Biology subjects

Kundu, S.

Publications and source records attributed to Kundu, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Bacteria-powered living materials enable coral larval settlement

The global decline of coral reefs calls for new strategies to rapidly restock coral populations and maintain ecosystem functions and services. Low recruitment success on degraded reefs hampers coral sexual propagation and contributes to limited genetic diversity and reef resilience. Here, we introduce a living bacteria-powered reef ink (Brink) for assisted coral recruitment. Brink can be rapidly applied to restoration substrates via photopolymerization, and it has been formulated to cultivate two settlement-inducing bacterial strains (Cellulophaga lytica and Thalassotalea euphylliae). Settlement assays performed with broadcast spawning (Montipora capitata) and brooding (Pocillopora acuta) Indo-Pacific corals showed that Brink-coated substrates increased settlement >5-fold compared to uncoated control substrates. Brink can be applied as a coating or 3D bioprinted, leading to various potential applications for integration with reef engineering. Our approach underscores the potential of using functional living materials for augmented ecosystem engineering and reef rehabilitation. SynopsisThis study introduces a functional and sustainable bacteria-powered living material that enhances coral settlement, promoting coral reef rehabilitation and ecosystem resilience.

bioengineering↗

bioRxiv2024

A consensus variant-to-function score to functionally prioritize variants for disease

Identifying and functionally characterizing causal disease variants in genome-wide association studies remains a pressing challenge. Here, we construct a consensus variant-to-function (cV2F) score that assigns a single value to each common single-nucleotide variant in the genome, and helps to predict and characterize causal disease variants. The cV2F score leverages features reflecting variant-level experimentally and computationally predicted function (e.g. allelic imbalance and sequence-based deep learning models) and element-level function (e.g. predicted enhancers), and learns optimal combinations of features by training a gradient boosting model on GWAS fine-mapping results. The cV2F-annotated variants attained an AUPRC of 0.822 at identifying held-out fine-mapped variants. Variants with high cV2F scores are highly enriched for heritability (14.2x, s.e. 0.5) across 66 diseases/traits, are uniquely informative for disease heritability, and are highly predictive of variants implicated by reporter assays; cV2F substantially outperforms previous variant-to-function scores using all of these metrics. GWAS fine-mapping of 110 diseases/traits informed by cV2F identified 14.3% more confidently fine-mapped (PIP > 0.95) variants than non-functionally informed fine-mapping. We further constructed tissue/cell line-specific cV2F scores that prioritize variants based on regulatory potential in specific tissues/cell lines, attaining high heritability enrichment for tissue-related diseases/traits (15.6x, s.e. 2.3) while providing independent information (average correlation of 0.27 with the primary cV2F score). We highlight examples of GWAS loci for which cV2F pinpoints causal variants with high confidence and elucidates their functional role.

genetics↗

bioRxiv2024

Explicitly Acquired Interrelations Among Mental Schema Reduces Cognitive Load and Facilitates Emergence of Novel Responses in Mice and Artificial Neural Networks.

Mammalian brain has evolved to infer from past experiences and elicit context relevant novel behavioural responses hitherto unexpressed by the animal. However, little is known about how prior knowledge influences the emergence of such responses. Remarkably, the brain not only arrives at these responses through logical inferences based on previous leanings, but also acquire new related information, without causing catastrophic interference. Mental schemas have often been proposed as the framework for this phenomenon. In this study, using mice as a model animal, we show that schematic networks not only enhance the cognitive load handling capacity (CLHC) and prevent catastrophic interference, but also facilitate the generation of novel, contextually relevant responses. Interestingly, when the animals were trained in a paradigm that did not invoke the pre-formed mental schema, we observed neither an enhancement to CLHC nor a generation of novel context relevant responses. Based on the principles of mental schemas discovered in our animal experiments, we developed a biologically plausible artificial neural network (ANN) that avoids catastrophic interference and captures the learning properties observed in our experiments. The custom architecture of this ANN enables it to generate responses similar to those of animals in novel scenarios. Significance StatementLittle is known about the role of mental schemas in preventing memory interference--a process in which overlapping or similar memories hinder the acquisition and retention of related information. In this study, we demonstrate that mental schemas enhance cognitive load handling capacity and improve the ability to solve novel but related problems. Using mice as a model, we show that the mere existence of a mental schema is not enough for improved cognitive load handling; instead, the relationship between existing and new information must be explicitly established during the learning process. Based on these findings, we developed a minimalistic artificial neural network (ANN) that effectively mimics this behaviour. These insights pave the way for developing more efficient learning and teaching strategies.

