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Results for “Complement C4b-Binding Protein”

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At least 19 recordsLinked to original sources
PubMed1992

Expression and characterization of recombinant human protein S in heterologous cells--studies of the interaction of amino acid residues leu-608 to glu-612 with human C4b-binding protein.

Mouse C127 epithelioid cells were genetically engineered to produce biologically active gamma-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of gamma-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully gamma-carboxylated and fully beta-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gln, respectively and this variant was designated C variant.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Binding of anticoagulant vitamin K-dependent protein S to platelet-derived microparticles.

Vitamin K-dependent protein S is an anticoagulant plasma protein serving as cofactor to activated protein C in degradation of coagulation factors Va and VIIIa on membrane surfaces. In addition, it forms a noncovalent complex with complement regulatory protein C4b-binding protein (C4BP), a reaction which inhibits its anticoagulant function. Both forms of protein S have affinity for negatively charged phospholipids, and the purpose of the present study was to elucidate whether they bind to the surface of activated platelets or to platelet-derived microparticles. Binding of protein S to human platelets stimulated with various agonists was examined with FITC-labeled monoclonal antibodies and fluorescence-gated flow cytometry. Protein S was found to bind to membrane microparticles which formed during platelet activation but not to the remnant activated platelets. Binding to microparticles was saturable and maximum binding was seen at approximately 0.4 microM protein S. It was calcium-dependent and reversed after the addition of EDTA. Inhibition experiments with monoclonal antibodies suggested the gamma-carboxyglutamic acid containing module of protein S to be involved in the binding reaction. An intact thrombin-sensitive region of protein S was not required for binding. The protein S-C4BP complex did not bind to microparticles or activated platelets even though it bound to negatively charged phospholipid vesicles. Intact protein S supported binding of both protein C and activated protein C to microparticles. Protein S-dependent binding of protein C/activated protein C was blocked by those monoclonal antibodies against protein S that inhibited its cofactor function. In conclusion, we have found that free protein S binds to platelet-derived microparticles and stimulates binding of protein C/activated protein C.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Regulation of C4b-binding protein gene expression by the acute-phase mediators tumor necrosis factor-alpha, interleukin-6, and interleukin-1.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. During an acute-phase response, we have shown that hepatic levels of murine C4BP mRNA are elevated 2.5-fold while rat liver C4BP gene expression exhibits a 4-fold induction. Furthermore, a survey of different mouse tissues showed that during acute inflammation C4BP gene expression was confined to the liver. To gain a better understanding of the acute-phase regulation of C4BP gene expression we utilized the rat hepatoma cell line FAO in which tumor necrosis factor-alpha (TNF-alpha) produced a 2.7-fold induction of C4BP mRNA levels. In the absence of TNF-alpha, interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) had little effect on C4BP gene expression but when all three cytokines were used together a synergistic 4-fold induction of C4BP mRNA levels was observed. In contrast the synthetic glucocorticoid dexamethasone inhibited TNF-alpha-induced C4BP gene expression. Cycloheximide-mediated inhibition of inducible C4BP gene expression demonstrated the requirement for ongoing protein synthesis. Rapid induction of C4BP mRNA levels by TNF-alpha and IL-6 (within 1 h) and the observation that stimulation was inhibited by actinomycin D provided evidence that regulation of C4BP gene expression during the acute-phase response is regulated at the transcriptional level. Isolation of a genomic clone extending into the 5' regulatory region of the rat C4BP gene enabled us to identify the major transcriptional start site and putative response elements through which TNF-alpha, IL-6, IL-1 alpha, and dexamethasone may exert their effects on C4BP gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Reaction↗
PubMed1992

Complete structure of the murine C4b-binding protein gene and regulation of its expression by dexamethasone.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. A murine genomic library was screened, and five clones were selected that covered the remaining four exons in the 5'-region of the C4BP gene. Together with previous work (Barnum, S. R., Kristensen, T., Chaplin, D. D., Seldin, M. F., and Tack, B. F. (1989) Biochemistry 28, 8312-8317), the entire C4BP gene has now been shown to be 23 kilobases (kb) long and comprised of 10 exons ranging in size from 86 to 442 base pairs (bp). Primer extension analysis revealed the major transcription start site to be 46 bp upstream of the published cDNA start site. Northern blot analysis of RNA isolated from several mouse tissues demonstrated that the C4BP gene is expressed in a liver-specific manner. Several regions homologous to known response elements were identified upstream of the C4BP gene including a strong hepatocyte nuclear factor 1 binding site and four putative glucocorticoid response elements. Furthermore, dexamethasone increased C4BP mRNA and protein levels in the mouse liver cell line, NMuLi. The stimulation of C4BP gene expression was rapid and independent of protein synthesis. These results suggest dexamethasone induction of the C4BP gene is a primary response and therefore a transcriptional effect. Inhibition of the dexamethasone effect on C4BP by actinomycin D supports this theory. These studies also provide evidence that, for optimal induction of the C4BP gene, the glucocorticoid receptor complex may cooperatively interact with accessory transcription factors. It is likely that stimulation of C4BP gene expression by dexamethasone may allude to a mechanism by which glucocorticoids exert their anti-inflammatory effects.

