title: Factor VIII C1 domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake.
abstract: The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent "hot spot" on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091-2104 and 2157-2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092-2093 and 2158-2159. Spike 2092-2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158-2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092-2093 and 2158-2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology.
authors.affiliation: From the Department of Plasma Proteins, Sanquin Research, 1066 CX Amsterdam, The Netherlands.
authors.forename: Esther
authors.initials: E
authors.lastname: Bloem
authors.forename: Maartje
authors.initials: M
authors.lastname: van den Biggelaar
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Wroblewska
authors.forename: Jan
authors.initials: J
authors.lastname: Voorberg
authors.forename: Johan H
authors.initials: JH
authors.lastname: Faber
authors.forename: Marianne
authors.initials: M
authors.lastname: Kjalke
authors.forename: Henning R
authors.initials: HR
authors.lastname: Stennicke
authors.forename: Koen
authors.initials: K
authors.lastname: Mertens
authors.forename: Alexander B
authors.initials: AB
authors.lastname: Meijer
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D041961:Deuterium Exchange Measurement; D004705:Endocytosis; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D026502:LDL-Receptor Related Proteins / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D009154:Mutation; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D026502:LDL-Receptor Related Proteins; D010718:Phosphatidylserines; D014841:von Willebrand Factor; D005169:Factor VIII
keywords: Endocytosis; Enzyme Kinetics; Epitope Mapping; Factor VIII; Mass Spectrometry (MS); Phospholipid; Surface Plasmon Resonance (SPR); von Willebrand Factor