From expected to actual barriers and facilitators when implementing a new screening tool: A qualitative study applying the Theoretical Domains Framework.

AIM AND OBJECTIVES: To identify determinants for using a new screening tool to identify older patients eligible for targeted nurse-led intervention, as perceived by healthcare professionals implementing the tool, and to examine how these perceptions changed over time. DESIGN: A cross-sectoral longitudinal qualitative study based on semi-structured interviews with healthcare professionals in a Danish hospital and two collaborating municipalities. METHODS: In three focus groups, seven single interviews and a workshop, we examined the healthcare professionals' perceptions of and attitudes towards the new screening tool before, during and after the implementation. The Theoretical Domains Framework was used to identify the healthcare professionals' perception of barriers and facilitators, followed by content analysis. The results were further discussed using the COM-B system as an analytic framework. This qualitative study is reported according to the Consolidated Criteria for Reporting Qualitative Studies (COREQ) checklist. RESULTS: 'Professional role', 'Goals' and 'Environmental context' were the domains most talked about by the healthcare professionals across the three time points. The content analysis identified four determinants for using the new screening tool:Making time for the project, External motivation and management, Expectations and reality, and Professional identity. The healthcare professionals' perception of the determinants changed during the implementation, influencing their behaviour and, consequently, the implementation's sustainability. CONCLUSION: Perception of barriers and facilitators to the interventions were time- and context-sensitive. Beliefs and motivational factors changed during the project, which points out the importance of following implementation processes systematically to understand the outcome of an intervention. RELEVANCE FOR CLINICAL PRACTICE: Perceptions and attitudes towards a new initiative may change over time, emphasising the importance of following barriers and facilitators during the implementation of an intervention and working with an implementation plan that can be adapted along the way.

Take-Home Naloxone Kits: Attitudes and Likelihood-Of-Use Outcomes from a European Survey of Potential Overdose Witnesses.

BACKGROUND: Injectable naloxone is already provided as take-home naloxone (THN), and new concentrated intranasal naloxone is now being introduced in Europe. Despite evidence of the effectiveness and cost-effectiveness of THN, little is known about the attitudes of key target populations: people who use opioids (PWUO), family/friends, and staff. We examined the acceptability of different naloxone devices (ampoule, prefilled syringe, and concentrated nasal spray) across 5 European countries. OBJECTIVES: The aim of this study was to compare THN target groups (PWUO vs. family/friends vs. staff) in their past rates of witnessed overdose and THN administration (as indicators of future use), current THN device preference, and THN carriage on the day of survey. METHOD: Cross-sectional survey of respondents (age ≥18) in addiction treatment, harm reduction, and recovery services in Denmark, England, Estonia, Norway, and Scotland. A purpose-developed questionnaire (59 items) was administered in the local language electronically or in a pen-and-paper format. RESULTS: Among n = 725 participants, 458 were PWUO (63.2%), 214 staff (29.5%), and 53 (7.3%) family members. The groups differed significantly in their likelihood-of-future THN use (p < 0.001): PWUO had the highest rate of previously witnessing overdoses (352; 77.7%), and staff members reported the highest past naloxone use (62; 30.1%). Across all groups, most respondents (503; 72.4%) perceived the nasal spray device to be the easiest to use. Most reported willingness to use the spray in an overdose emergency (508; 73.5%), followed by the prefilled syringe (457; 66.2%) and ampoules (64; 38.2%). Average THN carriage was 18.6%, ranging from 17.4% (PWUO) to 29.6% (family members). CONCLUSION: Respondents considered the concentrated naloxone nasal spray the easiest device to use. Still, most expressed willingness to use the nasal spray as well as the prefilled syringe in an overdose emergency. Carriage rates were generally low, with fewer than 1 in 5 respondents carrying their THN kit on the day of the survey.

Dialogue is a prerequisite for the nurse-patient relationship in nutritional care: A secondary analysis using the fundamentals of care framework.

