Factor Xa Generation by Computational Modeling: An Additional Discriminator to Thrombin Generation Evaluation

Factor (f)Xa is a critical enzyme in blood coagulation that is responsible for the initiation and propagation of thrombin generation. Previously we have shown that analysis of computationally generated thrombin profiles is a tool to investigate hemostasis in various populations. In this study, we evaluate the potential of computationally derived time courses of fXa generation as another approach for investigating thrombotic risk. Utilizing the case (n = 473) and control (n = 426) population from the Leiden Thrombophilia Study and each individual's plasma protein factor composition for fII, fV, fVII, fVIII, fIX, fX, antithrombin and tissue factor pathway inhibitor, tissue factor-initiated total active fXa generation was assessed using a mathematical model. FXa generation was evaluated by the area under the curve (AUC), the maximum rate (MaxR) and level (MaxL) and the time to reach these, TMaxR and TMaxL, respectively. FXa generation was analyzed in the entire populations and in defined subgroups (by sex, age, body mass index, oral contraceptive use). The maximum rates and levels of fXa generation occur over a 10- to 12- fold range in both cases and controls. This variation is larger than that observed with thrombin (3–6 fold) in the same population. The greatest risk association was obtained using either MaxR or MaxL of fXa generation; with an ∼2.2 fold increased risk for individuals exceeding the 90th percentile. This risk was similar to that of thrombin generation(MaxR OR 2.6). Grouping defined by oral contraceptive (OC) use in the control population showed the biggest differences in fXa generation; a >60% increase in the MaxR upon OC use. FXa generation can distinguish between a subset of individuals characterized by overlapping thrombin generation profiles. Analysis of fXa generation is a phenotypic characteristic which may prove to be a more sensitive discriminator than thrombin generation among all individuals

Anticoagulants and the Propagation Phase of Thrombin Generation

The view that clot time-based assays do not provide a sufficient assessment of an individual's hemostatic competence, especially in the context of anticoagulant therapy, has provoked a search for new metrics, with significant focus directed at techniques that define the propagation phase of thrombin generation. Here we use our deterministic mathematical model of tissue-factor initiated thrombin generation in combination with reconstructions using purified protein components to characterize how the interplay between anticoagulant mechanisms and variable composition of the coagulation proteome result in differential regulation of the propagation phase of thrombin generation. Thrombin parameters were extracted from computationally derived thrombin generation profiles generated using coagulation proteome factor data from warfarin-treated individuals (N = 54) and matching groups of control individuals (N = 37). A computational clot time prolongation value (cINR) was devised that correlated with their actual International Normalized Ratio (INR) values, with differences between individual INR and cINR values shown to derive from the insensitivity of the INR to tissue factor pathway inhibitor (TFPI). The analysis suggests that normal range variation in TFPI levels could be an important contributor to the failure of the INR to adequately reflect the anticoagulated state in some individuals. Warfarin-induced changes in thrombin propagation phase parameters were then compared to those induced by unfractionated heparin, fondaparinux, rivaroxaban, and a reversible thrombin inhibitor. Anticoagulants were assessed at concentrations yielding equivalent cINR values, with each anticoagulant evaluated using 32 unique coagulation proteome compositions. The analyses showed that no anticoagulant recapitulated all features of warfarin propagation phase dynamics; differences in propagation phase effects suggest that anticoagulants that selectively target fXa or thrombin may provoke fewer bleeding episodes. More generally, the study shows that computational modeling of the response of core elements of the coagulation proteome to a physiologically relevant tissue factor stimulus may improve the monitoring of a broad range of anticoagulants

Disease Detection by Ultrasensitive Quantification of Microdosed Synthetic Urinary Biomarkers

