A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

A recent chemical footprinting study in our laboratory suggested that region 1803–1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems...

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Published in:The Journal of biological chemistry 2013-09, Vol.288 (36), p.26105-26111
Main Authors: Bloem, Esther, Meems, Henriet, van den Biggelaar, Maartje, Mertens, Koen, Meijer, Alexander B.
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Binding Sites
Enzyme Kinetics
Factor IX - chemistry
Factor IX - genetics
Factor IX - metabolism
Factor VIII
Factor VIII - chemistry
Factor VIII - genetics
Factor VIII - metabolism
Factor VIIIa - chemistry
Factor VIIIa - genetics
Factor VIIIa - metabolism
Humans
Mutagenesis, Site-Directed
Mutation, Missense
Protein Stability
Protein Structure
Protein Structure and Folding
Protein Structure, Tertiary
Surface Plasmon Resonance (SPR)
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Summary:A recent chemical footprinting study in our laboratory suggested that region 1803–1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803–1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803–1810 and 1811–1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811–1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811–1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803–1810 and FVIII/FV 1811–1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811–1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811–1818 contributes to FIXa binding. Both regions 1803–1810 and 1811–1818 contribute to FVIIIa stability. Background: Factor VIII (FVIII) region 1803–1818 may bind activated factor IX (FIXa) or contribute to the stability of activated factor VIII (FVIIIa). Results: Replacing 1803–1810 or 1811–1818 with the homologous regions of factor V decreases FVIIIa stability. FVIII variant F1816A exhibits reduced FIXa binding and FVIIIa stability. Conclusion: FVIII region 1803–1818 is critical for cofactor function. Significance: Novel insight is obtained about FVIII cofactor function.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M113.500884