Storage of Factor VIII Variants with Impaired von Willebrand Factor Binding in Weibel-Palade Bodies in Endothelial Cells

BACKGROUND: Point mutations resulting in reduced factor VIII (FVIII) binding to von Willebrand factor (VWF) are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood. METHODOLOGY/PRINCIPAL FINDINGS: We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser) to severe (Tyr1680Phe, Ser2119Tyr) VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH. CONCLUSIONS: Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo

The Shear Stress-Induced Transcription Factor KLF2 Affects Dynamics and Angiopoietin-2 Content of Weibel-Palade Bodies

BACKGROUND: The shear-stress induced transcription factor KLF2 has been shown to induce an atheroprotective phenotype in endothelial cells (EC) that are exposed to prolonged laminar shear. In this study we characterized the effect of the shear stress-induced transcription factor KLF2 on regulation and composition of Weibel-Palade bodies (WPBs) using peripheral blood derived ECs. METHODOLOGY AND PRINCIPAL FINDINGS: Lentiviral expression of KLF2 resulted in a 4.5 fold increase in the number of WPBs per cell when compared to mock-transduced endothelial cells. Unexpectedly, the average length of WPBs was significantly reduced: in mock-transduced endothelial cells WPBs had an average length of 1.7 µm versus 1.3 µm in KLF2 expressing cells. Expression of KLF2 abolished the perinuclear clustering of WPBs observed following stimulation with cAMP-raising agonists such as epinephrine. Immunocytochemistry revealed that WPBs of KLF2 expressing ECs were positive for IL-6 and IL-8 (after their upregulation with IL-1β) but lacked angiopoietin-2 (Ang2), a regular component of WPBs. Stimulus-induced secretion of Ang2 in KLF2 expressing ECs was greatly reduced and IL-8 secretion was significantly lower. CONCLUSIONS AND SIGNIFICANCE: These data suggest that KLF2 expression leads to a change in size and composition of the regulated secretory compartment of endothelial cells and alters its response to physiological stimuli

Von Willebrand factor targets IL-8 to Weibel-Palade bodies in an endothelial cell line

Vascular endothelial cells are able to store the chemotactic cytokine interleukin-8 (IL-8) in specialized storage vesicles, Weibel-Palade bodies, together with von Willebrand factor (VWF) and P-selectin. We investigated whether VWF plays a role in the sorting of IL-8 into these organelles. We examined the effect of VWF expression on IL-8 targeting in an endothelial cell line (EC-RF24). This cell line has retained the typical phenotypic characteristics of primary endothelial cells but has lost the capacity to produce VWF in appreciable amounts. EC-RF24 cells were retrovirally transduced with a vector encoding a VWF-green fluorescent protein chimera (VWF-GFP). This approach enables direct visualization of the cellular distribution and secretory behavior of the VWF-GFP hybrid. Expression of VWF-GFP resulted in the generation of Weibel-Palade body-like organelles as shown by the colocalization of VWF-GFP and P-selectin. VWF-GFP expressing EC-RF24 cells also showed significant colocalization of VWF-GFP with IL-8 in these storage vesicles. Live cell imaging revealed that the number of VWF-GFP-containing granules decreased upon cell stimulation. These observations indicate that VWF plays an active role in sequestering IL-8 into Weibel-Palade bodie

ADAMTS-13, von Willebrand factor and related parameters in severe sepsis and septic shock

