Platelet Plasma Membrane Research Articles

Nanodomains enriched in arachidonic acid promote P2Y12 receptor oligomerization in the platelet plasma membrane

Nanodomains enriched in arachidonic acid promote P2Y12 receptor oligomerization in the platelet plasma membrane

GSDME-mediated pyroptosis contributes to chemotherapy-induced platelet hyperactivity and thrombotic potential

AbstractThrombotic complications due to platelet hyperreactivity are a major cause of death in patients undergoing chemotherapy. However, the underlying mechanisms are not fully understood. Herein, using human platelets and platelets from mice lacking gasdermin E (GSDME), we showed that GSDME is functionally expressed in anucleate platelets, and that GSDME-mediated pyroptosis, a newly identified form of cell death in mammalian nucleated cells, contributes to platelet hyperactivity in cisplatin-based chemotherapy. Cisplatin or etoposide activates caspase-3 to cleave GSDME, thereby releasing the N-terminal fragment of GSDME (GSDME-N) toward the platelet plasma membrane, subsequently forming membrane pores and facilitating platelet granule release. This eventually promotes platelet hyperactivity and thrombotic potential. We identified flotillin-2, a scaffold protein, as a GSDME-N interactor that recruits GSDME-N to the platelet membrane. Loss of GSDME protects mice from cisplatin-induced platelet hyperactivity. Our results provide evidence that targeting GSDME-mediated pyroptosis could reduce thrombotic potential in chemotherapy.

Ticagrelor increases its own potency at the P2Y12 receptor by directly changing the plasma membrane lipid order in platelets.

Although the amphiphilic nature of the widely used antithrombotic drug Ticagrelor is well known, it was never considered as a membranotropic agent capable of interacting with the lipid bilayer in a receptor-independent way. In this study, we investigated the influence of Ticagrelor on plasma membrane lipid order in platelets and if this modulates the potency of Ticagrelor at the P2Y12 receptor. We combined fluorescent in situ, in vitro and in silico approaches to probe the interactions between the plasma membrane of platelets and Ticagrelor. The influence of Ticagrelor on the lipid order of the platelet plasma membrane and large unilamellar vesicles was studied using the advanced fluorescent probe NR12S. Furthermore, the properties of model lipid bilayers in the presence of Ticagrelor were characterized by molecular dynamics simulations. Finally, the influence of an increased lipid order on the dose-response of platelets to Ticagrelor was studied. Ticagrelor incorporates spontaneously into lipid bilayers and affects the lipid order of the membranes of model vesicles and isolated platelets, in a nontrivial composition and concentration-dependent manner. We showed that higher plasma membrane lipid order in platelets leads to a lower IC50 value for Ticagrelor. It is shown that membrane incorporation of Ticagrelor increases its potency at the P2Y12 receptor, by increasing the order of the platelet plasma membrane. A novel dual mechanism of Ticagrelor action is suggested that combines direct binding to P2Y12 receptor with simultaneous modulation of receptor-lipid microenvironment.

Platelet-Rich Plasma-Embedded Porous Polycaprolactone Film with a Large Surface Area for Effective Hemostasis.

Uncontrollable and widespread bleeding caused by surgery or sudden accidents can lead to death if not treated with appropriate hemostasis. To prevent excessive life-threatening bleeding, various hemostatic agents based on polymeric biomaterials with various additives for accelerated blood coagulation have been adopted in clinical fields. In particular, platelet-rich plasma (PRP), which contains many blood coagulation factors that can accelerate blood clot formation, is considered as one of the most effective hemostatic additives. We investigated a PRP-embedded porous film using discarded (expired) PRP and a film with a leaf-stacked structure (FLSS), as a hemostatic agent to induce rapid hemostasis. The film, which contained an LSS on one side (PCL-FLSS), was fabricated by a simple heating-cooling technique using tetraglycol and polycaprolactone (PCL) film. Activated PRP was obtained by the thawing of frozen PRP at the end of its expiration date (the platelet cell membrane is disrupted during the freezing and thawing of PRP, thus releasing various coagulation factors) and embedded in the PCL-FLSS (PRP-FLSS). From in vitro and in vivo experiments using a rat hepatic bleeding model, it was recognized that PRP-FLSS is not only biocompatible but also significantly accelerates blood clotting and thus prevents rapid bleeding, probably due to a synergistic effect of the sufficient supply of various blood coagulants from activated PRP embedded in the LSS layer and the large surface area of the LSS itself. The study suggests that PRP-FLSS, a combination of a porous polymer matrix with a unique morphology and discarded biofunctional resources, can be an advanced hemostatic agent as well as an upcycling platform to avoid the waste of biofunctional resources.

