Rapid and automated screening of carbapenemase- and ESBL-producing Gram-negative bacteria from rectal swabs using chromogenic agar media and the ScanStation device.

The ScanSation 100 device (Interscience, France) is an incubator allowing real-time detection of bacterial colony growth by frequently imaging agar plates over time, counting CFU, and detecting colony color. This study evaluated its performance for the early detection of carbapenemase-producing bacteria (CPB) and extended-spectrum ß-Lactamase-producing bacteria (ESBL-PB) from rectal swabs inoculated on CHROMagar mSuperCARBA and ESBL media, respectively. Rectal screening ESwabs collected from patients admitted to Grenoble University Hospital between January and June 2021 were analyzed. After inoculation, chromogenic media were incubated for 24 h in the automaton, with image acquisition every 30 min. ScanStation results were compared to visual observations of the plates after 24 h of incubation. In total, 501 rectal swabs were tested. ScanStation showed 100% positive percent agreement (PPA) for the detection of CPB and ESBL-PB, whereas the PPA of color categorization ranged between 45% and 100%. Negative percent agreement (NPA) ranged between 70% and 98%. Negative predictive values (NPVs) were 100% for both bacterial groups, whereas positive predictive values (PPVs) were 70.3% for CPB and 81.0% for ESBL-PB. Importantly, real-time screening allowed detection of the first suspected colony within 10-14 h of growth, on average, whereas visual observation is usually only performed once a day after 18-24 h of incubation. Our study demonstrates the potential use of early images to accelerate the detection of CPB and ESBL-PB and implement effective and timely infection control measures. IMPORTANCE The ScanStation 100 device is an incubator able to follow the real-time growth of bacterial colonies on agar plates through digital imaging, allowing users to sort plates according to the presence or absence of colonies, and to distinguish their color using four numeric color filters. Real-time screening shows that first colony detection is possible much earlier (after 10-14 h of growth, on average), whereas visual observation is usually performed only once a day after 18-24 h of incubation. The ScanStation device, combined with chromogenic agar media, is an efficient automated screening method to accelerate the detection of Gram-negative multidrug-resistant bacteria in laboratories that do not have access to larger laboratory automation systems. Our study shows that setting the image acquisition to one or two early images may allow for the detection of positive samples that were inoculated in the morning, by the end of the working day.

Biomarker-Driven Developments in the Context of the New Regulatory Framework for Companion Diagnostics in the European Union.

The new In Vitro Diagnostic Regulation (EU) 2017/746 (IVDR) introduces important changes in the EU legal framework for companion diagnostics (CDx), including a new risk-based classification system for in vitro diagnostic tests (IVDs), a first legal definition for CDx and enhanced involvement of notified bodies in the conformity assessment and certification process of CDx. The IVDR also establishes an important link between the assessment of a CDx and the corresponding medicinal product by requiring the notified body to seek a scientific opinion from the medicines regulator on the suitability of the CDx for use with the concerned medicinal product(s) before issuing an IVD certificate. Whereas the IVDR aims at establishing a robust regulatory framework for IVDs, it is also associated with several challenges, such as insufficient capacity of notified bodies and readiness of manufacturers. To ensure timely access for patients to essential IVDs, a progressive roll-out for this new legislation has been introduced. In addition, the new consultation process for CDx requires increased collaboration and alignment of assessments performed by the different stakeholders involved in this process. The European Medicines Agency (EMA) and notified bodies are currently building experience based on the first CDx consultation procedures that have been submitted from January 2022 onward. In the current article, we describe the new European regulatory framework for certification of CDx and highlight several challenges for medicine and CDx co-development. In addition, we briefly touch upon the interplay between the Clinical Trial Regulation (EU) No. 536/2014 (CTR) and the IVDR.

