Anti-C1s monoclonal antibody BIVV009 in late antibody-mediated kidney allograft rejection-results from a first-in-patient phase 1 trial.

The classical pathway (CP) of complement may contribute to the pathogenesis of antibody-mediated rejection (ABMR). Selective CP blockade may be a promising strategy to counteract rejection. The objective of this first-in-patient phase 1b trial was to evaluate the safety/tolerability and CP-blocking potential of 4 weekly doses (60 mg/kg) of the anti-C1s antibody BIVV009 in complement-mediated disorders. Here we describe the results in a cohort of 10 stable kidney transplant recipients (median of 4.3 years posttransplantation) with late active ABMR and features of CP activation, such as capillary C4d or complement-fixing donor-specific antibodies (DSA). During 7 weeks follow-up, no severe adverse events were reported, and BIVV009 profoundly inhibited overall and DSA-triggered CP activation in serum. Five of 8 C4d-positive recipients turned C4d-negative in 5-week follow-up biopsies, while another 2 recipients showed a substantial decrease in C4d scores. There was, however, no change in microcirculation inflammation, gene expression patterns, DSA levels, or kidney function. In conclusion, we demonstrate that BIVV009 effectively blocks alloantibody-triggered CP activation, even though short-course treatment had no effect on indices of activity in late ABMR. This initial trial provides a valuable basis for future studies designed to clarify the therapeutic value of CP blockade in transplantation. ClinicalTrials.gov NCT#02502903.

Legionella feeleii: an unusual organism associated with cutaneous infection in an immunocompromised patient.

We report a 23-year-old immunocompromised woman who, following cardiac transplantation, presented with an unusual cutaneous eruption. She developed a widespread pustular rash, systemic symptoms and a high temperature with raised inflammatory markers. The diagnosis was reached when a skin biopsy was cultured onto Legionella agar (buffered charcoal yeast extract) and Legionella feeleii was isolated. The patient was treated with 6 weeks of moxifloxacin and her cutaneous lesions gradually resolved. Cutaneous Legionella infections are uncommon and usually affect immunocompromised patients.

3D Imaging of a Dislocation Loop at the Onset of Plasticity in an Indented Nanocrystal.

Structural quality and stability of nanocrystals are fundamental problems that bear important consequences for the performances of small-scale devices. Indeed, at the nanoscale, their functional properties are largely influenced by elastic strain and depend critically on the presence of crystal defects. It is thus of prime importance to be able to monitor, by noninvasive means, the stability of the microstructure of nano-objects against external stimuli such as mechanical load. Here we demonstrate the potential of Bragg coherent diffraction imaging for such measurements, by imaging in 3D the evolution of the microstructure of a nanocrystal exposed to in situ mechanical loading. Not only could we observe the evolution of the internal strain field after successive loadings, but we also evidenced a transient microstructure hosting a stable dislocation loop. The latter is fully characterized from its characteristic displacement field. The mechanical behavior of this small crystal is clearly at odds with what happens in bulk materials where many dislocations interact. Moreover, this original in situ experiment opens interesting possibilities for the investigation of plastic deformation at the nanoscale.

Effect of the Anti-C1s Humanized Antibody TNT009 and Its Parental Mouse Variant TNT003 on HLA Antibody-Induced Complement Activation-A Preclinical In Vitro Study.

The classic pathway (CP) of complement is believed to significantly contribute to alloantibody-mediated transplant injury, and targeted complement inhibition is currently considered to be a promising approach for preventing rejection. Here, we investigated the mode of action and efficacy of the humanized anti-C1s monoclonal antibody TNT009 and its parental mouse variant, TNT003, in preclinical in vitro models of HLA antibody-triggered CP activation. In flow cytometric assays, we measured the attachment of C1 subcomponents and C4/C3 split products (C4b/d, C3b/d) to HLA antigen-coated flow beads or HLA-mismatched aortic endothelial cells and splenic lymphocytes. Anti-C1s antibodies profoundly inhibited C3 activation at concentrations >20 µg/mL, in both solid phase and cellular assays. While C4 activation was also prevented, this was not the case for C1 subcomponent attachment. Analysis of serum samples obtained from 68 sensitized transplant candidates revealed that the potency of inhibition was related to the extent of baseline CP activation. This study demonstrates that anti-C1s antibodies TNT009 and TNT003 are highly effective in blocking HLA antibody-triggered complement activation downstream of C1. Our results provide the foundation for clinical studies designed to investigate the potential of TNT009 in the treatment or prevention of complement-mediated tissue injury in sensitized transplant recipients.