Personalized SO2 Prodrug for pH-Triggered Gas Enhancement in Anti-Tumor Radio-Immunotherapy.

The inhibition of the immune response in the tumor microenvironment by therapy regimens can impede the eradication of tumors, potentially resulting in tumor metastasis. As a non-invasive therapeutic method, radiotherapy is utilized for tumor ablation. In this study, we aimed to improve the therapeutic impact of radiotherapy and trigger an immune response by formulating a benzothiazole sulfinate (BTS)-loaded fusion liposome (BFL) nanoplatform, which was then combined with radiotherapy for anti-cancer treatment. The platelet cell membrane, equipped with distinctive surface receptors, enables BFL to effectively target tumors while evading the immune system and adhering to tumor cells. This facilitates BFL's engulfment by cancer cells, subsequently releasing BTS within them. Following the release, the BTS produces sulfur dioxide (SO2) for gas therapy, initiating the oxidation of intracellular glutathione (GSH). This process demonstrates efficacy in repairing damage post-radiotherapy, thereby achieving effective radiosensitization. It was revealed that an immune response was triggered following the enhanced radiosensitization facilitated by BFL. This approach facilitated the maturation of dendritic cell (DC) within lymph nodes, leading to an increase in the proportion of T cells in distant tumors. This resulted in significant eradication of primary tumors and inhibition of growth in distant tumors. In summary, the integration of personalized BFL with radiotherapy shows potential in enhancing both tumor immune response and the elimination of tumors, including metastasis.

Biomimetic Platelet-Cloaked Nanoparticles for the Delivery of Anti-Inflammatory Curcumin in the Treatment of Atherosclerosis.

Atherosclerosis still represents a major driver of cardiovascular diseases worldwide. Together with accumulation of lipids in the plaque, inflammation is recognized as one of the key players in the formation and development of atherosclerotic plaque. Systemic anti-inflammatory treatments are successful in reducing the disease burden, but are correlated with severe side effects, underlining the need for targeted formulations. In this work, curcumin is chosen as the anti-inflammatory payload model and further loaded in lignin-based nanoparticles (NPs). The NPs are then coated with a tannic acid (TA)- Fe (III) complex and further cloaked with fragments derived from platelet cell membrane, yielding NPs with homogenous size. The two coatings increase the interaction between the NPs and cells, both endothelial and macrophages, in steady state or inflamed status. Furthermore, NPs are cytocompatible toward endothelial, smooth muscle and immune cells, while not inducing immune activation. The anti-inflammatory efficacy is demonstrated in endothelial cells by real-time quantitative polymerase chain reaction and ELISA assay where curcumin-loaded NPs decrease the expression of Nf-κb, TGF-β1, IL-6, and IL-1β in lipopolysaccharide-inflamed cells. Overall, due to the increase in the cell-NP interactions and the anti-inflammatory efficacy, these NPs represent potential candidates for the targeted anti-inflammatory treatment of atherosclerosis.

Impact of lipid emulsions in parenteral nutrition on platelets: a literature review.

Lipid emulsions are essential components of parenteral nutrition solutions that provide energy and essential fatty acids. The complexity of the formulations of lipid emulsions may lead to adverse outcomes such as platelet reactivity and changes in platelet aggregation and related coagulation. Platelets are responsible for haemostasis; they activate and demonstrate morphological changes upon extracellular factors to maintain blood fluidity and vascular integrity. Although parenteral nutrition lipid emulsions are generally found safe with regard to modulation of platelet activity, studies are still accumulating. Thus, this review aims to investigate platelet-related changes by parenteral nutrition lipid emulsions in human studies. Studies have pointed out patients at risk of bleeding and increased platelet aggregation responses due to the administration of lipid emulsions. Lipid emulsions may further benefit patients at high risk of thrombosis due to anti-thrombotic effects and should be cautiously used in patients with thrombocytopenia. The reported platelet-related changes might be associated with the fatty acid change in the plasma membranes of platelets following changes in platelet synthesis and plasma levels of eicosanoids. In conclusion, studies investigating platelets and parenteral nutrition should be supported to minimize the adverse effects and to benefit from the potential protective effects of parenteral nutrition lipid emulsions.

