Tn-seq of the Candida glabrata reference strain CBS138 reveals epigenetic plasticity, structural variation, and intrinsic mechanisms of resistance to micafungin.

C. glabrata is an opportunistic pathogen that can resist common antifungals and rapidly acquire multidrug resistance. A large amount of genetic variation exists between isolates, which complicates generalizations. Portable Tn-seq methods can efficiently provide genome-wide information on strain differences and genetic mechanisms. Using the Hermes transposon, the CBS138 reference strain and a commonly studied derivative termed 2001 were subjected to Tn-seq in control conditions and after exposure to varying doses of the clinical antifungal micafungin. The approach revealed large differences between these strains, including a 131 kb tandem duplication and a variety of fitness differences. Additionally, both strains exhibited up to 1000-fold increased transposon accessibility in subtelomeric regions relative to the BG2 strain, indicative of open subtelomeric chromatin in these isolates and large epigenetic variation within the species. Unexpectedly, the Pdr1 transcription factor conferred resistance to micafungin through targets other than CDR1 . Other micafungin resistance pathways were also revealed including mannosyltransferase activity and biosynthesis of the lipid precursor sphingosine, the drugging of which by SDZ 90-215 or myriocin enhanced the potency of micafungin in vitro . These findings provide insights into complexity of the C. glabrata species as well as strategies for improving antifungal efficacy. Summary: Candida glabrata is an emerging pathogen with large genetic diversity and genome plasticity. The type strain CBS138 and a laboratory derivative were mutagenized with the Hermes transposon and profiled using Tn-seq. Numerous genes that regulate innate and acquired resistance to an important clinical antifungal were uncovered, including a pleiotropic drug resistance gene (PDR1) and a duplication of part of one chromosome. Compounds that target PDR1 and other genes may augment the potency of existing antifungals.

Calcineurin-dependent contributions to fitness in the opportunistic pathogen Candida glabrata.

The protein phosphatase calcineurin is vital for the virulence of the opportunistic fungal pathogen Candida glabrata. The host-induced stresses that activate calcineurin signaling are unknown, as are the targets of calcineurin relevant to virulence. To potentially shed light on these processes, millions of transposon insertion mutants throughout the genome of C. glabrata were profiled en masse for fitness defects in the presence of FK506, a specific inhibitor of calcineurin. Eighty-seven specific gene deficiencies depended on calcineurin signaling for full viability in vitro both in wild-type and pdr1∆ null strains lacking pleiotropic drug resistance. Three genes involved in cell wall biosynthesis (FKS1, DCW1, FLC1) possess co-essential paralogs whose expression depended on calcineurin and Crz1 in response to micafungin, a clinical antifungal that interferes with cell wall biogenesis. Interestingly, 80% of the FK506-sensitive mutants were deficient in different aspects of vesicular trafficking, such as endocytosis, exocytosis, sorting, and biogenesis of secretory proteins in the endoplasmic reticulum (ER). In response to the experimental antifungal manogepix that blocks GPI-anchor biosynthesis in the ER, calcineurin signaling increased and strongly prevented cell death independent of Crz1, one of its major targets. Comparisons between manogepix, micafungin, and the ER-stressing tunicamycin reveal a correlation between the degree of calcineurin signaling and the degree of cell survival. These findings suggest that calcineurin plays major roles in mitigating stresses of vesicular trafficking. Such stresses may arise during host infection and in response to antifungal therapies.IMPORTANCECalcineurin plays critical roles in the virulence of most pathogenic fungi. This study sheds light on those roles in the opportunistic pathogen Candida glabrata using a genome-wide analysis in vitro. The findings could lead to antifungal developments that also avoid immunosuppression.

Redefining pleiotropic drug resistance in a pathogenic yeast: Pdr1 functions as a sensor of cellular stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (CYB5, SSK1, SSK2, HOG1, TRP1). A bZIP transcription repressor of mitochondrial function (CIN5) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata. Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. IMPORTANCE Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1-a key determinant of fluconazole resistance-is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

Redefining Pleiotropic Drug Resistance in a Pathogenic Yeast: Pdr1 Functions as a Sensor of Cellular Stresses in Candida glabrata.