BACKGROUND: Providing good nutritional care is complex as it goes beyond assessing and ensuring the patients' dietary needs. So far, nutritional research has mainly focused on establishing evidence for the nutritional treatment, while less attention has been on the complexity of providing nutritional care. The Fundamentals of Care (FoC) describes five elements (focus, knowledge, anticipate, evaluate and trust) essential for establishing a nurse-patient relationship as a foundation for quality care. By studying how these elements shape nutritional care and dialogue, we can explore and describe the complexity of nutritional care. AIM: By using the FoC framework as an analytic framework, this study explores how the nurse-patient relationship shapes the nutritional care of orthopaedic patients. METHOD: This study is a secondary analysis using deductive content analysis of interviews with patients undergoing major orthopaedic surgery, nursing staff and observations of interactions between nursing staff and patients. The core dimension of the FoC framework, 'Establishment of relationship,' was used as an analytic framework. RESULT: The nurses perceived serving meals and providing nutritional supplements as an essential part of the nutritional care. Still, the nutritional care was organised as a routine task to be less time-consuming. Appropriate care was initiated when the nursing staff explored patients´ food preferences. When the nursing staff failed to familiarise themselves with the patient's preferences, the patients interpreted nutritional care as unrelated to their needs, resulting in a lack of trust. CONCLUSION: The need for efficiency within nutritional care must not compromise the patients' need for dialogue with the nurse. Establishing a trusting relationship between nurses and patients prevents nutritional care from becoming a routine task unrelated to the patients' needs.

Surveillance life and the shaping of 'genetically at risk' chronicities in Denmark.

Today, in the field of hereditary colorectal cancer in Denmark, more than 40,000 identified healthy individuals with an increased risk of cancer are enrolled in a surveillance program aimed at preventing cancer from developing, with numbers still growing. What this group of healthy individuals has in common is lifelong regular interaction with a healthcare system that has traditionally been geared towards treating the acutely and chronically ill. In this article, we explore how people living with an inherited elevated risk of colorectal cancer orient themselves towards their families' and their own predispositions as well as the lifelong surveillance trajectories that they have embarked upon - what we call surveillance life. Unlike prior critiques of predictive genetic testing as generative of 'pre-patients' or 'pre-symptomatically ill', we suggest that for those enrolled in lifelong surveillance programmes in welfare state Denmark, the relevance of risk fluctuates according to certain moments in life, e.g. at family reunions, when a close relative falls ill, in the time leading up to a surveillance colonoscopy or when enduring the procedures themselves. As such, rather than characterising surveillance life in terms of 'living with chronic risk' we show how 'genetically at risk' chronicities take shape as persons come to terms with a disease that possibly awaits them leading them to recalibrate familial bonds and responsibilities while leading lives punctuated by regular medical check-ups.

"It is two worlds" cross-sectoral nurse collaboration related to care transitions: A qualitative study.

AIMS AND OBJECTIVES: To explore how the hospital and home care nurses talk about and experience cross-sectoral collaboration related to the transitional care of frail older patients. BACKGROUND: Effective communication and collaboration between nurses involved in care transition are crucial for a safe patient handover. Organisational systems to support cross-sectoral collaboration have been developed but do not always promote the intended dialogue and precise and useful exchange of information. Other factors may also be of importance to an effective and constructive cross-sectoral nurse collaboration. DESIGN: A qualitative design using thematic analysis. METHODS: Data were extracted from 24 focus group interviews conducted with registered nurses from eight hospital wards and six municipalities and a total of 165 hr of observations conducted in three hospital wards and three municipalities. The study was reported according to the COREQ guidelines. RESULTS: The perception of nursing and care differed across sectors. The nurses expressed having shared goals for the patients-however, these goals derived from different values, the perception of nursing and approach to the patients. The lack of knowledge of each other's working conditions created assumptions and preconceptions, which affected communication and collaboration related to planning and executing care transition negatively. CONCLUSIONS: The nurses perceived the hospital and home care as" two worlds". The collaboration between the nurses was characterised by insufficient communication and preconceptions rather than concrete knowledge and different cultures and professional identities. It can be questioned whether cross-sectoral collaboration between nurses should be characterised as interprofessional rather than intraprofessional collaboration as the features of the nurses' collaboration. RELEVANCE TO CLINICAL PRACTICE: Organisational and political systems should recognise that nurses in different sectors are taking care of various aspects of nursing when planning on policies to support cross-sectoral collaboration. More possibilities for nurses across sectors to meet should be made available.

Nurses' perspectives on how an e-message system supports cross-sectoral communication in relation to medication administration: A qualitative study.