Effects of antenatal exercise in overweight and obese pregnant women on maternal and perinatal outcomes: a randomised controlled trial.

OBJECTIVE: To assess whether antenatal exercise in overweight/obese women would improve maternal and perinatal outcomes. DESIGN: Two-arm parallel randomised controlled trial. SETTING: Home-based intervention in Auckland, New Zealand. POPULATION AND SAMPLE: Pregnant women with body mass index ≥25 kg/m(2) . METHODS: Participants were randomised to a 16-week moderate-intensity stationary cycling programme from 20 weeks of gestation, or to a control group with no exercise intervention. MAIN OUTCOME MEASURES: Primary outcome was offspring birthweight. Perinatal and maternal outcomes were assessed, with the latter including weight gain, aerobic fitness, quality of life, pregnancy outcomes, and postnatal body composition. Exercise compliance was recorded with heart rate monitors. RESULTS: Seventy-five participants were randomised in the study (intervention 38, control 37). Offspring birthweight (adjusted mean difference 104 g; P = 0.35) and perinatal outcomes were similar between groups. Aerobic fitness improved in the intervention group compared with controls (48.0-second improvement in test time to target heart rate; P = 0.019). There was no difference in weight gain, quality of life, pregnancy outcomes or postnatal maternal body composition between groups. However, compliance with exercise protocol was poor, with an average of 33% of exercise sessions completed. Sensitivity analyses showed that greater compliance was associated with improved fitness (increased test time (P = 0.002), greater VO2 peak (P = 0.015), and lower resting heart rate (P = 0.014)), reduced postnatal adiposity (reduced fat mass (P = 0.007) and body mass index (P = 0.035)) and better physical quality of life (P = 0.034). CONCLUSIONS: Maternal non-weight-bearing moderate-intensity exercise in pregnancy improved fitness but did not affect birthweight or clinical outcomes. TWEETABLE ABSTRACT: Moderate-intensity exercise in overweight/obese pregnant women improved fitness but had no clinical effects.

Discrete structures in continuum descriptions of defective crystals.

I discuss various mathematical constructions that combine together to provide a natural setting for discrete and continuum geometric models of defective crystals. In particular, I provide a quite general list of 'plastic strain variables', which quantifies inelastic behaviour, and exhibit rigorous connections between discrete and continuous mathematical structures associated with crystalline materials that have a correspondingly general constitutive specification.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro.

Complex regulation of the TIR1/AFB family of auxin receptors.

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.

Non-recurrent SEPT9 duplications cause hereditary neuralgic amyotrophy.

BACKGROUND: Genomic copy number variants have been shown to be responsible for multiple genetic diseases. Recently, a duplication in septin 9 (SEPT9) was shown to be causal for hereditary neuralgic amyotrophy (HNA), an episodic peripheral neuropathy with autosomal dominant inheritance. This duplication was identified in 12 pedigrees that all shared a common founder haplotype. METHODS AND RESULTS: Based on array comparative genomic hybridisation, we identified six additional heterogeneous tandem SEPT9 duplications in patients with HNA that did not possess the founder haplotype. Five of these novel duplications are intragenic and result in larger transcript and protein products, as demonstrated through reverse transcription-PCR and western blotting. One duplication spans the entire SEPT9 gene and does not generate aberrant transcripts and proteins. The breakpoints of all the duplications are unique and contain regions of microhomology ranging from 2 to 9 bp in size. The duplicated regions contain a conserved 645 bp exon within SEPT9 in which HNA-linked missense mutations have been previously identified, suggesting that the region encoded by this exon is important to the pathogenesis of HNA. CONCLUSIONS: Together with the previously identified founder duplication, a total of seven heterogeneous SEPT9 duplications have been identified in this study as a causative factor of HNA. These duplications account for one third of the patients in our cohort, suggesting that duplications of various sizes within the SEPT9 gene are a common cause of HNA.

Evaluation of NT-proBNP to predict outcomes in advanced heart failure.

