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

Abstract

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 lightelectron 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 ultralarge 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 FVIIIcovered VWF strings, and impairs recruitment of platelets. Apparently, intracellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings. (Blood. 2011; 118(22):5947-5956) IntroductionWeibel-Palade bodies (WPBs) are secretory organelles specific for vascular endothelial cells with a typical elongated shape of 100 to 200 nm in diameter and up to 5 m in length. 1 These organelles are characterized by striations running along the longitudinal axis consisting of condensed von Willebrand factor (VWF). Stimulation with agonists, such as thrombin and epinephrine, releases VWF to arrests bleeding by recruiting blood platelets to sites of vascular perturbation. 2 VWF is synthesized as a prepropolypeptide of 2813 residues that is cleaved into a propeptide (D1-D2) and mature VWF monomer (DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK) of approximately 250 kDa. 3 VWF forms multimers through C-and N-terminal disulfide linkage. [4][5][6] Multimers assemble into long, slightly twisted tubules that determine the typical cigar-shaped appearance of WPBs. [7][8][9] On fusion of WPBs with the plasma membrane, VWF tubules are exposed to physiologic pH. This results in rapid disassembly of VWF tubules, which is presumably triggered by dissociation of propeptide from DЈ-D3 regions at neutral pH. 8,10 The released VWF multimers assemble into extended string-like structures on the surface of endothelial cells onto which platelets adhere. [10][11][12] The tubular organization of VWF is thought to be essential for orderly secretion of long VWF strings without tangling. 13 Besides recruiting platelets to sites of vascular damage, VWF functions as a chaperone for factor VIII (FVIII), a cofactor for activated factor IX in the factor Xase complex. It is thus far uncl...

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

“…This suggests that round WPBs are equally well released when compared to elongated WPB. This is in line with previous observations that WPBs containing FVIII, leading to the formation of spherical WPBs, can still release their cargo (Van den Biggelaar et al, 2009;Bouwens et al, 2011). Spherical WPBs in canine endothelial cells bearing the p.Tyr87Ser mutation, are rapidly releasing their cargo upon stimulation with appropriate secretagogues (Haberichter et al, 2005).…”

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

“…TEM samples were processed as described. 23 Correlated fluorescence and electron micrographs were overlaid in Adobe Photoshop CS6 (Adobe Inc, San Jose, CA) using the nuclei in the area of interest to align the two images. For electron tomography, 10-nm gold fiducials were added to the samples.…”

Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease

“…Immunofluorescence microscopy showed that VWF was stored by BOECs in elongated organelles ( Figure 1B) that displayed internal striations under electron microscopy (data not shown). 25 Furthermore, these organelles also stored the WPB-specific membrane protein P-selectin ( Figure 1B). These data indicate that VWF was stored in authentic WPB.…”

“…24 It has been suggested that BOECs provide a useful system to study the release of WPB and the formation of VWF strings. 25 In addition, morphology of WPB in the BOECs derived from a single patient with VWD has been characterized in a recent study. 17 The aim of the current study was to further explore the feasibility of using BOECs as an endothelial cell model to study the pathogenic nature of VWF mutations at the molecular cellular level.…”