The PLA1 epitope on platelet GPIIIa has a sulfhydryl-dependent conformation and is dependent on a leucine33/proline33 polymorphism. Monoclonal antibody LK-4 differentiates PLA1/PLA1 from PLA2/PLA2 platelet lysates on solid phase enzyme-linked immunosorbent assay (ELISA), as well as immunoblot. To determine whether LK-4 reacts at or near the binding site(s) for human anti-PLA1, nine such antibodies labs) (six neonatal; three posttransfusion) were examined in the presence and absence of LK-4 for binding to platelets, as well as rGPIIIa 1-66, a recombinant glutathione S- transferase fusion peptide. All nine human Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-dependent manner, employing both solid phase ELISA, as well as flow cytometry. Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibited by LK-4. IC50's for inhibition of binding of anti- PLA1 to rGPIIIa 1-66 varied from 8 to 160 μg/mL (5 x 10-8 - 1 x 10-6 mol/L). However, IC50's for LK-4 inhibition of binding to platelets was strikingly different. Six of the nine Abs had IC50's of 1 to 10 μg/mL (8- fold to 16-fold greater inhibition than with rGPIIIa 1-66), whereas three neonatal Abs bad IC50's of 380 to 1,013 μg/mL (6-fold to 48-fold less inhibition than with rGPIIIa 1-66). Similar results were noted with intact GPIIIa, rGPIIIa 1-66 blocked the binding of anti-PLA1 Abs to platelets end served to segregate the nine patients into two groups: a sensitive group of anti-PLA1 Abs from six patients in which binding to platelets was progressively inhibited by increasing concentrations of rGPIIIa 1-66 with inhibition at 1 μg/mL of 18% and inhibition at 256 μg/mL of 78%; a second resistant group of three anti-PLA1 Abs from three patients in which inhibition was first noted at 16 μg/mL of 4% with 35% inhibition at 256 μg/mL. Thus, LK-4 binds to GPIIIa at the 1-66 N-terminal region, inhibits binding of anti-PLA1 Ab to platelets, and segregates anti-PLA1 Abs into two groups. These data are compatible with two or more receptor sites for anti- PLA1 Ab: one that is present on rGPIIIa 166 and sensitive to LK-4 inhibition, another that is present on rGPIIIa 1-66, as well as other site(s) on platelet GPIIIa and insensitive to inhibition.
|Original language||English (US)|
|Number of pages||7|
|State||Published - Nov 1 1996|
ASJC Scopus subject areas
- Cell Biology