Receptor Aggregation Research Articles

Phosphatidylserine-blocking nanoparticles inhibit thrombosis without increased bleeding in mice

BackgroundPhosphatidylserine (PS) is a procoagulant phospholipid enriched on surfaces of activated vascular cells including platelets, endothelium, monocytes, and microvesicles. As a molecular driver of thrombosis accessible to drug blockade, PS is an attractive pharmacologic target for modulating thrombogenesis, with potentially reduced bleeding risk compared to anticoagulant and antiplatelet therapies. ObjectivesTest antithrombotic capabilities of a liposomal formulation, Zn-dipicolylamine cyanine-3[22,22]/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (molar ratio, 3:97), designated as DPAL, which we previously described binds selectively to PS-enriched cell surfaces, compared with effects on bleeding, in mouse models. MethodsPS-dependent DPAL binding to human and murine platelets was tested in vitro. Thrombosis and bleeding after DPAL intravenous administration were tested in C57Bl/6J mice following FeCl3 carotid arterial injury and tail tip amputation, respectively. Incorporation in hemostatic clots was investigated in the cremaster muscle laser injury model. Toxicity was tested by direct exposure to human endothelial cell cultures. ResultsDPAL bound agonist-stimulated, PS-positive human and murine platelets, blocked by Annexin V or Ano6 deletion, which ablate PS exposure. DPAL prolonged prothrombin time, but did not prevent thrombin-induced fibrinogen receptor activation or aggregation, nor alter blood cell counts including platelets. Following arteriolar laser injury, DPAL bound wound surfaces and edges without destabilizing plugs. DPAL dose-dependently blocked FeCl3-induced arterial thrombosis but did not substantially increase bleeding, or induce endothelial cell death. ConclusionDPAL reduces thrombogenesis with minimal effects on bleeding in mouse models via selective binding to PS. DPAL may support novel approaches to modulate pathogenic thrombin generation with improved safety profiles in multiple contexts.

Stored platelet hemostatic phenotype and function is not altered when donors are on testosterone replacement therapy.

Critical shortages in the national blood supply have led to a re-evaluation of previously overlooked donor sources for blood products. As a part of that effort, red blood cells collected from therapeutic phlebotomy of donors on testosterone replacement therapy (TRT) have been conditionally approved for transfusion. However, platelets from TRT donors are not currently approved for use due to limited data on effects of supraphysiologic testosterone on recipient safety and platelet function. The objective of this study was to provide a comprehensive profile of phenotype and function in platelets from TRT and control donors. Platelets in plasma were collected from TRT and control donors (N = 10 per group; age- and sex-matched) and stored at room temperature for 7 days. On storage Day 1 (D1) and Day 7 (D7), platelet products were analyzed for platelet count, metabolic parameters (i.e., glucose, lactate, mitochondrial function), surface receptor expression, aggregation, thrombin generation, and thrombus formation under physiological flow conditions. TRT donor platelets were not significantly different than control donor platelets in terms of count, surface phenotype, metabolic function, ability to aggregate, thrombin generation, or ability to form occlusive thrombus under arterial flow regimes. Both groups were similar to each other by D7, but had significantly lost hemostatic function compared to D1. Platelets derived from donors undergoing TRT have similar phenotypic and functional profiles compared to those derived from control donors. This suggests that therapeutic phlebotomy of TRT donors may provide a useful source for platelet products.

Bioinspired zwitterionic lysine glycopolymers: Enhancing wound repair through microenvironment modulation for bacterial elimination and optimal immunoregulation

