Stasis Promotes Erythrocyte Adhesion to von Willebrand Factor.

OBJECTIVE: Venous thromboembolism is a major contributor to global disease burden. Leukocytes and platelets initiate thrombogenesis on blood stasis and initiate the formation of a fibrin, VWF (von Willebrand factor), and neutrophil extracellular trap scaffold for erythrocytes. However, there is little knowledge on how erythrocytes become stably incorporated into this scaffold. Recently, we described the adhesion of calcium-loaded erythrocytes to endothelial-derived VWF strings. Because VWF is part of the scaffold of venous thrombi, we questioned whether reduced flow or stasis promotes the adhesion of normal erythrocytes to VWF and whether venous thrombi show evidence of erythrocyte-VWF interactions. APPROACH AND RESULTS: In the present work, we perfused, under controlled shear conditions, washed, normal erythrocytes over surface-immobilized plasma and extracellular matrix proteins and showed that normal erythrocytes specifically bind to VWF. The interaction between erythrocytes and VWF significantly increased when the wall shear stress was reduced. Next, we investigated whether erythrocyte-VWF interactions support the structure of venous thrombi. High-resolution immunofluorescence imaging of human venous thrombi showed a striking pattern between erythrocytes, VWF, and fibrin, which suggests that VWF plays a supporting role, linking erythrocytes to fibrin in the thrombus. CONCLUSIONS: Our data suggest that erythrocyte retention in venous thrombi is mediated by erythrocyte-VWF or erythrocyte-VWF-fibrin interactions. Targeting erythrocyte retention could be a new strategy in the treatment or prevention of venous thrombosis.

Content delivery to newly forming Weibel-Palade bodies is facilitated by multiple connections with the Golgi apparatus.

Weibel-Palade bodies (WPBs) comprise an on-demand storage organelle within vascular endothelial cells. It's major component, the hemostatic protein von Willebrand factor (VWF), is known to assemble into long helical tubules and is hypothesized to drive WPB biogenesis. However, electron micrographs of WPBs at the Golgi apparatus show that these forming WPBs contain very little tubular VWF compared with mature peripheral WPBs, which raises questions on the mechanisms that increase the VWF content and facilitate vesicle growth. Using correlative light and electron microscopy and electron tomography, we investigated WPB biogenesis in time. We reveal that forming WPBs maintain multiple connections to the Golgi apparatus throughout their biogenesis. Also by volume scanning electron microscopy, we confirmed the presence of these connections linking WPBs and the Golgi apparatus. From electron tomograms, we provided evidence that nontubular VWF is added to WPBs, which suggested that tubule formation occurs in the WPB lumen. During this process, the Golgi membrane and clathrin seem to provide a scaffold to align forming VWF tubules. Overall, our data show that multiple connections with the Golgi facilitate content delivery and indicate that the Golgi appears to provide a framework to determine the overall size and dimensions of newly forming WPBs.

Correlative light microscopy and electron tomography to study Von Willebrand factor exocytosis from vascular endothelial cells.

Revealing the ultrastructure and function of fluorescently labeled cellular components by correlative light and electron microscopy (CLEM) facilitates the study of structure-function relationships in complex biological processes. Given the diversity of available fluorescent tags, light microscopy is ideal for monitoring dynamic cellular processes, while electron microscopy reveals the morphological context of structures at high resolution. Endothelial cells lining the blood vessel wall contain storage organelles called Weibel-Palade bodies (WPBs), which contain tubules of densely packed helical spirals of the blood coagulation protein Von Willebrand factor (VWF). Exocytosis of WPBs is triggered upon vascular damage and results in the transformation of stored tubular VWF into secreted extracellular VWF. Upon exocytosis, VWF rearranges into long filamentous strings to recruit platelets from the blood. During this secretion process, large intracellular VWF exocytosis structures are formed called secretory pods. Here, we describe a CLEM method used to study the relationship between the secretory pod and secreted VWF where confocal microscopy on whole cells was combined with serial electron tomography on chemically fixed, plastic-embedded sections. We show that the combination of these two well-established microscopy modalities provides a robust and generic CLEM method suitable for the characterization of VWF secretion sites.

Storage and secretion of naturally occurring von Willebrand factor A domain variants.

Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.

Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies.

In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.

Multigranular exocytosis of Weibel-Palade bodies in vascular endothelial cells.

Regulated exocytosis of Weibel-Palade bodies (WPBs) is a pivotal mechanism via which vascular endothelial cells initiate repair in response to injury and inflammation. Several pathways have been proposed to enable differential release of bioactive molecules from WPBs under different pathophysiologic conditions. Due to the complexity, many aspects of WPB biogenesis and exocytosis are still poorly understood. Herein, we have investigated the regulated exocytosis of the major WPB constituent, von Willebrand Factor (VWF), which upon its release forms strings of up to several millimeters long that capture circulating platelets and thereby initiate the formation of a haemostatic plug. Using correlative, fluorescence, and electron microscopic imaging techniques, we provide evidence that multigranular exocytosis is an important pathway for VWF release in secretagogue-challenged human umbilical vein endothelial cells. A novel membrane-delimited structure (secretory pod) was identified as the site of WPB coalescence and VWF exocytosis. Clathrin-coated profiles present on the secretory pods suggested remodeling via compensatory membrane retrieval. Small, 30- to 40-nm cytoplasmic vesicles (nanovesicles) mediated the fusion of WPBs with secretory pods. Multigranular exocytosis may facilitate VWF string formation by pooling the content of multiple WPBs. In addition, it may provide a novel mechanism for the differential release of WPB cargo.