Production and characterization of transformed B-lymphocytes expressing the membrane defect of Scott syndrome.

Scott syndrome is a bleeding disorder associated with an isolated defect in expression of membrane coagulant activity by stimulated platelets. This defect represents a decrease in platelet membrane binding sites for coagulation factors Va and VIIIa, reflecting diminished surface exposure of phosphatidylserine (PS). To gain insight into the cellular and genetic basis for this disorder, B-lymphocytes from a patient with Scott syndrome and from normal donors were immortalized by EBV-transformation, and tested for their capacity to expose plasma membrane PS in response to the Ca2+ ionophore, A23187. Upon incubation with A23187, EBV-lymphoblasts derived from normal donors consistently induced surface expression of PS in > 70% of all cells, as detected by membrane association of the PS-binding proteins, factor Va or annexin V. PS exposure in these cells was maximal after 5 min, and saturated at < 100 microM external free [Ca2+]. By contrast, < 30% of Scott syndrome lymphoblasts exposed PS, and saturation was not observed at > 1 mM external free [Ca2+]. Single-cell clones derived from the Scott lymphoblasts all exhibited a diminished response to A23187 comparable with that of the parental cells, suggesting that all lymphocytes from this patient share this membrane abnormality. Hybridomas prepared by fusion of Scott lymphoblasts with the myeloma cell line UC-LUC showed responses to Ca2+ ionophore comparable to those observed for normal lymphoblasts and for hybridomas prepared by fusion of normal lymphoblasts with UC-LUC. This correction of the Scott abnormality suggests possible complementation of an aberrant gene(s) responsible for this disorder.