Unique surface‐exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding

Abstract

Background:The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surfaceexposed elements involved in VWF and FIXa interaction.Objective: The aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding. Methods:Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays.Results: Five surface-exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. Conclusion:The surface-exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle. K E Y W O R D Sfactor VIII, factor IXa, kinetics, surface plasmon resonance, Von Willebrand factor | 365 PRZERADZKA Et Al.

Molecular determinants of the factor VIII/von Willebrand factor complex revealed by BIVV001 cryo-electron microscopy

“…In addition to interactions with the FVIII-a3 peptide, VWF-D9-TIL9 makes extensive interactions with FVIII-C1 and FVIII-A3, consistent with previous studies on FVIII/VWF, [4][5][6]51 burying 987Å 2 and 386Å 2 of surface area, respectively. The VWF-TIL9/FVIII-C1 and VWF-TIL9/FVIII-A3 interfaces appear to be driven largely by Van der Waals interactions and shape complementarity (Figure 5A-B).…”

Protein residue network analysis reveals fundamental properties of the human coagulation factor VIII

“…We used the FVIII RIN to identify the immediate neighboring residues of the binding sites interacting with FIXa (Refs. 41 – 45 ), FX (Refs. 46 – 48 ), thrombin 49 , 50 , von Willebrand Factor 45 , 51 – 57 , and the phospholipid membrane 51 , 53 , 54 .…”

“… 41 – 45 ), FX (Refs. 46 – 48 ), thrombin 49 , 50 , von Willebrand Factor 45 , 51 – 57 , and the phospholipid membrane 51 , 53 , 54 . These binding sites were identified in the last 3 decades using site-directed mutagenesis, synthetic peptides, competition experiments, and detailed binding and enzyme kinetic assays (to this date, no complete FVIII structure in complex with other coagulation factors was determined).…”

Isolated Variable Domains of an Antibody Can Assemble on Blood Coagulation Factor VIII into a Functional Fv-like Complex

“…ScFv KM33 was shown to have an epitope on the FVIII C1 domain [ 22 , 23 ]. The C1 domain is involved in many FVIII interactions including those within the tenase complex, platelets, VWF, and FVIII clearance receptors such as low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) [ 22 , 24 , 25 , 26 , 27 , 28 ]. When bound to FVIII, KM33 inhibits its activity, cellular uptake, and interactions with VWF, LDLR, and LRP1 [ 23 , 24 , 29 , 30 ].…”