AIMS AND OBJECTIVES: To describe nurses' perspectives on how an e-message system supports communication between hospital and home care nurses in relation to medication administration. BACKGROUND: Medication administration is a complex process involving a high risk of error. Unclear communication and lack of updated information about medication have been identified as the leading cause of medication errors during care transitions. E-message systems are progressively being used to improve information exchange across sectors, yet little is known about their usage for exchange of medication information between nurses. DESIGN: A qualitative design using content analysis. METHODS: Focus group interviews were conducted with nurses from eight hospital wards and six municipalities (n = 79). Participant observations were conducted in three hospital wards and in three municipalities (165 hr). RESULTS: The e-message system did not provide the information needed by the home care nurses to ensure safe medication administration after discharge due to (1) limitations of the system, (2) hospital nurses' lack of knowledge about home care nurses' information needs and (3) hospital nurses' lack of access to medication information. Due the home care nurses' perception of responsibility, they bypassed the e-message system to get the information. CONCLUSIONS: The home care nurses perceived themselves as the last link in the safety chain. As such, access to adequate information was essential for the nurses to ensure safe medication administration; however, the e-message system did not support exchange of the information needed. RELEVANCE TO CLINICAL PRACTICE: E-message systems must meet home care nurses' need for information to ensure that safe medication administration does not depend on the knowledge of the individual nurse. Moreover, other system factors that influence interprofessional collaboration and nurses' cross-sectoral communication must be addressed.

Stuck in tradition-A qualitative study on barriers for implementation of evidence-based nutritional care perceived by nursing staff.

AIMS AND OBJECTIVES: To explore the barriers for nutritional care as perceived by nursing staff at an acute orthopaedic ward, aiming to implement evidence-based nutritional care. BACKGROUND: Previous studies indicate that nurses recognise nutritional care as important, but interventions are often lacking. These studies show that a range of barriers influence the attempt to optimise nutritional care. Before the implementation of evidence-based nutritional care, we examined barriers for nutritional care among the nursing staff. DESIGN: Qualitative study. METHODS: Four focus groups with thirteen members of the nursing staff were interviewed between October 2013-June 2014. The interview guide was designed according to the Theoretical Domains Framework. The interviews were analysed using qualitative content analysis. RESULTS: Three main categories emerged: lacking common practice, failing to initiate treatment and struggling with existing resources. The nursing staff was lacking both knowledge and common practice regarding nutritional care. They felt they protected patient autonomy by accepting patient's reluctance to eat or getting a feeding tube. The lack of nutritional focus from doctors decreased the nursing staffs focus leading to nonoptimal nutritional treatment. Competing priorities, physical setting and limited nutritional supplements were believed to hinder nutritional care. CONCLUSION: The results suggest that nutritional care is in a transitional state from experience- to evidence-based practice. Barriers for nutritional care are grounded in lack of knowledge among nursing staff and insufficient collaboration between nursing staff and the doctors. There is a need for nutritional education for the nursing staff and better support from the organisation to help nursing staff provide evidence-based nutritional care. RELEVANCE TO CLINICAL PRACTICE: This study contributes with valuable knowledge before the implementation of evidence-based nutritional care. The study provides an understanding of barriers for nutritional care and presents explanations to why nutritional care has failed to become an integrated part of the daily treatment and care.

Nurses' perception of how an e-message system influences cross-sectoral communication: A qualitative study.

AIM: To investigate hospital and home care nurses' experiences on how an e-message system influences cross-sectoral communication 2 years after introduction. BACKGROUND: Cross-sectoral communication is identified as the main barrier for high quality in transitional care. An e-message system was introduced to ensure dialogue and precise and useful information exchange. METHODS: Nurses from one hospital and six collaborating municipalities were included. Semi-structured focus group interviews and participation observation was conducted and data were analysed using content analysis. RESULTS: The e-message system was used in both sectors but did not promote cross-sectoral dialogue. The home care nurses expressed distrust in the information from the hospital. The hospital nurses' intention was to provide relevant and accurate information but their main focus was to fulfil the standards within the system rather than the quality of the information exchanged. CONCLUSIONS: The e-message system supports a one-way information flow, rather than the intended loop of information exchange and dialogue. IMPLICATIONS FOR NURSING MANAGEMENT: Nurse managers should recognise that although an e-message system is being used in a clinical setting, it may not support exchange of high-quality information and dialogue. Focus must be on both standards as well as quality when new systems are introduced.

