Corrigendum to Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction [Research and Practice in Thrombosis and Haemostasis Volume 8, Issue 1, January 2024, 102322].

[This corrects the article DOI: 10.1016/j.rpth.2024.102322.].

Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction.

Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction. Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays. Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected. Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.

RhoGAP6 interacts with COPI to regulate protein transport.

RhoGAP6 is the most highly expressed GTPase-activating protein (GAP) in platelets specific for RhoA. Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of δ-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and δ-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of δ-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable δ-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6/δ-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and δ-COP binding, however, neither δ-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6/δ-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and δ-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets.

Interaction between Engineered Pluronic Silica Nanoparticles and Bacterial Biofilms: Elucidating the Role of Nanoparticle Surface Chemistry and EPS Matrix.

Nanoparticles (NPs) are considered a promising tool in the context of biofilm control. Many studies have shown that different types of NPs can interfere with the bacterial metabolism and cellular membranes, thus making them potential antibacterial agents; however, fundamental understanding is still lacking on the exact mechanisms involved in these actions. The development of NP-based approaches for effective biofilm control also requires a thorough understanding of how the chosen nanoparticles will interact with the biofilm itself, and in particular with the biofilm self-produced extracellular polymeric matrix (EPS). This work aims to provide advances in the understanding of the interaction between engineered fluorescent pluronic silica (PluS) nanoparticles and bacterial biofilms, with a main focus on the role of the EPS matrix in the accumulation and diffusion of the particles in the biofilm. It is demonstrated that particle surface chemistry has a key role in the different lateral distribution and specific affinity to the biofilm matrix components. The results presented in this study contribute to our understanding of biofilm-NP interactions and promote the principle of the rational design of smart nanoparticles as an important tool for antibiofilm technology.

Synthesis and self-assembly of curcumin-modified amphiphilic polymeric micelles with antibacterial activity.

BACKGROUND: The ubiquitous nature of bacterial biofilms combined with the enhanced resistance towards antimicrobials has led to the development of an increasing number of strategies for biofilm eradication. Such strategies must take into account the existence of extracellular polymeric substances, which obstruct the diffusion of antibiofilm agents and assists in the maintenance of a well-defended microbial community. Within this context, nanoparticles have been studied for their drug delivery efficacy and easily customised surface. Nevertheless, there usually is a requirement for nanocarriers to be used in association with an antimicrobial agent; the intrinsically antimicrobial nanoparticles are most often made of metals or metal oxides, which is not ideal from ecological and biomedical perspectives. Based on this, the use of polymeric micelles as nanocarriers is appealing as they can be easily prepared using biodegradable organic materials. RESULTS: In the present work, micelles comprised of poly(lactic-co-glycolic acid) and dextran are prepared and then functionalised with curcumin. The effect of the functionalisation in the micelle's physical properties was elucidated, and the antibacterial and antibiofilm activities were assessed for the prepared polymeric nanoparticles against Pseudomonas spp. cells and biofilms. It was found that the nanoparticles have good penetration into the biofilms, which resulted in enhanced antibacterial activity of the conjugated micelles when compared to free curcumin. Furthermore, the curcumin-functionalised micelles were efficient at disrupting mature biofilms and demonstrated antibacterial activity towards biofilm-embedded cells. CONCLUSION: Curcumin-functionalised poly(lactic-co-glycolic acid)-dextran micelles are novel nanostructures with an intrinsic antibacterial activity tested against two Pseudomonas spp. strains that have the potential to be further exploited to deliver a secondary bioactive molecule within its core.

Enzyme-Functionalized Mesoporous Silica Nanoparticles to Target Staphylococcus aureus and Disperse Biofilms.

BACKGROUND: Staphylococcus aureus biofilms pose a unique challenge in healthcare due to their tolerance to a wide range of antimicrobial agents. The high cost and lengthy timeline to develop novel therapeutic agents have pushed researchers to investigate the use of nanomaterials to deliver antibiofilm agents and target biofilm infections more efficiently. Previous studies have concentrated on improving the efficacy of antibiotics by deploying nanoparticles as nanocarriers. However, the dispersal of the extracellular polymeric substance (EPS) matrix in biofilm-associated infections is also critical to the development of novel nanoparticle-based therapies. METHODS: This study evaluated the efficacy of enzyme-functionalized mesoporous silica nanoparticles (MSNs) against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) biofilms. MSNs were functionalized with the enzyme lysostaphin, which causes cell lysis of S. aureus bacteria. This was combined with two other enzyme functionalized MSNs, serrapeptase and DNase I which will degrade protein and eDNA in the EPS matrix, to enhance eradication of the biofilm. Cell viability after treatment with enzyme-functionalized MSNs was assessed using a MTT assay and CLSM, while crystal violet staining was used to assess EPS removal. RESULTS: The efficacy of all three enzymes against S. aureus cells and biofilms was significantly improved when they were immobilized onto MSNs. Treatment efficacy was further enhanced when the three enzymes were used in combination against both MRSA and MSSA. Regardless of biofilm maturity (24 or 48 h), near-complete dispersal and killing of MRSA biofilms were observed after treatment with the enzyme-functionalized MSNs. Disruption of mature MSSA biofilms with a polysaccharide EPS was less efficient, but cell viability was significantly reduced. CONCLUSION: The combination of these three enzymes and their functionalization onto nanoparticles might extend the therapeutic options for the treatment of S. aureus infections, particularly those with a biofilm component.

A high throughput method to investigate nanoparticle entrapment efficiencies in biofilms.

