C4b-binding Protein In Plasma Research Articles

LPS-Toll-Like Receptor-Mediated Signaling on Expression of Protein S and C4b-Binding Protein in the Liver

Protein S (PS), mainly synthesized in hepatocytes and endothelial cells, plays a critical role as a cofactor of anticoagulant activated protein C (APC). PS activity is regulated by C4b-binding protein (C4BP), structurally composed of seven α-chains (C4BPα) and a β-chain (C4BPβ). In this paper, based primarily on our previous studies, we review the lipopolysaccharide (LPS)-induced signaling which affects expression of PS and C4BP in the liver. Our in vivo studies in rats showed that after LPS injection, plasma PS levels are significantly decreased, whereas plasma C4BP levels first are transiently decreased after 2 to 12 hours and then significantly increased after 24 hours. LPS decreases PS antigen and mRNA levels in both hepatocytes and sinusoidal endothelial cells (SECs), and decreases C4BP antigen and both C4BPα and C4BPβ mRNA levels in hepatocytes. Antirat CD14 and antirat Toll-like receptor (TLR)-4 antibodies inhibited LPS-induced NFκB activation in both hepatocytes and SECs. Furthermore, inhibitors of NFκB and MEK recovered the LPS-induced decreased expression of PS in both cell types and the LPS-induced decreased expression of C4BP in hepatocytes. These data suggest that the LPS-induced decrease in PS expression in hepatocytes and SECs and LPS-induced decrease in C4BP expression in hepatocytes are mediated by MEK/ERK signaling and NFκB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism.

Protein S deposition at placenta: a possible role of protein S other than anticoagulation

Protein S is an antithrombotic cofactor for protein C that also has multifunctional anti-inflammatory, cellular protective, apoptotic and mitogenic properties. Protein S levels are thought to decrease during pregnancy, but the underlying mechanism remains unknown. We compared protein S concentrations throughout normal pregnancy with those of nonpregnant women and measured plasma C4b-binding protein levels in nonpregnant women and in pregnant women at the 40th gestational week. We also examined protein S and C4b-binding protein in the placenta by immunohistochemical staining at early (20th gestational week) and late (40th gestational week) stages of pregnancy. Plasma protein S activity and free protein S-antigen levels significantly decreased from the 10th gestational week and total protein S antigen decreased from the 20th. C4b-binding protein levels between pregnant and nonpregnant women did not significantly differ. The stainable portion of protein S was located at the fetomaternal interface, particularly at degenerative villi. C4b-binding protein was weakly stained at the same areas as protein S. Neither protein S nor C4b-binding protein were stained at normal villi. These results indicated that protein S can protect or restore damaged villi via a physiological effect in addition to its anticoagulation properties.

Regulatory mechanisms of C4b-binding protein (C4BP)alpha and beta expression in rat hepatocytes by lipopolysaccharide and interleukin-6.

C4b-binding protein (C4BP), a multimeric protein structurally composed of alpha chains (C4BPalpha) and a beta chain (C4BPbeta), regulates the anticoagulant activity of protein S (PS). Patients with sepsis have increased levels of plasma C4BP, which appears to be induced by interleukin (IL)-6. However, it is not fully understood how lipopolysaccharide (LPS) and IL-6 affect the plasma C4BP antigen level and C4BPalpha and C4BPbeta expression in hepatocytes. To assess the effect of LPS and IL-6 on plasma C4BP, PS-C4BP complex levels, PS activity, and C4BP expression by rat liver in vivo and on C4BP expression by isolated rat hepatocytes in vitro. Plasma C4BP antigen level transiently decreased from 2 to 12 h after LPS (2 mg kg(-1)) injection, and then it abruptly increased up to 24 h after LPS injection. Plasma C4BP antigen level increased until 8 h after IL-6 (10 microg kg(-1)) injection, and then gradually decreased up to 24 h after IL-6 injection. LPS significantly decreased the protein and mRNA expression of both C4BPalpha and C4BPbeta in rat hepatocytes, and this effect was inhibited by NFkappaB and MEK/ERK inhibitors. IL-6 mediated increase in C4BPbeta expression in rat hepatocytes, which leads to increased plasma PS-C4BP complex level and to decreased plasma PS activity, was inhibited by inhibition of STAT-3. LPS decreases both C4BPalpha and C4BPbeta expression via the NFkappaB and MEK/ERK pathways, whereas IL-6 specifically increases C4BPbeta expression via the STAT-3 pathway, causing an increase in plasma PS-C4BP complex, and thus decreasing the anticoagulant activity of PS.