BACKGROUND: Insufficient control of von Willebrand factor (VWF) multimer size as a result of severely deficient ADAMTS-13 activity results in thrombotic thrombocytopenic purpura associated with microvascluar thrombosis and platelet consumption, features not seldom seen in severe sepsis and septic shock. METHODS: ADAMTS-13 activity and VWF parameters of 40 patients with severe sepsis or septic shock were compared with those of 40 healthy controls of the same age and gender and correlated with clinical findings and sepsis outcome. RESULTS: ADAMTS-13 activity was significantly lower in patients than in healthy controls [median 60% (range 27-160%) vs. 110% (range 63-200%); P < 0.001]. VWF parameters behaved reciprocally and both VWF ristocetin cofactor activity (RCo) and VWF antigen (VWF:Ag) were significantly (P < 0.001) higher in patients compared with controls. Neither ADAMTS-13 activity nor VWF parameters correlated with disease severity, organ dysfunction or outcome. However, a contribution of acute endothelial dysfunction to renal impairment in sepsis is suggested by the significantly higher VWF propeptide and soluble thrombomodulin levels in patients with increased creatinine values as well as by their strong positive correlations (creatinine and VWF propeptide r(s) = 0.484, P < 0.001; creatinine and soluble thrombomodulin r(s) = 0.596, P < 0.001). CONCLUSIONS: VWF parameters are reciprocally correlated with ADAMTS-13 activity in severe sepsis and septic shock but have no prognostic value regarding outcom

Adjuvant activity on human cells in vitro

Efficient vaccines against intracellular microbes or tumors will be based on innovative adjuvants able to induce efficient activation of dendritic cells. Indeed, natural or synthetic products activating Toll-like receptors (TLR) on dendritic cells (DCs) are currently in development for this purpose. Herein, we describe in vitro assays on human cells which might be useful for the preclinical screening and assessment of potential DC activators.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Rate, extent and concentration dependence of histamine-evoked Weibel–Palade body exocytosis determined from individual fusion events in human endothelial cells

The rate, concentration dependence and extent of histamine-evoked Weibel–Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45 ± 0.26 (s.d., n = 144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0 ± 4.42 s (n = 9 cells) at 0.3 μm histamine and 1.57 ± 0.21 s (n = 15 cells) at 100 μm histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20 ± 0.16 WPB s−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 Ca2+ indicating that the initial burst of secretion is driven by Ca2+ release from internal stores, but sustained exocytosis required external Ca2+. 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

Aftiphilin and γ-Synergin Are Required for Secretagogue Sensitivity of Weibel-Palade Bodies in Endothelial Cells

Formation of secretory organelles requires the coupling of cargo selection to targeting into the correct exocytic pathway. Although the assembly of regulated secretory granules is driven in part by selective aggregation and retention of content, we recently reported that adaptor protein-1 (AP-1) recruitment of clathrin is essential to the initial formation of Weibel-Palade bodies (WPBs) at the trans-Golgi network. A selective co-aggregation process might include recruitment of components required for targeting to the regulated secretory pathway. However, we find that acquisition of the regulated secretory phenotype by WPBs in endothelial cells is coupled to but can be separated from formation of the distinctive granule core by ablation of the AP-1 effectors aftiphilin and γ-synergin. Their depletion by small interfering RNA leads to WPBs that fail to respond to secretagogue and release their content in an unregulated manner. We find that these non-responsive WPBs have density, markers of maturation, and highly multimerized von Willebrand factor similar to those of wild-type granules. Thus, by also recruiting aftiphilin/γ-synergin in addition to clathrin, AP-1 coordinates formation of WPBs with their acquisition of a regulated secretory phenotype

Direct and Indirect Influence of Parental Bedrock on Streambed Microbial Community Structure in Forested Streams▿

A correlative study was performed to determine if variation in streambed microbial community structure in low-order forested streams can be directly or indirectly linked to the chemical nature of the parental bedrock of the environments through which the streams flow. Total microbial and photosynthetic biomass (phospholipid phosphate [PLP] and chlorophyll a), community structure (phospholipid fatty acid analysis), and physical and chemical parameters were measured in six streams, three located in sandstone and three in limestone regions of the Bankhead National Forest in northern Alabama. Although stream water flowing through the two different bedrock types differed significantly in chemical composition, there were no significant differences in total microbial and photosynthetic biomass in the sediments. In contrast, sedimentary microbial community structure differed between the bedrock types and was significantly correlated with stream water ion concentrations. A pattern of seasonal variation in microbial community structure was also observed. Further statistical analysis indicated dissolved organic matter (DOM) quality, which was previously shown to be influenced by geological variation, correlated with variation in bacterial community structure. These results indicate that the geology of underlying bedrock influences benthic microbial communities directly via changes in water chemistry and also indirectly via stream water DOM quality