The commercial use of nanoparticles has increased in recent years due to their unique characteristics, including high surface area, modifiable shape and surface charge and size-dependent properties. Consequently, a greater number of nanomaterials are now being released into the environment and inevitably interact with the natural ecosystem. Bacterial biofilms have the potential to capture and retain nanoparticles, however the factors determining the specific nanoparticle entrapment efficiencies of biofilms are not yet fully understood. Based on fluorescent intensity measurements we developed a simple and straightforward method that allowed the entrapment of different silica nanoparticles by two Pseudomonas strains to be quantified. It was determined that, regardless of nanoparticle size or surface functionalisation, Pseudomonas putida biofilms showed enhanced entrapment efficiencies compared to Pseudomonas fluorescens biofilms. It was also noted that both biofilms showed a higher entrapment capacity towards positively charged NPs. The method developed has the potential to be utilized for high throughput biofilm screening studies in order to develop a new understating of the relationship between nanoparticle characteristics and its uptake by bacterial biofilms.

Surface functionalization-dependent localization and affinity of SiO2 nanoparticles within the biofilm EPS matrix.

The contribution of the biofilm extracellular polymeric substance (EPS) matrix to reduced antimicrobial susceptibility in biofilms is widely recognised. As such, the direct targeting of the EPS matrix is a promising biofilm control strategy that allows for the disruption of the matrix, thereby allowing a subsequent increase in susceptibility to antimicrobial agents. To this end, surface-functionalized nanoparticles (NPs) have received considerable attention. However, the fundamental understanding of the interactions occurring between engineered NPs and the biofilm EPS matrix has not yet been fully elucidated. An insight into the underlying mechanisms involved when a NP interacts with the EPS matrix will aid in the design of more efficient NPs for biofilm control. Here we demonstrate the use of highly specific fluorescent probes in confocal laser scanning microscopy (CLSM) to illustrate the distribution of EPS macromolecules within the biofilm. Thereafter, a three-dimensional (3D) colocalization analysis was used to assess the affinity of differently functionalized silica NPs (SiNPs) and EPS macromolecules from Pseudomonas fluorescens biofilms. Results show that both the charge and surface functional groups of SiNPs dramatically affected the extent to which SiNPs interacted and localized with EPS macromolecules, including proteins, polysaccharides and DNA. Hypotheses are also presented about the possible physicochemical interactions which may be dominant in EPS matrix-NP interactions. This research not only develops an innovative CLSM-based methodology for elucidating biofilm-nanoparticle interactions but also provides a platform on which to build more efficient NP systems for biofilm control.

Enhancing curcumin's solubility and antibiofilm activity via silica surface modification.

Bacterial biofilms are microbial communities in which bacterial cells in sessile state are mechanically and chemically protected against foreign agents, thus enhancing antibiotic resistance. The delivery of active compounds to the inside of biofilms is often hindered due to the existence of the biofilm extracellular polymeric substances (EPS) and to the poor solubility of drugs and antibiotics. A possible strategy to overcome the EPS barrier is the incorporation of antimicrobial agents into a nanocarrier, able to penetrate the matrix and deliver the active substance to the cells. Here, we report the synthesis of antimicrobial curcumin-conjugated silica nanoparticles (curc-NPs) as a possibility for dealing with these issues. Curcumin is a known antimicrobial agent and to overcome its low solubility in water it was grafted onto the surface of silica nanoparticles, the latter functioning as nanocarrier for curcumin into the biofilm. Curc-NPs were able to impede the formation of model P. putida biofilms up to 50% and disrupt mature biofilms up to 54% at 2.5 mg mL-1. Cell viability of sessile cells in both cases was also considerably affected, which is not observed for curcumin delivered as a free compound at the same concentration. Furthermore, proteomics of extracted EPS matrix of biofilms grown in the presence of free curcumin and curc-NPs revealed differences in the expression of key proteins related to cell detoxification and energy production. Therefore, curc-NPs are presented here as an alternative for curcumin delivery that can be exploited not only to other bacterial strains but also to further biological applications.

Ratiometric Imaging of the in Situ pH Distribution of Biofilms by Use of Fluorescent Mesoporous Silica Nanosensors.

Biofilms are communities of microorganisms enclosed in a self-generated matrix of extracellular polymeric substances. While biofilm recalcitrance and persistence are caused by several factors, a reduction in antimicrobial susceptibility has been closely associated with the generation of pH gradients within the biofilm structure. Cells embedded within the biofilm create a localized acidic microenvironment, which is unaffected by the external pH. Therefore, pH monitoring is a promising approach for understanding the complexities of a three-dimensional heterogeneous biofilm. A fluorescent pH nanosensor was designed through the synthesis of mesoporous silica nanoparticles (47 ± 5 nm diameter) conjugated to a pH-sensitive dye (fluorescein) and a pH-insensitive dye (rhodamine B) as an internal standard (dye-MSNs). The fluorescence intensity of fluorescein (IF) reduced significantly as the pH was decreased from 8.5 to 3.5. In contrast, the fluorescence intensity of rhodamine B (IR) remained constant at any pH. The ratio of IF/IR produced a sigmoidal curve with respect to the pH, in a working pH range between 4.5 and 7.5. Dye-MSNs enabled the measurement of pH gradients within Pseudomonas fluorescens WCS 365 biofilm microcolonies. The biofilms showed spatially distinct low-pH regions that were enclosed into large clusters corresponding to high-cell-density areas. Also present were small low-pH areas that spread indistinctly throughout the microcolony caused by the mass transfer effect. The lowest detected pH within the inner core of the microcolonies was 5.1, gradually increasing to a neutral pH toward the exterior of the microcolonies. The dye-MSNs were able to fully penetrate the biofilm matrix and allowed a quantitative ratiometric analysis of pH gradients and distribution throughout the biofilm, which was independent of the nanoparticle concentration.