Search PubMed⌕ Search

Biomedical subjects

P J Sims

Publications and source records attributed to P J Sims.

At least 37 records · Page 2Linked to original sources
PubMed1997

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

Phospholipid (PL) scramblase is a plasma membrane protein that mediates accelerated transbilayer migration of PLs upon binding Ca2+, facilitating rapid mobilization of phosphatidylserine to the cell surface upon elevation of internal Ca2+. In patients with Scott syndrome, a congenital bleeding disorder related to defective expression of membrane coagulant activity, circulating blood cells show decreased cell surface exposure of phosphatidylserine at elevated cytosolic [Ca2+], implying an underlying defect or deficiency of PL scramblase. To gain insight into the molecular basis of this disorder, we compared PL scramblase in Scott erythrocyte membranes to those of normal controls. Whereas membranes of Scott cells were unresponsive to Ca2+-induced activation of PL scramblase at neutral pH, apparently normal PL scramblase activity was induced at pH < 6.0. After extraction with octylglucoside, a membrane protein was isolated from the Scott cells which exhibited normal PL scramblase activity when reconstituted in vesicles with exogenous PLs. Like PL scramblase from normal erythrocytes, PL scramblase from Scott erythrocytes was maximally activated either by addition of Ca2+ (at pH 7.4) or by acidification to pH < 6.0, and similar apparent affinities for Ca2+ and rates of transbilayer transfer of PLs were observed. This suggests that the defect in Scott syndrome is related to an altered interaction of Ca2+ with PL scramblase on the endofacial surface of the cell membrane, due either to an intrinsic constraint upon the protein preventing interaction with Ca2+ in situ, or due to an unidentified inhibitor or cofactor in the Scott cell that is dissociated by detergent.

Blood Coagulation Disorders↗
PubMed1997

Complement C5b-9 increases plasminogen binding and activation on human endothelial cells.

Deposition of the terminal complement proteins (C5b-9) on human endothelial cells can result in cell lysis or nonlytic alterations of cell function including procoagulant responses. Because regulation of fibrinolysis is a central endothelial function and because C9 contains a carboxyl-terminal lysine similar to other proteins that bind and facilitate activation of plasminogen (PG), the effects of complement injury on PG binding and activation on these cells were investigated. Activation of complement through deposition of C5b67 complexes on endothelial cells resulted in a small increase (approximately 20%) in PG binding. Incorporation of C8 into C5b-8 resulted in no further increase in binding; however, specific 125I-PG binding was increased by approximately 100% after C5b-9 deposition. Moreover, PG was found to bind specifically to C7 and C9. The PG bound to endothelial cells after C5b-9 deposition was readily activated by tissue-type plasminogen activator (TPA). In a cell-free system, complement C9 and a synthetic peptide composed of the 20 carboxyl-terminal amino acids of C9 enhanced PG activation by TPA. Removal of the carboxyl-terminal lysine of C9 abolished the enhancement of PG activation without diminishing PG binding. We conclude that membrane C9 may comprise a binding site for PG and serve to enhance activation of this zymogen by TPA. These findings suggest that immune injury to the endothelium may enhance both the fibrin-generating and fibrinolytic capacity of the vessel wall.

Binding Sites↗
PubMed1996

Elimination of potential sites of glycosylation fails to abrogate complement regulatory function of cell surface CD59.

CD59 is a glycosylphosphatidylinositol-anchored membrane glycoprotein that serves as the principle cellular inhibitor of the C5b-9 membrane attack complex (MAC) of human complement. Approximately 50% of the total apparent mass of CD59 is attributable to glycosylation of a single Asn (Asn18). The deduced amino acid sequences of CD59 homologues identified in Old and New World primates as well as in rat reveal that the motif for N-linked glycosylation at the residue corresponding to Asn18 of human CD59 is invariably conserved, despite considerable sequence divergence elsewhere in the protein. Such conservation suggests that the post-translational modification at Asn18 has importance for either expression or normal function of CD59 at the cell surface. In this study, we specifically examined how deletion or transposition of the site of N-linked glycosylation in the CD59 polypeptide affects its MAC inhibitory function. Our data demonstrate that the inhibitory potency of CD59 is unaffected when glycosylation is transposed from Asn18 to another site in the polypeptide. Furthermore, we show that CD59 retains normal MAC regulatory function when mutated to eliminate all potential sites for N-linked glycosylation. These data suggest that the MAC inhibitory function of CD59 is entirely provided by residues exposed at the surface of the core polypeptide and that this core structure is not influenced by glycosylation at Asn18.

Amino Acid Sequence↗
PubMed1996

Isolation of an erythrocyte membrane protein that mediates Ca2+-dependent transbilayer movement of phospholipid.

