Quantification of Extracellular DNA Network Abundance and Architecture within Streptococcus gordonii Biofilms Reveals Modulatory Factors.

Extracellular DNA (eDNA) is an important component of biofilm matrix that serves to maintain biofilm structural integrity, promotes genetic exchange within the biofilm, and provides protection against antimicrobial compounds. Advances in microscopy techniques have provided evidence of the cobweb- or lattice-like structures of eDNA within biofilms from a range of environmental niches. However, methods to reliably assess the abundance and architecture of eDNA remain lacking. This study aimed to address this gap by development of a novel, high-throughput image acquisition and analysis platform for assessment of eDNA networks in situ within biofilms. Utilizing Streptococcus gordonii as the model, the capacity for this imaging system to reliably detect eDNA networks and monitor changes in abundance and architecture (e.g., strand length and branch number) was verified. Evidence was provided of a synergy between glucans and eDNA matrices, while it was revealed that surface-bound nuclease SsnA could modify these eDNA structures under conditions permissive for enzymatic activity. Moreover, cross talk between the competence and hexaheptapeptide permease systems was shown to regulate eDNA release by S. gordonii. This novel imaging system can be applied across the wider field of biofilm research, with potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit. IMPORTANCE Extracellular DNA (eDNA) is critical for maintaining the structural integrity of many microbial biofilms, making it an attractive target for the management of biofilms. However, our knowledge and targeting of eDNA are currently hindered by a lack of tools for the quantitative assessment of eDNA networks within biofilms. Here, we demonstrate use of a novel image acquisition and analysis platform with the capacity to reliably monitor the abundance and architecture of eDNA networks. Application of this tool to Streptococcus gordonii biofilms has provided new insights into how eDNA networks are stabilized within the biofilm and the pathways that can regulate eDNA release. This highlights how exploitation of this novel imaging system across the wider field of biofilm research has potential to significantly advance interrogation of the mechanisms by which the eDNA network architecture develops, how it can influence biofilm properties, and how it may be targeted for therapeutic benefit.

TEG PlateletMapping assay results may be misleading in the presence of cold stored platelets.

BACKGROUND: Viscoelastic tests (VETs) are used widely to monitor hemostasis in settings such as cardiac surgery. There has also been renewed interest in cold stored platelets (CSPs) to manage bleeding in this setting. CSPs are reported to have altered hemostatic properties compared to room temperature platelets (RTPs), including activation of GPIIb/IIIa. We investigated whether the functional differences between CSP and RTP affected the performance of the PlateletMapping VET on the TEG 5000 and 6s analyzer. METHOD: Platelet concentrates were divided equally into CSP (stored at 4°C ± 2°C) and RTP (stored at 22°C ± 2°C) fractions. Whole blood was treated to induce platelet dysfunction (WBIPD) by incubating with anti-platelet drugs (1.0 µM ticagrelor and 10 µM aspirin) or by simulating cardiopulmonary bypass. WBIPD samples were then mixed with 20% by volume of CSPs or RTPs to model platelet transfusion before analysis using the PlateletMapping VET. RESULTS: Addition of CSPs to WBIPD increased the PlateletMapping MAFIBRIN and MAADP parameters with the TEG 5000 analyzer (both p < 0.0001 compared to addition of buffer alone). This effect was not observed with RTPs. The differential effect of CSPs on the MAFIBRIN corrected after pre-incubation with the GPIIb/IIIa antagonist tirofiban and was quantitatively less with the PlateletMapping test for the TEG 6s analyzer which contains the GPIIb/IIa antagonist abciximab. DISCUSSION: The PlateletMapping MAFIBRIN and MAADP test results may be misleadingly high with CSPs, particularly with the TEG 5000 analyzer, most likely due to constitutive activation of GPIIb/IIIa on CSPs during storage. TEG PlateletMapping results should be interpreted with caution following CSP transfusion.

PIK3R3 is a candidate regulator of platelet count in people of Bangladeshi ancestry.