neuroscience↗

bioRxiv2024

Water-saving GC-MC model captures temporally differential enzymatic and transporter activities during C3-CAM transition

CAM (Crassulacean Acid Metabolism) plants reduce the water loss through transpiration in arid environments (Wickell et al., 2021), using an alternative pathway of carbon assimilation. To ensure food security, engineering CAM into C3 plants can be achieved by inverting the stomatal rhythm and the timing of major CO2 uptake from day to night. Identification of the metabolic enzymes and intra-cellular transporters, present in both C3 and CAM but having different differential temporal activities throughout the diel cycle (Yang et al., 2015; Heyduk et al., 2019) and quantitative estimations of the flux distributions along the biochemical trajectory of C3-to-CAM transition may help us to achieve the goal. Here, we simulate a constraint-based combined metabolic model of guard cell (GC) and mesophyll cell (MC), linking temporal fluctuations of temperature (T) and relative humidity (RH) throughout the diurnal cycle with osmolyte accumulation dependent stomatal opening, CO2 uptake and transpirational water loss. Starting with C3 metabolism, gradual increase in water-use efficiency (WUE) captures several known and new differential activities of metabolic enzymes, transporters, sugar-malate cycle etc. in GC and MC during C3-to-CAM transition.

systems biology↗

bioRxiv2024

Majority-LCL: Towards Malaria Cell Detection using Label Contrastive Learning and Majority voting Ensembling

Malaria is a contagious disease caused by Plasmodium, a group of single-celled parasites, and is most commonly transmitted by an infected female Anopheles mosquito. More than 40 percent of the global population is at risk, with approximately 219 million reported cases and around 435000 deaths recorded in 2017 alone. Despite the availability of several advanced diagnostic tools, accurate malaria diagnosis remains challenging in resource-constrained settings, where microscopists often struggle to improve diagnostic accuracy. Deep learning-based cell image classification helps reduce incorrect diagnostic conclusions by enabling automated analysis. This research aims to improve diagnostic accuracy by classifying malaria-infected cells using a majority voting ensemble framework combined with triplet loss aided label contrastive learning. Experimental results demonstrate the effectiveness of the proposed method on microscopic cell images in terms of accuracy, precision, recall, and other evaluation metrics.

bioinformatics↗

bioRxiv2024

RAF2Net: Automated grading of Renal cell Carcinoma utilizing Attention-enhanced deep learning models through Feature Fusion

It is anticipated that the number of instances of kidney cancer will continue to rise globally, which motivates changes to the current diagnostic framework in order to address emerging issues. Renal cell carcinoma (RCC) accounts for 80-85% of all renal tumors and is the most common kind of kidney cancer. Based on kidney histopathology images, this study presented a completely automated, robust, and computationally efficient Renal Cell Carcinoma Grading Network (RAF 2Net). Our suggested model incorporates 3 different Mobilenet backbones with intelligent feature fusion. Moreover, the attention blocks help us give more importance to the important pixels, which are majorly responsible for classification. For comparison purposes, Similar tests were conducted using transfer learning methods with pre-trained ImageNet weights and deep learning models created from scratch. To show the efficacy of the suggested method, we have computed evaluation parameters like Accuracy, Precision, F score, Recall, Confusion Matrix, and TSNE. Based on the provided KMC dataset, the experimental result demonstrates that the proposed RAF 2Net outperforms the nine most recent classification methods regarding prediction Accuracy, Recall, Precision, and F score with a value greater than 92%.