CCP1–4 of the C4b-binding protein α-chain are required for factor I mediated cleavage of complement factor C3b

C4b-binding protein (C4BP) is a potent regulator of the complement system because it strongly inhibits the classical pathway of complement. Furthermore, C4BP serves as a cofactor to factor I (FI) in the cleavage of fluid phase C3b and can, therefore, influence the alternative pathway of complement. The major form of C4BP in plasma consists of seven identical α-chains and one β-chain. Both types of subunits are composed of complement control protein (CCP) domains, eight such domains make up one α-chain. To elucidate the structural requirements for the interaction between C3b and the α-chain, nineteen recombinant C4BP variants were used: six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally four variants in which double alanine residues were introduced between CCPs. We found that C4BP requires all four N-terminal CCPs of the α-chain, with CCP2 and 3 being the most important, to act as a cofactor in the cleavage of C3b. Also, a cluster of positively charged amino acids on the interface between CCP1 and 2 is involved in the binding. Compared to the interaction with C4b, we conclude that binding of C3b to C4BP requires larger molecular surface on C4BP. We found that C4BP was able to act as cofactor in degradation of surface bound C3b and to accelerate decay of alternative C3-convertase. However, in both cases 1000-fold molar excess of C4BP over factor H (FH), well known inhibitor of the alternative pathway, was required to obtain the same effect.

Binding of human complement component C4b-binding protein (C4BP) to Streptococcus pyogenes involves the C4b-binding site.

A key step in the elimination of invading pathogens from the body is the covalent binding of complement proteins C3b and C4b to their surface. However, many pathogens have evolved mechanisms to avoid the complement system of the host. Understanding how these mechanisms work may lead to more efficacious forms of therapy. Here we provide an insight into the molecular basis of how Streptococcus pyogenes binds human plasma C4b-binding protein (hC4BP), a complement regulatory molecule that may decrease C3b and C4b deposition on the streptococcal surface. We show that streptococcal surface molecules bind to a site on hC4BP that is indistinguishable from the C4b binding site. This site involves multiple binding surfaces that span short consensus repeats 1 to 3 of the alpha-chain of hC4BP. We propose that hC4BP is bound to the bacterial surface because the streptococcal surface molecules involved in the interaction mimic human C4b epitopes.

Impairments of the protein C system and fibrinolysis in infection-associated stroke.

Infection/inflammation appears to be an important predisposing risk factor for brain infarction, but little is known regarding underlying molecular mechanisms. We examined the hypothesis that patients with brain infarction preceded by infection/inflammation within 1 week could be identified by a distinctive procoagulant laboratory profile characterized by abnormalities in the protein C system and endogenous fibrinolysis. We performed a case-control study examining the relationship between preceding systemic infectious/inflammatory syndromes and selected immunohematologic variables in 36 patients with acute brain infarction and 81 control subjects (community control subjects [n = 47] and hospitalized nonstroke neurological patient controls [n = 34]). The stroke group had a lower mean level of the circulating antithrombotic enzyme activated protein C (APC) (4.33 +/- 0.34% [log-transformed percentage of control value, mean +/- SD]) than community control subjects (4.51 +/- 0.27%, P < .02) or hospitalized neurological patient controls (4.57 +/- 0.31%, P < .005). The lowest circulating APC levels were found in the stroke group with antecedent infection/inflammation within 1 week preceding index brain infarction (4.23 +/- 0.4%, n = 12). Within the stroke group, circulating APC levels were inversely related to IgG isotype anticardiolipin antibody titers (r = -.55, P < .001). Only the stroke group with infection/inflammation within 1 week had elevated plasma C4b binding protein compared with control subjects (141 +/- 61% versus 112 +/- 44%, P < .05). Stroke patients with antecedent infection/inflammation had a distinctively lower ratio of active tissue plasminogen activator to plasminogen activator inhibitor (0.11 +/- 0.04, n = 9) than other stroke patients (0.19 +/- 0.06, n = 9, P < .01) and control subjects (0.22 +/- 0.16, n = 17, P < .02). Impairments in the protein C pathway and endogenous fibrinolysis may contribute to the increased risk for brain infarction after recent (< or = 1 week) infection/inflammation. A decrease in the circulating anticoagulant APC may be related to elevated antiphospholipid antibody titers.