Copper-Based Single-Atom Nanozyme System Mimicking Platelet Cells for Enhancing the Outcome of Radioimmunotherapy.

Radiotherapy is an indispensable part of the multidisciplinary treatment of breast cancer (BC). Due to the potential for serious side effects from ionizing radiation in the treatment of breast cancer, which can adversely affect the patient's quality of life, the radiation dose is often limited. This limitation can result in an incomplete eradication of tumors. In this study, biomimetic copper single-atom catalysts (platelet cell membrane camouflaging, PC) were synthesized with the aim of improving the therapeutic outcomes of radiotherapy for BC. Following guidance to the tumor site facilitated by the platelet cell membrane coating, PC releases a copper single-atom nanozyme (SAzyme). This SAzyme enhances therapeutic effects by generating reactive oxygen species from H2O2 and concurrently inhibiting the self-repair mechanisms of cancer cells through the consumption of intracellular glutathione (GSH) within the tumor microenvironment. PC-augmented radiotherapy induces immunogenic cell death, which triggers an immune response to eradicate tumors. With the excellent biocompatibility, PC exhibited precise tumor-targeting capabilities. Furthermore, when employed in conjunction with radiotherapy, PC showed impressive tumor elimination results through immunological activation. Remarkably, the tumor suppression rate achieved with PC-enhanced radiotherapy reached an impressive 93.6%. Therefore, PC presents an innovative approach for designing radiosensitizers with tumor-specific targeting capabilities, aiming to enhance the therapeutic impact of radiotherapy on BC.

An Ultrasmall Cu/Cu2O Nanoparticle-Based Diselenide-Bridged Nanoplatform Mediating Reactive Oxygen Species Scavenging and Neuronal Membrane Enhancement for Targeted Therapy of Ischemic Stroke.

Ischemic stroke is one of the major causes of death and disability worldwide, and an effective and timely treatment of ischemic stroke has been a challenge because of the narrow therapeutic window and the poor affinity with thrombus of the thrombolytic agent. In this study, rPZDCu, a multifunctional nanoparticle (NP) with the effects of thrombolysis, reactive oxygen species (ROS) scavenging, and neuroprotection, was synthesized based on an ultrasmall Cu4.6O NP, the thrombolytic agent rt-PA, and docosahexaenoic acid (DHA), which is a major component of the neuronal membrane. rPZDCu showed strong thrombus-targeting ability, which was achieved by the platelet cell membrane coating on the NP surface, and a good thrombolytic effect in both the common carotid artery clot model and embolic middle cerebral artery occlusion (MCAO) model of rats. Furthermore, rPZDCu exhibited a good escape from the phagocytosis of macrophages, effective promotion of the polarization of microglia, and efficient recovery of neurobiological and behavioral functions in the embolic MCAO model of rats. This is a heuristic report of (1) the Cu0/Cu+ NP for the treatments of brain diseases, (2) the integration of DHA and ROS scavengers for central nervous system therapies, and (3) diselenide-based ROS-responsive NPs for ischemic stroke treatments. This study also offers an example of cell membrane-camouflaged stimuli-responsive nanomedicine for brain-targeting drug delivery.

Unlocking the role of the B7-H4 polymorphism in psoriasis: Insights into methotrexate treatment outcomes: A prospective cohort study.

