Model systems of genetically modified platelets.

Although platelets are the smallest cells in the blood, they are implied in various processes ranging from immunology and oncology to thrombosis and hemostasis. Many large-scale screening programs, genome-wide association, and "omics" studies have generated lists of genes and loci that are probably involved in the formation or physiology of platelets under normal and pathologic conditions. This creates an increasing demand for new and improved model systems that allow functional assessment of the corresponding gene products in vivo. Such animal models not only render invaluable insight in the platelet biology, but in addition, provide improved test systems for the validation of newly developed anti-thrombotics. This review summarizes the most important models to generate transgenic platelets and to study their influence on platelet physiology in vivo. Here we focus on the zebrafish morpholino oligonucleotide technology, the (platelet-specific) knockout mouse, and the transplantation of genetically modified human or murine platelet progenitor cells in myelo-conditioned mice. The various strengths and pitfalls of these animal models are illustrated by recent examples from the platelet field. Finally, we highlight the latest developments in genetic engineering techniques and their possible application in platelet research.

Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood.

Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or alphaIIbbeta3 and GPIbalpha were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.

Folding properties of the hepatitis B core as a carrier protein for vaccination research.

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.

Dehydroascorbate uptake is impaired in the early response of Arabidopsis plant cell cultures to cadmium.

The balance between antioxidants, such as ascorbate (ASC) and glutathione, and oxidative reactive oxygen species (ROS) is known to play a pivotal role in the response of plant cells to abiotic stress. Here cell cultures of Arabidopsis thaliana were investigated with regard to their response to elevated levels of cadmium. At concentrations <100 microM, Cd induces a rapid and concentration-dependent H(2)O(2) accumulation. This response could be inhibited by diphenylene iodonium (DPI, 20 microM). Reverse transcription-PCR analysis of three RBOH (respiratory burst oxidase homologues) genes showed an increased transcription of RBOHF after 15 min. No change in ASC concentration was observed during the first 3 h after Cd addition. In contrast, glutathione levels completely diminished within 1 h. This drop could be attributed to an increase in phytochelatin 4. At the plasma membrane, Cd further induced a significant decrease in dehydroascorbate (DHA) uptake activity (up to 90% inhibition after 4 h). This decrease is not present when cells are treated with LaCl(3) before exposure to CdCl(2). LaCl(3) is a typical inhibitor of Ca channels and prevents Cd uptake in these cells as well as the Cd-induced ROS production. Therefore, these results appear to indicate that Cd uptake is a prerequisite for the change in DHA transport activity. However, DPI did not prevent the drop in DHA uptake activity present in Cd-treated Arabidopsis cells, indicating that this response seems to be independent of the Cd-induced H(2)O(2) production.