Elevation of intracellular Ca2+ in erythrocytes, platelets, and other cells initiates rapid redistribution of plasma membrane phospholipids (PL) between inner and outer leaflets, collapsing the normal asymmetric distribution. Consequently, phosphatidylserine and other lipids normally sequestered to the inner leaflet become exposed at the cell surface. This Ca2+-induced mobilization of phosphatidylserine to the surface of activated, injured, or apoptotic cells confers a procoagulant property to the plasma membrane, which promotes fibrin clotting and provides a signal for cell removal by the reticuloendothelial system. To identify the constituent of the membrane that mediates this Ca2+-dependent "PL scramblase" activity, we undertook purification and reconstitution of membrane component(s) with this activity from detergent extracts of erythrocyte ghosts depleted of cytoskeleton. Active fractions were identified by their capacity to mediate the Ca2+-dependent redistribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled PL between leaflets of reconstituted proteoliposomes. This PL scramblase activity co-eluted through multiple chromatographic steps with a single polypeptide of approximately 37 kDa, which was purified to apparent homogeneity as resolved by silver staining. The activity associated with this protein band was inactivated by trypsin. The isolated protein reconstituted in proteoliposomes mediated nonselective, bidirectional transport of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-PL between membrane leaflets, with half-maximal activation between 20 and 60 microM Ca2+ (saturation >100 microM), mimicking the Ca2+-dependent transbilayer lipid movement intrinsic to the erythrocyte membrane.

4-Chloro-7-nitrobenzofurazan↗
PubMed1996

Role of a disulfide-bonded peptide loop within human complement C9 in the species-selectivity of complement inhibitor CD59.

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective, and is most effective toward C9 derived from human or other primate plasma. The species-selective activity of CD59 was recently used to map the segment of human C9 that is recognized by this MAC inhibitor, using recombinant rabbit/human C9 chimeras that retain lytic function within the MAC [Husler, T., Lockert, D. H., Kaufman, K. M., Sodetz, J. M., & Sims, P. J. (1995) J. Biol. Chem. 270,3483-3486]. These experiments suggested that the CD59 recognition domain was contained between residues 334 and 415 in human C9. By analyzing the species-selective lytic activity of recombinant C9 with chimeric substitutions internal to this segment, we now demonstrate that the site in human C9 uniquely recognized by CD59 is centered on those residues contained between C9 Cys359/Cys384, with an additional contribution by residues C-terminal to this segment. Consistent with its role as a CD59 recognition domain, CD59 specifically bound a human C9-derived peptide corresponding to residues 359-384, and antibody (Fab) raised against this C9-derived peptide inhibited the lytic activity of human MAC. Mutant human C9 in which Ala was substituted for Cys359/384 was found to express normal lytic activity and to be fully inhibited by CD59. This suggests that the intrachain Cys359/Cys384 disulfide bond within C9 is not required to maintain the conformation of this segment of C9 for interaction with CD59.

Amino Acid Sequence↗
PubMed1996

Structure-function relationships of the complement regulatory protein, CD59.

CD59 (membrane inhibitor of reactive lysis, protectin) is a membrane protein whose functions include the inhibition of the insertion of the ninth component of complement into the target membrane. It belongs to a superfamily of proteins including Ly-6, elapid snake venom toxins, and urokinase receptor (UPAR); the members of the superfamily have a similar structure that includes four (in mammals five) disulfide bridges that maintain a three-dimensional conformation consisting of a central core, three finger-like "loops" extending from it and a small loop near the coboxyl end. We have used site directed mutagenesis to explore three aspects of the structure of CD59: 1) the role of the disulfide bridges in expression and function of the molecule; 2) the location of epitopes reacting with monoclonal antibodies to the molecule; and 3) the parts of the molecule that are critical to its function in inhibiting complement lysis. Mutant molecules in which the disulfides maintaining the finger-like loops (Cys3-Cys26, Cys19-Cys39, and Cys45-Cys63) were removed were not expressed on the cell surface. The mutation of the disulfide (Cys6-Cys13) resulted in no change in expression or function. The mutation of Cys64-Cys69 maintaining the small loop resulted in an expressed molecule with increased functional activity. The major epitope for 6 of 7 monoclonal antibodies was centered on Arg53 as the mutation 53Arg-->Ser resulted in a loss of interaction with these antibodies, as did the deletion of four nearby residues (Leu54-Asn57). The alteration 55Arg-->Ser resulted in loss of reactivity for some but not other antibodies. The reactivity with one monoclonal antibody, H19, was abrogated by the mutations 61Tyr-->Gly and 61Tyr-->Ala. Functional activity of the molecule was not adversely altered by mutations in the first and second loops; however, the 61Tyr-->Gly mutation was non-functional. The mutation of 61Tyr-->His diminished function but changes 61Tyr-->Ala and 61Tyr-->Phe had no effect on function. We conclude that the functional site of CD59 is located in this region of the molecule.