B7-H4 is a recently discovered member of B7 family that negatively regulates T-cell immunity, specifically Th1 and Th17 cell responses. However, its role in the pathogenesis of psoriasis has yet to be determined. This study aims to investigate the effect of B7-H4 polymorphism on the efficacy of methotrexate (MTX) and its mechanism in psoriasis. Four single nucleotide polymorphisms of B7-H4 were genotyped in 310 psoriatic patients who received 12-week MTX. The protein expression of B7-H4 in platelets was characterized using immunofluorescence staining, confocal laser scanning microscopy, and flow cytometry techniques. We found that GG genotype carriers of B7-H4 rs1935780 had a lower Psoriasis Area and Severity Index (PASI) 75 response rate and higher weight (p = 0.0245) and body mass index (p = 0.0185) than AA and AG genotype carriers. Multiple regression analysis showed that the PASI score at baseline (p = 0.01) and age at disease onset (p = 0.003) were positively correlated with PASI 75 response rate, while weight (p = 0.005) and the rs1935780 genotype (p = 0.003) were negatively associated with PASI 75 response rate. B7-H4 was expressed in the platelet plasma membrane and cytoplasm. Furthermore, the expression of B7-H4 protein in platelets was lower in good responders than in non-responders and was upregulated considerably after 12-week MTX or in vitro MTX stimulation in good responders. Collectively, these results demonstrate that psoriatic patients with GG genotype of B7-H4 rs1935780 had a poorer response to MTX. Low expression of B7-H4 protein in platelets correlated with better clinical outcomes of MTX in psoriasis.

Phosphoinositides take a central stage in regulating blood platelet production and function

Blood platelets are produced by megakaryocytes through a complex program of differentiation and play a critical role in hemostasis and thrombosis. These anucleate cells are the target of antithrombotic drugs that prevent them from clumping in cardiovascular disease conditions. Platelets also significantly contribute to various aspects of physiopathology, including interorgan communications, healing, inflammation, and thromboinflammation. Their production and activation are strictly regulated by highly elaborated mechanisms. Among them, those involving inositol lipids have drawn the attention of researchers. Phosphoinositides represent the seven combinatorially phosphorylated forms of the inositol head group of inositol lipids. They play a crucial role in regulating intracellular mechanisms, such as signal transduction, actin cytoskeleton rearrangements, and membrane trafficking, either by generating second messengers or by directly binding to specific domains of effector proteins. In this review, we will explore how phosphoinositides are implicated in controlling platelet production by megakaryocytes and in platelet activation processes. We will also discuss the diversity of phosphoinositides in platelets, their role in granule biogenesis and maintenance, as well as in integrin signaling. Finally, we will address the discovery of a novel pool of phosphatidylinositol 3-monophosphate in the outerleaflet of the plasma membrane of human and mouse platelets.

Antiplatelet Action of Chloramine Derivatives of Adenosine Phosphates and Their Chemical Activity in Relation to Sulfur-Containing Compounds.

We studied the properties of N6-chloroadenosine phosphates (ATP, ADP, and AMP chloramines) as compounds with potentially increased antiplatelet efficacy determined by their binding to the plasma membrane of platelets. Chloramine derivatives of ATP, ADP, and AMP do not differ in their optical absorption characteristics: their absorption spectra are in the range of 220-340 nm with a maximum at 264 nm. Chloramines of adenosine phosphates are characterized by high reactivity with respect to thiol compounds. In particular, the rate constants of the reaction of N6-chloroadenosine-5'-diphosphate with N-acetylcysteine, reduced glutathione, dithiothreitol, and cysteine reach 59,000, 250,000, 340,000, and 1,250,000 M-1×sec-1, respectively, and only 1.10±0.02 M-1×sec-1 with methionine. It has been found that N6-chloradenosine-5'-triphosphate is a strong inhibitor of platelet functions: it effectively suppresses ADP-induced cell aggregation (IC50 in the whole blood is 5 μM) and inhibits aggregation of preactivated platelets and induces dissociation of their aggregates.

Exploring the Role of PI3P in Platelets: Insights from a Novel External PI3P Pool

Phosphoinositides (PIs) play a crucial role in regulating intracellular signaling, actin cytoskeleton rearrangements, and membrane trafficking by binding to specific domains of effector proteins. They are primarily found in the membrane leaflets facing the cytosol. Our study demonstrates the presence of a pool of phosphatidylinositol 3-monophosphate (PI3P) in the outer leaflet of the plasma membrane of resting human and mouse platelets. This pool of PI3P is accessible to exogenous recombinant myotubularin 3-phosphatase and ABH phospholipase. Mouse platelets with loss of function of class III PI 3-kinase and class II PI 3-kinase α have a decreased level of external PI3P, suggesting a contribution of these kinases to this pool of PI3P. After injection in mouse, or incubation ex vivo in human blood, PI3P-binding proteins decorated the platelet surface as well as α-granules. Upon activation, these platelets were able to secrete the PI3P-binding proteins. These data sheds light on a previously unknown external pool of PI3P in the platelet plasma membrane that recognizes PI3P-binding proteins, leading to their uptake towards α-granules. This study raises questions about the potential function of this external PI3P in the communication of platelets with the extracellular environment, and its possible role in eliminating proteins from the plasma.

