How to perform aggregometry and lumi-aggregometry in mouse platelets

Light transmission aggregometry (LTA) and lumi-aggregometry are the gold standard platelet assays both clinically and for basic research. The availability of different strains of genetically modified mice, and mouse models of human disease means that often laboratories need to use mouse platelets in these assays. Overall, performing aggregometry and lumi-aggregometry with mouse platelets is similar to with human platelets, although methods need be adapted to accommodate their small size, reduced blood volume and different protein levels. This review aims to highlight these key considerations when planning aggregometry experiments with mouse platelets. These include the method of taking blood, including the use of anticoagulants, as well as the method of platelet preparation, and how to maximise yields. This review also covers how to maximise the number of aggregations that can be performed, both by understanding the minimum requirements of your aggregometer, or by considering new approaches. These include employing high throughput plate-based aggregometry (Optimul), or the use of TPO-mimetics to stimulate platelet production in mice to boost their platelet counts. Finally, phenotypic differences between mouse and human platelets, such as protein expression or sensitivity to agonists is discussed as an important consideration when planning experiments

2D‐Hexagonal Boron Nitride Screen‐Printed Bulk‐Modified Electrochemical Platforms Explored towards Oxygen Reduction Reactions

A low‐cost, scalable and reproducible approach for the mass production of screen‐printed electrode (SPE) platforms that have varying percentage mass incorporations of 2D hexagonal boron nitride (2D‐hBN) (2D‐hBN/SPEs) is demonstrated herein. These novel 2D‐hBN/SPEs are explored as a potential metal‐free electrocatalysts towards oxygen reduction reactions (ORRs) within acidic media where their performance is evaluated. A 5% mass incorporation of 2D‐hBN into the SPEs resulted in the most beneficial ORR catalysis, reducing the ORR onset potential by ca. 200 mV in comparison to bare/unmodified SPEs. Furthermore, an increase in the achievable current of 83% is also exhibited upon the utilisation of a 2D‐hBN/SPE in comparison to its unmodified equivalent. The screen‐printed fabrication approach replaces the less‐reproducible and time‐consuming dropcasting technique of 2D‐hBN and provides an alternative approach for the large‐scale manufacture of novel electrode platforms that can be utilised in a variety of application

Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: a novel mechanism of haemostasis

The Podoplanin-CLEC-2 axis is critical in mice for prevention of haemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A2 (TxA2). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of haemostasis in which podoplanin supporting platelet capture and activation at arteriolar rates of shear

A novel route for volume manufacturing of hollow braided composite beam structures

This work investigates the application of a rapid variothermal moulding process for direct processing of a braided thermoplastic commingled yarn. The process uses locally controllable, responsive tooling which provides opportunities for optimum part quality and significantly reduced cycle times compared with conventional processes. The proposed process was used to directly manufacture hollow beam structures from dry commingled braided preforms. It was demonstrated that the cycle time using the rapid process was reduced by more than 90% as compared to a conventional bladder moulding process, resulting in a total cycle time of 14 min. Additionally, initial three point flexure test results indicated an improvement in the mechanical performance of the resultant parts as compared to the benchmark

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the haematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other haematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that is dependent on CLEC-2 and Syk. These studies demonstrate that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behaviour in vitro

The relative influence of intellectual disabilities and autism on sensory impairments and physical disability:A whole‐country cohort of 5.3 million children and adults

Background: Intellectual disabilities and autism are lifelong and often co‐occur. Little is known on their extent of independent association with sensory impairments and physical disability. Methods: For Scotland's population, logistic regressions investigated age–gender‐adjusted odds ratios (OR) of associations, independently, of intellectual disabilities and autism with sensory impairments and physical disability. Results: 1,548,819 children/youth, and 3,746,584 adults. In children/youth, the effect size of intellectual disabilities and autism, respectively, was as follows: blindness (OR = 30.12; OR = 2.63), deafness (OR = 13.98; OR = 2.31), and physical disability (OR = 43.72; OR = 5.62). For adults, the effect size of intellectual disabilities and autism, respectively, was as follows: blindness (OR = 16.89; OR = 3.29), deafness (OR = 7.47; OR = 2.36), and physical disability (OR = 6.04; OR = 3.16). Conclusions: Intellectual disabilities have greater association with the population burden of sensory impairments/physical disability, but autism is also associated regardless of overlap with intellectual disabilities. These may impact further on communication limitations due to autism and intellectual disabilities, increasing complexity of assessments/management of other health conditions. Clinicians need to be aware of these important issues

IL-21 restricts T follicular regulatory T cell proliferation through Bcl-6 mediated inhibition of responsiveness to IL-2

T follicular regulatory (Tfr) cells control the magnitude and specificity of the germinal centre reaction, but how regulation is contained to ensure generation of high-affinity antibody is unknown. Here we show that this balance is maintained by the reciprocal influence of interleukin (IL)-2 and IL-21. The number of IL-2-dependent FoxP3+ regulatory T cells is increased in the peripheral blood of human patients with loss-of-function mutations in the IL-21 receptor (IL-21R). In mice, IL-21:IL-21R interactions influence the phenotype of T follicular cells, reducing the expression of CXCR4 and inhibiting the expansion of Tfr cells after T-cell-dependent immunization. The negative effect of IL-21 on Tfr cells in mice is cell intrinsic and associated with decreased expression of the high affinity IL-2 receptor (CD25). Bcl-6, expressed in abundance in Tfr cells, inhibits CD25 expression and IL-21-mediated inhibition of CD25 is Bcl-6 dependent. These findings identify a mechanism by which IL-21 reinforces humoral immunity by restricting Tfr cell proliferation

Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib

The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side-effects of ibrutinib. The second generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with Non-Hodgkin Lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide (CRP-XL) and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, while size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited SFKs and that SFKs have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of vWF and FVIII ex vivo by addition of intermediate purity FVIII (haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma vWF and FVIII