Induction of tissue factor activity in endothelial cells and monocytes by a modified form of albumin present in normal human plasma.

Molecules that induce tissue factor expression by responsive cells such as endothelial cells and monocytes may be important in the regulation of hemostasis and, perhaps, in mediating certain hemostatic disorders. A constituent of normal human plasma capable of inducing tissue factor activity in human endothelial cells and monocytes has been isolated and identified as a derivative of, or modification associated with albumin. Procoagulant albumin caused a concentration-dependent induction of tissue factor expression by human endothelial cells, but bovine endothelial cells were unresponsive. The dose-response curve developed a plateau phase, indicating that the capacity of endothelial cells to respond to the stimulus was finite. The maximum response induced by the procoagulant albumin was similar to that observed for maximally effective concentrations of endotoxin, interleukin-1, and tumor necrosis factor. Time-course studies showed that procoagulant albumin produced peak activity in 4 to 6 hours. Identification of a procoagulant form of albumin in normal human plasma suggests a potential role for this constituent in regulation of hemostasis.

Analysis of the effects of activation of the alternative pathway of complement on erythrocytes with an isolated deficiency of decay accelerating factor.

E from individuals with the Inab blood group phenotype have an isolated deficiency of decay accelerating factor (DAF, CD55). DAF is a glycosyl phosphatidylinositol anchored membrane protein that inhibits activation of both the classical and alternative pathways of complement. Deficiency of DAF from the E of paroxysmal nocturnal hemoglobinuria (PNH) is thought to contribute to their greater sensitivity to complement-mediated lysis. Unlike PNH E, however, Inab cells are not susceptible to acidified serum lysis, a process that is mediated through activation of the alternative pathway. This observation led us to hypothesize that membrane constituents other than DAF control susceptibility to acidified serum lysis. To investigate this hypothesis, Inab E were incubated in acidified serum, and hemolysis and C3 deposition (as a measure of alternative pathway activation) were quantitated. C3 deposition of Inab cells was approximately 20 times greater than normal, however, hemolysis was not observed. Inab E expressed a normal amount of membrane inhibitor of reactive lysis (MIRL, CD59), a glycosyl phosphatidylinositol anchored protein that is also deficient in PNH. When MIRL function was blocked with antibody, C3 deposition markedly increased, and 100% of the Inab cells hemolyzed in acidified serum. These studies demonstrate that susceptibility to acidified serum lysis is controlled primarily by MIRL, and that in addition to its regulatory affect on the membrane attack complex, MIRL also modulates the activity of the C3 convertase of the alternative pathway by a mechanism that remains to be determined.

Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum.

When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay-accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane-associated activation of the APC.

Does breastfeeding protect against sudden infant death syndrome?

Sudden Infant Death Syndrome (SIDS), the leading cause of infant death from one to six months in the developed world, strikes approximately two infants per 1000 live births in the U.S. The characteristics of the infants who die suddenly and unexpectedly are non-specific; none are universal except for the age distribution. Therefore, an infant is recognized to have died from SIDS only after thorough examination fails to demonstrate any other cause for the death. It is the purpose of this paper to review the most populat hypotheses of the causes of SIDS and try to explain through published scientific findings how breastfed infants appear to be protected from this condition. Many hypotheses have been proposed to explain SIDS. Some deficiencies/problems are related to the infant, such as a defect in sleep and/or breathing control, severe infant botulism, infections, reactions to immunizations, hypersensitivity to cow's milk, "maternal deprivation syndrome." Other causes are attributed to maternal circumstances, such as lower socioeconomic status, prenatal health, smoking, and the winter season. Additional suggestions of potential causes of SIDS include baby's thiamine deficiency, and hormonal and/or biochemical imbalance. The occurrence of most of these circumstances can be associated with a lack of breastfeeding. Because SIDS occurs much less frequently in breastfed infants, it is speculated that breastfeeding protects infants against SIDS. However, scientific literature lacks uniformity in the definitions of breastfeeding (whether partial and exclusive). This specification is necessary to select control infants to elucidate the well documented substantial lower rate of incidence of SIDS in breastfed babies.

Induction of the paroxysmal nocturnal hemoglobinuria phenotype in normal human erythrocytes: effects of 2-aminoethylisothiouronium bromide on membrane proteins that regulate complement.

To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement.

Analysis of the binding of human C3b to glycoproteins on rabbit and sheep erythrocytes.

To investigate the possibility that activation of the human alternative pathway of complement is influenced by cellular constituents that interact with C3b, the membrane proteins on rabbit and sheep erythrocytes that are associated with cell-bound human C3b have been analyzed. For both types of erythrocytes, activated C3b bound in a diffuse pattern via hydroxylamine-sensitive ester bonds. By using a homobifunctional crosslinker, a membrane component that has an apparent Mr of 35 kDa was shown to be noncovalently associated with C3b on sheep erythrocytes, but rabbit erythrocytes lacked a predominant C3b-associated protein. These studies suggest that regulation of human alternative pathway activity may be influenced by membrane glycoproteins that interact with cell-bound C3b.

Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell-associated protein.

The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol-specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.

Relationship between the membrane inhibitor of reactive lysis and the erythrocyte phenotypes of paroxysmal nocturnal hemoglobinuria.

