Platelet binding of randomized light chains paired with platelet autoantibody heavy-chain H44.
The heavy chain of GPIIb/IIIa-specific H44L4 was paired again with a library of more than 106 light chains derived from the original, unselected ITP patient A library, and 101 resorted clones were screened for platelet binding by flow cytometry. (A) Matrix illustrating the genetic composition of the single retrieved positive resorted clone (designated H44L125). For comparison, 20 (of the 100) randomly chosen negative clones (designated H44L126 through H44L145) and the original H44L4 antibody are tabulated. Numbers in shaded boxes represent mean fluorescent intensities. Note that the single positive platelet-binding clone comprises a light chain derived from the same Ig light-chain gene as the original L4 light chain (012/02), yet no other 012/02-encoded light chain (eg, L125-L128) conferred binding when paired with H44. (B) Sequence analysis of cohort of 012/02-encoded light chains retrieved in resorting experiment shows that light-chain L125, which reconstitutes platelet binding, may be clonally related to the original L4 light chain because of a distinctive VJ junction characterized by loss of an entire amino acid residue at position 95 (boldface region).