Filamin A in platelets: Bridging the (signaling) gap between the plasma membrane and the actin cytoskeleton.

Platelets are anucleate cells that are essential for hemostasis and wound healing. Upon activation of the cell surface receptors by their corresponding extracellular ligands, platelets undergo rapid shape change driven by the actin cytoskeleton; this shape change reaction is modulated by a diverse array of actin-binding proteins. One actin-binding protein, filamin A (FLNA), cross-links and stabilizes subcortical actin filaments thus providing stability to the cell membrane. In addition, FLNA binds the intracellular portion of multiple cell surface receptors and acts as a critical intracellular signaling scaffold that integrates signals between the platelet's plasma membrane and the actin cytoskeleton. This mini-review summarizes how FLNA transduces critical cell signals to the platelet cytoskeleton.

Engineered drug-loaded cellular membrane nanovesicles for efficient treatment of postsurgical cancer recurrence and metastasis

Cancer recurrence and metastasis are still common causes of postsurgery death in patients with solid tumors, suggesting that additional consolidation therapeutic strategies are necessary. We have previously found that oxaliplatin (OXA) treatment causes further up-regulation of CD155, which is abundantly expressed in tumors for resulting in increased sensitivity of cancer to anti-CD155 therapy. Here, we report O-TPNVs, which are TIGIT-expressing cell membrane and platelet cell membrane fusion nanovesicles (TPNVs) loaded with OXA. Platelet-derived membrane components enable O-TPNVs to target postsurgery wounds and interact with circulating tumor cells (CTCs). OXA directly kills residual tumor cells and CTCs, induces immunogenic cell death, and activates the immune system. TPNVs bind to CD155 on tumor cells, block the CD155/TIGIT pathway, and restore CD8+ T cell activity. In vivo analyses reveal that O-TPNVs achieve synergistic chemotherapeutic and immunotherapeutic effects, effectively inhibiting the recurrence and metastasis of triple-negative breast cancer (4T1) after surgery.

Biomimetic CuS nanoparticles for radiosensitization with mild photothermal therapy and GSH-depletion.

Due to its non-invasive and highly effective characteristics, radiotherapy has attracted significant interest in cancer treatment. However, radioresistance of solid tumors caused by a unique tumor microenvironment diminishes the therapeutic effect of cancer radiotherapy. To address this issue, we developed a nanoplatform for tumor-specific targeting to improve radiotherapy. Specifically, hollow CuS nanoparticles were decorated with the platelet cell membrane (PC), endowing this nanoplatform with the therapeutic property of navigating to the tumor region for glutathione (GSH)-depletion photothermal therapy. It was discovered that mild photothermal therapy mediated by PC ameliorated hypoxia in the tumor microenvironment. Meanwhile, GSH, which contributes to repairing radiotherapy-induced DNA double-strand breaks, was depleted by PC in an acidic microenvironment. Therefore, radioresistance could be diminished while cancer cell self-repair was prevented. At therapeutic doses, PC nanoparticles have negligible toxic effects on normal tissues. PC demonstrates promise for both in vivo and in vitro radiosensitization due to its GSH-depletion, photothermal efficiency, and tumor-specific properties.

Clinical Application of Epithelial Sodium Channel (ENaC) as a Biomarker for Arterial Hypertension.

Arterial hypertension (HTN) is a global public health concern and an important risk factor for cardiovascular diseases and renal failure. We previously reported overexpression of ENaC on the plasma membrane of human platelets is a hallmark of HTN. In this double-blinded study of an open population (n = 167), we evaluated the sensitivity and specificity of a diagnostic assay based on gold nanoparticles (AuNPs) conjugated to an antibody against epithelial sodium channel (ENaC) expressed on platelets, which is detected using a fluorescent anti-ENaC secondary antibody and spectrofluorometry. Using the cutoff value for the AuNP-anti-ENaC assay, we confirmed the diagnosis for 62.1% of patients with clinical HTN and detected 59.7% of patients had previously undiagnosed HTN. Although some shortcomings in terms of accurately discriminating healthy individuals and patients with HTN still need to be resolved, we propose this AuNP-anti-ENaC assay could be used for initial screening and early diagnosis to critically improve opportune clinical management of HTN.