Peripheral blood mononuclear cell tissue factor (F3 gene) transcript levels and circulating extracellular vesicles are elevated in severe coronavirus 2019 (COVID-19) disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with excessive coagulation, thrombosis, and mortality. OBJECTIVE: To provide insight into mechanisms that contribute to excessive coagulation in coronavirus 2019 (COVID-19) disease. PATIENTS/METHODS: Blood from COVID-19 patients was investigated for coagulation-related gene expression and functional activities. RESULTS: Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from severe COVID-19 patients revealed a 5.2-fold increase in tissue factor (TF [F3 gene]) transcript expression levels (P < .05), the trigger of extrinsic coagulation; a 7.7-fold increase in C1-inhibitor (SERPING1 gene; P < .01) transcript expression levels, an inhibitor of intrinsic coagulation; and a 4.4-fold increase in anticoagulant thrombomodulin (TM [THBD gene]) transcript expression levels (P < .001). Bulk RNA-seq analysis of sorted CD14+ monocytes on an independent cohort of COVID-19 patients confirmed these findings (P < .05). Indicative of excessive coagulation, 41% of COVID-19 patients' plasma samples contained high D-dimer levels (P < .0001); of these, 19% demonstrated extracellular vesicle TF activity (P = .109). COVID-19 patients' ex vivo plasma-based thrombin generation correlated positively with D-dimer levels (P < .01). Plasma procoagulant extracellular vesicles were elevated ∼9-fold in COVID-19 patients (P < .01). Public scRNA-seq data sets from bronchoalveolar lung fluid and our peripheral blood mononuclear cell scRNA-seq data show CD14+ monocytes/macrophages TF transcript expression levels are elevated in severe but not mild or moderate COVID-19 patients. CONCLUSIONS: Beyond local lung injury, SARS-CoV-2 infection increases systemic TF (F3) transcript levels and elevates circulating extracellular vesicles that likely contribute to disease-associated coagulation, thrombosis, and related mortality.

D-dimer predicts venous thromboembolism in multiple myeloma: a nested case-control study.

Background: Clinical risk assessment scores, such as IMPEDE VTE, can identify patients with multiple myeloma (MM) at high-risk of venous thromboembolism (VTE). Refinement of these scores, by including 1 or more biomarkers, could improve risk assessment. Objectives: We sought to determine the association between soluble P-selectin (sP-selectin) and D-dimer with VTE in MM. Methods: We identified 545 patients with newly diagnosed MM. Using a nested case-control design, we identified 38 cases of VTE within 6-months of MM treatment and 137 randomly selected controls. Using logistic regression, we examined the association between D-dimer and sP-selectin with VTE. We also analyzed the association after adjusting for IMPEDE VTE. Results: Each 1-point increase in IMPEDE VTE score was associated with a 27% increase in odds of VTE (odds ratio 1.27; 95% CI 1.08-1.51; c-statistic 0.61; 95% CI 0.51-0.71). There was no association between sP-selectin and VTE. Each one increase in natural log of D-dimer was associated with a 44% increase in odds of VTE, so we assigned points (ranging from -2 to +2) to D-dimer values and incorporated them into IMPEDE VTE, forming IMPEDED VTE. There was a 30% increase in odds of VTE per each 1-point increase in IMPEDED VTE (OR 1.30; 95% CI 1.12-1.52; c-statistic 0.65; 95% CI 0.55-0.75). Conclusion: Among patients with newly diagnosed MM starting chemotherapy, D-dimer was associated with increased odds of developing VTE within the subsequent 6-months. The addition of D-dimer to IMPEDE VTE-IMPEDED VTE-could improve prediction of VTE among patients with MM.

Doxycycline versus azithromycin for the treatment of anorectal Chlamydia trachomatis infection in women concurrent with vaginal infection (CHLAZIDOXY study): a multicentre, open-label, randomised, controlled, superiority trial.

