Hemato-oncological patients with chemotherapy-induced thrombocytopenia are a major recipient group of frequent platelet (PLT) transfusion. Prophylactic platelet transfusions are administered when platelet counts fall below 10 × 109 PLT/L, to prevent severe or fatal bleeding. However, these prophylactic platelet transfusions do not always result in the prevention of bleeding. Pre- or post-transfusion acquired dysfunction of donor platelets in this respect could play a role. We previously reported intrinsic and transfusion-dependent platelet alterations in hemato-oncological patients. In particular, the expression of relevant platelet receptors was affected in donor platelets after incubation with patient’s plasma, which could explain, at least in part, the variable efficacy of platelet transfusions in these patients. In the present manuscript we show that plasma from acute myeloid leukemia (AML) patients undergoing chemotherapy inhibits functionality of allogenic platelets. Further proteomic analysis allowed us to observe alterations in the composition of plasma samples, and to identify key plasma components which could be responsible for platelet function inhibition and explain bleeding in patients notwithstanding platelet transfusions. We anticipate that with the obtained results, platelet transfusion support can be further personalized in patients receiving chemotherapy and applications might expand to maximize the clinical efficacy of procedures such as bone marrow transplantation.

Snake venom is an ecologically critical functional trait, primarily applied for foraging and accordingly shaped by selective pressures. Recent insights underpinned the high variability of snake venoms down to the intraspecific level, with regional, ontogenetic, and seasonal variation being mostly investigated. In contrast, sex-based venom variation has received considerably less attention so far, and its influence on venom compositions is poorly described. Here, we compare venom profiles and bioactivity from pooled male and female samples of Central European adders (Vipera berus) to provide insights into potential sex-based venom variation in this species. Proteomics, paired with SDS-PAGE and RP-HPLC, revealed highly similar venom profiles. Likewise, phospholipases A2 and proteases activity profiling, as well as bioassays targeting the effects of venom on the coagulation cascade and the viability of different mammalian cell lines revealed similar activity spectra. Our results do not suggest a noteworthy extent of sex-based intraspecific venom variation in V. berus. We further discuss our data in light of the species' venom composition at larger geographic scales and its clinical relevance. This work contributes to a clearer framework for understanding venom biology in the world's most widespread medically relevant venomous snake.

ABSTRACT:Current antithrombotic therapies are effective in reducing thrombotic events but are limited by their associated risk of bleeding. Bruton tyrosine kinase (BTK) acts as a key signaling switch that drives platelet activation during thrombosis but is largely dispensable for routine hemostasis. It is an important nonredundant signaling mediator downstream of the glycoprotein VI and C-type lectin-like receptor 2 receptors and plays a key role in thrombosis with minimal involvement in hemostasis, making it an attractive antithrombotic target. Although BTK inhibitors effectively reduce thrombosis, their clinical use has been limited because of off-target effects. Protein degraders may overcome this limitation by enabling the ubiquitin proteasomal system to selectively target and degrade BTK. We here assessed the ability of the BTK degraders NX-2127 and NX-5948, currently in clinical trials for B-cell pathologies, to target platelet BTK for degradation. NX-2127 and NX-5948 induced concentration-dependent degradation of BTK in washed platelets, platelet-rich plasma, and whole blood. NX-5948 showed no hook effect and outperformed NX-2127 in potency, efficacy, and degradation kinetics. Proteomic analyses confirmed selective BTK degradation by NX-5948, with no evidence of major off-target effects. BTK degradation impaired collagen-related peptide (CRP)-mediated integrin αIIbβ3 activation, P-selectin expression, platelet aggregation, and in vitro thrombosis, with protease-activated receptor 1-mediated platelet function being left intact. Dosing mice with NX-5948 led to efficacious degradation of platelet BTK and impaired CRP-mediated, but not thrombin-mediated, ex vivo platelet function. In vivo, arterial thrombosis was markedly reduced, without an increase in bleeding time. Together, these results highlight NX-5948 as a potent, selective BTK degrader with antithrombotic potential and minimal hemostatic impact.