Bacterial infected wounds pose a significant threat to patient survival, necessitating prompt and effective therapeutic intervention. Biomaterials that are responsive to wound microenvironments, possess well-defined structures and are facile to fabricate are favored for fulfilling their mission of efficient wound repair. Drawing inspiration from the immunoregulatory properties of natural zwitterionic capsular polysaccharides (ZPS) and glucomannan (GM) as well as the biogenesis of nitric oxide (NO), this study introduces the zwitterionic lysine glycopolymers (ZLGs), which harmoniously integrate the antibacterial, anti-fouling and immunoregulatory functions into one entity, to effectively promote the healing of infected wounds without addition of any external cytokines or molecular drugs. The ZLGs, exhibiting excellent cytocompatibility, restore sustained NO biogenesis (∼7.1 μM) in wound environment and thus to clean bacteria fast and effectively (12 h clearance rate for S. aureus: 97 %; E. coli: 92 %). Notably, the cell-membrane affinitive ZLGs induce efficient aggregation of mannose receptors and polarize the macrophages to M2 phenotype via the PI3K-Akt/STAT3 pathway, resulting in a synergistic enhanced polarization efficiency increasement of 105 %, as compare to the cumulation of individual building modules. The bioinspired ZLGs accelerate the repair of S. aureus infected wounds in vivo, highlighting the significance of this multi-in-one construction strategy for chronic wound treatment.

Regulation of microtubule nucleation in mouse bone marrow-derived mast cells by ARF GTPase-activating protein GIT2.

Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

Synergistically Acting on Myostatin and Agrin Pathways Increases Neuromuscular Junction Stability and Endurance in Old Mice.

Sarcopenia is the primary cause of impaired motor performance in the elderly. The current prevailing approach to counteract such condition is increasing the muscle mass through inhibition of the myostatin system: however, this strategy only moderately improves muscular strength, not being able to sustain the innervation of the hypertrophic muscle per se, leading to a progressive worsening of motor performances. Thus, we proposed the administration of ActR-Fc-nLG3, a protein that combines the soluble activin receptor, a strong myostatin inhibitor, with the C-terminal agrin nLG3 domain. This compound has the potential of reinforcing neuro-muscular stability to the hypertrophic muscle. We previously demonstrated an enhancement of motor endurance and ACh receptor aggregation in young mice after ActR-Fc-nLG3 administration. Now we extended these observations by demonstrating that also in aged (2 years-old) mice, long-term administration of ActR-Fc-nLG3 increases in a sustained way both motor endurance and muscle strength, compared with ActR-Fc, a myostatin inhibitor, alone. Histological data demonstrate that the administration of this biological improves neuromuscular stability and fiber innervation maintenance, preventing muscle fiber atrophy and inducing only moderate hypertrophy. Moreover, at the postsynaptic site we observe an increased folding in the soleplate, a likely anatomical substrate for improved neurotransmission efficiency in the NMJ, that may lead to enhanced motor endurance. We suggest that ActR-Fc-nLG3 may become a valid option for treating sarcopenia and possibly other disorders of striatal muscles.

An Aptamer‐Functionalized DNA Circuit to Establish an Artificial Interaction between T Cells and Cancer Cells

AbstractNongenetic strategies that enable control over the cell‐cell interaction network would be highly desired, particularly in T cell‐based cancer immunotherapy. In this work, we developed an aptamer‐functionalized DNA circuit to modulate the interaction between T cells and cancer cells. This DNA circuit was composed of recognition‐then‐triggering and aggregation‐then‐activation modules. Upon recognizing target cancer cells, the triggering strand was released to induce aggregation of immune receptors on the T cell surface, leading to an enhancement of T cell activity for effective cancer eradication. Our results demonstrated the feasibility of this DNA circuit for promoting target cancer cell‐directed stimulation of T cells, which, consequently, enhanced their killing effect on cancer cells. This DNA circuit, as a modular strategy to modulate intercellular interactions, could lead to a new paradigm for the development of nongenetic T cell‐based immunotherapy.

In Situ Activation of the Receptor Aggregation for Cell Apoptosis by an AI-Au Intelligent Nanomachine via Tumor Extracellular Acidity.