Animals↗
PubMed1996

Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG peptide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.

Antibodies, Monoclonal↗
PubMed1995

The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions is not responsible for Ca2+-induced loss of phospholipid asymmetry in the human erythrocyte: a study in Scott syndrome, a disorder of calcium-induced phospholipid scrambling.

Elevation of cytoplasmic Ca2+ levels in human erythrocytes induces a progressive loss of membrane phospholipid asymmetry, a process that is impaired in erythrocytes from a patient with Scott syndrome. We show here that porcine erythrocytes are similarly incapable of Ca2+-induced redistribution of membrane phospholipids. Because a complex of phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+ has been proposed as the mediator of enhanced transbilayer movement of lipids (J Biol Chem 269:6347,1994), these cell systems offer a unique opportunity for testing this mechanism. Analysis of both total PIP2 content and the metabolic-resistant pool of PIP2 that remains after incubation with Ca2+ ionophore showed no appreciable differences between normal and Scott erythrocytes. Moreover, porcine erythrocytes were found to have slightly higher levels of both total and metabolic-resistant PIP2 in comparison with normal human erythrocytes. Although loading of normal erythrocytes with exogenously added PIP2 gave rise to a Ca2+-induced increase in prothrombinase activity and apparent transbilayer movement of nitrobenzoxadiazolyl (NBD)-phospholipids, these PIP2-loaded cells were also found to undergo progressive Ca2+-dependent cell lysis, which seriously hampers interpretation of these data. Moreover, loading Scott cells with PIP2 did not abolish their impaired lipid scrambling, even in the presence of a Ca2+-ionophore. Finally, artificial lipid vesicles containing no PIP2 or 1 mole percent of PIP2 were indistinguishable with respect to transbilayer movement of NBD-phosphatidylcholine in the presence of Ca2+. Our findings suggest that Ca2+-induced redistribution of membrane phospholipids cannot simply be attributed to the steady-state concentration of PIP2, and imply that such lipid movement is regulated by other cellular processes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

Identity of the segment of human complement C8 recognized by complement regulatory protein CD59.

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.

Amino Acid Sequence↗
PubMed1995

Chimeras of human complement C9 reveal the site recognized by complement regulatory protein CD59.

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex, thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective and is most effective toward C9 derived from human or other primate plasma. By contrast, rabbit C9, which can substitute for human C9 in the membrane attack complex, mediates unrestricted lysis of human cells. To identify the peptide segment of human C9 that is recognized by CD59, rabbit C9 cDNA clones were isolated, characterized, and used to construct hybrid cDNAs for expression of full-length human/rabbit C9 chimeras in COS-7 cells. All resulting chimeras were hemolytically active, when tested against chicken erythrocytes bearing C5b-8 complexes. Assays performed in the presence or absence of CD59 revealed that this inhibitor reduced the hemolytic activity of those chimeras containing human C9 sequence between residues 334-415, irrespective of whether the remainder of the protein contained human or rabbit sequence. By contrast, when this segment of C9 contained rabbit sequence, lytic activity was unaffected by CD59. These data establish that human C9 residues 334-415 contain the site recognized by CD59, and they suggest that sequence variability within this segment of C9 is responsible for the observed species-selective inhibitory activity of CD59.

Amino Acid Sequence↗
PubMed1995

Induction of cellular procoagulant activity by the membrane attack complex of complement.

In addition to their well-recognized role in immune defense, there is a growing recognition that the proteins of the complement system impact directly on vascular homeostatic mechanisms, evoking cellular responses that serve to both promote adherence of blood cells to the walls of blood vessels, and the formation of fibrin through the enzyme mechanisms of the coagulation system. This clot-promoting or 'procoagulant' activity initiated through the complement system entails both receptor-mediated as well as receptor-independent pathways of cell activation. In this review, we will focus specifically upon the role that is now thought to be played by the membrane attack complex of the complement system (MAC) in the induction of the procoagulant properties of human platelets and endothelium.

Blood Coagulation↗
PubMed1994

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

Amino Acid Sequence↗
PubMed1994

CD59 costimulation of T cell activation. CD58 dependence and requirement for glycosylation.