Background: Blood platelets are mediators of atherothrombotic disease and are regulated by complex sets of genes. Association studies in European ancestry populations have already detected informative platelet regulatory loci. Studies in other ancestries can potentially reveal new associations because of different allele frequencies, linkage structures, and variant effects. Objectives: To reveal new regulatory genes for platelet count (PLT). Methods: Genome-wide association studies (GWAS) were performed in 20,218 Bangladeshi and 9198 Pakistani individuals from the Genes & Health study. Loci significantly associated with PLT underwent fine-mapping to identify candidate genes. Results: Of 1588 significantly associated variants (P < 5 × 10-8) at 20 loci in the Bangladeshi analysis, most replicated findings in prior transancestry GWAS and in the Pakistani analysis. However, the Bangladeshi locus defined by rs946528 (chr1:46019890) did not associate with PLT in the Pakistani analysis but was in the same linkage disequilibrium block (r2 ≥ 0.5) as PLT-associated variants in prior East Asian GWAS. The single independent association signal was refined to a 95% credible set of 343 variants spanning 8 coding genes. Functional annotation, mapping to megakaryocyte regulatory regions, and colocalization with blood expression quantitative trait loci identified the likely mediator of the PLT phenotype to be PIK3R3 encoding a regulator of phosphoinositol 3-kinase (PI3K). Conclusion: Abnormal PI3K activity in the vessel wall is already implicated in the pathogenesis of atherothrombosis. Our identification of a new association between PIK3R3 and PLT provides further mechanistic insights into the contribution of the PI3K pathway to platelet biology.

Pseudohomozygous dysfibrinogenemia.

Hypodysfibrinogenemia (HD) is a heterogeneous disorder in which plasma fibrinogen antigen and function are both reduced but discordant. This report addresses the key clinical question of whether genetic analysis enables clinically useful subclassification of patients with HD. We report a new case and identify a further eight previously documented cases that have the laboratory features of HD but biallelic inheritance of quantitative and qualitative fibrinogen gene variants. The cases displayed both bleeding and thrombosis and sometimes had undetectable fibrinogen activity. In all cases, the predicted effect of the coinherited variants is reduced levels of circulating fibrinogen that is all dysfunctional. We propose the term pseudohomozygous dysfibrinogenemia for this subtype of recessively inherited HD that is distinct from the more commonly recognized monoallelic HD caused by a single fibrinogen gene variant.

Differential effects of direct factor IIa and factor Xa inhibitors in protein C-deficient plasma detected using thrombin generation and viscoelastometry assays.

INTRODUCTION: Protein C (PC) deficiency results in dysregulated thrombin generation and increases thrombosis risk. METHODS: In order to investigate the potential effects of anticoagulant drugs in PC deficiency, we evaluated the pharmacodynamic effect of selective direct factor (F) IIa inhibitors (dabigatran and argatroban), selective direct FXa inhibitors (rivaroxaban and apixaban) and an indirect FXa/FIIa inhibitor (enoxaparin) in commercial PC-deficient plasma using thrombin generation and viscoelastometry assays modified to reflect PC anticoagulant activity. RESULTS: Endogenous thrombin potential (ETP) and peak thrombin concentration (PTC) were increased in PC-deficient plasma but this corrected completely with PC concentrate. Inhibition of FIIa and FXa with the selective inhibitors also corrected the increased ETP and PTC but required high drug concentrations. There was sustained low-level thrombin generation in PC-deficient plasma with FXa inhibitors but not with FIIa inhibitors. Adding PC concentrate to PC-deficient plasma anticoagulated with dabigatran had little additional effect on ETP or PTC. In contrast, addition of even small quantities of PC concentrate to PC-deficient plasma anticoagulated with rivaroxaban further diminished ETP, primarily by abolishing sustained thrombin generation. In the viscoelastometry assay, the coagulation time was shortened and α-angle increased in PC-deficient plasma. These abnormalities reversed with both dabigatran and rivaroxaban. CONCLUSION: The selective direct FXa and FIIa inhibitors at high concentrations both counteracted the abnormal thrombin generation and clot formation observed in PC-deficient plasma, but with qualitative differences in their effects.