Assembly, Storage, and Secretion of Von Willebrand Factor.

Von Willebrand factor (VWF) plays a central role in hemostasis, and dysregulation of VWF can cause either bleeding or thrombosis. Endothelial cells assemble VWF multimers in two stages that occur in distinct intracellular compartments: proVWF subunits dimerize in the endoplasmic reticulum through “tail-to-tail” disulfide bonds between C-terminal CK domains, and the proVWF dimers form enormous multimers in the Golgi through “head-to-head” disulfide bonds between N-terminal D3 domains. The finished multimers are packaged into ordered tubules within storage organelles called Weibel-Palade bodies (WPB), and tubular packing is necessary for the secretion of VWF filaments that have normal hemostatic function. We have recreated VWF tubule assembly in vitro, starting with pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D’D3 domains. No other cellular proteins or components of WPB are needed. Assembly requires low pH and calcium ions, similar to conditions in the Golgi. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D’D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging. Secretion into the blood exposes VWF tubules to neutral pH conditions, releasing the constraints that maintain tubular packing and permitting the orderly unfurling of large VWF multimers. Some VWF multimers bind platelets and initiate the growth of platelet-rich thrombi. Under normal circumstances, these thrombi are limited in size by ADAMTS13, a metalloprotease that cleaves VWF multimers and releases the platelets. The absence of large VWF multimers causes bleeding that is typical of von Willebrand disease. Conversely, congenital or acquired deficiency of ADAMTS13 prevents the dissolution of VWF-platelet aggregates, which can cause the widespread microvascular thrombosis that characterizes thrombotic thrombocytopenic purpura (TTP). Interestingly, ADAMTS13 deficiency alone need not trigger TTP. Some patients persist for months or years without active disease but become ill whenever they suffer additional inflammatory stress associated with infection, surgery, or pregnancy. Thus, interactions between inflammatory mediators and endothelial cells can determine the course of VWF-dependent thrombosis. The mechanisms responsible for these phenomena remain poorly understood.