Evidence for restricted reactivity of ADAMDEC1 with protein substrates and endogenous inhibitors.

ADAMDEC1 is a proteolytically active metzincin metalloprotease displaying rare active site architecture with a zinc-binding Asp residue (Asp-362). We previously demonstrated that substitution of Asp-362 for a His residue, thereby reconstituting the canonical metzincin zinc-binding environment with three His zinc ligands, increases the proteolytic activity. The protease also has an atypically short domain structure with an odd number of Cys residues in the metalloprotease domain. Here, we investigated how these rare structural features in the ADAMDEC1 metalloprotease domain impact the proteolytic activity, the substrate specificity, and the effect of inhibitors. We identified carboxymethylated transferrin (Cm-Tf) as a new ADAMDEC1 substrate and determined the primary and secondary cleavage sites, which suggests a strong preference for Leu in the P1' position. Cys(392), present in humans but only partially conserved within sequenced ADAMDEC1 orthologs, was found to be unpaired, and substitution of Cys(392) for a Ser increased the reactivity with α2-macroglobulin but not with casein or Cm-Tf. Substitution of Asp(362) for His resulted in a general increase in proteolytic activity and a change in substrate specificity was observed with Cm-Tf. ADAMDEC1 was inhibited by the small molecule inhibitor batimastat but not by tissue inhibitor of metalloproteases (TIMP)-1, TIMP-2, or the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3). However, N-TIMP-3 displayed profound inhibitory activity against the D362H variants with a reconstituted consensus metzincin zinc-binding environment. We hypothesize that these unique features of ADAMDEC1 may have evolved to escape from inhibition by endogenous metalloprotease inhibitors.

Barriers and facilitators for implementing a new screening tool in an emergency department: A qualitative study applying the Theoretical Domains Framework.

AIM: The aim was to identify the factors that were perceived as most important as facilitators or barriers to the introduction and intended use of a new tool in the emergency department among nurses and a geriatric team. BACKGROUND: A high incidence of functional decline after hospitalisation for acute medical illness has been shown in the oldest patients and those who are physically frail. In Denmark, more than 35% of older medical patients acutely admitted to the emergency department are readmitted within 90 days after discharge. A new screening tool for use in the emergency department aiming to identify patients at particularly high risk of functional decline and readmission was developed. DESIGN: Qualitative study based on semistructured interviews with nurses and a geriatric team in the emergency department and semistructured single interviews with their managers. METHODS: The Theoretical Domains Framework guided data collection and analysis. Content analysis was performed whereby new themes and themes already existing within each domain were described. RESULTS: Six predominant domains were identified: (1) professional role and identity; (2) beliefs about consequences; (3) goals; (4) knowledge; (5) optimism and (6) environmental context and resources. The content analysis identified three themes, each containing two subthemes. The themes were professional role and identity, beliefs about consequences and preconditions for a successful implementation. CONCLUSIONS: Two different cultures were identified in the emergency department. These cultures applied to different professional roles and identity, different actions and sense making and identified how barriers and facilitators linked to the new screening tool were perceived. RELEVANCE FOR CLINICAL PRACTICE: The results show that different cultures exist in the same local context and influence the perception of barriers and facilitators differently. These cultures must be identified and addressed when implementation is planned.

ADAMDEC1 is a metzincin metalloprotease with dampened proteolytic activity.

ADAMDEC1 (Decysin-1) is a putative ADAM (a disintegrin and metalloprotease)-like metalloprotease with an unknown physiological role, selectively expressed in mature dendritic cells and macrophages. When compared with other members of the ADAM family, ADAMDEC1 displays some unusual features. It lacks the auxiliary cysteine-rich, EGF, and transmembrane domains, as well as the cytoplasmic tail. The active site of ADAMDEC1 is unique by being the only mammalian ADAM protease with a non-histidine zinc ligand, having an aspartic acid residue instead. Here we demonstrate that ADAMDEC1, despite these unique features, functions as an active metalloprotease. Thus, ADAMDEC1 is secreted as a mature, glycosylated, and proteolytically active metalloprotease, capable of cleaving macromolecular substrates. In the recombinant form, three of the four potential N-linked glycosylation sites are modified by carbohydrate attachment. Substitution of basic residues at the predicted proprotein convertase cleavage site blocks proprotein processing, revealing both specific ADAMDEC1-dependent and specific ADAMDEC1-independent cleavage of the prodomain. The pro-form of ADAMDEC1 does not have proteolytic activity, demonstrating that the prodomain of ADAMDEC1, like in other members of the ADAM family, confers catalytic latency. Interestingly, the proteolytic activity of mature ADAMDEC1 can be significantly enhanced when a canonical ADAM active site with three zinc-coordinating histidine residues is introduced.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model.

