Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin.
We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49-66. Surprisingly, binding avidity for GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99-102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution.
Li
Zongdong
Z
Department of Medicine, New York University School of Medicine, New York, New York 10016. Electronic address: liz04@med.nyu.edu.
Nardi
Michael A
MA
Department of Pediatrics, New York University School of Medicine, New York, New York 10016.
Wu
Jing
J
Department of Medicine, New York University School of Medicine, New York, New York 10016.
Pan
Ruimin
R
Department of Medicine, New York University School of Medicine, New York, New York 10016.
Zhang
Wei
W
Department of Medicine, New York University School of Medicine, New York, New York 10016.
Karpatkin
Simon
S
Department of Medicine, New York University School of Medicine, New York, New York 10016. Electronic address: simon.karpatkin@med.nyu.edu.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001792:Blood Platelets / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D039661:Integrin beta3 / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D015539:Platelet Activation; D010974:Platelet Aggregation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; C061248:ITGB3 protein, human; D039661:Integrin beta3; D019151:Peptide Library