BACKGROUND: Anorectal infections with Chlamydia trachomatis are commonly found in women. Although the efficacy of doxycycline and azithromycin is comparable in the treatment of urogenital infection, their efficacies toward anorectal infection remain unclear. We therefore aimed to compare a single dose of azithromycin with a 7-day course of doxycycline for the treatment of anorectal C trachomatis infection in women with concurrent vaginal infection. METHODS: We did a multicentre, open-label, randomised, controlled, superiority trial involving four sexually transmitted infection screening centres and three pregnancy termination centres in France. We included sexually active adult women (≥18 years) with a positive C trachomatis vaginal swab who agreed to provide self-collected anorectal swabs for C trachomatis detection. Participants were randomly assigned (1:1), using block sizes of six and eight and stratification by each investigating centre, to orally receive either azithromycin (a single 1-g dose, with or without food) or doxycycline (100 mg in the morning and evening at mealtimes for 7 days [ie, 100 mg of doxycycline twice per day for 7 days]). All laboratory staff who did the bacteriological analyses, but not the participants and the investigators, were masked to the treatment groups. The primary outcome was the microbiological anorectal cure rate defined as a C trachomatis-negative nucleic acid amplification test (NAAT) result in anorectal specimens 6 weeks after treatment initiation among women who had a baseline C trachomatis-positive anorectal NAAT result. The primary analysis was done in the modified intention-to-treat population, with multiple imputation, which included all women who underwent randomisation and had a C trachomatis-positive vaginal and anorectal NAAT result at baseline. Adverse events were reported in all women who underwent randomisation. This study is registered with ClinicalTrials.gov, number NCT03532464. FINDINGS: Between Oct 19, 2018, and April 17, 2020, we randomly assigned a total of 460 participants to either the doxycycline group (n=230) or the azithromycin group (n=230). Four (1%) of 460 participants were excluded because they refused to take doxycycline or were found to be ineligible after randomisation. Among the 456 participants, 357 (78%) had a concurrent C trachomatis-positive anorectal NAAT result at baseline; 184 (52%) of 357 were in the doxycycline group and 173 (48%) were in the azithromycin group (ie, the modified intention-to-treat population). Microbiological anorectal cure occurred in 147 (94%) of 156 participants in the doxycycline group (28 missing values) versus 120 (85%) of 142 in the azithromycin group (31 missing values; adjusted odds ratio with imputation of missing values 0·43 [95% CI 0·21-0·91]; p=0·0274). Reported adverse events possibly related to treatment were notified in 53 (12%) of 456 women: 24 (11%) of 228 in the doxycycline group and 29 (13%) of 228 in the azithromycin group. Gastrointestinal disorders were the most frequently occurring, in 43 (9%) of 456 women: 17 (8%) of 228 in the doxycycline group and 26 (11%) of 228 in the azithromycin group. INTERPRETATION: The microbiological anorectal cure rate was significantly lower among women who received a single dose of azithromycin than among those who received a 1-week course of doxycycline. This finding suggests that doxycycline should be the first-line therapy for C trachomatis infection in women. FUNDING: French Ministry of Health. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

The EU Response to the Presence of Nitrosamine Impurities in Medicines.

The unexpected detection of nitrosamine impurities in human medicines has recently seen global regulators act to understand the risks of these contaminations to patients and to limit their presence. Over 300 nitrosamines are known, many of which are highly potent mutagenic carcinogens. Regulators first became aware of the presence of nitrosamines in EU medicines in 2018, with reports of detection of N-nitroso-dimethylamine (NDMA) in valsartan from one manufacturer. A subsequent EU review of all valsartan medicines was triggered by the European Medicines Agency (EMA) and was later extended to other angiotensin receptor blockers/sartans. A separate review was also started for ranitidine medicines. This was followed by an EU-wide examination of the risk of presence of nitrosamines in all human medicines. This article reflects on the investigation of the EU regulatory network into the presence of nitrosamines and the scientific knowledge informing recommendations for developers on how to limit nitrosamines in medicines.

Evaluation of Rapid Sepsityper® protocol and specific MBT-Sepsityper module (Bruker Daltonics) for the rapid diagnosis of bacteremia and fungemia by MALDI-TOF-MS.

During bloodstream infections, rapid adaptation of empirical treatment according to the microorganism identified is essential to decrease mortality. The aim of the present study was to assess the microbiological performances of a new rapid version of the Sepsityper® kit (Bruker Daltonics) allowing identification of bacteria and yeast by MALDI-TOF mass spectrometry directly from positive blood cultures in 10 min and of the specific MBT-Sepsityper module for spectra analysis, designed to increase identification performance. Identification rates were determined prospectively on 350 bacterial and 29 fungal positive blood cultures, and compared to conventional diagnostic method. Our rapid diagnosis strategy (Rapid Sepsityper® protocol: one spot with and one without formic acid extraction step) combined to MBT-Sepsityper module provided 65.4%, 78.9% and 62% reliable identification to the species level of monomicrobial positive blood cultures growing respectively Gram-positive, Gram-negative bacteria or yeast. Importantly, identification rates of Gram-positive bacteria were higher in anaerobic than in aerobic bottles (77.8% vs 22.2%; p = 0.004), if formic acid extraction step was performed (60.8% vs 39.2%; p = 1.8e-6) and if specific MBT-Sepsityper module was used (76.2% vs 61.9%, p = 0.041) while no significant differences were observed for Gram-negative bacteria. For yeasts identification, formic acid extraction step improved rapid identification rate by 37.9% while the specific MBT-Sepsityper module increased overall performances by 38%, providing up to 89.7% reliable identification if associated with the standard Sepsityper® protocol. These performances, associated with a reduce turnaround time, may help to implement a rapid identification strategy of bloodstream infections in the routine workflow of microbiology laboratories.