Candida glabrata is a prominent opportunistic fungal pathogen of humans. The increasing incidence of C. glabrata infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes Hermes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 ( CYB5 , SSK1 , SSK2 , HOG1 , TRP1 ). A bZIP transcription repressor of mitochondrial function ( CIN5 ) positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in C. glabrata . Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. Importance: Candida glabrata is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1 - a key determinant of fluconazole resistance - is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.

Ontogeny, evolution and palaeogeographic distribution of the world's largest ammonite Parapuzosia (P.) seppenradensis (Landois, 1895).

The world's largest ammonite, Parapuzosia (P.) seppenradensis (Landois, 1895), fascinated the world ever since the discovery, in 1895, of a specimen of 1.74 metres (m) diameter near Seppenrade in Westfalia, Germany, but subsequent findings of the taxon are exceedingly rare and its systematic position remains enigmatic. Here we revise the historical specimens and document abundant new material from England and Mexico. Our study comprises 154 specimens of large (< 1 m diameter) to giant (> 1m diameter) Parapuzosia from the Santonian and lower Campanian, mostly with stratigraphic information. High-resolution integrated stratigraphy allows for precise cross-Atlantic correlation of the occurrences. Our specimens were analysed regarding morphometry, growth stages and stratigraphic occurrence wherever possible. Our analysis provides insight into the ontogeny of Parapuzosia (P.) seppenradensis and into the evolution of this species from its potential ancestor P. (P.) leptophylla Sharpe, 1857. The latter grew to shell diameters of about 1 m and was restricted to Europe in the early Santonian, but it reached the Gulf of Mexico during the late Santonian. P. (P.) seppenradensis first appears in the uppermost Santonian- earliest Campanian on both sides of the Atlantic. Initially, it also reached diameters of about 1 m, but gradual evolutionary increase in size is seen in the middle early Campanian to diameters of 1.5 to 1.8 m. P. (P.) seppenradensis is characterized by five ontogenetic growth stages and by size dimorphism. We therefore here include the many historic species names used in the past to describe the morphological and size variability of the taxon. The concentration of adult shells in small geographic areas and scarcity of Parapuzosia in nearby coeval outcrop regions may point to a monocyclic, possibly even semelparous reproduction strategy in this giant cephalopod. Its gigantism exceeds a general trend of size increase in late Cretaceous cephalopods. Whether the coeval increase in size of mosasaurs, the top predators in Cretaceous seas, caused ecological pressure on Parapuzosia towards larger diameters remains unclear.

Genome and transcriptome of a pathogenic yeast, Candida nivariensis.

We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.

A new comb-star (Asteroidea, Astropectinidae) from the Upper Triassic (Carnian) of China.

A new astropectinid asteroid, Eoastropecten sechuanensis gen. et sp. nov. is described from the Upper Triassic (Carnian) of Sechuan Province, China, based upon marginal ossicles. This occurrence extends the known fossil record of the family Astropectinidae back by 50 myr from the Lower Jurassic, and indicates that a significant part of the radiation of the neoasteroids probably took place around 240 Ma, only 12 myr after the P-T boundary extinction.

The fossil record of the asteroid (Echinodermata) family Chaetasteridae Sladen, 1889 and subfamily Hyalothricinae Fisher, 1911.

The Cretaceous asteroid Arthraster is referred to the living family Chaetasteridae on the basis of similarities in ossicular arrangement in the abactinal disc and arms. Chaetasterina, previously identified as a chaetasterid, is transferred to the family Asterinidae, subfamily Hyalothricinae, and constitutes the first fossil record of the subfamily.

Identification of Essential Genes and Fluconazole Susceptibility Genes in Candida glabrata by Profiling Hermes Transposon Insertions.