Epithelial and endothelial monolayers maintain homeostasis by adapting to physiological stimuli and injury through conversion processes that remain incompletely understood. Using human umbilical vein endothelial cell cultures (HUVECs), we elucidate how monolayer maturation and mechanotransduction-induced remodeling are molecularly regulated. Maturation involves reduced cell perimeter leading to increased junctional VE-cadherin that recruits junctional actin, integrins and vinculin to establish a quiescent, stable monolayer. Remarkably, we identify a previously unrecognized, rapid and reversible intermediate-state, marked by VE-cadherin linearization (clustering) and actomyosin relaxation via myosin light chain (MLC)-dephosphorylation, that emerges during mechanotransduction-induced activation, triggered by onset or shifts in shear stress-induced mechanical load. This novel tension-mediated intermediate state enhances junctional actin, integrin and vinculin recruitment, thereby strengthening barrier function while protecting endothelial cells from overstimulation and mechanical damage. MLC rephosphorylation dissolves junctional actin, forms stress fibers and induces the formation of "Junction-Associated-Intermittent-Lamellipodia" (JAIL), enabling cell shape change and arterial phenotype remodeling. Overall, junctional VE-cadherin concentration, together with mechanosensitive signaling that reduces actomyosin tension, governs actin recruitment, revealing a tension-sensitive, intermediate state that protects cells and primes endothelial remodeling. The data provide a broader model for endothelial mechanotransduction and stress adaptation.

Abstract The efficient and safe transfection of hematopoietic cells is a major hurdle that limits efficacy of therapeutic approaches like RNA-interference. We therefore used our modular EL ectrostatic A ntibody si R NA T argeted therapy platform (ELART) to develop nanocarriers decorated with antibodies for delivery of siRNA into hematopoietic CD20-, CD22-, or CD33-positive cells. To analyze internalization efficacy into tumor cells, we applied nanocarriers loaded with Cy3- or Cy5-labeled siRNA and reached nearly all target cells within 1–4 h. Exposure to Cy5-labeled non-functional siRNAs resulted in reduction of the mitochondrial membrane potential and reduced viability, as detected in tetramethylrhodamine methyl ester (TMRM) and CellTiter-Glo (CTG) assays. We concluded that with our modular nanocarrier system, we can transport cytotoxic agents such as cyanine dyes when bound to siRNA, as ELART nanocarriers safely complex anionic siRNA electrostatically and releases it intracellularly within the target cell. This proof-of-concept study shows that ELART nanocarriers can transport anti-cancer agents utilizing siRNA as carrier substance.

C-type lectins are the major non-enzymatic protein constituents in Viperidae venoms. Neuwlectin, a novel member of the C-type lectin family, was purified to homogeneity from Bothrops neuwiedi venom and characterized as a 32.2 kDa homodimer of 16.2 kDa subunits by MALDI-TOF. In this study, we investigated its effect on platelet aggregation triggered by cancer cells and on angiogenesis. Each subunit contains 135 amino acids with eight cysteine residues and shares high sequence similarity to homologous snake venom Ca2-dependent C-type lectins. Neuwlectin induced agglutination of rabbit and human erythrocytes, which was largely inhibited by EDTA, d-galactose, and d-lactose. Adhesion assays using plasma and extracellular matrix proteins evidenced preferential binding to fibrinogen in a concentration-dependent manner. Furthermore, neuwlectin binds to major platelet collagen receptor glycoprotein (GP) VI, and alphaV integrin, suggesting a role in thrombosis and metastasis. Notably, neuwlectin inhibited approximately 40% of washed platelet aggregation mediated by convulxin, and ∼50% aggregation promoted by MDA-MB-231 breast tumor cells. Although the lectin did not inhibit the platelet adhesion to different substrates, neuwlectin inhibits the adhesion of tumor cells to vitronectin. Its antiangiogenic effect was analyzed in the chick chorioallantoic membrane assay, where neuwlectin reduced vessel formation comparable to the VEGF-targeting monoclonal antibody bevacizumab. In conclusion, neuwlectin represents a promising model for the development of antimetastatic agents, acting through GPVI and alphaV integrin binding to inhibit platelet aggregation and angiogenesis.

The journal retracts the article titled “Structure–Activity Relationship of Synthetic Linear KTS-Peptides Containing Meta-Aminobenzoic Acid as Antagonists of α1β1 Integrin with Anti-Angiogenic and Melanoma Anti-Tumor Activities” [...]

We designed and synthesised an anionic small-molecular photosensitizer (aPSM-Cy3.5) for incorporation into electrostatic antibody targeted (ELART) vesicles, consisting of a protamine-coupled antibody as targeting unit, free protamine and aPSM-Cy3.5. These nanocarriers specifically internalize into different solid tumour cell lines. Upon illumination, the aPSM component initiates the production of reactive oxygen species (ROS). Tumour cells from lung, colorectal and pancreatic cancer with internalized aPSM show decreased growth in colony forming assay. This supports the development of systemically applicable anionic ROS inducers capable of specifically targeting tumour cells.