Polyvalent ligand-induced cell receptor aggregation is closely related to cell behavior regulation. At present, most of the means to induce receptor aggregation rely on external stimuli such as light, heat, and magnetic fields, which may cause side effects to normal cells. How to achieve receptor aggregation on the cancer cell surface to achieve cell apoptosis selectively is still a challenge. Therefore, by taking advantage of the unique property of cancer cells' slightly acidic microenvironment, an easy-to-use apoptosis-inducing mode for the in situ activation of cell surface nucleolin clustering has been developed, which not only opened a new channel for nucleolin receptor aggregation to regulate cell function and further development but also avoided damage to normal cells, providing a new strategy for tumor treatment. Dual functional ssDNA (AS1411 aptamer and pH-responsive I-strand sequence) was modified on the surface of gold nanoparticles (AuNPs) to fabricate AI-Au intelligent nanomachines. Then, the specific binding on cancer cells and aggregation of the nucleolin receptors can be achieved via the formation of an i-Motif structure among adjacent AuNPs under the acidic microenvironment. The result showed that AI-Au nanomachines mediated nucleolin cross-linking on the cell surface, resulting in a cytotoxic effect of approximately 60%. Experiments such as calcein-AM/PI staining, nuclear dye staining, and flow cytometry demonstrated that cell apoptosis became more evident with the increase of acidity under the cell surface microenvironment. Immunofluorescence imaging further confirmed the Cyt-c/caspase-3 apoptosis pathway induced by AI-Au nanomachines. The proposed strategy used for specific cancer cell apoptosis by the in situ activation of tumor cell membrane receptor aggregation is inexpensive and simple to use, which not only provides a new means of nucleolin receptor aggregation for regulating cell function but also offers a new strategy for tumor treatment with reduced side effect to normal cells. This work is significant for comprehending the ligand-induced receptor aggregation process and can lead to the development of a promising anticancer drug.

Capture and neutralization of SARS-CoV-2 and influenza virus by algae-derived lectins with high-mannose and core fucose specificities.

We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.

Direct electrical stimulation impacts on neuromuscular junction morphology on both stimulated and unstimulated contralateral soleus.

There is increasing evidence of crosstalk between organs. The neuromuscular junction (NMJ) is a peripheral chemical synapse whose function and morphology are sensitive to acetylcholine (ACh) release and muscle depolarization. In an attempt to improve our understanding of NMJ plasticity and muscle crosstalk, the effects of unilateral direct electrical stimulation of a hindlimb muscle on the NMJ were investigated in rats exposed long-term post-synaptic neuromuscular blockade. Sprague Dawley rats were subjected to post-synaptic blockade of neuromuscular transmission by systemic administration of α-cobrotoxin and mechanically ventilated for up to 8days and compared with untreated sham operated controls and animals exposed to unilateral chronic electrical stimulation 12h/day for 5 or 8days. NMJs produced axonal and glial sprouts (growth of processes that extend beyond the confines of the synapse defined by high-density aggregates of acetylcholine receptors [AChRs]) in response to post-synaptic neuromuscular blockade, but less than reported after peripheral denervation or pre-synaptic blockade. Direct electrical soleus muscle stimulation reduced the terminal Schwann cell (tSC) and axonal sprouting in both stimulated and non-stimulated contralateral soleus. Eight days chronic stimulation reduced (P<0.001) the number of tSC sprouts on stimulated and non-stimulated soleus from 6.7±0.5 and 6.9±0.5 sprouts per NMJ, respectively, compared with 10.3±0.9 tSC per NMJ (P<0.001) in non-stimulated soleus from rats immobilized for 8days. A similar reduction of axonal sprouts (P<0.001) was observed in stimulated and non-stimulated contralateral soleus in response to chronic electrical stimulation. RNAseq-based gene expression analyses confirmed a restoring effect on both stimulated and unstimulated contralateral muscle. The cross-over effect was paralleled by increased cytokine/chemokine levels in stimulated and contralateral unstimulated muscle as well as in plasma. Motor axon terminals and terminal Schwann cells at NMJs of rats subjected to post-synaptic neuromuscular blockade exhibited sprouting responses. These axonal and glial responses were likely dampened by a muscle-derived myokines released in an activity-dependent manner with both local and systemic effects.

Multi-Catcher Polymers Regulate the Nucleolin Cluster on the Cell Surface for Cancer Therapy.