Within the budding yeasts, the opportunistic pathogen Candida glabrata and other members of the Nakaseomyces clade have developed virulence traits independently from C. albicans and C. auris To begin exploring the genetic basis of C. glabrata virulence and its innate resistance to antifungals, we launched the Hermes transposon from a plasmid and sequenced more than 500,000 different semi-random insertions throughout the genome. With machine learning, we identified 1278 protein-encoding genes (25% of total) that could not tolerate transposon insertions and are likely essential for C. glabrata fitness in vitro Interestingly, genes involved in mRNA splicing were less likely to be essential in C. glabrata than their orthologs in S. cerevisiae, whereas the opposite is true for genes involved in kinetochore function and chromosome segregation. When a pool of insertion mutants was challenged with the first-line antifungal fluconazole, insertions in several known resistance genes (e.g., PDR1, CDR1, PDR16, PDR17, UPC2A, DAP1, STV1) and 15 additional genes (including KGD1, KGD2, YHR045W) became hypersensitive to fluconazole. Insertions in 200 other genes conferred significant resistance to fluconazole, two-thirds of which function in mitochondria and likely down-regulate Pdr1 expression or function. Knockout mutants of KGD2 and IDH2, which consume and generate alpha-ketoglutarate in mitochondria, exhibited increased and decreased resistance to fluconazole through a process that depended on Pdr1. These findings establish the utility of transposon insertion profiling in forward genetic investigations of this important pathogen of humans.

An engineered factor Va prevents bleeding induced by direct-acting oral anticoagulants by different mechanisms.

Control of bleeding with direct-acting oral anticoagulants (DOACs) remains an unmet clinical need. Activated superFactor V (superFVa) is an engineered activated protein C (APC)-resistant FVa variant with enhanced procoagulant activity resulting from an A2/A3 domain disulfide bond and was studied here for control of DOAC-induced bleeding. SuperFVa reversed bleeding induced by FXa inhibitors (rivaroxaban, apixaban), and the FIIa inhibitor dabigatran in BalbC mice. The blocking anti-protein C and APC [(A)PC] antibody SPC-54 also reduced FXa inhibitor induced bleeding similar to superFVa, whereas dabigatran-induced bleeding was not affected. This indicated that sufficient APC was generated to contribute to bleeding in the presence of FXa inhibitors, but not in the presence of dabigatran, suggesting that mechanisms contributing to bleeding differed for FXa and FIIa inhibitors. Despite different mechanisms contributing to bleeding, superFVa effectively reduced bleeding for all DOACs, indicating the versatility of superFVa's properties that contribute to its universal prohemostatic effects for DOAC associated bleeding. Supported by thrombin generation assays on endothelial cells in normal plasma spiked with DOACs and patient plasma anticoagulated with DOACs, 3 complementary mechanisms were identified by which superFVa achieved DOAC class-independent prohemostatic efficiency. These mechanisms are resistance to inactivation by APC, overcoming the FV activation threshold, and maximizing the efficiency of the prothrombinase complex when the available FXa is increased by FVIIa-based prohemostatics. In summary, it is this versatility of superFVa that delineates it from other prohemostatic agents as a promising class-independent rescue agent in bleeding situations associated with DOACs.

Comparing the utility of in vivo transposon mutagenesis approaches in yeast species to infer gene essentiality.

In vivo transposon mutagenesis, coupled with deep sequencing, enables large-scale genome-wide mutant screens for genes essential in different growth conditions. We analyzed six large-scale studies performed on haploid strains of three yeast species (Saccharomyces cerevisiae, Schizosaccaromyces pombe, and Candida albicans), each mutagenized with two of three different heterologous transposons (AcDs, Hermes, and PiggyBac). Using a machine-learning approach, we evaluated the ability of the data to predict gene essentiality. Important data features included sufficient numbers and distribution of independent insertion events. All transposons showed some bias in insertion site preference because of jackpot events, and preferences for specific insertion sequences and short-distance vs long-distance insertions. For PiggyBac, a stringent target sequence limited the ability to predict essentiality in genes with few or no target sequences. The machine learning approach also robustly predicted gene function in less well-studied species by leveraging cross-species orthologs. Finally, comparisons of isogenic diploid versus haploid S. cerevisiae isolates identified several genes that are haplo-insufficient, while most essential genes, as expected, were recessive. We provide recommendations for the choice of transposons and the inference of gene essentiality in genome-wide studies of eukaryotic haploid microbes such as yeasts, including species that have been less amenable to classical genetic studies.

