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PubMed1995

Shedding and enrichment of the glycolipid-anchored complement lysis inhibitor protectin (CD59) into milk fat globules.

Protectin (CD59) is a glycolipid-anchored inhibitor of the membrane attack complex (MAC) of human complement (C) that protects blood cells, endothelial cells and various epithelial cells from C-mediated lysis. Because of its activities protectin is a candidate molecule for use in the treatment of paroxysmal nocturnal haemoglobinuria or conditions where MAC causes tissue damage. Soluble, phospholipid-free forms of protectin have been isolated from human urine and produced in recombinant form, but they have only a relatively weak C lysis-inhibiting activity. In the present study we have looked for functionally active protectin in human breast milk. Milk is rich in fat droplets, milk fat globules (MFG), that are enveloped in a plasma membrane derived from secretory cells of the mammary gland. The membranes of MFG contain a variety of glycoproteins expressed by the mammary epithelial cells. Both immunofluorescence and immunoblotting analysis demonstrated that protectin was strongly expressed on human MFG. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, MFG protectin (CD59M) appeared as distinct bands with apparent molecular weights of 19,000-23,000 MW, similar to protectin extracted from MCF7 breast carcinoma cells. CD59M in breast milk was functionally active and had a glycophospholipid anchor, as judged by its ability to incorporate into guinea-pig erythrocytes and inhibit their lysis by human complement. These results indicate that functionally active protectin becomes enriched in MFG and imply that secretion of glycophospholipid-anchored molecules, e.g. into cow milk and colostrum, could be exploited as a means of producing bioactive molecules that need to be targeted into cell membranes.

CD59 Antigens↗
PubMed1995

Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII.

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Anticoagulants↗
PubMed1979

Activation of the classical complement pathway by nephritic factor bound to the alternative pathway C3/C5 convertase.

Nephritic Factor (NF), the potent alternative pathway activator, which is occasionally found in association with certain types of nephritis has recently been identified as an IgG class autoantibody specific for the C3 convertase (C3bB) of the alternative pathway. In these studies we have examined the possibility that the cell-bound NF-stabilized C3 convertase (EC3 bBNF) binds and activates the first component of the classical pathway of complement. EC3bBNF bound C1q, and the extent of binding was dependent upon the number of NF molecules bound per cell and decreased parallel to the dissociation and release of NF from the cells. Interaction of C1 with bound NF resulted in its activation as shown by the proteolytic conversion of proenzyme C1s to its activated form C1s. As was the case with C1q binding, C1 activation was dependent on the number of NF molecules bound per cell. Thus the NF-stabilized C3 convertase binds and activates C1.

Binding Sites↗
PubMed1995

Role of free protein S and C4b binding protein in regulating the coagulant response to Escherichia coli.

Previous studies showed that infusion of C4b-binding protein with sublethal Escherichia coli (E. coli) in the primate produced a consumptive coagulopathy followed by microvascular thrombosis and renal failure. The first objective of this study was to characterize the pathophysiology and mechanism of this phenomena following infusion of both these agents with emphasis on defining the role of free protein S. The second objective was to examine the relevance of this model to the hemolytic uremic syndrome. Infusion of C4b-binding protein alone reduced free protein S and decreased platelet concentration to 20% of baseline, whereas infusion of the C4b-binding protein/protein S complex did not. There was no activation of other inflammatory or coagulant factors. Infusion of sublethal E coli alone produced a transient inflammatory response with no reduction of free protein S. However, coinfusion of C4b-binding protein with sublethal E coli reduced free protein S and produced a thrombocytopenia, anemia, and a microvascular thrombotic response, whereas infusion of the C4b-binding protein/protein S complex with sublethal E coli did not. Studies comparing the effects of neutralizing (S-163) and nonneutralizing (S-145) antibodies with protein S coinfused with sublethal E coli produced similar contrasting results. Therefore, we concluded that neutralization of free protein S, and not some other property of C4b-binding protein influenced by protein S, accounted for this microvascular thrombotic response. This response is similar to the hemolytic uremic syndrome characterized by thrombocytopenia, anemia, shistocytosis, and renal glomerular thrombosis with uremia. Comparison of the respective renal histopathologic appearance supports this conclusion. This raises the possibility that inhibition of protein S activity (possibly by one of the forms of C4b-binding proteins) might be one of the factors contributing to microvascular thrombotic disorder, such as the hemolytic uremic syndrome.

Animals↗
PubMed1995

Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein.

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1994

Studies on the inflammatory-coagulant axis in the baboon response to E. coli: regulatory roles of proteins C, S, C4bBP and of inhibitors of tissue factor.

