−1 (n = 9) at 0.3 μM and 3.66 ± 0.45 WPB s −1 at 100 μM histamine (n = 15). These occurred 2-5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca 2+ indicating that the initial burst of secretion is driven by Ca 2+ release from internal stores, but sustained exocytosis required external Ca 2+ . Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μM), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s (n = 6), and a peak fusion rate of ∼10 WPB s −1 .

Exocytosis of specialized endothelial cell secretory organelles, Weibel-Palade bodies (WPBs), is thought to play an important role in regulating hemostasis and intravascular inflammation. The major WPB core proteins are Von Willebrand factor (VWF) and its propolypeptide (Proregion), constituting more than 95% of the content. Although the composition of the WPBs can be fine-tuned to include cytokines and chemokines (eg, interleukin-8 [IL-8] and eotaxin-3), it is generally assumed that WPB exocytosis is inextricably associated with secretion of VWF. Here we show that WPBs can undergo a form of exocyto-sis during which VWF and Proregion are retained while smaller molecules, such as IL-8, are released. Imaging individual WPBs containing fluorescent cargo molecules revealed that during weak stimulation approximately 25% of fusion events result in a failure to release VWF or Prore-gion. The WPB membrane protein P-selectin was also retained; however, the membrane tetraspannin CD63 was released. Accumulation or exclusion of ex-tracellular fluorescent dextran molecules ranging from 3 kDa to 2 mDa show that these events arise due to the formation of a fusion pore approximately 12 nm in diameter. The pore behaves as a molecular filter, allowing selective release of WPB core and membrane proteins. WPB exocytosis is not inextricably associated with secretion of VWF. (Blood. 2008;111: 5282-5290)

Endothelial cells are reported to contain several distinct populations of regulated secretory organelles, including Weibel-Palade bodies (WPBs), the tissue plasminogen activator (tPA) organelle, and the type-2 chemokine-containing organelle. We show that the tPA and type-2 organelles in human endothelial cells represent a single compartment primarily responsible for unstimulated secretion of tPA or, in cells exposed to interleukin-1␤ (IL-1␤), the cytokines IL-8, IL-6, monocyte chemoattractant protein-1 (MCP-1), and growth-regulated oncogene-␣ (GRO-␣). This compartment was distinct from WPBs in that it lacked detectable von Willebrand factor, P-selectin, Rab27a, or CD63 immunoreactivity, displayed no time-dependent decrease in intragranule pH, underwent detectable unstimulated exocytosis, and was very poorly responsive to Ca 2؉ -elevating secretagogues. WPBs could also contain tPA, and in IL-1␤-treated cells, IL-8, IL-6, MCP-1, and GRO-␣, and were the primary source for histamine or ionomycin-stimulated secretion of these molecules. However, analysis of the storage efficiency of cytokines and tPA revealed that all were very poorly stored compared with von Willebrand factor. The nonmammalian, nonsecretory protein EGFP, when expressed in the secretory pathway, also entered WPBs and had a storage efficiency similar to tPA and the cytokines tested. Based on these data, we proposed a revised model for storage and secretion of cytokines and tPA. (Blood. 2010;116(12):2183-2191) IntroductionSeveral studies suggest that endothelial cells (ECs) possess several distinct populations of regulated secretory organelles (RSOs) in which different subsets of bioactive peptides and proteins are stored, trafficked, and rapidly secreted in response to physiologic stimuli. These include (1) Weibel-Palade bodies (WPBs) whose major cargo protein is von Willebrand factor (VWF) 1 ; (2) a small punctate organelle, morphologically distinct from WPBs, lacking endogenous VWF immunoreactivity but containing the anticoagulant protein tissue plasminogen activator (tPA; the tPA organelle) 2-4 ; and (3) a small punctate organelle, reported to specifically contain the small chemotactic cytokines growthregulated oncogene-␣ (GRO-␣) and monocyte chemoattractant protein 1 (MCP-1) and termed the type-2 granule. 5 The presence of distinct populations of RSOs within the same cell is not uncommon 6-8 and may allow the stimulated release of diverse bioactive molecules to be differentially controlled. If distinct populations of RSOs do exist in ECs, then a careful examination of their properties would provide insights into how trafficking and secretion of specific groups of bioactive molecules are regulated.WPBs are the best characterized RSO of ECs. After their formation at the trans-Golgi network (TGN), WPBs accumulate in the cytoplasm and can remain within the cell for long periods of time (1-2 days). 9,10 By these criteria, we define WPBs as true storage organelles. Under resting conditions, WPBs undergo a very slow process of basal exocytosis, 10 undete...

Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pH o ) on the release of von Willebrand factor (VWF), the VWFpropolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pH o 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pH o range. At pH o 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pH o 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pH o 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pH o 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.Local acidosis is associated with inflammation and ischemia and can have significant effects on the normal function of cells, tissues, cellular, and blood components, in particularly those associated with the immune, vascular, and hemostatic systems (1-8). Endothelial cells regulate inflammatory, vascular and hemostatic responses through the secretion of a wide range of bioactive molecules from specialized secretory organelles, the Weibel-Palade bodies (WPBs). 3The major WPB core proteins are von Willebrand factor (VWF) and the VWF-propolypeptide (Proregion). VWF is synthesized as a pre-proprotein comprising an N-terminal signal peptide (pre-), and several distinct repeating structural domains (termed A, B, C, and D) arranged as D1-D2-DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (9). During translation, the signal peptide is removed to yield proVWF, which then undergoes disulfide-linked dimerization to produce proVWF dimers (10). The Proregion domains (D1-D2) are cleaved from the main peptide in the Golgi apparatus, and further disulfide bond formation produces VWF multimers. The two resulting proteins, VWF and Proregion are co-packaged into the WPB where they noncovalently associate to form ordered tubules in a pH-and Ca 2ϩ -dependent fashion (11). Other physiologically important WPB proteins include P-selectin, interle...

Not available.

Background Familial pseudohyperkalemia (FP) is characterized by an increased rate of potassium leakage in refrigerated red cells and is associated with the minor allele of the single nucleotide polymorphism rs148211042 (R723Q) in the ABCB6 gene. The study aims were to obtain the minor allele frequencies of ABCB6 variants and to measure supernatant potassium accumulation, and other red cell storage parameters, in red cell concentrates (RCC) from carriers of variant rs148211042 under standard blood bank conditions. Study Design Whole blood units were collected from 6 FP individuals and 11 controls and processed into RCC in additive solution. RCC were sampled and tested over cold storage for full blood count, extracellular potassium, glucose, lactate, microvesicle release, deformability, hemolysis, pH, adenosine triphosphate, and 2,3‐diphosphoglycerate. Results Screening of genotyped cohorts identified that variant rs148211042 is present in 1 in 394 British citizens of European ancestry. FP RCC had significantly higher supernatant potassium at all time points from day 3 onwards (p < .001) and higher mean cell volume (p = .032) than controls. The initial rate of potassium release was higher in FP RCC; supernatant potassium reached 46.0 (23.8–57.6) mmol/L (mean [range]) by day 5, increasing to 68.9 (58.8–73.7) mmol/L by day 35. Other quality parameters were not significantly different between FP RCC and controls. Conclusion These data suggest that if a blood donor has FP, reducing the RCC shelf‐life to 5 days may be insufficient to reduce the risk of hyperkalemia in clinical scenarios such as neonatal large volume transfusion.

The PI-treatment process did not increase red cell haemolysis or decrease ATP levels over storage. The lower haemolysis and supernatant potassium levels in treated RCC compared with control RCC were attributed to the exchange step. The effects of combining PI treatment and prion reduction were not more than additive when prion reduction precedes PI treatment.

Background Familial pseudohyperkalemia (FP) is a rare asymptomatic condition characterized by an increased rate of potassium leak from red blood cells (RBC) on refrigeration. Gamma irradiation compromises RBC membrane integrity and accelerates potassium leakage. Here, we compared the effect of irradiation, applied early or late in storage, on FP versus non‐FP RBC. Study Design Five FP and 10 non‐FP individuals from the National Institute for Health Research Cambridge BioResource, UK, and three FP and six non‐FP individuals identified by Australian Red Cross Lifeblood consented to the study. Blood was collected according to standard practice in each center, held overnight at 18–24°C, leucocyte‐depleted, and processed into red cell concentrates (RCC) in Saline Adenine Glucose Mannitol. On Day 1, RCC were split equally into six Red Cell Splits (RCS). Two RCS remained non‐irradiated, two were irradiated on Day 1 and two were irradiated on Day 14. RBCs were tested over cold storage for quality parameters. Results As expected, non‐irradiated FP RCS had significantly higher supernatant potassium levels than controls throughout 28 days of storage (p < .001). When irradiated early, FP RCS released potassium at similar rates to control. When irradiated late, FP RCS supernatants had higher initial post‐irradiation potassium concentration than controls but were similar to controls by the end of storage (14 days post‐irradiation). No other parameters studied showed a significant difference between FP and control. Discussion FP does not increase the rate of potassium leak from irradiated RBCs. Irradiation may cause a membrane defect similar to that in FP RBCs.