PRODUCTION AND CHARACTERIZATION OF TRANSFORMED B-LYMPHOCYTES EXPRESSING THE MEMBRANE DEFECT OF SCOTT SYNDROME

Scott syndrome is a bleeding disorder associated with an isolated defe ct in expression of membrane coagulant activity by stimulated platelet s. This defect represents a decrease in platelet membrane binding site s 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 wit h Scott syndrome and from normal donors were immortalized by EBV-trans formation, 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 sur face expression of PS in > 70% of all cells, as detected by membrane a ssociation of the PS-binding proteins, factor Va or annexin V. PS expo sure in these cells was maximal after 5 min, and saturated at < 100 mu M external free [Ca2+]. By contrast, < 30% of Scott syndrome lymphobl asts exposed PS, and saturation was not observed at > 1 mM external fr ee [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 shar e this membrane abnormality. Hybridomas prepared by fusion of Scott ly mphoblasts with the myeloma cell line UC-LUC showed responses to Ca2ionophore comparable to those observed for normal lymphoblasts and for hybridomas prepared by fusion of normal lymphoblasts with UC-LUC. Thi s correction of the Scott abnormality suggests possible complementatio n of an aberrant gene(s) responsible for this disorder.