Key Points Targeting of an anticlotting drug to activated platelets promises effective blood clot prevention without bleeding side effects.

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BackgroundThe induction of protein synthesis by exogenous delivery of coding synthetic mRNA in desired cells is an auspicious strategy in the fields of basic cell biology, regenerative medicine, treatment of diseases, and reprogramming of cells. Here, we produced modified messenger RNA (mRNA) with reduced immune activation potential and increased stability and performed transfection experiments with different cells, HEK293 cells, BJ fibroblasts, and endothelial cells (ECs).ResultsThe mRNA induced protein expression in cells was analyzed after transfection with different mRNA amounts and transfection reagents using flow cytometry. Different cell types showed different degrees of eGFP expression. HEK293 cells exhibited the highest eGFP expression compared to the BJ fibroblasts and ECs. However, the mRNA induced eGFP expression was detected in all cell types until 3 days after transfection. Already, the use of 0.5 μg of the synthesized mRNA led to the significant expression of eGFP in ECs. From all analyzed ECs approximately 87% were eGFP positive, which showed a high transfection efficiency.ConclusionsThe synthesis of stabilized mRNA and the high transfection efficiency will enable the mRNA engineering of ECs as well as other somatic cells. The delivery of synthetic exogenous mRNA into cells allows the transient expression of desired proteins, which would be normally not expressed by the cells.

Objective-Hypothermia is used in various clinical settings to inhibit ischemia-related organ damage. However, prothrombotic effects have been described as potential side effects. This study aimed to elucidate the mechanism of hypothermia-induced platelet activation and subsequent prothrombotic events and to develop preventative pharmacological strategies applicable during clinically used hypothermia. Methods and Results-Platelet function was investigated ex vivo and in vivo at clinically used hypothermia (28°C/18°C).Hypothermic mice demonstrated increased expression of platelet activation marker P-selectin, platelet-leukocyte aggregate formation, and thrombocytopenia. Intravital microscopy of FeCl 3 -injured murine mesenteric arteries revealed increased platelet thrombus formation with hypothermia. Ex vivo flow chamber experiments indicated increased platelet-fibrinogen adhesion under hypothermia. We show that hypothermia results in reduced ADP hydrolysis via reduction of CD39 (E-NTPDase1) activity, resulting in increased levels of ADP and subsequent augmented primary and secondary platelet activation. In vivo administration of ADP receptor P 2 Y 12 antagonists and recombinant soluble CD39 prevented hypothermia-induced thrombus formation and thrombocytopenia, respectively. Key Words: platelet receptor blockers Ⅲ platelets Ⅲ ADP Ⅲ CD39 Ⅲ hypothermia C ooling as a means of reducing oxygen requirements of organs was initially reported in 1950 by Bigelow et al 1 This concept was first implemented in cardiac surgery in 1952, when John Lewis and his team used total-body hypothermia for the closure of an atrial septal defect. 2 Since then, hypothermia has been routinely used in cardiac surgery as an adjunct to extracorporeal circulation (ECC) with the aim of protecting organs against ischemia-related damage. Temperatures applied are usually between 28°C and 32°C. Furthermore, during deep hypothermic circulatory arrest (which is used either in congenital cardiac surgery, for operations on the thoracic aorta in adults, or in neurosurgical operations for the treatment of cerebral aneurysms), temperatures lower than 20°C are used. Mild hypothermia is also used successfully in emergency and intensive care medicine to improve survival and neurological outcomes of patients after cardiac arrest. [3][4][5][6] Potential prothrombotic events of medically used hypothermia are of major concern. In fact, increased activation, aggregation, and sequestration of platelets and (micro)vascular thrombus formation have all been seen in both in vitro and in vivo settings at hypothermia. 4,[7][8][9][10][11][12][13][14][15][16][17] An increased platelet response to activation at temperatures less than 37°C could be considered part of human physiology. It has been proposed that platelets act as thermosensors, being less responsive to thrombogenic stimuli at the core body temperature of the central circulation, where coronary or cerebral thrombus formation could be lethal. However, at the lower temperatures of external body surfaces, the sites ...

The bacterial small heat shock protein IbpA protects client proteins from aggregation. Due to redundancy in the cellular chaperone network, deletion of the ibpA gene often leads to only a mild or no phenotypic defect. In this study, we show that a Pseudomonas putida ibpA deletion mutant has a severe growth defect under heat stress conditions and reduced survival during recovery revealing a critical role of IbpA in heat tolerance. Transcription of the ibpA gene depends on the alternative heat shock sigma factor σ32. Production of IbpA protein only at heat shock temperatures suggested additional translational control. We conducted a comprehensive structural and functional analysis of the 5′ untranslated regions of the ibpA genes from P. putida and Pseudomonas aeruginosa. Both contain a ROSE-type RNA thermometer that is substantially shorter and simpler than previously reported ROSE elements. Comprised of two hairpin structures only, they inhibit translation at low temperature and permit translation initiation after a temperature upshift. Both elements regulate reporter gene expression in Escherichia coli and ribosome binding in vitro in a temperature-dependent manner. Structure probing revealed local melting of the second hairpin whereas the first hairpin remained unaffected. High sequence and structure conservation of pseudomonal ibpA untranslated regions and their ability to confer thermoregulation in vivo suggest that short ROSE-like thermometers are commonly used to control IbpA synthesis in Pseudomonas species.

Synthetically modified mRNA is a unique bioactive agent, ideal for use in therapeutic applications, such as cancer vaccination or treatment of single-gene disorders. In order to facilitate mRNA transfections for future therapeutic applications, there is a need for the delivery system to achieve optimal transfection efficacy, perform with durable stability, and provide drug safety. The objective of our study was to comprehensively analyze the use of 3β-[N-(N',N'-dimethylaminoethane) carbamoyl](DC-Cholesterol)/dioleoylphosphatidylethanolamine (DOPE) liposomes as a potential transfection agent for modified mRNAs. Our cationic liposomes facilitated a high degree of mRNA encapsulation and successful cell transfection efficiencies. More importantly, no negative effects on cell viability or immune reactions were detected posttransfection. Notably, the liposomes had a long-acting transfection effect on cells, resulting in a prolonged protein production of alpha-1-antitrypsin (AAT). In addition, the stability of these mRNA-loaded liposomes allowed storage for 80 days, without the loss of transfection efficacy. Finally, comprehensive analysis showed that these liposomes are fully hemocompatible with fresh human whole blood. In summary, we present an extensive analysis on the use of DC-cholesterol/DOPE liposomes as mRNA delivery vehicles. This approach provides the basis of a safe and efficient therapeutic strategy in the development of successful mRNA-based drugs.

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