Animals↗
PubMed1992

Pig apolipoprotein R: a new member of the short consensus repeat family of proteins.

Apolipoprotein R (apoR) is a 23-kDa protein found on very low-density lipoprotein (VLDL), on chylomicrons, and in the d > 1.21 g/mL fraction of pig plasma. The plasma concentration of apoR is 5.1 micrograms/mL, with 11.5% of apoR found on VLDL. In vitro, apoR can transfer from the d > 1.21 g/mL infranatant onto artificial lipid emulsions or human chylomicrons but not onto human VLDL. An apoR cDNA was isolated from a pig liver lambda gt11 expression library. DNA sequence analysis of the apoR cDNA revealed 67% identity with the 3'-terminal region of human C4b-binding protein alpha-chain cDNA (C4BP alpha). C4BP alpha is a 70-kDa glycoprotein that regulates both the coagulation and the complement cascades. In plasma, C4BP alpha exists as disulfide-linked multimers consisting of seven C4BP alpha chains and a single C4BP beta chain. Like C4BP, apoR forms high molecular weight disulfide-linked complexes in plasma. However, unlike C4BP alpha, apoR complexes do not appear to contain C4BP beta. ApoR mRNA was detected in pig liver, spleen, lung, bone marrow, and lymph node, but was absent in intestine and white blood cells. This distribution is consistent with the production of apoR in terminally differentiated macrophages but not in blood monocytes. ApoR mRNA was not detected in RNA isolated from human liver or lung. ApoR may be a lipoprotein-borne regulator of either the coagulation or the complement cascades.

Amino Acid Sequence↗
PubMedDate not supplied

The effect of low-density lipoprotein apheresis on plasma thrombomodulating factors.

The authors have investigated the effects of double-membrane filtration with hollow fiber membranes made of different artificial materials and low-density lipoprotein (LDL)-adsorbent dextran sulfate cellulose (DS) columns on coagulation-related proteins and complement components to evaluate their hemocompatibility. All membrane filters showed similar sieving effects, which depended upon the molecular weights of the proteins. In the double-membrane filtration system, free protein S, protein C, antithrombin III, and alpha 1-antitrypsin were returned to the intracorporeal circulation, whereas alpha 2-macroglobulin and fibrinogen were almost completely removed from the circulation; C4b-binding protein (C4BP)-protein S complexes were also trapped by the second membrane, and in some instances were even blocked by the first membrane, if it was made of material that activated the classic pathway of the complement system. The DS column adsorbed C4BP-protein S complex, but free protein S was almost completely recovered in the eluate.

Antithrombin III↗
PubMed1992

Coagulation inhibitors in pulmonary cancer patients.

Pulmonary cancer patients are known to have an elevated risk to suffer from thromboembolic complications. Because hereditary deficiencies of coagulation inhibitors antithrombin III, protein C and protein S are known to cause thromboembolic events it was the aim of our study to search for acquired alterations of these proteins in pulmonary cancer patients. We could demonstrate antithrombin III and protein C to be within the normal range in patients suffering from pulmonary carcinoma. In contrast, in patients suffering from metastatic pulmonary carcinoma bound protein S was increased, while free protein S was significantly reduced. In some patients the decrease of free protein S was comparable to the diminution observed in hereditary protein S deficient patients. A high positive correlation was observed between C4b-binding protein and bound protein S, indicating C4b-binding protein to be a regulatory protein for the shift from free and anticoagulatory active to bound and anticoagulatory inactive protein S. In conclusion, the decrease of free protein S is one source for thromboembolic complications in pulmonary cancer patients. For interpretation of altered free protein S levels it is useful to measure C4b-binding protein.

Aged↗
PubMed1992

Meningococcaemia in an adult with hereditary C4b-binding protein deficiency: study of the variations of the protein S system.

Serial studies of the plasma protein C-protein S system were performed during the clinical course of a pregnant woman with meningococcaemia who recovered under therapy. The patient had limited purpura fulminans skin lesions and hereditary C4b-binding protein deficiency was suspected. This diagnosis was confirmed in the patient 1 year after delivery and also by family studies. During the meningococcaemia, an initial mild and transient acquired protein C deficiency was seen but no protein S deficiency was observed despite consumption of the latter protein. As C4b-binding protein partial deficiency is associated with high free protein S and protein S activity, this may have protected against acquired protein S deficiency during meningococcaemia.

Adult↗
PubMed1992

Protein C, protein S and C4b-binding protein in neonatal severe infection and septic shock.