AIMS: To determine which factors predict outcomes in a group of patients with advanced heart failure, and in particular if NT-proBNP provides additional clinical and prognostic information to other haemodynamic and biochemical data. METHODS AND RESULTS: Ninety-one patients were studied who were being evaluated for heart transplantation, with 166 assessments. The patients had advanced heart failure as determined by median cardiac index of 2.0 l/min/m(2), left ventricular end-diastolic diameter of 7.0 mm and levels of NT-proBNP of 2473 pg/ml. Median follow-up time was 359 days. Clinicians were blinded to NT-proBNP levels. NT-proBNP significantly correlated with cardiac index (R = -0.44, p < 0.001), right atrial pressure (R = 0.40, p < 0.001), pulmonary arterial wedge pressure (R = 0.38, p < 0.001) and albumin (R = -0.52, p < 0.001), and total bilirubin with right atrial pressure (R = 0.59, p < 0.001). Cardiac index was the most important independent predictor of outcome (p = 0.0001), although bilirubin (p = 0.001) and NT-proBNP (p < 0.05) were also significant. In patients with a 50% increase in NT-proBNP, 64% had adverse outcomes, whereas those in whom levels were stable, 22% had adverse outcomes (p < 0.05). CONCLUSION: Cardiac index is the primary independent predictor of outcome in advanced heart failure when haemodynamic deterioration is evident. In situations where invasive haemodynamics are not available, total bilirubin (reflecting hepatic congestion) and NT-proBNP (related to haemodynamics) also provide important prognostic information.

Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata.

It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce gamma-casein, a member of the casein gene family, only if they are maintained on floating collagen gels (Emerman, J.T., and D.R. Pitelka, 1977, In Vitro, 13:316-328). In this paper we show: (a) Cells on floating collagen gels secrete not only gamma-casein but also alpha 1-, alpha 2-, and beta-caseins. These are not secreted by cells on plastic and are secreted to only a very limited extent by cells on attached collagen gels. (b) The floating collagen gel regulates at the level of synthesis and/or stabilization of the caseins rather than at the level of secretion alone. Contraction of the floating gel is important in that cells cultured on floating glutaraldehyde cross-linked gels do not secrete any of the caseins. (c) The secretion of an 80,000-mol-wt protein, most probably transferrin, and a 67,000-mol-wt protein, probably butyrophilin, a major protein of the milk fat globule membrane are partially modulated by substrata. However, in contrast to the caseins, these are always detectable in media from cells cultured on plastic and attached gels. (d) Whey acidic protein, a major whey protein, is actively secreted by freshly isolated cells but is secreted in extremely limited quantities in cultured cells regardless of the nature of the substratum used. alpha-Lactalbumin secretion is also decreased significantly in cultured cells. (e) A previously unreported set of proteins, which may be minor milk proteins, are prominently secreted by the mammary cells on all substrata tested. We conclude that while the substratum profoundly influences the secretion of the caseins, it does not regulate the expression of every milk-specific protein in the same way. The mechanistic implications of these findings are discussed.

Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences.

Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)

Hormonal regulation of the polarized function and distribution of Na/H exchange and Na/HCO3 cotransport in cultured mammary epithelial cells.

