Structure and functional characterization of the B-domain and the full-length coagulation factor VIII

Abstract

Factor VIII (FVIII) is a large glycoprotein that serves as a non-enzymatic cofactor for activated Factor IX, facilitating the proteolytic activation of Factor X in the coagulation cascade. Deficiency of FVIII leads to Hemophilia-A (HA), a bleeding disorder. Synthesized as a single-chain protein (~300 kDa) by liver sinusoidal endothelial cells, FVIII undergoes processing to yield a mature protein with the domain organization A1-a1-A2-a2-B-a3-C1-C2. In circulation, FVIII exists as a heterodimer comprising a heavy chain (A1-A2-B) and a light chain (A3-C1-C2) and stabilized by divalent metal ions such as copper (Cu²⁺) and calcium (Ca²⁺). The B-domain, of approximately 1,000 amino acids, is the largest and most heavily glycosylated region of FVIII. Since the B-domain is cleaved off during the activation of FVIII, it is dispensable for cofactor activity as evidenced in recombinant FVIII products lacking B domain. Although not critical to FVIII activity, the B-domain plays a key but not fully understood role in FVIII synthesis, secretion, and intracellular stability. This B-domain deleted FVIII (BDD-FVIII), forms the basis for many recombinant FVIII products used in HA replacement therapies. The B-domain remains structurally uncharacterized, which poses a significant challenge to the complete understanding of FVIII biology. To bridge this gap, we employed an integrative hybrid approach to develop detailed structural models of glycosylated and furin-cleaved full-length FVIII (<em>Gly/FC-FL-FVIII</em>), including the B-domain. Leveraging the Nobel-recognized AlphaFold2 (AF2) predictions, we refined these models and validated them with biophysical data from atomic force microscopy (AFM) and cryo-electron microscopy (cryo-EM) analyses of recombinant and plasma-derived full-length FVIII proteins. We further extended our studies by modeling a full-length FVIII–von Willebrand Factor (vWF) complex, incorporating both the B-domain and D′-D3 domains (<em>FL-FVIII–vWF-D′-D3</em>), to explore the B-domain's interactions during intracellular processing. Our findings reveal that the B-domain functions predominantly as an independent domain that encircles the FVIII core. Structural models indicate that glycosylations contribute to protein stability and proper folding. Cryo-EM and AFM studies also revealed a globular structure of FL-FVIII, with vWF in a tail-like configuration. Structural models of the <em>FL-FVIII–vWF-D′-D3</em> complex suggest that furin-mediated cleavages allow the B-domain to unfold and facilitate critical interactions with vWF and other partners. Additionally, the B-domain appears to shield key FVIII epitopes, reducing immune recognition and mitigating premature activation. Further computational studies shed light on the B-domain's contribution to FVIII secretion, stability, and immune evasion. This study introduces a robust structural-functional model of the B-domain and FL-FVIII, offering novel insights into the B-domain's role in FVIII biology. These findings provide a valuable foundation for advancing FVIII-based therapeutic strategies, addressing challenges in HA treatment, and enhancing our understanding of FVIII biology.