in response 1] to soluble PS (dicaproyl-phosphatidylserine; C6PS), 2] to PS membranes, and 3] to FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa fluor 555) at FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites, a regulatory site (Reg site) at the N-terminus (previously identified as involving the Gla and EGFN domains) and a presumptive protein-recognition site in the catalytic domain (Prot site). Binding of C6PS to the regulatory site increased the inter-probe distance by ~ 3 Å, while saturation of both sites further increased the distance by ~ 6.4 Å. FXa binding to a membrane produced a smaller length increase (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane than when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS or PS-containing membranes bound to FXa, the overall change in length was comparable (~ 5.6–5.8 Å), indicating that C6PS and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analyzing structure-function relationships in FXa and in the FXa.FVa2 complex

Healthy Nebraska: Advancing Human Health and Developing Healthy Communities

Healthy Nebraska: Advancing Human Health and Developing Healthy Communities Every day, the Institute of Agriculture and Natural Resources (IANR) is putting together a wickedly complex puzzle, in which each faculty member, researcher, Extension educator, student, staff member, partner and stakeholder is a vitally important piece. As the pieces come together, we see a picture of the world in which IANR is making a meaningful difference in sustainable food, fuel, feed, and fiber production

Fresh takes on five health data sharing domains: Quality, privacy, equity, incentives, and sustainability

As entities around the world invest in repositories and other infrastructure to facilitate health data sharing, scalable solutions to data sharing challenges are needed. We conducted semi-structured interviews with 24 experts to explore views on potential issues and policy options related to health data sharing. In this Perspective, we describe and contextualize unconventional insights shared by our interviewees relevant to issues in five domains: data quality, privacy, equity, incentives, and sustainability. These insights question a focus on granular quality metrics for gatekeeping; challenge enthusiasm for maximalist risk disclosure practices; call attention to power dynamics that potentially compromise the patient's voice; encourage faith in the sharing proclivities of new generations of scientists; and endorse accounting for personal disposition in the selection of long-term partners. We consider the merits of each insight with the broad goal of encouraging creative thinking to address data sharing challenges

Real-Time Imaging of HIF-1α Stabilization and Degradation

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α

Creating a data resource: what will it take to build a medical information commons?

National and international public–private partnerships, consortia, and government initiatives are underway to collect and share genomic, personal, and healthcare data on a massive scale. Ideally, these efforts will contribute to the creation of a medical information commons (MIC), a comprehensive data resource that is widely available for both research and clinical uses. Stakeholder participation is essential in clarifying goals, deepening understanding of areas of complexity, and addressing long-standing policy concerns such as privacy and security and data ownership. This article describes eight core principles proposed by a diverse group of expert stakeholders to guide the formation of a successful, sustainable MIC. These principles promote formation of an ethically sound, inclusive, participant-centric MIC and provide a framework for advancing the policy response to data-sharing opportunities and challenges