PB0198 - Stable Binding to Phosphatidylserine-containing Membranes Requires Conserved Arginine Residues in Tandem C Domains of Blood Coagulation Factor VIII

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Co-Author(s)

SP

Shaun Peters

Graduate Student
Western Washington University
Bellingham, Washington, United States
KC

Kenneth Childers

Postdoctoral Researcher
Western Washington University
Bellingham, Washington, United States
CM

Corbin Mitchell

Graduate Student
Western Washington University
Bellingham, Washington, United States
SR

Steven Reese

Undergraduate
Western Washington University
Bellingham, Washington, United States
CM

Cristopher Mitchell

Undergraduate
Western Washington University
Bellingham, Washington, United States
SW

Serena Wo

Graduate Student
Western Washington University
Bellingham, Washington, United States
CS

Christopher Swanson

Graduate Student
Western Washington University
Bellingham, Washington, United States
CB

Caileen Brison

Undergraduate
Western Washington University
Bellingham, Washington, United States

Presenting Author(s)

PS

Paul C. Spiegel, Jr., PhD

Professor and Chair
Western Washington University
Bellingham, Washington, United States

Background: Blood coagulation factor VIII (fVIII) is activated by thrombin and binds to activated platelet surfaces with activated factor IX to form the intrinsic ‘tenase’ complex and promote blood coagulation. Previous structural and mutational studies of fVIII have identified the C1 and C2 domains in binding to negatively charged membrane surfaces through β-hairpin loops with solvent-exposed hydrophobic residues and a ring of positively charged basic residues. Several hemophilia A-associated mutations within the C domains are suggested to disrupt lipid binding, preventing formation of the intrinsic tenase complex.

Aims: In this study, we have aimed to generate recombinant C1, C2, and C1C2 constructs, along with hypothesis-driven and hemophilia A-associated mutants, to examine the membrane binding behavior for each domain. Additionally, we focused on elucidating the structural nature of phosphatidylserine binding at atomic resolution.

Methods: We devised a novel expression platform for generating recombinant C1, C2, and C1C2 domain constructs and performed site-directed mutagenesis of several charged residues proximal to the putative membrane binding region of each C domain. Membrane binding was measured with established ELISA methods, biolayer interferometry (BLI) with affinity-labeled lipid nanodiscs and a liposome sedimentation assay. Structure determination was performed with X-ray crystallography.

Results: Mutations to basic residues adjacent to the surface-exposed hydrophobic regions of C1 and C2 differentially disrupted membrane binding, with abrogation of binding occurring for mutations to conserved arginine residues in the C1 (R2163) and C2 (R2320) domains. We also determined the high-resolution X-ray crystal structure of the porcine fVIII C2 domain bound to o-phospho-L-serine, the polar headgroup of phosphatidylserine, which binds to a basic cleft and makes charge-charge contact with R2320.

Conclusion(s): We conclude that fVIII binds to phosphatidylserine-containing membranes at similar basic clefts containing conserved arginine residues through C domain modularity, where each C domain possesses modest electrostatic-dependent affinity, and tandem domains are required for high affinity binding.