bioinformatics↗

bioRxiv2024

Robust coexistence in ecological competitive communities

Darwin already recognized that competition is fiercest among conspecifics, a principle that later made intraspecific competition central to ecological theory through concepts such as niche differentiation and limiting similarity. Beyond shaping coexistence, strong intraspecific competition can also stabilize community dynamics by ensuring that populations return to equilibrium after disturbance. Here we investigate a more fundamental question: how intraspecific competition influences the very existence of a steady state (feasibility) in large random ecological communities dominated by competition. We show that, analogous to classical results on stability, there is a critical level of intraspecific competition above which a feasible steady state is guaranteed to exist. We derive a general expression for the probability of feasibility and prove that, asymptotically (as species number grows), the transition to stability occurs before the transition to feasibility with probability one. Thus, in large competitive communities, any feasible equilibrium is automatically stable. This ordering persists even when many species in the initial pool cannot coexist and extinctions occur: the dynamics prune the community, shifting feasibility and stability thresholds but never reversing their order. These results imply that large competitive communities generically converge to a globally stable equilibrium, making sustained oscillations or chaos unlikely--consistent with experimental observations.

ecology↗

bioRxiv2024

Lessons from combined metabolic model of mesophyll and guard cells

Understanding of overall metabolisms of guard cell (GC) and mesophyll cell (MC), their possible interactions and roles in stomatal movement will help in designing crop plants with higher water use efficiencies. Although, previous constraint based modelling and analyses correctly predicted some known metabolic patterns of isolated C3 MC, GC and Crassulacean Acid Metabolism (CAM) MC, quantitative estimations of combined metabolism of GC and MC and detailed analysis of GC metabolism in CAM are still awaiting. A six phase diel combined model of GC and MC is constructed integrating existing models of two different cell types with necessary modifications guided by known physiology. It is used to predict the similarities and dissimilarities of GC and MC metabolisms. In addition to previously predicted results, analysis of this new two-cell model successfully shows higher activities of some experimentally observed C4 like enzymes in GC than MC in C3, the possible storage patterns of the osmolytes like K+, malate2-, sucrose etc., in CAM GC. Results also show different patterns of starch synthesis and accumulation, ATP production and utilization in GC and MC of C3 and CAM. This combined model integrates stomatal opening, gaseous exchange and GC-MC metabolisms. It is a significant step towards understanding and quantitative estimation of the whole leaf tissue metabolism linking gaseous exchange with environment. Significance statementOur time-resolved combined metabolic model of guard and mesophyll cells integrates stomatal opening, gaseous exchange and cellular metabolisms in C3, CAM and partial CAM conditions; their analyses provide quantitative estimations of metabolic fluxes, ATP production and utilization, and different metabolic patterns of starch synthesis and accumulation of both the cells. It is a significant step towards understanding and quantitative estimation of the whole leaf tissue metabolism linking gaseous exchange with environment and phloem loading.

systems biology↗

bioRxiv2024

Design of a novel epitope-based tetravalent subunit vaccine against dengue virus: an immunoinformatics approach