Optimized MALDI TOF Mass Spectrometry Identification of Francisella tularensis Subsp. holarctica.

Francisella tularensis is a tier 1 agent causing the zoonosis tularemia. This highly infectious Gram-negative bacterium is occasionally isolated from human samples (especially blood samples) in routine clinical microbiology laboratories. A rapid and accurate method for identifying this pathogen is needed in order to optimize the infected patient's healthcare management and prevent contamination of the laboratory personnel. MALDI TOF mass spectrometry has become the gold standard for the rapid identification of most human pathogens. However, F. tularensis identification using such technology and commercially available databases is currently considered unreliable. Real-time PCR-based methods for rapid detection and accurate identification of F. tularensis are not available in many laboratories. As a national reference center for tularemia, we developed a MALDI TOF database allowing accurate identification of the species F. tularensis and its differentiation from the closely related neighbor species F. tularensis subsp. novicida and F. philomiragia. The sensitivity and specificity of this database were validated by testing 71 F. tularensis strains and 165 strains from 63 species not belonging to the Francisella genus. We obtained accurate identification at the species level and differentiation of all the tested bacterial strains. In particular, F. tularensis could be accurately differentiated from other small Gram-negative bacilli occasionally isolated from human samples, including species of the HACEK group and Brucella melitensis.

Evaluation of ID Fungi Plates Medium for Identification of Molds by MALDI Biotyper.

MALDI-TOF mass spectrometry (MS) identification of pathogenic filamentous fungi is often impaired by difficulties in harvesting hyphae embedded in the medium and long extraction protocols. The ID Fungi Plate (IDFP) is a novel culture method developed to address such difficulties and improve the identification of filamentous fungi by MALDI-TOF MS. We cultured 64 strains and 11 clinical samples on IDFP, Sabouraud agar-chloramphenicol (SAB), and ChromID Candida agar (CAN2). We then compared the three media for growth, ease of harvest, amount of material picked, and MALDI-TOF identification scores after either rapid direct transfer (DT) or a long ethanol-acetonitrile (EA) extraction protocol. Antifungal susceptibility testing and microscopic morphology after subculture on SAB and IDFP were also compared for ten molds. Growth rates and morphological aspects were similar for the three media. With IDFP, harvesting of fungal material for the extraction procedure was rapid and easy in 92.4% of cases, whereas it was tedious on SAB or CAN2 in 65.2% and 80.3% of cases, respectively. The proportion of scores above 1.7 (defined as acceptable identification) were comparable for both extraction protocols using IDFP (P = 0.256). Moreover, rates of acceptable identification after DT performed on IDFP (93.9%) were significantly higher than those obtained after EA extraction with SAB (69.7%) or CAN2 (71.2%) (P = <0.001 and P = 0.001, respectively). Morphological aspects and antifungal susceptibility testing were similar between IDFP and SAB. IDFP is a culture plate that facilitates and improves the identification of filamentous fungi, allowing accurate routine identification of molds with MALDI-TOF-MS using a rapid-extraction protocol.

Latest clinical recommendations on valproate use for migraine prophylaxis in women of childbearing age: overview from European Medicines Agency and European Headache Federation.

Migraine is a common and burdensome neurological condition which affects mainly female patients during their childbearing years. Valproate has been widely used for the prophylaxis of migraine attacks and is also included in the main European Guidelines. Previous (2014) European recommendations on limiting the use of valproate in women of childbearing age did not achieve their objective in terms of limiting the use of valproate in women of childbearing age and raising awareness regarding the hazardous effect of valproate to children exposed in utero. The teratogenic and foetotoxic effects of valproate are well documented, and more recent studies show that there is an even greater neurodevelopmental risk to children exposed to valproate in the womb. The latest 2018 European review from the European Medicines Agency, with the active participation of the European Headache Federation, concluded that not enough has been done to mitigate the risks associated with in utero exposure to valproate. The review called for more extensive restrictions to the conditions for prescribing, better public awareness, and a more effective education campaign in migrainous women.

Combining noninvasive genetics and a new mammalian sex-linked marker provides new tools to investigate population size, structure and individual behaviour: An application to bats.