Expression and characterization of a recombinant C4b-binding protein lacking the beta-chain.

C4b-binding protein (C4BP) is a high-molecular-mass glycoprotein which contains binding sites for complement component C4b, anti-coagulant vitamin K-dependent protein S and serum amyloid P component (SAP). The major form of C4BP in plasma is composed of seven identical alpha-chains and a single beta-chain. We have expressed full-length cDNA for the alpha-chain in a eukaryotic expression system and characterized functional properties of non-beta-chain-containing C4BP. During synthesis, recombinant alpha-chains polymerized into two different high-molecular-mass C4BP forms which were composed of seven or eight alpha-chains. Recombinant C4BP bound C4(H2O) (used instead of C4b) equally as well as native C4BP, functioned equally as well as factor I cofactor in the degradation of C4(H2O) and bound to SAP. In contrast, the recombinant C4BP did not bind protein S and therefore did not inhibit the ability of protein S to function as a cofactor to activated protein C. Tunicamycin treatment of the transfected cells prevented N-linked glycosylation, but did not affect polymerization of the alpha-chains into a high-molecular-mass C4BP. The non-glycosylated C4BP had comparable properties to glycosylated C4BP in several functional assays. These results demonstrate polymerization of C4BP alpha-chains to be independent both of the beta-chain and of the N-linked carbohydrates. Moreover, N-linked carbohydrates and the beta-chain were neither required for the ability of C4BP to bind C4b and to function as factor I cofactor nor for the interaction with SAP.

Molecular Analysis of the /5-Chain of Human C4B-Binding Protein

C4b-binding protein (C4BP) is a multimeric glycoprotein in plasma with important regulatory functions in the complement system. It occurs in two forms, as free protein and in a non-covalent bimolecular complex with the vitamin K-dependent protein S. Protein S is an important anticoagulant and enhances the rate of inactivation by activated protein C of blood coagulation factors, Va and VIIIa. Protein S bound to C4BP is inactive as an anticoagulant, indicating C4BP to have a regulatory function in the blood coagulation process. Approximately 50% of C4BP in plasma circulates in complex with protein S, but little has been known about as to how these proteins interact. This report describes the structure of C4BP and its relation to protein S binding. A novel C4BP subunit, designated the beta-chain, which in all likelihood contains the protein S binding site, has been identified, isolated and characterized. The major form of C4BP is composed of seven alpha-chains and one beta-chain, and the subunits are covalently linked by their carboxy-terminal regions giving the molecule a spider-like quaternary structure. A subpopulation of C4BP, which does not bind protein S, was found to lack the beta-chain. This provides support for the concept that the single protein S binding site is located on the beta-chain. The beta-chain is structurally related to the alpha-chain of C4BP, and both subunits belong to the superfamily of C3b/C4b-binding proteins. The genes coding for the alpha- and beta-chains of C4BP were found to be closely linked within a cluster of genes, coding for structurally related proteins, on the long arm of human chromosome 1.

Markedly Reduced Plasma Free Protein S Levels in Patients with Hemophagocytic Histiocytosis

In 3 patients with hemophagocytic histiocytosis (HH) accompanied by disseminated intravascular coagulation syndrome (DIC) and multiple organ failure (MOF), a markedly significant decrease of plasma free protein S (PS) levels was found with markedly raised plasma C4b-binding protein (C4bp) levels as compared to non-HH DIC patients with MOF. Moreover, a significant elevation of serum cytokines, interleukin-lβ and tumor necrosis factorα, was observed when compared to non-HH DIC patients with MOF. Plasma free PS was inversely correlated with these two cytokines, while plasma C4bp was positively correlated with them.These results imply that abnormal plasma PS distribution, that is, markedly reduced free PS with raised C4bp-PS complex which was closely associated with hypercytokinemia would contribute to the pathogenesis of DIC and/or MOF in HH.