Dengue imposes a profound global impact, with millions affected annually. Its transmission by Aedes mosquitoes poses significant challenges to combat, aggravated by urbanization and climate change. Despite efforts, no impeccable antiviral treatment exists to date, highlighting the urgency for a vaccine. Developing one encounters hurdles like the four distinctive serotypes of the virus and complex immune responses. In this study, we employed an immunoinformatics approach to design an epitope-based tetravalent subunit vaccine aimed at confronting all DENV serotypes. The study contemplates epitopes prediction, toxicity assessment, molecular docking, molecular dynamics (MD) simulations, immune simulations. On sequence retrieval, the epitopes were predicted and prioritized. The sequence of the finally designed vaccine was reached after a broad analysis of the antigenicity scores, serotype coverage, and population coverage. Human {beta}-defensin 3 has been added as an adjuvant to the core vaccine sequence that comprises 23 epitopes and linkers. Notably, the vaccine has the highest antigenicity (0.9319) and 97.35% population coverage worldwide. The molecular docking operations of the vaccine with toll-like receptor 2 (TLR2) and TLR4 showed promising interactions with lowest energies of -1240.5 kJ/mol (76 members) and -1393.3 kJ/mol (40 members), respectively. Molecular dynamics (MD) simulations were run up to 200 nanoseconds, and the complexes of the vaccine with TLR2 and TLR4 were found to be very stable and flexible. Moreover, in immune simulations, the vaccine evoked robust immune responses. These findings suggest that our vaccine outperforms any other Dengue vaccine developed to date. However, since this study was conducted through in silico methods, in vitro and in vivo validations are required to confirm the vaccine as a potential candidate for clinical trials. Author summaryWe developed a novel epitope-based tetravalent subunit vaccine against all dengue virus (DENV) serotypes using immunoinformatics. Our vaccine demonstrated high antigenicity (0.9319) and wide population coverage (97.35%). Molecular docking and dynamics simulations indicated strong interactions with immune receptors which is crucial for the vaccines activity inside human body. Moreover, immune simulations showed robust responses indicating proper immunity against DENV. Therefore, our vaccine offers a promising solution to dengue fever, pending further in vitro and in vivo validations for clinical trials. Notably, this is the first ever dengue vaccine with such high efficacy in terms of immunoinformatics and vaccinomic approaches.

bioinformatics↗

bioRxiv2024

The invasion phenotypes of glioblastoma depend on plastic and reprogrammable cell states

Glioblastoma (GBM), the most common primary brain cancer in adults, is characterized by rapid local invasion along diverse routes, such as infiltration of white matter tracts and penetration of perivascular spaces. We investigate the hypothesis that GBM invasion routes correlate with the transcriptional states of individual cells and identify regulators of route-specific invasion. Utilizing patient-derived GBM xenograft models, we integrate single-cell transcriptomics and spatial proteomics, revealing that mesenchymal and oligodendrocyte progenitor-like GBM cells migrate perivascularly, while neural progenitor and astrocyte-like GBM cells invade diffusely. Computational reconstruction identifies ANXA1 as a perivascular invasion driver and lineage-restricted transcription factors RFX4 and HOPX as drivers of diffuse invasion, predictive of patient survival. Genetic ablation of these genes alters invasion phenotypes and extends survival in xenografted mice, clarifying the role of cell states in GBM invasion, and highlighting potential therapeutic targets for selective invasion route targeting in GBM patients.

cancer biology↗

bioRxiv2024

Leukemia aggressiveness is driven by chromatin remodeling and expression changes of core regulators

Mechanisms driving the increase of cell growth in developing leukemia are not fully understood. We focused on epigenomic regulation of this process by analyzing the changes of chromatin marks and gene expression in leukemic cell clones as they progressed towards increased proliferation in a mouse model of acute myeloid leukemia (AML). This progression was characterized by gradual modulation of chromatin states and gene expression across the genome, with a surprising preferential trend of reversing the prior changes associated with the origins of leukemia. Our analyses of this modulation in independently developing clones predicted a small set of potential growth regulators whose transcriptomic and epigenomic progression was consistent between clones and maintained both in vivo and ex vivo. We selected three of these genes as candidates (Irx5 and Plag1 as growth suppressors and Smad1 as a driver) and successfully validated their causal growth effects by overexpression in leukemic cells. Public patient data confirmed expression levels of IRX5 and SMAD1 as markers of AML status and survival, suggesting that multiomic analysis of evolving clones in a mouse model is a valuable predictive approach relevant to human AML.

molecular biology↗

bioRxiv2024

Exposure to live saprophytic Leptospira before challenge with a pathogenic serovar prevents severe leptospirosis and promotes kidney homeostasis