Cell signal transduction mediated by cell surface ligand-receptor is crucial for regulating cell behavior. The oligomerization or hetero-aggregation of the membrane receptor driven by the ligand realizes the rearrangement of apoptotic signals, providing a new ideal tool for tumor therapy. However, the construction of a stable model of cytomembrane receptor aggregation and the development of a universal anti-tumor therapy model on the cellular surface remain challenging. This work describes the construction of a "multi-catcher" flexible structure GC-chol-apt-cDNA with a suitable integration of the oligonucleotide aptamer (apt) and cholesterol (chol) on a polymer skeleton glycol chitosan (GC), for the regulation of the nucleolin cluster through strong polyvalent binding and hydrophobic membrane anchoring on the cell surface. This oligonucleotide aptamer showed nearly 100-fold higher affinity than that of the monovalent aptamer and achieved stable anchoring to the plasma membrane for up to 6 hours. Moreover, it exerted a high tumor inhibition both in vitro and in vivo by activating endogenous mitochondrial apoptosis pathway through the cluster of nucleolins on the cell membrane. This multi-catcher nano-platform combines the spatial location regulation of cytomembrane receptors with the intracellular apoptotic signaling cascade and represents a promising strategy for antitumor therapy. This article is protected by copyright. All rights reserved.

Polyoxidovanadates [MoVIVV9O28]5- and [H2PtIVVV9O28]5- interact with CHO cell plasma membrane lipids causing aggregation and activation of a G protein-coupled receptor

Mono substituted heteropolyoxidovanadates, when compared to effects of a corresponding isopolyoxidovanadate (POV), were found to be more effective initiators of signal transduction by a G protein-coupled receptor (GPCR), specifically the luteinizing hormone receptor (LHR). Here we report that LHRs signal productively when CHO cells expressing the receptor are treated with two heteropolyoxidovanadates PtIV in monoplatino(IV)nonavanadate(V) ([H2PtVIVV9O28]5-, V9Pt), and MoIV in monomolybdo(VI)nonavanadate(V) (Mo[VIVV9O28]5-, V9Mo). Both substituted decavanadate derivatives were more effective than decavanadate which is more charged, has greater stability and forms the [V10O28]6- anion (V10) in cell culture medium at pH 7.4. For viable CHO cells expressing 10 k or 32 k LHR/cell and treated with 11 μM V9Pt and 13 μM V9Mo, mono substituted heteropolyoxidovanadates significantly decreased the packing of plasma membrane lipids for about 1 h. This brief change in membrane structure was accompanied by increased aggregation of LHR and cell signaling as indicated by increased intracellular levels of cAMP. More pronounced changes in lipid packing and LHR signaling were associated with short acting heteropolyoxidovanadates than with the more stable V10. When LHR was overexpressed, V9Pt and V9Mo had little or no effect on membrane lipid packing or receptor aggregation and the LHR was constitutively activated as indicated by elevated intracellular cAMP levels. Speciation of V9Pt and V9Mo in H2O and cell medium was monitored using 51V NMR spectroscopy and confirmed that V9Pt and V9Mo had greater effects on CHO cells despite decomposing more rapidly in the cell growth medium. Thus, under conditions that promote CHO cell growth, V9Pt and V9Mo, despite their smaller molecular charge and their reduced stability, favor LHR signaling over that induced by V10. Importantly, under the same experimental conditions, CHO cells treated with V9Pt and V9Mo do not exhibit as strong toxic effects observed for cells treated with the longer lived V10. In summary, unlike the longer lived V10 which is more growth inhibitory to cells, monosubstituted heteropolyoxidovanadates are more effective in transiently initiating signaling by a G protein-coupled receptor but, because of rapid hydrolysis, inhibit cell growth less.

Spatial Stochastic Model of the Pre-B Cell Receptor.

