Intracellular trafficking, localization, and mobilization of platelet-borne thiol isomerases

OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13dJinx mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.

Willingness-to-pay for a probabilistic flood forecast: a risk-based decision-making game

Probabilistic hydro-meteorological forecasts have over the last decades been used more frequently to communicate forecastuncertainty. This uncertainty is twofold, as it constitutes both an added value and a challenge for the forecaster and the user of the forecasts. Many authors have demonstrated the added (economic) value of probabilistic over deterministic forecasts across the water sector (e.g. flood protection, hydroelectric power management and navigation). However, the richness of the information is also a source of challenges for operational uses, due partially to the difficulty to transform the probability of occurrence of an event into a binary decision. This paper presents the results of a risk-based decision-making game on the topic of flood protection mitigation, called “How much are you prepared to pay for a forecast?”. The game was played at several workshops in 2015, which were attended by operational forecasters and academics working in the field of hydrometeorology. The aim of this game was to better understand the role of probabilistic forecasts in decision-making processes and their perceived value by decision-makers. Based on the participants’ willingness-to-pay for a forecast, the results of the game show that the value (or the usefulness) of a forecast depends on several factors, including the way users perceive the quality of their forecasts and link it to the perception of their own performances as decision-makers.