The delivery of exogenous agents can enable noninvasive disease monitoring, but existing low-dose approaches require complex infrastructure. In this paper, we describe a microdose-scale injectable formulation of nanoparticles that interrogate the activity of thrombin, a key regulator of clotting, and produce urinary reporters of disease state. We establish a customized single molecule detection assay that enables urinary discrimination of thromboembolic disease in mice using doses of the nanoparticulate diagnostic agents that fall under regulatory guidelines for “microdosing.”National Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award F32CA159496-02)Burroughs Wellcome Fund (Career Award at the Scientific Interface)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)David H. Koch Institute for Integrative Cancer Research at MIT (Frontier Research Program

Impact of the Resident Microbiota on the Nutritional Phenotype of Drosophila melanogaster

Background: Animals are chronically infected by benign and beneficial microorganisms that generally promote animal health through their effects on the nutrition, immune function and other physiological systems of the host. Insight into the host-microbial interactions can be obtained by comparing the traits of animals experimentally deprived of their microbiota and untreated animals. Drosophila melanogaster is an experimentally tractable system to study host-microbial interactions. Methodology/Principal Findings: The nutritional significance of the microbiota was investigated in D. melanogaster bearing unmanipulated microbiota, demonstrated by 454 sequencing of 16S rRNA amplicons to be dominated by the a-proteobacterium Acetobacter, and experimentally deprived of the microbiota by egg dechorionation (conventional and axenic flies, respectively). In axenic flies, larval development rate was depressed with no effect on adult size relative to conventional flies, indicating that the microbiota promotes larval growth rates. Female fecundity did not differ significantly between conventional and axenic flies, but axenic flies had significantly reduced metabolic rate and altered carbohydrate allocation, including elevated glucose levels. Conclusions/Significance: We have shown that elimination of the resident microbiota extends larval development and perturbs energy homeostasis and carbohydrate allocation patterns of of D. melanogaster. Our results indicate that th

Drosophila melanogaster as a Model Host for the Burkholderia cepacia Complex

Colonization with bacterial species from the Burkholderia cepacia complex (Bcc) is associated with fast health decline among individuals with cystic fibrosis. In order to investigate the virulence of the Bcc, several alternative infection models have been developed. To this end, the fruit fly is increasingly used as surrogate host, and its validity to enhance our understanding of host-pathogen relationships has been demonstrated with a variety of microorganisms. Moreover, its relevance as a suitable alternative to mammalian hosts has been confirmed with vertebrate organisms.The aim of this study was to establish Drosophila melanogaster as a surrogate host for species from the Bcc. While the feeding method proved unsuccessful at killing the flies, the pricking technique did generate mortality within the populations. Results obtained with the fruit fly model are comparable with results obtained using mammalian infection models. Furthermore, validity of the Drosophila infection model was confirmed with B. cenocepacia K56-2 mutants known to be less virulent in murine hosts or in other alternative models. Competitive index (CI) analyses were also performed using the fruit fly as host. Results of CI experiments agree with those obtained with mammalian models.We conclude that Drosophila is a useful alternative infection model for Bcc and that fly pricking assays and competition indices are two complementary methods for virulence testing. Moreover, CI results indicate that this method is more sensitive than mortality tests

Regulation of early steps of GPVI signal transduction by phosphatases: a systems biology approach

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation

Genome-Wide Patterns of Gene Expression during Aging in the African Malaria Vector Anopheles gambiae

The primary means of reducing malaria transmission is through reduction in longevity in days of the adult female stage of the Anopheles vector. However, assessing chronological age is limited to crude physiologic methods which categorize the females binomially as either very young (nulliparous) or not very young (parous). Yet the epidemiologically relevant reduction in life span falls within the latter category. Age-grading methods that delineate chronological age, using accurate molecular surrogates based upon gene expression profiles, will allow quantification of the longevity-reducing effects of vector control tools aimed at the adult, female mosquito. In this study, microarray analyses of gene expression profiles in the African malaria vector Anopheles gambiae were conducted during natural senescence of females in laboratory conditions. Results showed that detoxification-related and stress-responsive genes were up-regulated as mosquitoes aged. A total of 276 transcripts had age-dependent expression, independently of blood feeding and egg laying events. Expression of 112 (40.6%) of these transcripts increased or decreased monotonically with increasing chronologic age. Seven candidate genes for practical age assessment were tested by quantitative gene amplification in the An. gambiae G3 strain in a laboratory experiment and the Mbita strain in field enclosures set up in western Kenya under conditions closely resembling natural ones. Results were similar between experiments, indicating that senescence is marked by changes in gene expression and that chronological age can be gauged accurately and repeatedly with this method. These results indicate that the method may be suitable for accurate gauging of the age in days of field-caught, female An. gambiae