Monitoring wild populations is crucial for their effective management. Noninvasive genetic methods provide robust data from individual free-ranging animals, which can be used in capture-mark-recapture (CMR) models to estimate demographic parameters without capturing or disturbing them. However, sex- and status-specific behaviour, which may lead to differences in detection probabilities, is rarely considered in monitoring. Here, we investigated population size, sex ratio, sex- and status-related behaviour in 19 Rhinolophus hipposideros maternity colonies (Northern France) with a noninvasive genetic CMR approach (using faeces) combined with parentage assignments. The use of the DDX3X/Y-Mam sexual marker designed in this study, which shows inter- and intrachromosomal length polymorphism across placental mammals, together with eight polymorphic microsatellite markers, produced high-quality genetic data with limited genotyping errors and allowed us to reliably distinguish different categories of individuals (males, reproductive and nonreproductive females) and to estimate population sizes. We showed that visual counts represent well-adult female numbers and that population composition in maternity colonies changes dynamically during the summer. Before parturition, colonies mainly harbour pregnant and nonpregnant females with a few visiting males, whereas after parturition, colonies are mainly composed of mothers and their offspring with a few visiting nonmothers and males. Our approach gives deeper insight into sex- and status-specific behaviour, a prerequisite for understanding population dynamics and developing effective monitoring and management strategies. Provided sufficient samples can be obtained, this approach can be readily applied to a wide range of species.

Comparative analysis of the scope of European Union paediatric investigation plans with corresponding orphan designations.

BACKGROUND: Market forces may not be sufficient to stimulate research and development of medicines for small patient populations, such as children and patients with rare diseases. Both the European Union Orphan and Paediatric Regulations were introduced to address the unmet public health needs of these smaller patient populations through the use of incentives, rewards and obligations. Developers for new medicines for rare diseases must agree a paediatric investigation plan (PIP) or waiver with the European Medicines Agency's (EMA) Paediatric Committee (PDCO), and can also apply for an orphan designation (OD) from the EMA's Committee of Orphan Medicinal Products (COMP). The scope of both the OD and the PIP (or waiver) is defined by the agreed condition. OBJECTIVES: The aim of this study was to analyse the approach of PDCO and COMP in defining the appropriate condition for a PIP or OD, respectively, in order to investigate potential challenges in the paediatric development of orphan medicines which have to meet the requirements of both legislations. METHODS: A comparative analysis of PIP conditions and OD conditions was performed for medicines that have been reviewed by both Committees. RESULTS: We found that in the substantial majority of cases there is no divergence between the conclusions of COMP and PDCO with regard to the condition for which a medicine is to be developed. CONCLUSION: These findings demonstrate that a collaborative approach allows both Regulations to work synergistically to foster pharmaceutical development for rare diseases in childhood.

Professional's Perspectives on Care Management of Young People with Perinatally Acquired HIV during Transition: A Qualitative Study in Adult Care Setting.

BACKGROUND: Increasing numbers of young people with perinatally acquired HIV are surviving to adulthood. When they come of age, they leave pediatric services in which they were followed and have to be transferred to the adult health care system. Difficulties in adaptation to adult care and the numbers of young people lost to follow up after transfer to adult care have been reported. This transition phase and their retention in adult care are crucial in maintaining the clinical status of these young with HIV in adulthood. Our study aimed to explore how HIV professionals working in adult care perceive and adapt their practices to young people in transition. METHODS: Qualitative interviews were conducted with 18 health and social services professionals in hospitals or patient associations in France. A thematic analysis was conducted. RESULTS: Adult care professionals were found to be making a distinction between these young people and their patients who were infected during adulthood. On the basis of the healthcare teams' experience, a simplified categorization of these young people into four levels can be used: those "who have everything good"; those who have some deficiencies that must be addressed; those "who have everything bad"; and those lost to follow up. Professionals interviewed highlighted the difficulties they encountered with young people in transition. Three types of problematic situations were identified: problems of acceptance of the disease; communication problems; and problems of disorientation in the new care environment. CONCLUSIONS: Despite the lack of specific training or national policy recommendations for the integration of young people with perinatally acquired HIV into adult services, all the adult healthcare teams interviewed tried to adapt their practice to this population. The results suggested that professional involvement during transition should depend on the characteristics of the patient, not be limited to a single transition model and that a dedicated structure for transition care is not appropriate for all young people.

