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

Abstract

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, EBVlymphoblasts 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 ,uM 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. (J. Clin. Invest. 1994Invest. . 94:2237Invest. -2244

Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids.

“…The possibility of an autosomal recessive mode of inheritance of the Scott defect was suggested by recent evidence for partially reduced activity of PL scramblase in cells of other family members (28). In addition to platelets, this aberrant membrane phenotype was also demonstrated in erythrocytes and leukocytes obtained from these patients, and the aberrant Scott phenotype was shown to propagate in cultured lymphoblasts derived by EBV-transformation of the patients' B cells (28,31). Consistent with evidence for an underlying recessive gene defect, hybridoma clones produced by fusion of defective Scott lymphoblasts with wild-type cells were all found to exhibit normal PL scramblase activity (31).…”

“…The plasma membranes of these cells contain normal amounts of PS and other PLs, and exhibit normal aminophospholipid translocase activity (24,29). Although Scott syndrome was originally described as an isolated platelet disorder, it is now clear that other blood cells, including RBC and lymphocytes, are also affected, and there is now evidence that this disorder is related to a defective gene that selectively affects membrane PL scrambling (28,30,31). The molecular defect giving rise to Scott syndrome remains to be elucidated.…”

The role of phosphatidylserine in recognition of apoptotic cells by phagocytes

“…Abnormalities in the function of this protein in vivo mediate the bleeding disorder known as Scott syndrome, in which failure of coagulation results from lack of PS expression on the surface of activated platelets (e.g. see Sims et al, 1989;Kojima et al, 1994;Stout et al, 1997).…”

Phosphatidylserine, a death knell

“…Artificially elevating Ca 2+ using ionophore similarly fails to activate the scramblase in Scott platelets, erythrocytes and lymphocytes. 138,139 But activation of the scramblase in apoptotic Scott lymphocytes proceeds normally, implying that the physiological Ca signaling pathway utilized by activated platelets is not the pathway used by apoptotic lymphocytes (P Williamson et al, submitted).…”