Structural and functional requirements for von Willebrand factor function

Von Willebrand factor (VWF) is an adhesive, multi-functional, multimeric protein with multiple domains harboring binding sites for collagen and platelet glycoprotein receptors mediating its role in primary hemostasis. In secondary hemostasis VWF is important as a transport protein that protects coagulation factor VIII (FVIII) from degradation. Its functional domains enable VWF to bind to the injured vessel wall, to recruit platelets to the site of injury by adhesion and aggregation and to bind FVIII. VWF functions in the circulation are shear-dependent and some of them are strictly correlating with its multimer size. Employing, e.g. shear flow assays (Figure 1A), cone and plate aggregometry (Figure 1B), immunofluorescence (Figure 1C) and multimer analysis (Figure 1D) we are investigating effects of genetic variations of VWF on string formation, platelet binding, cleavage by ADAMTS13, and multimer biosynthesis.

Figure 1. Methodological approaches to investigate shear dependent and shear independent functions of VWF. (A) VWF string detection assay under shear flow. HUVEC cells were perfused at 5 dyne/cm2 shear stress and stimulated with histamine. VWF strings were visualized by binding of GPIbα-beads and imaged with phase contrast microscopy [1]. (B) View of platelet deposition after cone and plate aggregometry (CPA). The image shows platelet aggregate formation in whole blood of a control individual. The sample was tested employing CPA at 400 rpm for 2 min, stained with May-Grünwald solution and subsequently pictures were taken and analyzed by an internal Image analyzer (Impact R). (C) Immunofluorescent staining of endogenous VWF in HUVEC cells. Nuclei were stained by DAPI. (D) Multimer analyses of VWF mutants associated with different von Willebrand disease phenotypes [2]. 

  (1) Kraus E, Obser T, Kraus K, Oyen F, Schneppenheim R, Brehm MA. Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13. Throm. Res., 2014 Aug 28.  
  (2) Schneppenheim R, Budde U. von Willebrand factor: the complex molecular genetics of a multidomain and multifunctionalprotein. J Thromb Haemost 2011; 9 (Suppl. 1): 209–215