INHIBITION OF BINDING OF ANTI-PLA(1) ANTIBODIES TO PLATELETS WITH MONOCLONAL-ANTIBODY LK-4 - EVIDENCE FOR MULTIPLE PLA(1) RECEPTOR-SITES ONPLATELET GPIIIA

The PLA(1) epitope on platelet GPIIIa has a sulfhydryl-dependent confo rmation and is dependent on a leucine(33)/proline(33) polymorphism. Mo noclonal antibody LK-4 differentiates PLA(1)/PLA(1) from PLA(2)/PLA(2) platelet lysates on solid phase enzyme-linked immunosorbent assay (EL ISA), as well as immunoblot. To determine whether LK-4 reacts at or ne ar the binding site(s) for human anti-PLA(1), nine such antibodies (Ab s) (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 hum an Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-de pendent manner, employing both solid phase ELISA, as well as flow cyto metry, Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibi ted by LK-4. IC50's for inhibition of binding of anti-PLA(1) to rGPIII a 1-66 varied from 8 to 160 mu g/mL (5 x 10(-8) - 1 x 10(-6) mol/L), H owever, IC,,'s for LK-4 inhibition of binding to platelets was strikin gly different. Six of the nine Abs had IC50's of 1 to 10 mu g/mL (8-fo ld to 16-fold greater inhibition than with rGPIIIa 1-66), whereas thre e neonatal Abs had IC50's of 380 to 1,013 mu g/mL (6-fold to 48-fold l ess inhibition than with rGPIIIa 1-66). Similar results were noted wit h intact GPIIIa. rGPIIIa 1-66 blocked the binding of anti-PLA(1) Abs t o platelets and served to segregate the nine patients into two groups: a sensitive group of anti-PLA(1) Abs from six patients in which bindi ng to platelets was progressively inhibited by increasing concentratio ns of rGPIIIa 1-66 with inhibition at 1 mu g/mL of 18% and inhibition at 256 mu g/ml of 78%; a second resistant group of three anti-PLA(1) A bs from three patients in which inhibition was first noted at 16 mu g/ mL of 4% with 35% inhibition at 256 mu g/mL. Thus, LK-4 binds to GPIII a at the 1-66 N-terminal region, inhibits binding of anti-PLA(1) Ab to platelets, and segregates anti-PLA(1) Abs into two groups. These data are compatible with two or more receptor sites for anti-PLA(1) Ab: on e that is present on rGPIIIa 1-66 and sensitive to LK-4 inhibition, an other that is present on rGPIIIa 1-66, as well as other site(s) on pla telet GPIIIa and insensitive to inhibition. (C) 1996 by The American S ociety of Hematology.