Co-Trafficking of Coagulation Factor VIII with Von Willebrand Factor Alters the Macromolecular Structure Inside Secretory Weibel-Palade Bodies

Abstract 325

The liver is generally recognized as the major site of coagulation factor (F)VIII synthesis. However, there is now increasing evidence that FVIII can also be synthesized in specific endothelial cells where it is stored with its natural carrier protein von Willebrand factor (VWF) in the Weibel-Palade bodies (WPBs). WPBs have a typical cigar-shaped appearance that most likely originates from the macromolecular organization of VWF multimers into tubules. The tubular storage of VWF is thought to be essential for orderly secretion of VWF strings during activation of endothelial cells. Recently we have shown that expression of FVIII with VWF changes the WPB morphology to spherical vesicles. This finding suggests alterations in the biochemical properties of stored VWF. We now studied in detail the effect of FVIII co-expression on the VWF molecule using a combination of innovative techniques, including correlative light-electron microscopy (CLEM), and live-cell fluorescence microscopy under flow conditions. Analysis of human blood outgrowth endothelial cells (BOECs) expressing human B-domain deleted FVIII-GFP by CLEM revealed that FVIII containing WPBs were electron-dense, spherical structures. These structures contained disorganized short VWF tubules, which was confirmed in 3D by electron tomography. Double immunogold labelling with VWF and GFP antibodies showed that the spherical FVIII containing structures were always positive for VWF. These observations imply that FVIII blocks the expansion of VWF tubules, possibly by binding to the N-terminal VWF domains. As the N-terminal domains are also implicated in the formation of multimers, we therefore investigated whether FVIII affects VWF multimer size. Indeed, multimer analysis showed that VWF secreted by FVIII-GFP transduced BOECs was multimerized to a lesser extent when compared to VWF secreted by non-transduced BOECs. The combined absence of high molecular weight (HMW) VWF multimers and long VWF tubules made us question whether these cells could still release ultra-large VWF (UL-VWF) strings. UL-VWF strings play a key role in bleeding arrest, as platelets adhere to the released VWF string which ultimately leads to the formation of a platelet plug. We examined the release of UL-VWF strings under shear stress from BOECs expressing FVIII-GFP employing live-cell confocal imaging. This technique allowed us to follow FVIII release during exocytosis of WPBs in real-time as well. When we stimulated FVIII-transduced BOECs with histamine, these cells were equally able to release VWF strings as non-transduced BOECs. Although spherical WPBs lacked long VWF tubules and did not secrete HMW multimers, released VWF strings were of similar length as strings secreted by non-transduced BOECs. Surprisingly, released VWF strings were completely covered with FVIII which remained attached to the strings throughout the whole experiment. Another remarkable observation was that platelet binding to the FVIII-covered VWF strings was almost completely absent. We hypothesize that FVIII either shields the A1 domain for platelet binding or causes a conformational change in the VWF strings that prevents platelets from binding to the strings. Our results demonstrate that FVIII co-trafficking with VWF has a major impact on properties of VWF as it reduces the degree of multimerization, shortens tubules and prevents platelets from adhering to strings. This leads us to the conclusion that the macromolecular structure of VWF is considerably altered when FVIII is present in WPBs.