The time course for development of polarized function and morphological distribution of pH regulatory mechanisms has been examined in a mouse mammary epithelial cell line (31EG4). Monolayers grown on permeable supports had tight junctions when grown 3-4 days in the presence of the lactogenic hormones dexamethasone (D, a synthetic glucocorticoid) and insulin (I), or in D, I, and prolactin (P), but there were no tight junctions in the absence of D. Microspectrofluorimetry of the pH-sensitive dye BCECF was used to measure pH (pHi) in cells mounted in a two-sided perfusion chamber to distinguish pH regulatory activity at the apical and basolateral membranes. Na/H exchange was assayed as the Na-dependent, amiloride-sensitive component of pHi recovery from an acid load induced by a pulse of NH3/NH4-containing solution. When monolayers were grown 3-4 d in the presence of P, D, and I, Na/H exchange was restricted to the basolateral membrane. In contrast, in the absence of P, Na/H exchange was present on both the apical and basolateral membranes. After 5-6 days, in the presence or absence of P, Na/H exchange was present only on the basolateral membrane. An antibody to the NHE-1 isoform of the Na/H exchanger was used to determine its morphological distribution. In all hormone conditions the antibody recognized a protein of approximately 110 kD (Western blot), and confocal immunofluorescence microscopy of this antibody and of an anti-ZO-1 (the marker of the tight junctions) antibody showed that the morphological distribution of the Na/H exchanger was similar to the functional distribution under all hormonal treatments. In addition, a putative Na/HCO3 cotransport system was monitored as a Na-dependent, amiloride-insensitive pHi recovery mechanisms that was inhibited by 200 microM H2DIDS. After treatment with D+I (but not with I alone) cotransport appeared exclusively on the basolateral membrane, and the polarized expression of this transporter was not altered by P. We conclude that when mammary cells are grown in D+I-containing media, the Na/H exchanger is expressed initially (i.e., after 3-4 d) on both the apical and basolateral membranes and later (5-6 d) on only the basolateral membrane. P (in the presence of D+I) selectively speeds this polarization, which is determined by polarized distribution of the exchanger to the apical and/or basal membrane and not by the activation and/or inactivation of transporters.(ABSTRACT TRUNCATED AT 400 WORDS)

Derivation and validation of diagnostic criteria for chronic inflammatory demyelinating polyneuropathy.

To develop diagnostic criteria for chronic inflammatory demyelinating polyneuropathy (CIDP), a retrospective series of patients' records diagnosed by sexpert consensus as CIDP or other chronic polyneuropathies were analyzed. Classification and regression tree analysis was applied to 150 patients to derive a classification rule. According to the rule, diagnosis of CIDP required that a patient have a chronic non-genetic polyneuropathy, progressive for at least eight weeks, without a serum paraprotein and either 1) recordable compound muscle action potentials in > or =75% of motor nerves and either abnormal distal latency in >50% of nerves or abnormal motor conduction velocity in >50% of nerves or abnormal F wave latency in >50% of nerves; or 2) symmetrical onset of motor symptoms, symmetrical weakness of four limbs, and proximal weakness in > or =1 limb. When validated in 117 patients, the rule had 83% sensitivity (95% confidence interval 69%-93%) and 97% specificity (95% confidence interval 89%-99%) and performed better than published criteria.

Rare occurrence of a bigeye tuna Thunnus obesus in British waters, with notes on other occurrences of sub-tropical tunas.

A freshly dead bigeye tuna Thunnus obesus was washed ashore near Burry Port, Wales (51 degrees 40' N; 4 degrees 15' W) in August, 2006. This is only the third occasion that the species has been observed in British waters, and is the largest and most northerly recorded specimen.

Smoking after cardiac transplantation.

Although smoking cessation is a prerequisite prior to listing for cardiac transplantation, some patients return to smoking after recovery. We have covertly assessed the smoking habits of our cardiac transplant recipients (with ethical approval) since 1993 by measuring urinary cotinine: a level of >500 ng/mL signifying continued tobacco use. We retrospectively analyzed survival, causes of death and the development of graft coronary artery disease (GCAD) with respect to the number of positive and negative cotinine levels. One hundred four of 380 (27.4%) patients tested positive for active smoking at some point posttransplant, and 57 (15.0%) tested positive repeatedly. Smokers suffered significantly more deaths due to GCAD (21.2% vs. 12.3%, p < 0.05), and due to malignancy (16.3% vs. 5.8%, p < 0.001). In univariate analysis, smoking after heart transplantation shortened median survival from 16.28 years to 11.89 years. After correcting for the effects of pretransplant smoking in time-dependent multivariate analysis, posttransplant smoking remained the most significant determinant of overall mortality (p < 0.00001). We conclude that tobacco smoking after cardiac transplantation significantly impacts survival by accelerating the development of graft vasculopathy and malignancy. We hope that this information will deter cardiac transplant recipients from relapsing, and intensify efforts in improving cessation rates.

Multicenter experience of ABO-incompatible pediatric cardiac transplantation.