Susceptibility to hemolysis initiated by activated cobra venom factor (CoF) complexes is a characteristic that distinguishes the most complement-sensitive type III erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) from the intermediately sensitive type II and the normally sensitive type I cells. Recently we isolated a membrane constituent from normal erythrocytes that inhibits CoFBb-initiated hemolysis, and this protein was designated membrane inhibitor of reactive lysis (MIRL). To investigate the molecular basis of the variability in complement sensitivity among PNH erythrocytes, the surface expression of MIRL and decay accelerating factor (DAF) on the three phenotypes of PNH was quantified immunochemically. Both complement regulatory proteins were markedly deficient on the erythrocytes from a patient with predominately type III cells. The erythrocytes from patients with a majority of either type II or I cells were also significantly deficient in both MIRL and DAF. While cytofluorometric analysis confirmed the quantitative deficiencies, segregation of erythrocytes into discrete subpopulations that expressed either no MIRL or normal amounts of MIRL was not observed. The results of immunoprecipitation studies were consistent with quantitative, but not qualitative abnormalities of MIRL and DAF. Selective removal of the sensitive erythrocytes indicated that approximately 20% of the normal amount of MIRL is sufficient to protect cells from CoF-initiated lysis. These studies suggest that relatively subtle quantitative differences in membrane complement regulatory proteins underlie the variability in complement sensitivity of PNH erythrocytes.

Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

The observation that type III erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are susceptible to hemolysis initiated by activated cobra venom factor complexes (CoFBb), whereas normal erythrocytes are resistant, implies that the PNH III cells are deficient in a membrane constituent that regulates this process. To isolate the inhibitory factor from normal erythrocytes, membrane proteins were first extracted with butanol and then subjected to sequential anion exchange, hydroxylapatite, and hydrophobic chromatography. Analysis by SDS-PAGE and silver stain of the inhibitory fractions showed a single band corresponding to a protein with an apparent Mr of 18 kD. PNH erythrocytes were incubated with incremental concentrations of the radiolabeled protein and then washed. In a dose-dependent fashion, the protein incorporated into the cell membrane and inhibited CoFBb-initiated lysis. This protein inhibitor functioned by restricting the assembly of the membrane attack complex at the level of C7 and C8 incorporation. By using a monospecific antibody to block the function of the inhibitor, it was shown that normal erythrocytes are rendered susceptible to CoFBb-initiated hemolysis. Analysis by Western blot of membrane proteins revealed that PNH III erythrocytes are deficient in the 18-kD protein. By virtue of its molecular weight and inhibitory activity, the 18-kD protein appears to be discrete from other previously described erythrocyte membrane proteins that regulate complement. These studies also indicate that the susceptibility of PNH III erythrocytes to reactive lysis is causally related to a deficiency of the 18-kD membrane inhibitor.

Vitronectin (S protein) is associated with platelets.

Vitronectin is a plasma glycoprotein that has regulatory activity in the complement and the coagulation systems, in cell-cell and cell-substrate interactions, and in monocyte/macrophage function. Because of its potential to participate in several of the processes of inflammation and repair, the association of vitronectin with platelets was investigated. Immunochemical studies demonstrated that the majority of the platelet associated vitronectin was intracellular, while a relatively modest amount was localized to the ectoplasmic portion of the plasma membrane. Analysis by Western blot showed that the electrophoretic mobility of platelet associated vitronectin was indistinguishable from that of vitronectin isolated from plasma. In response to thrombin, approximately 1 microgram of vitronectin was released into the supernate of 10(9) platelets, while somewhat less than one-half of the total platelet vitronectin remained cell associated. The binding of vitronectin to platelets was investigated by comparing the capacity of unlabelled vitronectin and fibronectin to inhibit binding of radiolabelled fibronectin to thrombin stimulated platelets. On a weight basis, inhibition by the two proteins was equivalent, suggesting that vitronectin competes with fibronectin for binding to platelet glycoprotein IIb/IIIa. These results demonstrate that vitronectin is a platelet specific protein which, because of its multifunctional properties, may participate in physiological and pathophysiological events associated with thrombosis and haemostasis.

Characterization of the enhanced susceptibility of paroxysmal nocturnal hemoglobinuria erythrocytes to complement-mediated hemolysis initiated by cobra venom factor.

When whole serum C is activated by cobra venom factor complexes (CoFBb), paroxysmal nocturnal hemoglobinuria (PNH) III E (the most C-sensitive type) are hemolyzed, but normal and PNH II E (the intermediately sensitive type) are not. Previous studies have shown that after exposure to CoFBb and serum, PNH III E bind relatively large amounts of the trimolecular C complex, C5b67, whereas normal and PNH II E bind virtually none. In the studies reported herein, we have observed that when normal and PNH III E are incubated with isolated C5, C6, and 125I-C7 in the presence CoFBb, the normal E bind more C5b-7 than the PNH cells. When C7-deficient serum is included in the reaction mixture, however, the PNH E are once again observed to bind much greater amounts of C5b-7. These observations suggest that plasma and membrane factors act in concert to restrict the assembly of the trimolecular C5b-7 complex on human E. PNH III E appear to be deficient in the membrane component of this inhibitory system.