Rapid progress has been made in the exciting field of secretome research in health and disease. The tumor secretome, which is a significant proportion of the tumor proteome, is secreted into the extracellular space to promote intercellular communication and thus tumor progression. Among the many molecules of the secretome, integrins and matrix metalloproteinase 14 (MMP14) stand out as the interplay of adhesion and proteolysis drives invasion. Integrins serve as mechanosensors that mediate the contact of cells with the scaffold of the extracellular matrix and are significantly involved in the precise positioning and activity control of the membrane-bound collagenase MMP14. As a secretome proteinase, MMP14 influences and modifies the secretome itself. While integrins and MT-MMPs are membrane bound, but can be released and are therefore border crossers between the cell surface and the secretome, the extracellular matrix is not constitutively cell-bound, but its binding to integrins and other cell receptors is a stringently regulated process. To understand the mutual interactions in detail, we first summarize the structure and function of MMP14 and how it is regulated at the enzymatic and cellular level. In particular, the mutual interactions between integrins and MMP14 include the proteolytic cleavage of integrins themselves by MMP14. We then review the biochemical, cell biological and physiological effects of MMP14 on the composition and associated functions in the tumor secretome when either bound to the cell membrane, or located on extracellular microvesicles, or as a proteolytically shed non-membrane-bound ectodomain. Novel methods of proteomics, including the analysis of extravesicular vesicles, and new methods for the quantification of MMP14 will provide new research and diagnostic tools. The proteolytic modification of the tumor secretome, especially by MMP14, may bring an additional aspect to tumor secretome studies and will have an impact on the diagnosis and most likely also on the therapy of cancer patients.

Almost every cell of a multicellular organism is in contact with the extracellular matrix (ECM), which provides the shape and mechanic stability of tissue, organs and the entire body. At the molecular level, cells contact the ECM via integrins. Integrins are transmembrane cell adhesion molecules that connect the ECM to the cytoskeleton, which they bind with their extracellular and intracellular domains. Cysteine residues are abundant in both integrin subunits α and β. If pairwise oxidized into disulfide bridges, they stabilize the folding and molecular structure of the integrin. However, despite the oxidative environment of the extracellular space, not all pairs of cysteines in the extracellular integrin domains are permanently engaged in disulfide bridges. Rather, the reversible and temporary linkage of cystine bridges of these cysteine pairs by oxidation or their reductive cleavage can cause major conformational changes within the integrin, thereby changing ligand binding affinity and altering cellular functions such as adhesion and migration. During recent years, several oxidoreductases and thiol isomerases have been characterized which target such allosteric disulfide bridges. This outlines much better, albeit not comprehensively, the role that such thiol switches play in the redox regulation of integrins. The platelet integrin αIIbβ3 is the best examined example so far. Mostly referring to this integrin, this review will provide insights into the thiol switch-based redox regulation of integrins and the known effects of their allosteric disulfide bridges on conformational changes and cell functions, as well as on the machinery of redox-modifying enzymes that contribute to the redox regulation of cell contacts with the ECM.

Environmental stimuli, including the exposure to ultraviolet (UV)-B light, are known to play a role in the modulation of immune-mediated mechanisms in multiple sclerosis (MS). In experimental autoimmune encephalomyelitis (EAE), we have shown that UV-B irradiation ameliorates disease outcome by regulatory T cells (Treg) expansion. Moreover, the UV-B-mediated induction of Treg numbers was also observed in MS. The aryl hydrocarbon receptor (AhR) can be activated by environmental factors including UV-B-induced photoproducts of tryptophan. Thus, we investigated the role of AhR during the transmission of UV-B irradiation. Therefore, wild-type (WT) and AhR-deficient mice (AhR-/-) were irradiated with UV-B light and immunized with myelin oligodendrocyte glycoprotein (MOG)-peptide. In WT mice it was shown that UV-B irradiation reduces EAE symptoms by Treg expansion. This effect was abrogated in animals with AhR deficiency. To better understand the underlying mechanisms of AhR regulation, we used mice with a deletion of AhR specifically in different subsets of antigen-presenting cells (APC) that have been shown to mediate the expansion of Treg. Interestingly, we could show that the AhR activation in murine cutaneous APC was sufficient to switch APC from a stimulatory into a regulatory phenotype, and moreover, responsible for APC cell maturation and migration into regional lymph nodes. Thus, our data indicate that AhR activation in APC might be required for UV-B-mediated immunosuppression during MOG-induced EAE. Hence, activation of AHR in tissue-resident APC, potentially by low-dose UV-B irradiation, might be beneficial as an adjuvant treatment in inflammatory or degenerative diseases of the central nervous system.