Survival and proliferation of immature B lymphocytes requires expression and tonic signaling of the pre-B cell receptor (pre-BCR). This low level, ligand-independent signaling is likely achieved through frequent, but short-lived, homo interactions. Tonic signaling is also central in the pathology of precursor B acute lymphoblastic leukemia (B-ALL). In order to understand how repeated, transient events can lead to sustained signaling and to assess the impact of receptor accumulation induced by the membrane landscape, we developed a spatial stochastic model of receptor aggregation and downstream signaling events. Our rule- and agent-based model builds on previous mature BCR signaling models and incorporates novel parameters derived from single particle tracking of pre-BCR on surfaces of two different B-ALL cell lines, 697 and Nalm6. Live cell tracking of receptors on the two cell lines revealed characteristic differences in their dimer dissociation rates and diffusion coefficients. We report here that these differences affect pre-BCR aggregation and consequent signal initiation events. Receptors on Nalm6 cells, which have a lower off-rate and lower diffusion coefficient, more frequently form higher order oligomers than pre-BCR on 697 cells, resulting in higher levels of downstream phosphorylation in the Nalm6 cell line.

The Functional Properties and Physiological Roles of Signal-Transducing Adaptor Protein-2 in the Pathogenesis of Inflammatory and Immune Disorders.

Adaptor molecules play a crucial role in signal transduction in immune cells. Several adaptor molecules, such as the linker for the activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76), are essential for T cell development and activation following T cell receptor (TCR) aggregation, suggesting that adaptor molecules are good therapeutic targets for T cell-mediated immune disorders, such as autoimmune diseases and allergies. Signal-transducing adaptor protein (STAP)-2 is a member of the STAP family of adaptor proteins. STAP-2 functions as a scaffold for various intracellular proteins, including BRK, signal transducer, and activator of transcription (STAT)3, STAT5, and myeloid differentiation primary response protein (MyD88). In T cells, STAP-2 is involved in stromal cell-derived factor (SDF)-1α-induced migration, integrin-dependent cell adhesion, and Fas-mediated apoptosis. We previously reported the critical function of STAP-2 in TCR-mediated T cell activation and T cell-mediated autoimmune diseases. Here, we review how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases in order to develop novel STAP-2-targeting therapeutic strategies.

MMP7 cleavage of amino-terminal CD95 death receptor switches signaling toward non-apoptotic pathways

CD95 is a death receptor that can promote oncogenesis through molecular mechanisms that are not fully elucidated. Although the mature CD95 membrane receptor is considered to start with the arginine at position 17 after elimination of the signal peptide, this receptor can also be cleaved by MMP7 upstream of its leucine at position 37. This post-translational modification occurs in cancer cells but also in normal cells such as peripheral blood leukocytes. The non-cleaved CD95 amino-terminal region consists in a disordered domain and its in silico reconstitution suggests that it might contribute to receptor aggregation and thereby, regulate the downstream death signaling pathways. In agreement with this molecular modeling analysis, the comparison of CD95-deficient cells reconstituted with full-length or N-terminally truncated CD95 reveals that the loss of the amino-terminal region of CD95 impairs the initial steps of the apoptotic signal while favoring the induction of pro-survival signals, including the PI3K and MAPK pathways.

Sorting nexin 17 increases low-density lipoprotein receptor-related protein 4 membrane expression: A novel mechanism of acetylcholine receptor aggregation in myasthenia gravis.

Myasthenia gravis (MG) is characterized by autoimmune damage to the postsynaptic membrane of the neuromuscular junction (NMJ) with impaired postsynaptic acetylcholine receptor (AChR) aggregation. Low-density lipoprotein receptor-related protein 4 (LRP4) plays an important role in AChR aggregation at endplate membranes via the Agrin–LRP4–muscle-specific receptor tyrosine kinase (MuSK) cascade. Sorting nexin 17 (SNX17) regulates the degradation and recycling of various internalized membrane proteins. However, whether SNX17 regulates LRP4 remains unclear. Therefore, we examined the regulatory effects of SNX17 on LRP4 and its influence on AChR aggregation in MG. We selected C2C12 myotubes and induced LRP4 internalization via stimulation with anti-LRP4 antibody and confirmed intracellular interaction between SNX17 and LRP4. SNX17 knockdown and overexpression confirmed that SNX17 promoted MuSK phosphorylation and AChR aggregation by increasing cell surface LRP4 expression. By establishing experimental autoimmune MG (EAMG) mouse models, we identified that SNX17 upregulation improved fragmentation of the AChR structure at the NMJ and alleviated leg weakness in EAMG mice. Thus, these results reveal that SNX17 may be a novel target for future MG therapy.