Previous studies demonstrated that Leptospira biflexa, a saprophytic species, triggers innate immune responses in the host during early infection. This raised the question of whether these responses could suppress a subsequent challenge with pathogenic Leptospira. We inoculated C3H/HeJ mice with a single or a double dose of L. biflexa before challenge with a pathogenic serovar, L. interrogans serovar Copenhageni FioCruz (LIC). Pre-challenge exposure to L. biflexa did not prevent LIC dissemination and colonization of the kidney. However, it rescued weight loss and mouse survival thereby mitigating disease severity. Unexpectedly, there was correlation between rescue of overall health (weight gain, higher survival, lower kidney fibrosis marker ColA1) and higher shedding of LIC in urine. This stood in contrast to the L. biflexa unexposed LIC challenged control. Immune responses were dominated by increased frequency of effector T helper (CD4+) cells in spleen, as well as significant increases in serologic IgG2a. Our findings suggest that exposure to live saprophytic Leptospira primes the host to develop Th1 biased immune responses that prevent severe disease induced by a subsequent challenge with a pathogenic species. Thus, mice exposed to live saprophytic Leptospira before facing a pathogenic serovar may withstand infection with far better outcomes. Furthermore, a status of homeostasis may have been reached after kidney colonization that helps LIC complete its enzootic cycle. SignificancePrevious evidence of host innate immunity induced by live saprophytic Leptospira in mice led us to posit that these responses might mitigate leptospirosis severity upon a subsequent challenge with a pathogenic serovar. In this study, we validated our hypothesis. This is important for development of novel strategies to control leptospirosis and for understanding the epidemiologic risk factors of this and other infectious diseases transmitted by direct contact between pathogen and host. Unexpectedly, these studies also show that there is a correlation between kidney health after L. interrogans infection (less fibrosis marker ColA1) and higher shedding of this spirochete in urine. This suggests that a status of homeostasis may be reached after kidney colonization by L. interrogans that helps the spirochete fulfill its enzootic cycle.

immunology↗

bioRxiv2024

Translation coupled mRNA-decay is a function of both structural and codon level characteristics

The majority of mRNA degradation occurs co-translationally. Several works in the past elucidated the role of codon composition in regulating co-translational mRNA decay. Integration of mRNA sequence, structure and ribosomal density unravels common regulatory factors of translational and degradation and helps in understanding the intricate association between these two important processes. Co-translational degradation is a two-step process, involving translational stalling and mRNA release for degradation. Our findings highlight the role of Codon Adaptation Index, a sequence-level feature that serves as the primary determinant of translation rates facilitating transcript release from translational machinery upon stalling. Concurrently, cellular endonucleases targeting Internal Unstructured Segments, facilitating easy degradation of the stalled mRNA transcripts, influencing their half-lives across the genome and over evolutionary timescales.

bioinformatics↗

bioRxiv2024

Characterisation and comparison of semen microbiota and sperm function in men with infertility, recurrent miscarriage, or proven fertility

Several studies have associated seminal microbiota abnormalities with male infertility but have yielded differing results owing to their limited sizes or depths of analyses. The semen microbiota during recurrent pregnancy loss (RPL) has not been investigated. Comprehensively assessing the seminal microbiota in men with reproductive disorders could elucidate its potential role in clinical management. We used semen analysis, terminal-deoxynucleotidyl-transferase-mediated-deoxyuridine-triphosphate-nick-end-labelling, Comet DNA fragmentation, luminol ROS chemiluminescence and metataxonomic profiling of semen microbiota by16S rRNA amplicon sequencing in this prospective, cross-section study to investigate composition and bacterial load of seminal bacterial genera and species, semen parameters, reactive oxidative species (ROS), and sperm DNA fragmentation in men with reproductive disorders and proven fathers. 223 men were enrolled included healthy men with proven paternity (n=63), the male partners in a couple encountering RPL (n=46), men with male factor infertility (n=58), and the male partners of couples unexplained infertility (n=56). Rates of high sperm DNA fragmentation, elevated ROS and oligospermia were more prevalent in the study group compared with control. In all groups, semen microbiota clustered into three major genera-dominant groups (1, Streptococcus; 2, Prevotella; 3, Lactobacillus and Gardnerella); no species clusters were identified. Group 2 had the highest microbial richness (P<0.001), alpha-diversity (P<0.001), and bacterial load (P<0.0001). Overall bacterial composition or load has not found to associate with semen analysis, ROS or DNA fragmentation. Whilst, global perturbation of the seminal microbiota is not associated with male reproductive disorders, men with unidentified seminal Flavobacterium are more likely to have abnormal seminal analysis. Future studies may elucidate if Flavobacterium reduction has therapeutic potential.

