The
integrin alpha IIb beta 3 (GPIIb-IIIa complex) of blood platelets mediates platelet aggregation by binding adhesive
proteins which form bridges between activated cells. This same process is implicated in arterial
thrombosis. The goal of our research is to take B-lymphocytes from patients possessing inhibitory
antibodies to GPIIb-IIIa and develop technology permitting their production ex vivo. Starting point is the peripheral blood from two patients with Glanzmann's
thrombasthenia, an inherited disorder in which platelets lack these complexes, and where high titre
antibodies to GPIIb-IIIa have formed following contact with normal platelets after transfusion and/or pregnancy. We describe a strategy of in vitro stimulation to overcome the following constraints: (i) peripheral blood contains a low concentration of
antigen-reactive specific B-cells, and (ii) the circulating B-cells are arrested in a phase in which additional stimuli are required to induce
antigen-specific clonal activation. Optimal conditions involve the use of a combination of
growth factors, polyclonal activators and soluble GPIIb-IIIa prior to the fusion of activated B-cells with either (a) the murine myeloma cell line X63 Ag 8,653 or (b) the heteromyeloma cell line SPM4-0. In this way, we have obtained several cell lines secreting
antibodies specific for the GPIIb-IIIa complex. Our next aim is to rescue the relevant human immunoglobulin genes from these hybridoma cells.