Summary.  Background:  Low‐density lipoprotein (LDL) receptor family members contribute to the cellular uptake of factor VIII. How von Willebrand factor fits into this endocytic pathway has remained poorly understood. Objectives:  It has been suggested that macrophages contribute to the clearance of the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. We now assessed the mechanisms of uptake employing human monocyte‐derived macrophages. Methods:  A confocal microscopy study was employed to study the uptake by monocyte‐derived macrophages of a functional green fluorescent FVIII‐GFP derivative in the presence and absence of VWF. Results:  The results revealed that FVIII‐GFP is internalized by macrophages. We found that FVIII‐GFP co‐localizes with LDL receptor‐related protein (LRP), and that the LRP antagonist Receptor Associated Protein (RAP) blocks the uptake of FVIII‐GFP. However, FVIII‐GFP was not detected in the macrophages in the presence of VWF, suggesting that the FVIII‐VWF complex is not internalized by these cells at all. Apart from static conditions, we also investigated the effect of shear stress on the uptake of FVIII‐GFP in presence of VWF. Immunofluorescence studies demonstrated that VWF does not block endocytosis of FVIII‐GFP under flow conditions. Moreover, VWF itself was also internalized by the macrophages. Strikingly, in the presence of RAP, endocytosis of FVIII‐GFP and VWF was inhibited. Conclusion:  The results show that shear stress is required for macrophages to internalize both constituents of the FVIII‐VWF complex.

Background: von Willebrand factor (VWF) protects factor VIII (FVIII) from rapid clearance and degradation. Results: Mass spectrometric footprinting revealed that FVIII protects Lys-773 and the N-terminal Ser-764 of VWF from chemical modification. VWF(S764A) showed increased and VWF(K773A) showed decreased FVIII binding. Conclusion:The N terminus of VWF is critical for FVIII binding. Significance: This study sheds new light on the mechanism of FVIII-VWF complex assembly.

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2248 Factor VIII (FVIII) circulates in a tight complex with its carrier protein von Willebrand factor (VWF). Activation of FVIII results in the dissociation of the FVIII-VWF complex after which FVIII can perform its role in the coagulation cascade. In the complex with VWF, FVIII is protected from rapid clearance from the circulation. Individuals with a mutation in VWF that impairs the ability of VWF to bind FVIII can therefore have a bleeding disorder caused by a low plasma level of FVIII. Mature VWF contains multiple domains of which the N-terminal D'-D3 domains have been shown to comprise the FVIII binding site. Detailed information about amino acid regions in VWF that contribute to the direct interaction with FVIII is, however, lacking. In the present study we have employed a chemical footprinting approach to identify amino acid regions of VWF that are involved in binding FVIII. To this end, the lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII. VWF was subsequently cleaved into peptides employing chymotrypsin. The identity of the peptides and whether or not they contained a modified lysine residue was assessed by nanoLC mass spectrometry. The results showed that the lysine residues of almost all identified peptides were modified to the same extent upon incubation of VWF with FVIII or activated FVIII. However, lysine residue 773 in the N-terminal peptide comprising the residues 766-SCRPPMVKL-774 was protected from chemical modification in the presence of FVIII. In addition, a peptide was identified in which the free amine group of serine 764 at the start of the D' domain was also differentially modified in the presence of FVIII or activated FVIII. We next studied the structure of a molecular model of the D' domain that was obtained by comparative homology modeling. Structure analysis revealed that the N-terminal region 764–773 is situated at the tip of the D' domain and that the amino acid residues Ser764 and Lys773 are in close proximity. This observation combined with the results obtained with the chemical footprinting approach implies that the residues Ser764 and Lys773 at the N-terminus of VWF are directly involved in the FVIII-VWF complex formation. Alternatively, upon binding of FVIII, there is a conformational change in this N-terminal region resulting into a differential accessibility of these residues for chemical modification. To further investigate on this issue, we constructed recombinant VWF variants in which the lysine residue 773 and the serine residue at position 764 were replaced by alanines. The variants Ser764Ala, Lys773Ala and WT-VWF were expressed in 293 cells and purified. The binding of Ser764Ala and Lys773Ala to FVIII was evaluated employing surface plasmon resonance (SPR) analysis. The data revealed that the N-terminal region of the VWF D' domain modulates the interaction with FVIII. The contribution of Ser764 and Lys733 was mainly reflected in the dissociation kinetics of the complex. We also assessed the association of the VWF variants to FVIII in a solid phase binding assay. In addition, we evaluated to what extent the VWF variants can compete with WT-VWF for binding FVIII. The results were in agreement with the findings obtained with SPR analysis, and demonstrated a modulatory role of the residues 764 and 773. Taken together, our data reveal that the residues Ser764 and Lys773 at the N-terminus of mature VWF contribute to the affinity of the FVIII-VWF complex. Disclosures: No relevant conflicts of interest to declare.