[Adolescent medicine: conduct in the context of familial, social and/or scholastic breakdown(s)]. / Médecine de l'adolescent: conduite à tenir dans le cadre du concept de rupture.

Adolescent medicine is not a specialty but constitutes a specific clinical practice. The majority of teenagers go through adolescence without necessarily requiring systematic medical monitoring For others, it is a period that reveals past and/or present events that can combine with one another and be a source of morbidity: post-traumatic symptoms, familial dysfunction, behavioral problems unmasking a psychiatric disorder, onset of chronic disease... Medical follow-up of adolescents brings to light unique clinical profiles reflecting the existence of familial, social and/or scholastic rupture(s). Herein, through the introduction of the concept of rupture, we describe the principals of pragmatic medical management. This approach emphasizes that, first and foremost, the teenager must be reassured as to the proper functioning of his/her body, so that, in parallel or secondarily, psychotherapy can be envisaged. This strategy, while providing a guide for an individual physician, demonstrates that, in certain cases, he/she can work in tandem with a multidisciplinary hospital unit, in which all care functions can be mobilized around the youth.

Factor V, tissue factor pathway inhibitor, and east Texas bleeding disorder.

In a report reading like a fascinating detective story, Vincent and colleagues crack the mysterious case of east Texas bleeding disorder. They show that affected individuals have a mutation in exon 13 of the coagulation F5 gene that causes increased expression of an alternatively spliced transcript, which encodes a previously unrecognized factor V (FV) isoform they call FV-short. This FV isoform lacks a large portion of the B domain of FV, which is normally released upon the proteolytic activation of FV by thrombin and binds tightly to the coagulation regulator tissue factor pathway inhibitor-α (TFPIα). This interaction leads to an approximately 10-fold increase in the level of TFPIα circulating in plasma and a resultant anticoagulant effect that produces a hemorrhagic diathesis.

[Can narrative medicine be an answer to patient physician relationship teaching according to students' demand in medical education curricula?]. / L'enseignement de la médecine narrative peut-il être une réponse à l'attente de formation des étudiants à la relation médecin-malade ?

INTRODUCTION: Coming from literature and medicine and medical humanities north American seminars, narrative medicine has applied narratology for analyzing patients' discourse and has been taught during a decade. METHODS: At Paris Descartes School of Medicine a twenty-hour narrative medicine elective program including whole class lectures and writing and reading small group exercises for second year medical students has been assessed using satisfaction questionnaires. RESULTS: Although several students were uncomfortable with the first writing and reading exercises, the whole satisfaction scores demonstrate that this new program is very well appreciated even when students did not choose this program because they were interested with the patient physician relationship. These results have been confirmed when all students state this program should be continued and when half of them state this program should be offered to more students or made mandatory. DISCUSSION: The primary focus on literary characteristics of patients' and physicians' discourses, without ignoring psychoanalysis theory, has shown to be safe for young students. Writing exercises are encouraged but not mandatory, and reading is optional if ever they feel embarrassed after producing their own texts. Narrative medicine impact on students' attitudes and behaviors has now to be assessed before implementing new educational programs.

Tissue factor pathway inhibitor: structure-function.

TFPI is a multivalent, Kunitz-type proteinase inhibitor, which, due to alternative mRNA splicing, is transcribed in three isoforms: TFPIalpha, TFPIdelta, and glycosyl phosphatidyl inositol (GPI)-anchored TFPIbeta. The microvascular endothelium is thought to be the principal source of TFPI and TFPIalpha is the predominant isoform expressed in humans. TFPIalpha, apparently attached to the surface of the endothelium in an indirect GPI-anchor-dependent fashion, represents the greatest in vivo reservoir of TFPI. The Kunitz-2 domain of TFPI is responsible for factor Xa inhibition and the Kunitz-1 domain is responsible for factor Xa-dependent inhibition of the factor VIIa/tissue factor catalytic complex. The anticoagulant activity of TFPI in one-stage coagulation assays is due mainly to its inhibition of factor Xa through a process that is enhanced by protein S and dependent upon the Kunitz-3 and carboxyterminal domains of full-length TFPIalpha. Carboxyterminal truncated forms of TFPI as well as TFPIalpha in plasma, however, inhibit factor VIIa/tissue factor in two-stage assay systems. Studies in gene-disrupted mice demonstrate the physiological importance of TFPI.

TFPIß is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes.

Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIß mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIß protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIß based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIß.