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.

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.

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.