In addition to the TCR-CD3 complex, T lymphocytes can be activated via another surface glycoprotein, the CD2 molecule. CD58 is the principal ligand for human CD2; CD59 and CD48 are two additional, low affinity ligands that have been defined for CD2. In this study, we have explored the role of CD59 in T cell activation. We have expressed human rCD58 and rCD59 molecules in Chinese hamster oocytes (CHO), and tested paraformaldehyde-treated transfectants for the ability to promote proliferation of and IL-2 secretion from PBMC and human purified T cells. We have shown that CD59 enhanced CD58-dependent T cell proliferation and IL-2 secretion in the presence of suboptimal concentration of PHA or a submitogenic combination of stimulatory anti-CD2 mAbs, presence of suboptimal concentration of PHA or a submitogenic combination of stimulatory anti-CD2 mAbs, T11-2 + T11-3. CD59-dependent costimulation was dependent on several factors including the level of co-expression of CD58, the ratio of CHO cell transfectants to T cells added in culture, the concentration of mitogen, and also donor-dependent differences. As expected, CD59 costimulation of CD58-dependent T cell proliferation was inhibited by Abs directed against CD59, CD58, and CD2. In our hands, the CD59 molecule itself, in the absence of CD58, was unable to support proliferation alone even in the presence of exogenous recombinant IL-1, IL-2, or IL-6. Finally, the ability of CD59 to enhance CD58-dependent T cell responses was shown to be dependent on N-glycosylation of CD59 at amino acid Asn18.

Animals↗
PubMed1994

Protection of porcine aortic endothelial cells from complement-mediated cell lysis and activation by recombinant human CD59.

Discordant xenogeneic organ transplantation is a potential solution to the critical shortage of suitable donor organs. However, clinical application of xenotransplantation with physiologically suitable organs such as those from the pig, is currently limited by the lack of agents to prevent antibody and complement-mediated hyperacute rejection of the transplanted organ. We have used retrovirus-mediated gene transfer to express the terminal complement inhibitor protein, human CD59, in neonatal porcine aortic endothelial cells (nPAEC). Human CD59 was constitutively expressed in nPAECs at levels similar to that of native CD59 in human umbilical vein endothelial cells. The protein was tethered to the cell surface by a glycosyl-phosphatidylinositol anchor, as demonstrated by its removal following treatment with phosphatidylinositol-specific phospholipase C. In a model of antibody-dependent complement activation, nPAECs expressing human CD59 were protected from membrane pore formation and cell lysis by complement derived from either human or baboon sera. Conversely, nPAECs expressing CD59 were not protected from lysis by rabbit or dog complement, indicating that recombinant CD59 retained its species-restricted inhibitory activity. Additionally, CD59 expressed on nPAECs inhibited the C5b-9-dependent generation of membrane prothrombinase activity. Collectively, these data establish that stable expression of human CD59 on xenotypic (porcine) endothelial cells renders these cells resistant to both the cytolytic and procoagulant effects of human complement. We propose that expression of recombinant human CD59 on porcine donor organs may prevent complement-mediated lysis and activation of endothelial cells that leads to hyperacute rejection.

Amino Acid Sequence↗
PubMed1994

Serum concentrations of erythromycin after intravenous infusion in preterm neonates treated for Ureaplasma urealyticum infection.

Erythromycin is receiving renewed attention as an alternative for treatment of neonatal infections caused by Ureaplasma urealyticum because of recently proved abilities of this organism to produce systemic disease in this population. Although erythromycin has been used clinically for almost 40 years, very little is known about its activity in the preterm neonate. Fourteen neonates, birth weights < or = 1500 g and < or = 15 days of age, from whom U. urealyticum was isolated from the lower respiratory tract were randomized to receive erythromycin lactobionate either 25 or 40 mg/kg/day in four divided doses at 6-hour intervals scheduled for a total of 10 days. Blood samples collected at multiple time points after initial and steady state doses were assayed for erythromycin by liquid chromatography. Minimal inhibitory concentrations (MICs) of erythromycin for the U. urealyticum isolates were determined. MICs ranged from 0.031 to 2 micrograms/ml; MIC90 = 2 micrograms/ml. Serum erythromycin concentrations met or exceeded most MICs, with peak values of 3.05 to 3.69 and 1.92 to 2.9 micrograms/ml for the 40- and 25-mg/kg/day dosage groups, respectively. Pharmacokinetic parameters were calculated after the initial dose and at steady state for both dosage groups and compared. No adverse effects thought to be related to administration of erythromycin were observed. These preliminary findings showed that erythromycin is well-tolerated, has favorable pharmacokinetic activity in the preterm neonate and should be further investigated for treatment of ureaplasmal infections.

Bacteriological Techniques↗
PubMed1994

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.

Annexin A5↗
PubMed1993

Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane-binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b-9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9-treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

Adult↗