Although ABO blood group incompatible cardiac transplantation in neonates and infants reduces waiting list mortality without compromising outcome, the technique has not been adopted by all centers, and to date Toronto remains the only center to have published results from a large case series. We present a review of ABO-incompatible heart transplantation in the United Kingdom (UK) where current recipient selection criteria differ somewhat from those used in the United States (US) and Canada. Between February 2000 and November 2006, 21 ABO-incompatible cardiac transplants were performed in children aged 2-40 months (median 10.0). Immunosuppression followed standard regimens. Pretransplant donor-specific isohemagglutinins of >1:4, (the UNOS cutoff), were present in five patients and reduced by plasma exchange. After transplantation, 19/21 recipients demonstrated persisting deficiency of donor-specific isohemagglutinins. Significant donor-specific isohemagglutinins levels were detected repeatedly in 2/21 recipients who have shown no clinical or biopsy evidence of rejection. All recipients survive without retransplantation and there have been no episodes of humoral rejection. We conclude it is possible for other centers to replicate the excellent results achieved in Toronto and that ABO-incompatible transplantation may be performed successfully in some patients beyond infancy with established isohemagglutinin production providing preoperative antibody removing strategies are used.

Outcomes of lung transplantation for cystic fibrosis in a large UK cohort.

BACKGROUND: Lung transplantation is an important option to treat patients with advanced cystic fibrosis (CF) lung disease. The outcomes of a large UK cohort of CF lung transplantation recipients is reported. METHODS: Retrospective review of case notes and transplantation databases. RESULTS: 176 patients with CF underwent lung transplantation at our centre. The majority (168) had bilateral sequential lung transplantation. Median age at transplantation was 26 years. Diabetes was common pretransplantation (40%). Polymicrobial infection was common in individual recipients. A diverse range of pathogens were encountered, including the Burkholderia cepacia complex (BCC). The bronchial anastomotic complication rate was 2%. Pulmonary function (forced expiratory volume in 1 s % predicted) improved from a pretransplantation median of 0.8 l (21% predicted) to 2.95 l (78% predicted) at 1 year following transplantation. We noted an acute rejection rate of 41% within the first month. Our survival values were 82% survival at 1 year, 70% at 3 years, 62% at 5 years and 51% at 10 years. Patients with BCC infection had poorer outcomes and represented the majority of those who had a septic death. Data are presented on those free from these infections. Bronchiolitis obliterans syndrome (BOS) and sepsis were common causes of death. Freedom from BOS was 74% at 5 years and 38% at 10 years. Biochemical evidence of renal dysfunction was common although renal replacement was infrequently required (<5%). CONCLUSION: Lung transplantation is an important therapeutic option in patients with CF even in those with more complex microbiology. Good functional outcomes are noted although transplantation associated morbidities accrue with time.

Mitral valve dysfunction and repair following orthotopic heart transplantation: a case report.

Mitral valve dysfunction after orthotopic heart transplantation may cause symptoms refractory to medical therapy. In this report, we present a patient who underwent mitral annuloplasty for severe symptomatic mitral valve insufficiency 9 years after heart transplantation, and we critically appraise the literature available for mitral valve dysfunction in this setting. Mitral valve repair, when feasible, should be considered for mitral insufficiency after transplantation to improve functional status and reduce the risk of retransplantation--this is particularly prudent in view of chronic donor shortage.

Mouse embryogenesis requires the tissue factor extracellular domain but not the cytoplasmic domain.

Recent studies indicate that tissue factor (TF) acts in embryogenesis, metastasis, and angiogenesis. Three independent groups showed that targeted disruption of the murine TF (mTF) gene results in 90% lethality of mTF null embryos at embryonic days 9. 5-10.5. We have demonstrated that expression of wild-type human TF (hTF) from a minigene rescues the embryonic lethality of mTF null embryos. To investigate the role of TF in embryogenesis, we made mutant hTF minigenes whose products either bound FVII/VIIa at a reduced level or lacked the cytoplasmic domain. Two independent transgenic lines expressing the hTF extracellular domain mutant failed to rescue the embryonic lethality of mTF null embryos, suggesting that FVII/VIIa binding by TF, proteolytic activity by the TF/FVIIa complex, or both were required for embryogenesis. In contrast, two transgenic lines expressing the hTF cytoplasmic domain mutant rescued the embryonic lethality of mTF null embryos, indicating that the cytoplasmic domain of TF was not required for embryogenesis. We propose that TF/FVIIa-dependent extracellular protease activity is required for embryogenesis.