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Balancing life with an increased risk of cancer: lived experiences in healthy individuals with Lynch syndrome.

Possibilities to undergo predictive genetic testing for cancer have expanded, which implies that an increasing number of healthy individuals will learn about cancer predisposition. Knowledge about how an increased risk of disease influences life in a long-term perspective is largely unknown, which led us to explore lived experiences in healthy mutation carriers with Lynch syndrome. Individual interviews were subjected to descriptive phenomenological analysis. Four constitutions, namely, family context, interpretation and transformation, approach to risk and balancing life at risk were identified and formed the essence of the phenomenon "living with knowledge about risk." Family context influences how experiences and knowledge are interpreted and transformed into thoughts and feelings, which relates to how risk is approached and handled. The constitutions influence each other in a dynamic relationship and create a balancing act between anxiety and worry and feelings of being safe and in control.

National Experiences from 30 Years of Provider-Mediated Cascade Testing in Lynch Syndrome Families-The Danish Model.

Cascade genetic testing and surveillance reduce morbidity and mortality in Lynch syndrome. However, barriers to conveying information about genetic disorders within families result in low uptake of genetic testing. Provider-mediated interventions may increase uptake but raise legal and ethical concerns. We describe 30 years of national experience with cascade genetic testing combining family- and provider-mediated contact in Lynch syndrome families in the Danish Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Register. We aimed to estimate the added value of information letters to family members in Lynch syndrome families (provider-mediated contact) compared to family members not receiving such letters and thus relying on family-mediated contact. National clinical practice for cascade genetic testing, encompassing infrastructure, legislation, acceptance, and management of the information letters, is also discussed. Cascade genetic testing resulted in 7.3 additional tests per family. Uptake of genetic testing was 54.4% after family-mediated and 64.9% after provider-mediated contact, corresponding to an odds ratio of 1.8 (p < 0.001). The uptake of genetic testing was highest in the first year after diagnosis of Lynch syndrome in the family, with 72.5% tested after provider-mediated contact. In conclusion, the Danish model combining family- and provider-mediated contact can increase the effect of cascade genetic testing.

Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells.

The Warburg effect is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production, as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain wildtype growth rate while producing negligible lactate. Further characterization of Warburg-null CHO cells showed a compensatory increase in oxygen consumption, a near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. These cells remained amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining product glycosylation. Thus, the ability to eliminate the Warburg effect is an important development for biotherapeutic production and provides a tool for investigating a near-universal metabolic phenomenon.

Sense of coherence and self-concept in Lynch syndrome.

OBJECTIVE: Most individuals who learn about hereditary cancer manage well, but identification of subgroups who find this knowledge burdening would allow psychosocial intervention. The objective of the study was to assess sense of coherence (SOC) in individuals with Lynch syndrome with comparison to a general population and correlation to self-concept. METHODS: A total of 345 individuals with Lynch syndrome completed the 13-item SOC scale and the 20-item Lynch syndrome self-concept scale. SOC scores were compared to a general Danish population and were correlated to self-concept estimates in individuals with Lynch syndrome. Characteristics of subgroups with adverse scores were described. RESULTS: Individuals with Lynch syndrome reported SOC scores similar to the general population. SOC and self-concept correlated well with a correlation coefficient of -0.51. Subsets with convergent and divergent scores, which may reflect different effects from knowledge about hereditary cancer, were identified. CONCLUSION: Individuals with Lynch syndrome report SOC scores similar to the general population. SOC and self-concept correlate well but allow identification of subset who report adverse outcome and may be relevant for targeted intervention.

Tralokinumab Effectively Disrupts the IL-13/IL-13Rα1/IL-4Rα Signaling Complex but Not the IL-13/IL-13Rα2 Complex.

Tralokinumab, a fully human mAb specifically targeting the IL-13 cytokine, has demonstrated clinical efficacy and safety in patients with moderate-to-severe atopic dermatitis. Tralokinumab binds IL-13 with high affinity, which prevents the interaction of IL-13 with IL-13Rα1 and subsequent signaling. Similarly, tralokinumab-bound IL-13 cannot bind to IL-13Rα2, a proposed decoy receptor that is reported to bind IL-13 with extraordinarily high affinity. It has however not been fully elucidated to what extent tralokinumab interferes with the endogenous regulation of IL-13 through IL-13Rα2. In this mechanistic study, we used biophysical, biochemical, and cellular assays to investigate the effect of tralokinumab on the interaction between IL-13 and IL-13Rα1 and IL-13Rα2, respectively, as well as the effects on IL-13Rα2-mediated IL-13 internalization. We demonstrate that IL-13Rα2 binds IL-13 with exceptionally high affinity and that tralokinumab is unable to displace IL-13 from IL-13Rα2. In contrast to this, tralokinumab is able to disrupt the IL-13/IL-13Rα1 and IL-13Rα1/IL-13/IL-4Rα complex. Furthermore, we demonstrate that whereas the IL-13/tralokinumab complex is unable to bind IL-13Rα2, any IL-13 that is not bound by tralokinumab (i.e., free IL-13) can be bound by IL-13Rα2 and subsequently internalized, regardless of the presence of tralokinumab. In summary, our study indicates that tralokinumab does not interfere with endogenous IL-13Rα2-mediated regulation of free IL-13.

The endothelial protein C receptor supports tissue factor ternary coagulation initiation complex signaling through protease-activated receptors.

Protease-activated receptor (PAR) signaling is closely linked to the cellular activation of the pro- and anticoagulant pathways. The endothelial protein C receptor (EPCR) is crucial for signaling by activated protein C through PAR1, but EPCR may have additional roles by interacting with the 4-carboxyglutamic acid domains of procoagulant coagulation factors VII (FVII) and X (FX). Here we show that soluble EPCR regulates the interaction of FX with human or mouse tissue factor (TF)-FVIIa complexes. Mutagenesis of the FVIIa 4-carboxyglutamic acid domain and dose titrations with FX showed that EPCR interacted primarily with FX to attenuate FX activation in lipid-free assay systems. In human cell models of TF signaling, antibody inhibition of EPCR selectively blocked PAR activation by the ternary TF-FVIIa-FXa complex but not by the non-coagulant TF-FVIIa binary complex. Heterologous expression of EPCR promoted PAR1 and PAR2 cleavage by FXa in the ternary complex but did not alter PAR2 cleavage by TF-FVIIa. In murine smooth muscle cells that constitutively express EPCR and TF, thrombin and FVIIa/FX but not FVIIa alone induced PAR1-dependent signaling. Although thrombin signaling was unchanged, cells with genetically reduced levels of EPCR no longer showed a signaling response to the ternary complex. These results demonstrate that EPCR interacts with the ternary TF coagulation initiation complex to enable PAR signaling and suggest that EPCR may play a role in regulating the biology of TF-expressing extravascular and vessel wall cells that are exposed to limited concentrations of FVIIa and FX provided by ectopic synthesis or vascular leakage.

The functional role of the autolysis loop in the regulation of factor X upon hemostatic response.

BACKGROUND: The regulation of factor X (FX) is critical to maintain the balance between blood coagulation and fluidity. OBJECTIVES: To functionally characterize the role of the FX autolysis loop in the regulation of the zymogen and active form of FX. METHODS: We introduced novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. The activity and inhibition of recombinant FX variants was quantified in pure component assays. The in vitro thrombin generation potential of the FX variants was evaluated in FX-depleted plasma. RESULTS: The factor VIIa (FVIIa)-mediated activation and prothrombin activation was reduced, presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, whereas ATIII inhibition in the presence of the heparin-analog Fondaparinux was modestly increased by 2- to 15-fold compared with wild-type. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FXa variants. CONCLUSION: The autolysis loop participates in all aspects of FX regulation. In plasma-based assays, a modest decrease in FX activation rate appeared to knock down the procoagulant response even when down regulation of FXa activity by inhibitors was reduced.