Hypofibrinolysis in diabetes: a therapeutic target for the reduction of cardiovascular risk

An enhanced thrombotic environment and premature atherosclerosis are key factors for the increased cardiovascular risk in diabetes. The occlusive vascular thrombus, formed secondary to interactions between platelets and coagulation proteins, is composed of a skeleton of fibrin fibres with cellular elements embedded in this network. Diabetes is characterised by quantitative and qualitative changes in coagulation proteins, which collectively increase resistance to fibrinolysis, consequently augmenting thrombosis risk. Current long-term therapies to prevent arterial occlusion in diabetes are focussed on anti-platelet agents, a strategy that fails to address the contribution of coagulation proteins to the enhanced thrombotic milieu. Moreover, antiplatelet treatment is associated with bleeding complications, particularly with newer agents and more aggressive combination therapies, questioning the safety of this approach. Therefore, to safely control thrombosis risk in diabetes, an alternative approach is required with the fibrin network representing a credible therapeutic target. In the current review, we address diabetes-specific mechanistic pathways responsible for hypofibrinolysis including the role of clot structure, defects in the fibrinolytic system and increased incorporation of anti-fibrinolytic proteins into the clot. Future anti-thrombotic therapeutic options are discussed with special emphasis on the potential advantages of modulating incorporation of the anti-fibrinolytic proteins into fibrin networks. This latter approach carries theoretical advantages, including specificity for diabetes, ability to target a particular protein with a possible favourable risk of bleeding. The development of alternative treatment strategies to better control residual thrombosis risk in diabetes will help to reduce vascular events, which remain the main cause of mortality in this condition

Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host–Microbe Model System

Drosophila melanogaster is emerging as an important model of non-pathogenic host–microbe interactions. The genetic and experimental tractability of Drosophila has led to significant gains in our understanding of animal–microbial symbiosis. However, the full implications of these results cannot be appreciated without the knowledge of the microbial communities associated with natural Drosophila populations. In particular, it is not clear whether laboratory cultures can serve as an accurate model of host–microbe interactions that occur in the wild, or those that have occurred over evolutionary time. To fill this gap, we characterized natural bacterial communities associated with 14 species of Drosophila and related genera collected from distant geographic locations. To represent the ecological diversity of Drosophilids, examined species included fruit-, flower-, mushroom-, and cactus-feeders. In parallel, wild host populations were compared to laboratory strains, and controlled experiments were performed to assess the importance of host species and diet in shaping bacterial microbiome composition. We find that Drosophilid flies have taxonomically restricted bacterial communities, with 85% of the natural bacterial microbiome composed of only four bacterial families. The dominant bacterial taxa are widespread and found in many different host species despite the taxonomic, ecological, and geographic diversity of their hosts. Both natural surveys and laboratory experiments indicate that host diet plays a major role in shaping the Drosophila bacterial microbiome. Despite this, the internal bacterial microbiome represents only a highly reduced subset of the external bacterial communities, suggesting that the host exercises some level of control over the bacteria that inhabit its digestive tract. Finally, we show that laboratory strains provide only a limited model of natural host–microbe interactions. Bacterial taxa used in experimental studies are rare or absent in wild Drosophila populations, while the most abundant associates of natural Drosophila populations are rare in the lab