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Interrelation between mononuclear phagocyte system (MPS) activation and hemostatic abnormality was investigated in 3 patients with hemophagocytic histiocytic syndrome (HH), a representative clinical model of MPS proliferative disorders, accompanied by disseminated intravascular coagulation syndrome (DIC) and multiple organ failure (MOF). A markedly significant decrease of plasma free protein S (PS) levels was found with markedly raised plasma C4b-binding protein (C4bp) levels as compared to non-HH DIC patients with MOF. And also an extreme increase of plasma plasminogen activator inhibitor 1 (PAI-1) and von Willebrand factor antigen (vWF: Ag) levels was found. Moreover, a significant elevation of serum cytokines, especially interleukin-1β and tumor necrosis factor α, was observed when compared to non-HH DIC patients with MOF. Plasma free PS was inversely correlated with these cytokines, while each of C4bp, PAI-1 and vWF: Ag was positively correlated with them.These results imply that markedly reduced plasma free PS and extremely increased plasma PAT-1 and vWF: Ag, which were possibly caused by the modulation of liver cells or vascular endothelial cells by these increased cytokines, would contribute to the pathogenesis of hemostatic abnormalities, particularly DIC and/or MOF in HH.

Characterization of a synthetic peptide that inhibits the interaction between protein S and C4b-binding protein

Protein S is unique among the vitamin K-dependent proteins found in blood plasma because it is a cofactor rather than a zymogen of a serine protease. Instead of a trypsin-like domain, protein S contains a domain that has sequence homology with steroid binding proteins. In order to understand the function of this structural domain, peptides have been synthesized with amino acid sequences that are homologous between human protein S and rat androgen binding protein. Two peptides, corresponding to amino acids 400-407 (PINPRLDG) and 605-614 (GVQLDLDEAI) of the protein S sequence have been tested for their effects on protein S function. Neither peptide altered the clotting of bovine or human plasma. The peptide GVQLDLDEAI enhanced the anticoagulant activity of human-activated protein C in human plasma while the peptide PINPRLDG had no effect. The peptide GVQLDLDEAI was observed to inhibit the binding of protein S to C4b-binding protein in plasma, resulting in increased concentrations of free protein S. GVQLDLDEAI was also observed to enhance the disassociation of the protein S.C4b-binding protein complex when purified complex was used. Finally, C4b-binding protein was observed to bind to GVQLDLDEAI. These results suggest that the carboxyl-terminal region of protein S, which contains the sequence GVQLDLDEAI, is involved in the interaction between protein S and C4b-binding protein.

Familial Protein S Deficiency With a Variant Protein S Molecule in Plasma and Platelets

AbstractA protein S deficient family presenting a variant protein S molecule in plasma and platelets is described. The propositus, age 20, and two brothers suffered from venous thrombotic disease. The propositus, the only family member studied while taking oral anticoagulants, had a protein S antigen (ag) level of 17% and undetectable activity. As demonstrated by immunoblotting both the propositus and one clinically affected brother (42% ag, 7% activity) presented variant protein S molecules of 65,000 molecular weight (mol wt) while the other clinically affected brother (64% ag, 11% activity) had only protein S with normal electrophoretic mobility of 70,000 mol wt. The mother had normal protein S levels (93% ag, 100% activity) but had both normal and variant protein S molecules and based on her functional protein S data a normal anticoagulant activity of the variant molecule is suggested. One asymptomatic but protein S deficient sister (68% ag, 9% activity) as well as the asymptomatic protein S deficient father (59% ag, 10% activity) had only protein S molecules of 70,000 mol wt. The variant protein S bound to C4b-binding protein in plasma, and differed from normal protein S in carbohydrate content. Platelets of each family member contained the same immunoblotting pattern of normal and variant protein S forms as found in plasma, consistent with the hypothesis that protein S gene expression involves codominant expression of two alleles that is similar in cells that control the synthesis of both platelet and plasma forms of protein S.