Enhanced SLAMF7 Homotypic Interactions by Elotuzumab Improves NK Cell Killing of Multiple Myeloma.

Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, and CD319), which is highly expressed on multiple myeloma (MM) cells, natural killer (NK) cells, and subsets of other leukocytes. By engaging with FcγRIIIA (CD16), Elo promotes potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) toward SLAMF7+ MM tumor cells. Relapsed/refractory MM patients treated with the combination of Elo, lenalidomide, and dexamethasone have improved progression-free survival. We previously showed that Elo enhances NK cell activity via a costimulation mechanism, independent of CD16 binding. Here, we further studied the effect of Elo on cytotoxicity of CD16-negative NK-92 cells. Elo, but not other SLAMF7 antibodies, uniquely enhanced cytotoxicity mediated by CD16-negative NK-92 cells toward SLAMF7+ target cells. Furthermore, this CD16-independent enhancement of cytotoxicity required expression of SLAMF7 containing the full cytoplasmic domain in the NK cells, implicating costimulatory signaling. The CD16-independent costimulation by Elo was associated with increased expression of NKG2D, ICAM-1, and activated LFA-1 on NK cells, and enhanced cytotoxicity was partially reduced by NKG2D blocking antibodies. In addition, an Fc mutant form of Elo that cannot bind CD16 promoted cytotoxicity of SLAMF7+ target cells by NK cells from most healthy donors, especially if previously cultured in IL2. We conclude that in addition to promoting NK cell-mediated ADCC (CD16-dependent) responses, Elo promoted SLAMF7-SLAMF7 interactions in a CD16-independent manner to enhance NK cytotoxicity toward MM cells.

Auxin-Inducible Depletion of the Essentialome Suggests Inhibition of TORC1 by Auxins and Inhibition of Vrg4 by SDZ 90-215, a Natural Antifungal Cyclopeptide.

Gene knockout and knockdown strategies have been immensely successful probes of gene function, but small molecule inhibitors (SMIs) of gene products allow much greater time resolution and are particularly useful when the targets are essential for cell replication or survival. SMIs also serve as lead compounds for drug discovery. However, discovery of selective SMIs is costly and inefficient. The action of SMIs can be modeled simply by tagging gene products with an auxin-inducible degron (AID) that triggers rapid ubiquitylation and proteasomal degradation of the tagged protein upon exposure of live cells to auxin. To determine if this approach is broadly effective, we AID-tagged over 750 essential proteins in Saccharomyces cerevisiae and observed growth inhibition by low concentrations of auxin in over 66% of cases. Polytopic transmembrane proteins in the plasma membrane, Golgi complex, and endoplasmic reticulum were efficiently depleted if the AID-tag was exposed to cytoplasmic OsTIR1 ubiquitin ligase. The auxin analog 1-napthylacetic acid (NAA) was as potent as auxin on AID-tags, but surprisingly NAA was more potent than auxin at inhibiting target of rapamycin complex 1 (TORC1) function. Auxin also synergized with known SMIs when acting on the same essential protein, indicating that AID-tagged strains can be useful for SMI screening. Auxin synergy, resistance mutations, and cellular assays together suggest the essential GMP/GDP-mannose exchanger in the Golgi complex (Vrg4) as the target of a natural cyclic peptide of unknown function (SDZ 90-215). These findings indicate that AID-tagging can efficiently model the action of SMIs before they are discovered and can facilitate SMI discovery.

Augmentation of Human Monocyte Responses to Lipopolysaccharide by the Protein S and Mer/Tyro3 Receptor Tyrosine Kinase Axis.