Halide ion directed templation effect of quadruple-stranded helicates

The allosteric effect, described as biological enzymes’ conformation change to accommodate specific substrates, plays an important role in metabolic regulation. However, well-organized aggregation of synthetic receptors into high-order supramolecular structures with different conformations and functions in response to a variety of external stimuli presents a formidable challenge. Herein, a series of quadruple-stranded helicates are created through the templation effect of halide ions in a library of metallo-macrocycles. Single-crystal X-ray diffraction unambiguously confirms that the four intertwined strands are bridged with halide ions via eight robust C–H···X¯ hydrogen bonds, four anion-π interactions, and two electrostatic contacts. The binding of I¯ results in a D 2 -symmetric helicate, while the Br¯ or Cl¯ adducts display asymmetric infrastructures. Furthermore, the templation effect exhibits non-thermodynamically controlled, high selectivity toward I¯ due to its nucleophilicity toward breaking up partial coordination bonds. • Halide ions create a templation effect, leading to quadruple-stranded helicates • Multiple non-covalent interactions support the quadruple-stranded helicates • The templation effect exhibits unusual nucleophilic selectivity toward halide ions • Over 90% of I¯ ions wereseparated in the presence of other competing halide anions Liu and Jang et al. report halide ion directed (I¯, Br¯, and Cl¯) templation effects, leading from metallo-macrocycles to quadruple-stranded helicates. Multiple non-covalent interactions co-stabilized the assemblies, which is reminiscent of allosteric proteins in nature.

Cold-stored platelet function is not significantly altered by agitation or manual mixing.

Cold storage of platelets (CS-PLT), results in better maintained hemostatic function compared to room-temperature stored platelets (RT-PLT), leading to increased interest and use of CS-PLT for actively bleeding patients. However, questions remain on best storage practices for CS-PLT, as agitation of CS-PLT is optional per the United States Food and Drug Administration. CS-PLT storage and handling protocols needed to be determined prior to upcoming clinical trials, and blood banking standard operating procedures need to be updated accordingly for the release of units due to potentially modified aggregate morphology without agitation. We visually assessed aggregate formation, then measured surface receptor expression (GPVI, CD42b (GPIbα), CD49 (GPIa/ITGA2), CD41/61 (ITGA2B/ITGB3; GPIIB/GPIIIA; PACI), CD62P, CD63, HLAI), thrombin generation, aggregation (collagen, adenosine diphosphate [ADP], and epinephrine activation), and viscoelastic function (ExTEM, FibTEM) in CS-PLT (Trima collection, 100% plasma) stored for 21 days either with or without agitation (Phase 1, n=10 donor-paired units) and then without agitation with or without daily manual mixing to minimize aggregate formation and reduce potential effects of sedimentation (Phase 2, n=10 donor-paired units). Agitation resulted in macroaggregate formation, whereas no agitation caused film-like sediment. We found no substantial differences in CS-PLT function between storage conditions, as surface receptor expression, thrombin generation, aggregation, and clot formation were relatively similar between intra-Phase storage conditions. Storage duration and not condition impacted phenotype and function. CS-PLT can be stored with or without agitation, and with or without daily mixing and standard metrics of hemostatic function will not be significantly altered.