microbiology↗

bioRxiv2023

NPZfc: An ecological relation based fish catch prediction model using Artificial Neural Network

Quantifying interactions of organisms of the various trophic levels is very important in understanding the dynamics of aquatic ecosystems. With regards to fish, as both ecologically and commercially important parts of an ecosystem, predicting their catch in relation to primary producers provides insight into sustainable management. The present paper describes a novel model NPZfc, for nutrients, phytoplankton, zooplankton and fishes, which can predict planktivorous fish catch. Unlike the existing models which deal with the interactions within the system through mathematical equilibrium, the proposed model uses artificial neural network (ANN) to automatically learn inter-dependencies between different related variables and predict the fish catch of a water body. The efficiency of the model was increased by refining the input variables. Here biomass of plankton species population (phyto-plankton and zooplankton) were specifically selected from feeding ecology studies of target fish species as input variable. The study of two of the commercially important fish species, Etroplus suratensis and Nematalosa nasus in Chilika lagoon showed that the model can predict with high accuracy from limited input data. The root mean square error (RMSE) is found to be very satisfactory, ranging from 3.53% to 11.5% for E. suratensis and from 1.63% to 2.22% for N. nasus. Higher accuracy and better predictive ability with a smaller dataset makes this ANN-based NPZfc model more conducive.

ecology↗

bioRxiv2023

Transcriptomics and chromatin accessibility in multiple African population samples

Mapping the functional human genome and impact of genetic variants is often limited to European-descendent population samples. To aid in overcoming this limitation, we measured gene expression using RNA sequencing in lymphoblastoid cell lines (LCLs) from 599 individuals from six African populations to identify novel transcripts including those not represented in the hg38 reference genome. We used whole genomes from the 1000 Genomes Project and 164 Maasai individuals to identify 8,881 expression and 6,949 splicing quantitative trait loci (eQTLs/sQTLs), and 2,611 structural variants associated with gene expression (SV-eQTLs). We further profiled chromatin accessibility using ATAC-Seq in a subset of 100 representative individuals, to identity chromatin accessibility quantitative trait loci (caQTLs) and allele-specific chromatin accessibility, and provide predictions for the functional effect of 78.9 million variants on chromatin accessibility. Using this map of eQTLs and caQTLs we fine-mapped GWAS signals for a range of complex diseases. Combined, this work expands global functional genomic data to identify novel transcripts, functional elements and variants, understand population genetic history of molecular quantitative trait loci, and further resolve the genetic basis of multiple human traits and disease.

genomics↗

bioRxiv2023

Potential use of Sodium Butyrate (SB) as an anti-virulence agent against Vibrio cholerae targeting ToxT virulence protein.