Resolution of the inflammatory response requires coordinated regulation of pro- and anti-inflammatory mediator production, together with clearance of recruited inflammatory cells. Many different receptors have been implicated in phagocytosis of apoptotic cells (efferocytosis), including Mer, a receptor tyrosine kinase that can mediate recognition and subsequent internalization of apoptotic cells. In this manuscript, we examine the expression and function of the Tyro3/Axl/Mer (TAM) family of receptors by human monocytes. We demonstrate that the Mer ligand, protein S, binds to the surface of viable monocytes via phosphatidylserine-dependent and -independent mechanisms. Importantly, we have identified a novel role for receptor tyrosine kinase signaling in the augmentation of monocyte cytokine release in response to LPS. We propose that low-level phosphatidylserine exposure on the plasma membrane of viable monocytes allows protein S binding that leads to TAM-dependent augmentation of proinflammatory cytokine production. Our findings identify a potentially important role for TAM-mediated signaling during the initiation phase of inflammation.

Sensitive in vivo Visualization of Breast Cancer Using Ratiometric Protease-activatable Fluorescent Imaging Agent, AVB-620.

With the goal of improving intraoperative cancer visualization, we have developed AVB-620, a novel intravenously administered, in vivo fluorescent peptide dye conjugate that highlights malignant tissue and is optimized for human use. Matrix metalloproteinases (MMPs) hydrolyze AVB-620 triggering tissue retention and a ratiometric fluorescence color change which is visualized using camera systems capable of imaging fluorescence and white light simultaneously. AVB-620 imaging visualizes primary tumors and demonstrated high in vivo diagnostic sensitivity and specificity (both >95%) for identifying breast cancer metastases to lymph nodes in two immunocompetent syngeneic mouse models. It is well tolerated and single-dose toxicology studies in rats determined a no-observed-adverse-effect-level (NOAEL) at >110-fold above the imaging and estimated human dose. Protease specificity and hydrolysis kinetics were characterized and compared using recombinant MMPs. To understand the human translation potential, an in vitro diagnostic study was conducted to evaluate the ability of AVB-620 to differentiate human breast cancer tumor from healthy adjacent tissue. Patient tumor tissue and healthy adjacent breast tissue were homogenized, incubated with AVB-620, and fluorogenic responses were compared. Tumor tissue had 2-3 fold faster hydrolysis than matched healthy breast tissue; generating an assay sensitivity of 96% and specificity of 88%. AVB-620 has excellent sensitivity and specificity for identifying breast cancer in mouse and human tissue. Significant changes were made in the design of AVB-620 relative to previous ratiometric protease-activated agents. AVB-620 has pharmaceutical properties, fluorescence ratio dynamic range, usable diagnostic time window, a scalable synthesis, and a safety profile that have enabled it to advance into clinical evaluation in breast cancer patients.

Heparin Binding to an Engineered Virus-like Nanoparticle Antagonist.

The anticoagulant activity of heparin administered during medical interventions must be reversed to restore normal clotting, typically by titrating with protamine. Given the acute toxicity associated with protamine, we endeavored to generate safer heparin antagonists by engineering bacteriophage Qß virus-like particles (VLPs) to display motifs that bind heparin. A particle bearing a single amino acid change from wild-type (T18R) was identified as a promising candidate for heparin antagonism. Surface potential maps generated through molecular modeling reveal that the T18R mutation adds synergistically to adjacent positive charges on the particle surface, resulting in a large solvent-accessible cationic region that is replicated 180 times over the capsid. Chromatography using a heparin-sepharose column confirmed a strong interaction between heparin and the T18R particle. Binding studies using fluorescein-labeled heparin (HepFL) resulted in a concentration-dependent change in fluorescence intensity, which could be perturbed by the addition of unlabeled heparin. Analysis of the fluorescence data yielded a dissociation constant of approximately 1 nM and a 1:1 binding stoichiometry for HepFL:VLP. Dynamic light scattering (DLS) experiments suggested that T18R forms discrete complexes with heparin when the VLP:heparin molar ratios are equivalent, and in vitro clotting assays confirmed the 1:1 binding stoichiometry as full antagonism of heparin is achieved. Biolayer interferometry and backscattering interferometry corroborated the strong interaction of T18R with heparin, yielding Kd ∼ 1-10 nM. These biophysical measurements further validated T18R, and VLPs in general, for potential clinical use as effective, nontoxic heparin antagonists.