The incidence of colorectal cancer (CRC) and the associated mortality in CRC patients have been rising in recent years. Quiescin sulfhydryl oxidase 1 (QSOX1), a secreted disulfide catalyst essential for extracellular matrix (ECM) assembly, is upregulated in several tumors (e.g. pancreatic, breast, and lung cancer), often correlating with aggressive tumor phenotypes and worse prognosis. In contrast, colorectal and hepatocellular carcinoma specimens show significant downregulation of QSOX1 compared to normal or adjacent tissue. Recognizing cancer as a heterocellular tissue where stromal cell types are crucial to tumor behavior, we evaluated by immunohistochemistry stromal and epithelial QSOX1 expression in 140 CRC cases (mean age: 64 years, 56

BACKGROUND AND PURPOSE:The C-type lectin-like receptor-2 (CLEC-2) is a platelet receptor for the endogenous ligand podoplanin. This interaction contributes to several (patho)physiological processes, such as lymphangiogenesis, preservation of blood and lymphatic vessel integrity organ development, and tumour metastasis. Activation of CLEC-2 leads to the phosphorylation of its cytoplasmic hemITAM domain and initiates a signalling cascade involving the kinase Syk. The aim of this study was to identify and characterise a novel small molecule inhibitor of CLEC-2. EXPERIMENTAL APPROACH:An AlphaScreen-based high-throughput screening was used to identify a small molecule inhibitor of the CLEC-2-podoplanin interaction. Binding site interactions were assessed using in silico modelling. Functional assays, including light transmission aggregometry, platelet spreading and phosphorylation assays, were used to evaluate the effect of a small molecule on CLEC-2-mediated platelet activation. KEY RESULTS:A total of 18,476 small molecules were screened resulting in 14 candidates. Following secondary screening, one novel small molecule, MAS9, was taken forward for further characterisation. The binding sites of MAS9 to CLEC-2 were predicted to share binding sites with the CLEC-2 ligands podoplanin and rhodocytin. MAS9 inhibited CLEC-2-mediated platelet aggregation, spreading and signalling. MAS9 also resulted in inhibited fibrinogen binding. CONCLUSION AND IMPLICATIONS:MAS9 inhibits CLEC-2-mediated aggregation, platelet spreading and signalling, showing selectivity of CLEC-2 inhibition over GPVI. This study paves the way for future preclinical assays to test the potential of MAS9 as a novel therapeutic tool to treat pathologies such as thromboinflammation and cancer.

BACKGROUND:ATP-binding cassette A1 (ABCA1) is a membrane-associated cholesterol efflux pump that is crucial for high-density lipoprotein (HDL) biogenesis and cellular cholesterol homeostasis. Pathogenic variants in the ABCA1 gene cause Tangier disease (TD), a rare autosomal recessive disorder characterized by nearly absent HDL in plasma and cholesteryl ester accumulation in tissue macrophages. Clinical manifestations vary among patients with TD, including splenomegaly, thrombocytopenia, and cardiovascular disease (CVD), with no clear association with specific ABCA1 pathogenic variants. Thrombocytopenia is attributed to hypersplenism-mediated platelet clearance; however, there is controversy regarding platelet production and function in TD. OBJECTIVE:To identify and functionally characterize the suspected alteration in ABCA1, and to study platelet production and function in a Spanish family with HDL deficiency and thrombocytopenia. METHODS:Lipid and apolipoprotein profile analyses, next-generation/Sanger sequencing, in vitro ABCA1 expression and cholesterol efflux assays, primary megakaryocyte differentiation cultures, and platelet functional studies were performed. RESULTS:We identified a novel variant, ABCA1:NM_005502.4c.3306del:p.(Ile1103Serfs*16), which results in a truncated protein with defective apolipoprotein AI-dependent cholesterol efflux. Mild to severe thrombocytopenia and splenomegaly were observed in homozygous carriers; however, there was no clinical history of CVD. Platelet degranulation was overtly normal, although a distinct aggregation profile was observed in ABCA1:p.(Ile1103Serfs*16) carriers. Impaired megakaryocyte differentiation associated with aberrant accumulation of neutral lipids in megakaryocytes was observed in primary cell cultures from homozygous carriers. CONCLUSION:The ABCA1:NM_005502.4c.3306del:p.(Ile1103Serfs*16) variant causes TD. Our findings suggest that thrombocytopenia in TD is not merely due to platelet clearance but also a consequence of ineffective megakaryopoiesis due to ABCA1 dysfunction.