ABSTRACTCholera, a diarrhoeal disease caused by gram-negative bacterium Vibrio cholerae remains a global health threat in developing countries owing to its high transmissibility and increase in antibiotic resistance. The current issue is to overcome the problem of resistance by antimicrobial therapy. There is a need for alternative strategies with an emphasis on anti-virulent approaches to alter the outcome of bacterial infections. Vibrio cholerae causes cholera by secreting virulence factors in the intestinal epithelial cells. Virulence factors help in cholera toxin production and colonisation during infection. Here, we show that sodium butyrate (SB), a small molecule, had no effect on bacterial viability but was effective in suppressing the virulence attributes of V. cholerae. The production of cholera toxin (CT) was downregulated in a standard V. cholerae El Tor strain and two clinical isolates when grown in presence of sodium butyrate. Analysis of mRNA and protein levels further demonstrated that sodium butyrate reduced the expression of the ToxT-dependent virulence genes like tcpA and ctxAB. DNA-protein interaction assays conducted at cellular (ChIP) and in in vitro conditions (EMSA) indicated that sodium butyrate weakens the binding between ToxT and its downstream promoter DNA, likely by blocking DNA binding. Furthermore, the efficacy of sodium butyrate was confirmed by showing its anti-virulence activity and tissue damage recovery in animal models. Collectively, these findings suggest that sodium butyrate (SB) has the potential to be developed as an anti-virulence agent against V. cholerae in place of conventional antibiotics or as an adjunctive therapy to combat cholera. IMPORTANCEThe world has been facing an upsurge in cholera cases since 2021 with a similar trend continuing into 2022 with over 29 countries reporting cholera outbreaks (World Health Organization 16 December 2022 Disease Outbreak News; Cholera - Global situation). Treatment of cholera involves oral rehydration therapy coupled with antibiotics to reduce the duration of the illness. However, over the last few years, there has been indiscriminate use of antibiotics that contributed largely to the reservoir of antibiotic-resistant strains. In this study, we have addressed the problem of antibiotic resistance by targeting virulence factors. The screening of several compounds led to the identification of a small molecule, sodium butyrate that inhibits the virulence cascade in V. cholerae. We demonstrated that (i) sodium butyrate intervened with ToxT protein-DNA binding and subsequently affected the expression of ToxT-regulated virulence genes (ctxAB and tcpA) (ii) Sodium Butyrate is a potential therapeutic candidate for development of novel antimicrobial agents.

microbiology↗

bioRxiv2023

Protection of β2GPI Deficient Mice from Thrombosis Reflects a Defect in PAR3-facilitated Platelet Activation

BackgroundAntibodies to {beta}2-glycoprotein I ({beta}2GPI) cause thrombosis in antiphospholipid syndrome, however the role of {beta}2GPI itself in regulation of coagulation pathways in vivo is not well understood. MethodsWe developed {beta}2GPI-deficient mice (Apoh-/-) by deleting exon 2 and 3 of Apoh using CRISPR/Cas9 and compared the propensity of wild-type (WT) and Apoh-/-mice to develop thrombosis using rose bengal and FeCl3-induced carotid thrombosis, laser-induced cremaster arteriolar injury, and inferior vena cava (IVC) stasis models. We also compared tail bleeding times and assessed platelet activation in WT and Apoh-/- mice in the absence and presence of exogenous {beta}2GPI. ResultsCompared to WT littermates, Apoh-/- mice demonstrated a prolonged time to occlusion of the carotid artery after exposure to rose bengal or FeCl3, and reduced platelet and fibrin accumulation in cremasteric arterioles after laser injury. Similarly, significantly smaller thrombi were retrieved from the IVC of Apoh-/-mice 48 hours after IVC occlusion. The activated partial thromboplastin time (aPTT) and prothrombin time, as well as aPTT reagent- and tissue factor-induced thrombin generation times using plasma from Apoh-/- and WT mice revealed no differences. However, we observed significant prolongation of tail bleeding in Apoh-/- mice, and reduced P-selectin expression and binding of fibrinogen to the activated 2b{beta}3 integrin on platelets from these mice after stimulation with low thrombin concentrations; these changes were reversed by exogenous {beta}2GPI. An antibody to PAR3 blocked thrombin-induced activation of WT, but not Apoh-/- platelets, as well as the ability of {beta}2GPI to restore the activation response of Apoh-/- platelets to thrombin. {beta}2GPI deficiency did not affect platelet activation by a PAR4-activator peptide, or ADP. ConclusionsIn mice, {beta}2GPI may mediate procoagulant activity by enhancing the ability of PAR3 to present thrombin to PAR4, promoting platelet activation at low thrombin concentrations. Key PointsO_LI{beta}2GPI deficient mice are protected from experimental arterial, venous, and microvascular thrombosis. C_LIO_LI{beta}2GPI deficient mice display prolonged tail bleeding times and reduced PAR3-facilitated platelet activation by low concentrations of thrombin. C_LI

cell biology↗