Introduction to a review series on von Willebrand factor, factor VIII, and factor IX

This review series focuses on 3 essential hemostatic proteins: von Willebrand factor (VWF), factor VIII (FVIII), and factor IX (FIX). The functional roles of these 3 proteins are interconnected: VWF is required for the recruitment of platelets to a developing hemostatic plug, but also serves as a chaperone for FVIII in the circulation; FVIII is released from VWF upon activation to FVIIIa, becoming an essential cofactor in the intrinsic factor X-ase complex; and FIXa binds to FVIIIa in the presence of calcium and an appropriate membrane surface rapidly activates factor X to factor Xa. A deficiency state of any one of these proteins is associated with hemorrhagic complications, and a therapeutic option available for each disorder involves the use of purified proteins, recombinant or purified from plasma, to prevent or treat bleeding complications. Since the initial purification and characterization of VWF, FVIII, and FIX, multiple studies have contributed to our understanding of the structure of each protein, either in isolation or in contact with other molecules. These structural studies have provided important insights into the synthesis, functionality, intramolecular and intermolecular interactions, disease states, immunogenicity, and other essential functions, and this knowledge is being applied to the development of new therapeutics.

This review series includes the following papers.

• Peter J. Lenting, Cécile V. Denis, and Olivier D. Christophe, “How unique structural adaptations support and coordinate the complex function of von Willebrand factor”

• Benjamin J. Samelson-Jones, Bhavya S. Doshi, and Lindsey A. George, “Coagulation factor VIII: biological basis of emerging hemophilia A therapies”

• Mettine H. A. Bos, Rianne E. van Diest, and Dougald M. Monroe, “Blood coagulation factor IX: structural insights impacting hemophilia B therapy”

The review by Lenting et al covers the important insights that have been made through a variety of studies into how VWF interacts with multiple different molecules, addressing how the timing of these interactions is impacted by shear, which can thereby impact the function. VWF is a large protein consisting of heterogeneously sized multimers assembled by intermolecular disulfide bridges of a multidomain monomer. FVIII binds to the D′D3 region of VWF with high affinity, protecting it from rapid clearance and proteolytic degradation. Type 2N von Willebrand disease is associated with mutations in the D′D3 region that decrease the affinity of VWF for FVIII. Other intermolecular interactions with VWF that are important for hemostasis include the shear-dependent binding of VWF to platelet surface glycoprotein Ibα through the A1 domain, shear-independent binding to collagen I/III through the A3 domain, and shear-independent binding to integrin αIIb/β3 through an RGD (arginyl-glycyl-aspartic acid) motif in the C4 domain. An essential regulatory mechanism impacting VWF function involves the binding of the metalloprotease ADAMTS13 to the VWF A2 domain when it unfolds, a process that occurs more easily with larger multimers. The absence of ADAMTS13 is associated with the development of thrombotic thrombocytopenic purpura (TTP), and caplacizumab, a bivalent nanobody that binds to the VWF A1 domain, is used in the treatment of TTP by blocking the interaction between large multimers and platelet glycoprotein Ibα.

The review by Samelson-Jones et al covers FVIII, addressing the structural studies that inform our understanding of its synthesis and secretion, cofactor activity, regulation, and immunogenicity. The expression and secretion of FVIII from mammalian cells are decreased by several mechanisms, and the authors explore how targeted mutagenesis can be applied to develop new variants with improved expression and longer half-lives in circulation. An improved understanding of how FVIIIa interacts with FIXa, enhancing its cofactor activity, has led to the development of several variants with enhanced specific activity that may be able to offset lower expression levels. In addition, targeted mutations have been used to decrease the inactivation of FVIII, providing improved hemostatic activity in animal models of FVIII deficiency. The authors also address the important issue that any modifications to FVIII introduced to improve hemostatic efficacy need to be balanced against the possibility of enhanced immunogenicity. An improved understanding of T-cell and B-cell epitopes may enable the development of variants with reduced immunogenicity.

The review by Bos et al completes the triptych and provides a state-of-the-art update on FIX, reviewing structure-function research and providing an overview of FIX bioengineering strategies to develop better therapeutics. The authors review how the FIXa active site is uniquely regulated compared with other vitamin K-dependent proteases, with optimal activity achieved only when it is bound to its cofactor, FVIIIa, and substrate, factor X. They also describe the important intermolecular connections that are necessary to assemble the intrinsic factor X-ase complex, including interactions of the enzyme with its cofactor and the membrane surface. This understanding sets the stage for an elegant discussion of FIX modifications that extend the half-life of the protein, enhance activity, and reduce the inactivation of the activated molecule. The authors also introduce the potential role for FIX distributed in the extravascular space, as well as describe several nontraditional strategies for mimicking cofactor function, such as developing a FIXa that is modified to activate factor X in the absence of FVIIIa.

This review series provides a comprehensive update on these 3 hemostatic proteins, reviewing the structural discoveries that have led to a detailed understanding of the molecules and the development of novel therapeutic approaches. We are setting the stage for exciting discoveries and developments in the years ahead!