HRASKO/NRASKO double knockout mice exhibit exceedingly high rates of perinatal lethality due to respiratory failure caused by a significant lung maturation delay. The few animals that reach adulthood have a normal lifespan, but present areas of atelectasis mixed with patches of emphysema and normal tissue in the lung.
Eight double knockout and eight control mice were analyzed using micro-X-ray computerized tomography and a Small Animal Physiological Monitoring system. Tissues and samples from these mice were analyzed using standard histological and Molecular Biology methods and the significance of the results analyzed using a Student´s T-test.
The very few double knockout mice surviving up to adulthood display clear craniofacial abnormalities reminiscent of those seen in RASopathy mouse models, as well as thrombocytopenia, bleeding anomalies, and reduced platelet activation induced by thrombin. These surviving mice also present heart and spleen hyperplasia, and elevated numbers of myeloid-derived suppressor cells in the spleen. Mechanistically, we observed that these phenotypic alterations are accompanied by increased KRAS-GTP levels in heart, platelets and primary mouse embryonic fibroblasts from these animals.
Our data uncovers a new, previously unidentified mechanism capable of triggering a RASopathy phenotype in mice as a result of the combined removal of HRAS and NRAS.
© 2024. The Author(s).

Ageing of the immune system is characterized by decreased lymphopoiesis and adaptive immunity, and increased inflammation and myeloid pathologies1,2. Age-related changes in populations of self-renewing haematopoietic stem cells (HSCs) are thought to underlie these phenomena3. During youth, HSCs with balanced output of lymphoid and myeloid cells (bal-HSCs) predominate over HSCs with myeloid-biased output (my-HSCs), thereby promoting the lymphopoiesis required for initiating adaptive immune responses, while limiting the production of myeloid cells, which can be pro-inflammatory4. Ageing is associated with increased proportions of my-HSCs, resulting in decreased lymphopoiesis and increased myelopoiesis3,5,6. Transfer of bal-HSCs results in abundant lymphoid and myeloid cells, a stable phenotype that is retained after secondary transfer; my-HSCs also retain their patterns of production after secondary transfer5. The origin and potential interconversion of these two subsets is still unclear. If they are separate subsets postnatally, it might be possible to reverse the ageing phenotype by eliminating my-HSCs in aged mice. Here we demonstrate that antibody-mediated depletion of my-HSCs in aged mice restores characteristic features of a more youthful immune system, including increasing common lymphocyte progenitors, naive T cells and B cells, while decreasing age-related markers of immune decline. Depletion of my-HSCs in aged mice improves primary and secondary adaptive immune responses to viral infection. These findings may have relevance to the understanding and intervention of diseases exacerbated or caused by dominance of the haematopoietic system by my-HSCs.
© 2024. The Author(s), under exclusive licence to Springer Nature Limited.

Pseudoexfoliation syndrome (PEX) is characterized by the accumulation of abnormal extracellular matrix material in ocular and non-ocular tissues, including blood vessel walls. Clot-forming dysfunction might be responsible for venous thrombosis in PEX. We investigated global coagulation, the proteome, and functions of platelets in PEX patients and aimed to determine prognostic biomarkers for thrombosis risk in PEX. Peripheral blood was collected from PEX and retinal vein occlusion (RVO) patients, and age-sex matched controls. Viscoelastic hemostasis was evaluated by rotational thromboelastometry (ROTEM). Platelet markers (CD41, CD42, CD61, and CD62p) and endothelial markers (P-selectin, E-selectin, and von Willebrand factor) were investigated by flow cytometry and ELISA, respectively. The platelet proteome was analyzed by 2D fluorescence difference gel electrophoresis followed by mass spectrometry. Clot formation time (CFT) is significantly reduced in PEX patients compared to the controls (p < 0.05). P-selectin levels were higher in PEX patients than in controls (p < 0.05); E-selectin and von Willebrand factor remained unchanged. The monitorization of CFT by ROTEM, and soluble P-selectin, may help assess thrombotic risk in PEX patients. Proteomic analysis revealed differential expression of Profilin-1 in platelets. Profilin-1 regulates the stability of actin-cytoskeleton and may contribute to impaired platelet hemostatic functions. Increased P-selectin levels together with impaired coagulation dynamics might be responsible for the thrombotic events in PEX disease.

Ayurveda is considered to be one of the most ancient forms of medicine still practiced. The Ayurvedic preparation Raudra Rasa and its derivatives have been widely employed against cancer since the 12th century, but the effect of these traditional formulations on platelet function and signaling has not previously been examined. Here we demonstrate that Raudra Rasa and its derivatives significantly reduce thrombin-induced integrin activation and granule secretion in platelets, as observed by reduced PAC-1 binding and P-selectin externalization, respectively. These formulations also inhibited thrombin-stimulated phosphatidylserine exposure, mitochondrial reactive oxygen species generation, and mitochondrial transmembrane potential in platelets. Consistent with the above, Raudra Rasa significantly reduced thrombin-induced tyrosine phosphorylation of the platelet proteins, as well as phosphorylation of the enzymes AKT and GSK-3β. In summary, Raudra Rasa inhibits agonist-mediated platelet activation without affecting cell viability, suggesting it may have therapeutic potential as an anti-platelet/anti-thrombotic agent.
© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Platelet transfusions are essential for managing bleeding and hemostatic dysfunction and could be expanded as a cell therapy due to the multifunctional role of platelets in various diseases. Creating these cell therapies will require modifying transfusable donor platelets to express therapeutic proteins. However, there are currently no appropriate methods for genetically modifying platelets collected from blood donors. Here, we describe an approach using platelet-optimized lipid nanoparticles containing mRNA (mRNA-LNP) to enable exogenous protein expression in human and rat platelets. Within the library of mRNA-LNP tested, exogenous protein expression did not require nor correlate with platelet activation. Transfected platelets retained hemostatic function and accumulated in regions of vascular damage after transfusion into rats with hemorrhagic shock. We expect this technology will expand the therapeutic potential of platelets.