Selective factor VIII activation by the tissue factor-factor VIIa-factor Xa complex.

Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.

Activation-resistant homozygous protein C R229W mutation causing familial perinatal intracranial hemorrhage and delayed onset of thrombosis.

INTRODUCTION: We describe a family with two first-degree cousins who presented with similar phenotypes characterized by neonatal intracranial hemorrhage and subsequent onset of thrombosis. PATIENTS/METHODS: We enrolled the two affected patients, five unaffected family members and fifty-five normal controls. Clinical, laboratory, and radiological characteristics of patients were obtained. Exome sequencing was performed for the older affected child. PROC c.811 C>T was genotyped by PCR in patients, family members, and controls. Protein C amidolytic activity and antigen were measured using the STACHROM® protein C kit and ELISAs. To define functional abnormalities caused by the patients' mutation, recombinant wildtype protein C and its mutants R229W, R229Q and R229A were studied. RESULTS: For the two cousins, protein C amidolytic activity was 61% and 59% and antigen was 57% and 73% (nl 70-140%), respectively. Exome sequencing revealed a homozygous variant in exon 9 of the protein C (PROC) gene c.811 C>T (R229W). The R229W mutation is located in the calcium binding loop of protein C's protease domain that mediates thrombomodulin interactions. Recombinant R229W-protein C mutant was strikingly defective in rate of activation by thrombin: thrombomodulin, suggesting an in vivo deficit in these children for generation of activated protein C. CONCLUSIONS: These cases emphasize that protein C and activated protein C are important in maintaining the integrity of the brain vascular endothelium in humans. Moreover, routine protein C assays utilizing snake venom protease fail to detect protein C mutants that are resistant to thrombin:thrombomodulin activation.

Safety, Stability and Pharmacokinetic Properties of (super)Factor Va, a Novel Engineered Coagulation Factor V for Treatment of Severe Bleeding.

PURPOSE: Activated (super)Factor V ((super)FVa) is a novel engineered FV with excellent prohemostatic efficacy. (Super)FVa has three APC cleavage site mutations and an interdomain disulfide bond. Stability, pharmacokinetics, and immunogenic and thrombogenic potential are reported here. METHODS: Stability and circulating half-life were determined after incubation in buffer and human plasma, and after injection into FVIII-deficient mice. Immunogenicity potential was assessed by B- and T-cell specific epitope prediction and structural analysis using surface area and atomic depth computation. Thrombogenic potential was determined by quantification of lung fibrin deposition in wild-type mice after intravenous injection of (super)FVa (200 U/kg), recombinant human (rh) Tissue Factor (0.4-16 pmol/kg), rhFVIIa (3 mg/kg) or saline. RESULTS: FVa retained full activity over 30 h in buffer, the functional half-life in human plasma was 4.9 h, and circulating half-life in FVIII-deficient mice was ~30 min. Predicted immunogenicity was not increased compared to human FV. While rh Tissue Factor, the positive control, resulted in pronounced lung fibrin depositions (mean 121 µg/mL), (super)FVa did not (6.7 µg/mL), and results were comparable to fibrin depositions with rhFVIIa (7.6 µg/mL) or saline (5.6 µg/mL). CONCLUSION: FVa has an appropriate safety and stability profile for further preclinical development as a prohemostatic against severe bleeding.

Improved coagulation and haemostasis in haemophilia with inhibitors by combinations of superFactor Va and Factor VIIa.

Bypassing inhibitors in haemophilia patients is limited to activated (a) Factor(F)VII products. We introduced "FVa activity augmentation" as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilising properties of superFVa and recombinant human (rh)FVIIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in haemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. SuperFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e. g. 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalise clot lysis times could be reduced by 100-fold (e. g. from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In haemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of haemophilia with inhibitors and it merits further evaluation for new bypassing strategies.