Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A

Abstract

A normal hemostatic response to vascular injury requires both factor VIII (FVIII) and von Willebrand factor (VWF). In plasma, VWF and FVIII normally circulate as a noncovalent complex, and each has a critical function in the maintenance of hemostasis. Furthermore, the interaction between VWF and FVIII plays a crucial role in FVIII function, immunogenicity, and clearance, with VWF essentially serving as a chaperone for FVIII. Several novel recombinant FVIII (rFVIII) therapies for hemophilia A have been in clinical development, which aim to increase the half-life of FVIII (∼12 hours) and reduce dosing frequency by utilizing bioengineering techniques including PEGylation, Fc fusion, and single-chain design. However, these approaches have achieved only moderate increases in half-life of 1.5- to 2-fold compared with marketed FVIII products. Clearance of PEGylated rFVIII, rFVIIIFc, and rVIII-SingleChain is still regulated to a large extent by interaction with VWF. Therefore, the half-life of VWF (∼15 hours) appears to be the limiting factor that has confounded attempts to extend the half-life of rFVIII. A greater understanding of the interaction between FVIII and VWF is required to drive novel bioengineering strategies for products that either prolong the survival of VWF or limit VWF-mediated clearance of FVIII.

Figure 1

Details of the sites of synthesis and clearance of VWF and FVIII. Although the synthesis of VWF has long been known to be the vascular endothelium and megakaryocytes, the location of FVIII expression has only recently been confirmed in some types of endothelial cell: in fenestrated forms of endothelium (liver sinusoidal endothelium and glomerular endothelium), in lymphatic endothelium, and in some high endothelial venules. In most forms of endothelium, VWF and FVIII are not coexpressed. Clearance of VWF and FVIII occurs most frequently as a complex, in the sinusoids of the liver and spleen where a range of lectin and scavenger receptors expressed on macrophages, sinusoidal endothelium, and hepatocytes bind to and internalize the 2 proteins. Protein clearance is influenced by factors such as shear, desialylation, and protein sequence variants. SCARA5, scavenger receptor class A, member 5.

Figure 2

Dynamic equilibrium between VWF and FVIII and details of the VWF-FVIII association under normal conditions of synthesis, secretion, and clearance. Whereas the vast majority of VWF circulates as an FVIII-free protein in the circulation, the opposite is true for FVIII with 95% to 98% being in complex with VWF. Although of relatively high affinity (K_D, 0.2 nM), complex formation is characterized by a temperature-sensitive highly dynamic equilibrium, with rapid association and dissociation rate constants (2-4 × 10⁶ M⁻¹ s⁻¹ and 0.3-6 × 10⁻³ s⁻¹), respectively. The influence of shear on the VWF-FVIII association and configuration is unresolved but may play a role in modulating clearance and immunogenicity.

Figure 3

VWF-D′ solution structure. FVIII and VWF-D′ domain structures. Residues where substitutions affected or would be expected to affect binding affinity are shown in spherical representation (blue, positively charged; red, negatively charged; brown, hydrophobic; turquoise, neutral). (A) Hydrophobic FVIII-C2 residues M2199, F2200, L2251, and L2252 interact with VWF, whereas flanking surface-exposed residues R2215, R2220, and K2249 make this FVIII-C2 region positively charged. Five noncysteine hemophilic FVIII-C1 domain amino acid substitutions that affected FVIII-VWF binding (http://www.factorviii-db.org/) are indicated. The cleavage site for the FVIII light-chain (LC) acidic peptide is also indicated. VWF-D′ is oriented with its flexible TIL′ domain approaching FVIII. (B) VWF-D′ is oriented here with its rigid E domain at the top. Noncysteine amino acids whose substitutions are associated with type 2N VWD are shown in spherical representation, and those affecting charged side chains are labeled. Disulfide bonds are shown in orange stick representation.