Assembly of von Willebrand factor tubules with in vivo helical parameters requires A1 domain insertion

Abstract

The von Willebrand factor (VWF) glycoprotein is stored in tubular form in Weibel-Palade bodies (WPBs) prior to secretion from endothelial cells into the bloodstream. The organization of VWF in the tubules promotes formation of covalently linked VWF polymers and enables orderly secretion without polymer tangling. Recent studies have described the high-resolution structure of helical tubular cores formed in vitro by the D1D2 and D´D3 amino-terminal protein segments of VWF. Here we show that formation of tubules with the helical geometry observed for VWF in intracellular WPBs requires also the VWA1 (A1) domain. We reconstituted VWF tubules from segments containing the A1 domain and discovered it to be inserted between helical turns of the tubule, altering helical parameters and explaining the increased robustness of tubule formation when A1 is present. The conclusion from this observation is that the A1 domain has a direct role in VWF assembly, along with its known activity in hemostasis post-secretion.

VWD domain stabilization by autocatalytic Asp‐Pro cleavage

“…The experimental structure and top‐ranking predicted model had all‐atom root mean square deviation (RMSD) values of 1.12 Å for the isolated C8 domain and 1.07 Å for the VWD region spanning H4084 to N4123 and V4134 to C4255, which excludes the hairpin loop between β‐strands β5 and β6 because its conformation is affected by interaction with C8. Despite the agreement within domains, four of the five top‐ranking AlphaFold2 models positioned the C8 domain against one side of the β‐sandwich as observed previously in D assembly structures (Anderson et al, 2022 ; Dong et al, 2019 ; Javitt et al, 2019 , 2020 , 2022 ; Reznik et al, 2022 ). Interestingly, in the prediction ranked third, the C8 domain was dissociated from the outside of the β‐sandwich and instead interacted with the VWD loop, analogous to the crystal structure.…”

“…The most striking feature of the FCGBP D10 segment structure was the relative orientation of the VWD and C8 domains. In other D assembly structures (Anderson et al, 2022;Dong et al, 2019;Javitt et al, 2019Javitt et al, , 2020Javitt et al, , 2022Reznik et al, 2022), the C8 domain packs against the outside of one of the β-sheets in the VWD domain β-sandwich (Figure 2a). Specifically, two helices in the C8 domain interact with β-strands β1, β2, and β3, and in some cases β-strand β14, in the VWD domain, using mainly polar and some hydrophobic interactions.…”

“…The four D assembly domains are named the von Willebrand factor type D (VWD), C8, TIL, and E domains (Figure 1a ) (Nilsson et al, 2014 ). VWF and mucin structures have been determined using x‐ray crystallography and cryo‐electron microscopy (cryo‐EM), revealing a compact arrangement of the VWD, C8, and TIL domains (Figure 1b ) (Anderson et al, 2022 ; Dong et al, 2019 ; Javitt et al, 2019 , 2020 , 2022 ; Reznik et al, 2022 ). The E domain projects out of the globular VWD/C8/TIL structure and appears to provide flexibility to the junctions between tandem D assemblies in VWF and mucins (Reznik et al, 2022 ).…”

Altered Storage and Function of von Willebrand Factor in Human Cardiac Microvascular Endothelial Cells Isolated from Recipient Transplant Hearts

“…The formation of rod-shaped WPBs is driven by the assembly of tubule-like structures in VWF pro-peptide dimers and the D'D3 domains of mature VWFs [13,15,17,18,31]. This process requires a low pH and high Ca environment [13,31].…”

“…Elevated levels of VWF are a known risk factor for coronary heart disease, ischemic stroke and sudden death [9][10][11][12]. VWF is stored within WPBs as helical tubules that enable the packing of the protein at high concentrations [13][14][15][16][17][18]. The orderly arrangement of VWF tubules gives the WPB its distinctive rod shape [14] and is thought to be important for the correct deployment of the protein upon WPB exocytosis, whereupon it forms long (hundreds of microns), extracellular, string-like structures that efficiently capture platelets from solution under flow [19][20][21][22].…”