The role of CYP2C19 genotyping to guide antiplatelet therapy following ischemic stroke or transient ischemic attack

ABSTRACT Introduction Clopidogrel is an antiplatelet agent recommended for secondary prevention of ischemic stroke (IS) and transient ischemic attack (TIA). Conversion of clopidogrel to its active metabolite by hepatic cytochrome P450-2C19 (CYP2C19) is essential for the inhibition of the P2Y12 receptor and subsequent platelet aggregation to prevent thrombotic events. CYP2C19 is highly polymorphic, with over 30 loss of function (LoF) alleles. This review considers whether there is sufficient data to support genotype guided antiplatelet therapy after stroke. Areas covered A systematic literature review retrieved articles, which describe the interaction between CYP2C19 genotype and clinical outcomes following IS or TIA when treated with clopidogrel. The review documents efforts to identify optimal antiplatelet regimens and explores the value genotype guided antiplatelet therapy. The work outlines the contemporary understanding of clopidogrel metabolism and appraises evidence linking CYP2C19 LoF variants with attenuated platelet inhibition and poorer outcomes. Expert opinion There is good evidence that CYP2C19 LoF allele carriers of Han-Chinese ancestry have increased risk for further vascular events following TIA or IS when treated with clopidogrel. The evidence base is less certain in other populations. The expansion of pharmacogenetics into routine clinical practice will facilitate further research and help tailor other aspects of secondary prevention.

Abstract 5287: Aggregation of HER2 receptors on the tumor cell surface induced by a novel anti-HER2 bispecific antibody led to enhanced tumor cell growth inhibition

Abstract Trastuzumab, an anti-HER2 monoclonal antibody developed by Roche, is now a standard of care for treating HER2 overexpressing cancers. However, patients treated with trastuzumab generally develop resistance to the drug within a year after initiation of the treatment. Pertuzumab, another anti-HER2 monoclonal antibody developed by Roche, targets the domain 2 of the HER2 excellular domain (ECD) instead of the domain 4 to which trastuzumab binds and demonstrates enhanced antitumor activities when combined with trastuzumab. Here, we developed a tetravalent bispecific antibody (BsAb) which targets the domain 2 and 4 of the HER2 ECD simultaneously. Using time-lapse microscopy, we showed that the BsAb induced aggregation of HER2 on NCI-N87 tumor cells, possibly indicating that the receptors could be crosslinked by the BsAb intermolecularly. Furthermore, the tetravalent BsAb induced aggregation faster than did the bivalent BsAb. Importantly, the tetravalent BsAbs exhibited superior tumor cell growth inhibition to that of the two parental monoclonal antibodies alone or combined. In line with the aggregation rates, the tetravalent BsAb was also more potent in inhibiting tumor cell growth than that of the bivalent BsAb. Thus we provide a piece of evidence that the BsAbs targeting two distinct domains of HER2 simultaneously might induce the receptor aggregation which might associate with the enhanced inhibition of tumor cell growth. Citation Format: Lan Deng, Jie Zhao, Xiaoqing Meng, Aotian Xiao, Zhenping Zhu, Haomin Huang. Aggregation of HER2 receptors on the tumor cell surface induced by a novel anti-HER2 bispecific antibody led to enhanced tumor cell growth inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5287.

Changes in expression of signal transduction-related genes, and formation of aggregates of GPER1 and OXTR receptors in mucopolysaccharidosis cells

Mucopolysaccharidoses (MPS) are inherited metabolic diseases caused by storage of glycosaminoglycans (GAGs), however, various modulations of the course of these diseases were identified recently due to impairment of different cellular processes. Here, using transcriptomic analyses in cells derived from patients suffering from eleven types of MPS, we demonstrated that expression of dozens to hundreds of genes coding for proteins involved in signal transduction processes is significantly changed in MPS cell relative to controls. When studying membrane estrogen receptor 1 (GPER1) and oxytocin receptor (OXTR) in more detail, we unexpectedly found formation of aggregates of GPER1 in MPS I, and those of OXTR in both MPS I and MPS II cells. The presence of these aggregates did not correlate with levels of expression of GPER1 and OXTR genes and levels of corresponding gene products. On the other hand, the aggregates disappeared in cells treated with enzymes which are otherwise deficient in MPS I and MPS II, causing efficient degradation of GAGs. We demonstrated that GPER1 and OXTR aggregates might be formed due to interactions with GAGs rather than arising from changes of levels of these proteins in cells.