The baboon model of E. coli sepsis illustrates three concepts with respect to the host response and vascular endothelium. First, the endothelium is the primary target. E. coli sepsis is an acute inflammatory disease of the vascular endothelium. Second, the endothelium is not a passive target. Initially it regulates both the inflammatory and coagulopathic aspects of E. coli sepsis through membrane associated regulatory receptor/plasma protein assemblies including protein C/thrombomodulin, activated protein C/protein S, C4bBP/protein S, tissue factor pathway inhibitor/Xa, antithrombin III/glycosaminoglycans. Third, when overridden by inflammatory events, the endothelium can change its anticoagulant phenotype and mount a massive procoagulant fibrinolytic counter-attack on its luminal side through the expression of tissue factor and release of tissue plasminogen activator. Fourth, again when overridden by inflammatory events, the endothelium can change its antioxidant phenotype and produce a "distal" tissue hypoxia on its abluminal side through induction of free radical generation and peroxidation of mitochondrial lipid membranes of those tissues with high metabolic rates. It has become increasingly clear that the so-called anticoagulant systems which act on the proximal factors of the clotting cascade (protein C, TFPI, AT-III, PGI2) also attenuate the amplification of the inflammatory response. Aspects of the mechanism by which this occurs are coming to light. This includes the attenuation of Il-6 response by TFPI and the attenuation of the complement effects by C4bBP/PS. The specifics of these observations in the E. coli sepsis model will be reviewed.

Animals↗
PubMed1994

Differences in protein S and C4b-binding protein levels in different groups of patients with antiphospholipid antibodies.

Total protein S (tPS), free protein S (fPS) and C4b-binding protein (C4b-BP) were measured by immunological assays in 73 patients with antiphospholipid (aPL) antibodies, in order to determine whether the previously reported abnormalities in PS levels in this group of patients could be related to the presence of lupus anticoagulant (LA) or anticardiolipin (aCL) antibodies. As compared with the normal controls (n = 44), the authors found a significant decrease of tPS, fPS and C4b-BP in 45 LA(+)aCL(+) patients (P < 0.001), a decrease of tPS (P < 0.001), fPS and C4b-BP (P < 0.01) in eight LA(-)aCL(+) patients and a decrease of only fPS (P < 0.05) in 20 LA(+)aCL(-) patients. There was no difference in the levels of tPS, fPS and C4b-BP between LA(+)aCL(+) and LA(-)aCL(+) patients. In contrast, the LA(+)aCL(+) patients had lower values of tPS, fPS and C4b-BP than LA(+)aCL(-) patients (P < 0.05). In some patients, protein S activity (PSact) was also measured and a high correlation was observed between fPS antigen and PSact (r = 0.93, P < 0.001). The data show that the presence of aCL antibodies is associated with a probably acquired deficiency of PS and C4b-BP. On the other hand, in LA patients without a CL antibodies, the fPS deficiency is unrelated to an increase in C4b-BP levels and may be due to abnormal binding of PS to C4b-BP.

Abortion, Habitual↗
PubMed1994

Rabbit plasma, unlike its human counterpart, contains no complex between protein S and C4b-binding protein.

In human plasma, the anticoagulant vitamin K-dependent protein S exists in two molecular forms, as free protein and complexed to C4b-binding protein (C4BP), a complement regulatory protein. It has been suggested that rabbit plasma also contains two forms of protein S and that the interaction between protein S and C4BP in rabbits can be modulated by synthetic peptides corresponding to a sequence (residues 605-614) in the carboxy-terminal part of protein S. In this report, we provide results which challenge the conclusion that rabbit plasma contains the complexed form of protein S. The two forms of protein S in human plasma were separated by gel filtration chromatography on Sephacryl S-300 and the presence of protein S in the various fractions analyzed by Western blotting using a monoclonal antibody (HPS 21) directed against the gamma-carboxyglutamic acid rich module of human protein S. This antibody, which was found to cross-react with rabbit protein S on Western blotting, was used in affinity purification of protein S from rabbit plasma as well as of recombinant rabbit protein S. HPS 21 specifically recognized protein S in rabbit plasma and did not cross-react with the other vitamin K-dependent plasma proteins. To elucidate whether rabbit plasma contained two forms of protein S, rabbit plasma was subjected to gel-filtration chromatography followed by Western blotting of the fractions with monoclonal antibody HPS 21. Protein S was found only in fractions eluting at a position corresponding to that of free protein S.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Studies of the interaction between human protein S and human C4b-binding protein using deletion variants of recombinant human protein S.

Human protein S interacts noncovalently with human C4b-binding protein (C4BP). We have studied this interaction using deletion variants of recombinant human protein S. Two deletion variants were constructed by restriction enzyme digestion and in vitro site-specific mutagenesis of the human protein S cDNA. The variants were stably expressed in C127 cells. Recombinant proteins were purified using Fast Flow Q anion-exchange chromatography. The activated protein C (APC) cofactor activity, C4BP binding properties and reactivity to different monoclonal antibodies against human protein S were examined. The first variant (E variant), which has a deletion of the third epidermal growth factor (EGF)-like domain (deletion of exon VII, corresponding to amino acid residues ASP-160 to Asp-202) expresses normal APC cofactor activity in a plasma system. This activity was inhibited by the addition of purified C4BP. The second variant (L variant), which has a deletion of the C-terminal loop of the sex hormone binding globulin (SHBG)-like domain (deletion of exon XV, corresponding to amino acid residues Asp-583 to Ser-635) also expresses normal APC cofactor activity in plasma. This activity could only be partially inhibited by the addition of purified C4BP. Binding of the recombinant proteins to C4BP was studied in a system using purified proteins. The E variant binds to C4BP with the same affinity similar as recombinant wild type protein S (apparent Kd approximately 10(-10) M). The L variant, however, shows a markedly reduced affinity for binding to C4BP (apparent Kd approximately 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