Familial protein S deficiency with a variant protein S molecule in plasma and platelets

A protein S deficient family presenting a variant protein S molecule in plasma and platelets is described. The propositus, age 20, and two brothers suffered from venous thrombotic disease. The propositus, the only family member studied while taking oral anticoagulants, had a protein S antigen (ag) level of 17% and undetectable activity. As demonstrated by immunoblotting both the propositus and one clinically affected brother (42% ag, 7% activity) presented variant protein S molecules of 65,000 molecular weight (mol wt) while the other clinically affected brother (64% ag, 11% activity) had only protein S with normal electrophoretic mobility of 70,000 mol wt. The mother had normal protein S levels (93% ag, 100% activity) but had both normal and variant protein S molecules and based on her functional protein S data a normal anticoagulant activity of the variant molecule is suggested. One asymptomatic but protein S deficient sister (68% ag, 9% activity) as well as the asymptomatic protein S deficient father (59% ag, 10% activity) had only protein S molecules of 70,000 mol wt. The variant protein S bound to C4b-binding protein in plasma, and differed from normal protein S in carbohydrate content. Platelets of each family member contained the same immunoblotting pattern of normal and variant protein S forms as found in plasma, consistent with the hypothesis that protein S gene expression involves codominant expression of two alleles that is similar in cells that control the synthesis of both platelet and plasma forms of protein S.

Low total protein S antigen but high protein S activity due to decreased C4b-binding protein in neonates

Protein S, a vitamin K-dependent cofactor for activated protein C, exists in normal adult plasma in a free anticoagulantly active form and in an inactive form complexed to C4b-binding protein. Immunologic and functional levels of protein S and C4b-binding protein in plasma were determined for 20 newborn infants and compared with adult normal pooled plasma. Total protein S antigen levels averaged 23%, similar to other vitamin K-dependent plasma proteins. However, the protein S anticoagulant activity was 74% of that of adult normal plasma. This apparent discrepancy of activity to antigen was shown to be due to low or undetectable levels of C4b-binding protein, which results in the presence of most if not all of protein S in its free and active form. The relatively high level of anticoagulantly active protein S in infants may enhance the potential of the protein C pathway, thereby minimizing risks of venous thrombosis in this group.

Low Total Protein S Antigen but High Protein S Activity due to Decreased C4b-Binding Protein in Neonates

Protein S, a vitamin K-dependent cofactor for activated protein C, exists in normal adult plasma in a free anticoagulantly active form and in an inactive form complexed to C4b-binding protein. Immunologic and functional levels of protein S and C4b-binding protein in plasma were determined for 20 newborn infants and compared with adult normal pooled plasma. Total protein S antigen levels averaged 23%, similar to other vitamin K-dependent plasma proteins. However, the protein S anticoagulant activity was 74% of that of adult normal plasma. This apparent discrepancy of activity to antigen was shown to be due to low or undetectable levels of C4b-binding protein, which results in the presence of most if not all of protein S in its free and active form. The relatively high level of anticoagulantly active protein S in infants may enhance the potential of the protein C pathway, thereby minimizing risks of venous thrombosis in this group.

Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S.

C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9 X 10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1 X 10(3) M-1 . S-1 and 1.8 X 10(-4)-4.5 X 10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7 X 10(-7) M was calculated for the C4b-binding protein-protein S interaction.

High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein.

Protein S, a recently described vitamin K-dependent plasma protein, is shown to exist in two forms in plasma--free protein and in complex with C4b-binding protein. C4b-binding protein is involved in the regulation of the rate of complement activation. A major proportion of C4b-binding protein in plasma is in complex with protein S. The complex is a major and previously unrecognized component of the group of plasma proteins that adsorbs to barium citrate. The complex dissociates in the presence of NaDodSO4, indicating that C4b-binding protein and protein S are held together by noncovalent bonds. Uncomplexed C4b-binding protein was purified from the supernatant after barium citrate adsorption. On NaDodSO4/polyacrylamide gels without reduction, it appeared to have a slightly faster migration rate than the C4b-binding protein dissociated from the complex with protein S. After reduction, the subunits of the two forms of C4b-binding protein appeared to have identical molecular weights. Furthermore, there is an equilibrium between free and bound protein S in plasma. The role of protein S in the complex is unknown.