Design principles of bioinspired interfaces for biomedical applications in therapeutics and imaging.

In the past two decades, we have witnessed rapid developments in nanotechnology, especially in biomedical applications such as drug delivery, biosensing, and bioimaging. The most commonly used nanomaterials in biomedical applications are nanoparticles, which serve as carriers for various therapeutic and contrast reagents. Since nanomaterials are in direct contact with biological samples, biocompatibility is one of the most important issues for the fabrication and synthesis of nanomaterials for biomedical applications. To achieve specific recognition of biomolecules for targeted delivery and biomolecular sensing, it is common practice to engineer the surfaces of nanomaterials with recognition moieties. This mini-review summarizes different approaches for engineering the interfaces of nanomaterials to improve their biocompatibility and specific recognition properties. We also focus on design strategies that mimic biological systems such as cell membranes of red blood cells, leukocytes, platelets, cancer cells, and bacteria.

Targeted delivery of fat extract by platelet membrane-cloaked nanocarriers for the treatment of ischemic stroke

BackgroundOur previous studies suggest that human fat extract (FE) contains a variety of angiogenic factors and may provide an alternative treatment option for stroke. However, the therapeutic effect is largely limited due to its short half-life, and inaccurate targeting.ResultsHerein, we leverage the targeting abilities of platelets (PLTs) to the lesion area of stroke and Arg-Gly-Asp (RGD) peptides to the angiogenic blood vessels to develop a biomimetic nanocarrier that capable of delivering FE precisely to treat stroke. The biomimetic nanocarriers are comprised of FE-encapsulated PLGA (poly(lactic-co-glycolic acid)) core enclosed by RGD peptides decorated plasma membrane of PLTs, namely RGD-PLT@PLGA-FE. We found that RGD-PLT@PLGA-FE not only targeted damaged and inflamed blood vessels but also achieved rapid accumulation in the lesion area of ischemic brain. In addition, RGD-PLT@PLGA-FE kept a sustained release behavior of FE at the lesion site, effectively increased its half-life and promoted angiogenesis and neurogenesis with delivering neurotrophic factors including BDNF, GDNF and bFGF to the brain, that ultimately resulted in blood flow increase and neurobehavioral recovery.ConclusionsIn conclusion, our study provides a new strategy to design a biomimetic system for FE delivery and it is a promising modality for stroke therapy.Graphical

Maintaining flippase activity in procoagulant platelets is a novel approach to reducing thrombin generation

BackgroundDuring thrombosis, procoagulant platelets expose phosphatidylserine (PS), which enhances local thrombin generation. Reducing platelet PS exposure could be a novel anti‐thrombotic approach. PS is confined to the inner leaflet of the plasma membrane in unstimulated platelets by ATP‐dependent “flippase” activity. Ca2+ ionophores trigger all platelets to expose a high level of PS by activating a scramblase protein and inactivating the flippase. Although R5421 was previously shown to reduce Ca2+ ionophore‐induced PS exposure, its mechanism of action is unknown. ObjectivesTo determine the mechanism by which R5421 reduces platelet PS exposure. MethodsWashed human platelets were stimulated with the Ca2+ ionophore, A23187, to induce procoagulant platelet formation while bypassing proximal receptor signalling. Platelets PS exposure was detected using annexin V or lactadherin in flow cytometry. NBD (7‐nitro‐2‐1,3‐benzoxadiazol‐4‐yl)‐PS was used to assess scramblase and flippase activity. Thrombin generation was monitored using a fluorogenic substrate. Results and conclusionsR5421 reduced the extent of A23187‐stimulated platelet PS exposure, as demonstrated with annexin V or lactadherin binding. R5421 also maintained flippase activity in procoagulant platelets. Although R5421 appeared to inhibit scramblase activity in procoagulant platelets, it did not once the flippase had been inhibited, demonstrating that scramblase activity is not directly inhibited. Furthermore, R5421 inhibited the contribution of A23187‐stimulated platelets to thrombin generation. Together these data demonstrate that R5421 reduces the extent of PS exposure in procoagulant platelets by maintaining flippase activity. Maintaining flippase activity in procoagulant platelets is a novel and effective approach to reducing thrombin generation.