The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. However, it is very difficult to establish the real clinical benefits of these therapies, as the lack of characterization and detailed production methods of platelet-based bioproducts persists in the literature and precludes cross-study comparisons. We characterized the molecular composition and in vitro regenerative capacity of platelet-rich plasma (PRP) produced in a closed-system. Furthermore, we performed a parallel characterization on different PRP subfractions (plasma and plasma-free platelet lysate), identifying that the fractions containing platelet-derived cargo exert the most potent regenerative capacity. This observation led us to develop a method to obtain a platelet secretome highly enriched in growth factors, free of plasma and cellular components (PCT/IB2022/057936), with the aim of establishing a superior bioproduct. The molecular characterization of secretomes revealed agonist-dependent differences, which correlates with beneficial grades of regenerative capacity. Importantly, secretomes showed general superiority to PRP in vitro. We discuss the variables influencing the bioproduct quality (inter-donor variation, platelet source and processing methods). Finally, we propose that the characteristics of secretomes circumvents certain limitations of PRP (autologous vs allogeneic), and envision that optimizing post-processing protocols (nanoencapsulation, lyophilization), would allow their clinical application even beyond regenerative medicine. STATEMENT OF SIGNIFICANCE: The use and demand of platelet-based bioproducts in regenerative medicine is steadily increasing. However, it is very difficult to establish the real clinical benefits of these therapies, or to improve/personalize them, as the lack of characterization of the bioproducts and their production methods is a constant in the literature, reason that precludes cross-study comparisons. In the present manuscript, we provide a comprehensive molecular and functional characterization of platelet-based bioproducts and subfractions, including platelet rich plasma, plasma fractions and platelet secretomes produced with a methodology developed by our group. Our results show that the molecular composition of each fraction correlates with its regenerative capacity in vitro. Thus, a rigorous characterization of platelet-derived bioproducts will potentially allow universal use, customizing and new applications.

In the original publication [...].

To develop peptide drugs targeting integrin receptors, synthetic peptide ligands endowed with well-defined selective binding motifs are necessary. The snake venom KTS-containing disintegrins, which selectively block collagen α1β1 integrin, were used as lead compounds for the synthesis and structure–activity relationship of a series of linear peptides containing the KTS-pharmacophore and alternating natural amino acids and 3-aminobenzoic acid (MABA). To ensure a better stiffness and metabolic stability, one, two and three MABA residues, were introduced around the KTS pharmacophore motif. Molecular dynamics simulations determined that the solution conformation of MABA peptide 4 is more compact, underwent larger conformational changes until convergence, and spent most of the time in a single cluster. The peptides’ binding affinity has been characterized by an enzyme linked immunosorbent assay in which the most potent peptide 4 inhibited with IC50 of 324 ± 8 µM and 550 ± 45 µM the binding of GST-α1-A domain to collagen IV fragment CB3, and the cell adhesion to collagen IV using α1-overexpressor cells, respectively. Docking studies and MM-GBSA calculations confirmed that peptide 4 binds a smaller region of the integrin near the collagen-binding site and penetrated deeper into the binding site near Trp1. Peptide 4 inhibited tube formation by endothelial cell migration in the Matrigel angiogenesis in vitro assay. Peptide 4 was acutely tolerated by mice, showed stability in human serum, decreased tumor volume and angiogenesis, and significantly increased the survival of mice injected with B16 melanoma cells. These findings propose that MABA-peptide 4 can further serve as an α1β1-integrin antagonist lead compound for further drug optimization in angiogenesis and cancer therapy.

Immune cells are major players of the tumor microenvironment (TME), having profound effects on tumor development and metastatic progression. We present oscillating-gradient diffusion-weighted MRI (OGSE-DWI) as non-invasive imaging approach to monitor the intratumoral immune cell infiltrate, relying on size differences between cancer cells, T-cells and macrophages. By applying the Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED) model to sine-shaped OGSE-DWI, changes within the TME and its specific immune cell composition were monitored and compared in syngeneic murine breast cancer models with different degrees of malignancy during tumor progression, clodronate liposome-mediated depletion of macrophages and immune checkpoint inhibitor treatment.