Regular Article THROMBOSIS AND HEMOSTASIS Solution structure of the major factor VIII binding region on von Willebrand factor

• The high-resolution structure of the complex disulfidebonded TIL9E9 (D9) region of VWF is presented. • The major factor VIII binding site is localized around a flexible region on the TIL9 domain. Althoughmuchof the functionof vonWillebrand factor (VWF)hasbeen revealed, detailed insight into the molecular structure that enables VWF to orchestrate hemostatic processes, in particular factor VIII (FVIII) binding and stabilization in plasma, is lacking. Here, we present the high-resolution solution structure and structural dynamics of the D9 regionof VWF,which constitutes themajor FVIII bindingsite. D9 consists of 2 domains, trypsin-inhibitor–like (TIL9) and E9, of which the TIL9 domain lacks extensive secondary structure, is strikinglydynamicandharbors a cluster of pathologicalmutations leading to decreased FVIII binding affinity (type 2N von Willebrand disease [VWD]). This indicates that the backbonemalleability of TIL9 is important for its biological activity. The principal FVIII binding site is localized to a flexible, positively charged region on TIL9, which is supported by the rigid scaffold of the TIL9 and E9 domain b sheets. Furthermore, surfacechargemappingof theTIL9E9 structure reveals apotentialmechanism for theelectrostatically guided, high-affinity VWF×FVIII interaction.Our findingsprovidenovel insights intoVWF×FVIII complex formation, leading to a greater understanding of the molecular basis of the bleeding diathesis type 2N VWD. (Blood. 2014;123(26):4143-4151)