We have studied the behaviour of total protein S, free protein S, protein C and C4b-binding protein fifteen neonates with severe infections, eight with septic shock and in a group of ten healthy newborns. Protein C was decreased in shock and septic patients, but only the shock group showed significant differences compared to normal neonates. Total protein S was normal in both groups of patients, although free protein S had significantly lower values in shock and nonshock infants. C4b-binding protein was higher than normal in septic and shock patients compared to the control group. Decreased values of protein C and free protein S can be explained by the activation of coagulation and their subsequent consumption. On the other hand, the increased levels of C4b-binding protein can affect the distribution of protein S in plasma, producing a shift in protein S to the complexed inactive form. These findings can contribute to an increased risk of microthrombosis during neonatal sepsis.

Carrier Proteins↗
PubMed1992

Maternal coagulation inhibitors and the effects of cesarean delivery.

Maternal hypercoagulability in normal pregnancy results from significant increases in blood factors that promote thrombosis or decreases in factors that inhibit thrombosis, such as antithrombin III (AT-III) and proteins C and S. The precise role of these factors in puerperal hemostasis is not clear. In 10 normal, pregnant women at term undergoing scheduled repeat cesarean section, the percent activities of AT-III, proteins C and S, and C4b-binding protein were determined in peripheral venous blood preoperatively and in samples of uterine venous blood before the uterine incision was made and 5 and 15 minutes after placental delivery using the Laurell Rocket electroimmunodiffusion technique. The mean percent activities of AT-III (73%), protein S (81%) and C4b-binding protein (85%) were lower than those in nonpregnant controls, were similar in peripheral and uterine venous blood and were unchanged after placental delivery. These data suggest that such factors may not play an important role in acute uteroplacental hemostasis during normal pregnancy.

Antithrombin III↗
PubMed1992

The thrombotic diathesis associated with the presence of phospholipid antibodies may be due to low levels of free protein S.

PURPOSE: To determine if abnormalities in the protein C/protein S anticoagulant system exist in patients with phospholipid antibodies who had the primary clinical complaint of fetal wastage. PATIENTS AND METHODS: Eleven patients with fetal wastage and phospholipid antibodies were selected for study. Some patients also gave a history of previous thrombotic events related to oral contraceptives and/or pregnancy, but patients were not selected because of a history of clinical thrombosis. The levels of protein C (chromogenic assay), protein S (both free and bound) (Laurell rocket), and C4b-binding protein (Laurell rocket) were measured, and assays for the presence of antibodies against protein S or protein C were performed. RESULTS: Seven of the 11 patients were found to have low levels of free protein S. Total protein S and protein C levels were within the normal range in all patients. Antibodies to protein C and protein S were not found in any patient. These findings suggest that free protein S levels may be abnormally low in some patients with phospholipid antibodies. CONCLUSION: Free protein S levels are abnormally low in some patients with phospholipid antibodies, and this abnormality may be a factor contributing to the thrombotic diathesis associated with phospholipid antibodies.

Abortion, Habitual↗
PubMed1992

Recurrent venous thrombosis during warfarin treatment related to acquired protein S deficiency.

Failure of warfarin to prevent new thrombotic processes was observed in three patients with very low free protein S concentrations and high C4b-binding protein (C4bBP) concentrations, and in one patient with hereditary protein S deficiency. We suggest that an increase in C4bBP reduces the free Protein S level, and warfarin treatment causes an additional decrease of free protein S. The four patients presented indicate that such reductions are of clinical importance. Heparin seems preferable as an anticoagulant in this situation, as warfarin given alone is ineffective, or may even be harmful. In a group of pancreatic cancer patients with advanced disease, subnormal mean free protein S was found, whereas mean total protein S concentration, and mean C4bBP concentrations were significantly higher (p less than 0.01) than in healthy controls. These findings indicate that an increase in C4bBP may induce free protein S deficiency contributing to the increased thrombotic tendency in this group of patients. The correlation between free protein S and C4bBP was 0.11, (n.s.), between total protein S and C4bBP 0.73 (p less than 0.0001).

Adult↗
PubMed1992

Changes in serum lipoprotein(a) and C4b-binding protein levels after acute myocardial infarction.

We investigated changes in serum lipoprotein(a) (Lp(a)) and C4b-binding protein (C4bp) levels following acute myocardial infarction (AMI). Serum lipids, apolipoproteins, C-reactive protein (CRP), sialic acid and haptoglobin (Hp) were also measured and evaluated. The study involved 16 patients (11 men, 5 women, mean age 68.2 +/- 8.4 years) with AMI admitted within 12 h after the onset of illness. CRP rose sharply and immediately after the onset of AMI, reaching its maximum level on the 3rd day of illness. This was followed by temporary increases in sialic acid and Hp, both of which peaked on the 7th day of illness. The Apo A-I level was significantly lower on the 14th day, while the Apo B level was significantly lower on the 3rd day. Serum total cholesterol (T-Ch), HDL-Ch and LDL-Ch showed temporary decreases after the onset of illness. Serial examination of the serum of patients with AMI yielded the following findings; 1) Both Lp(a) and C4bp levels rose temporarily with similar patterns of variation, and 2) they took longer time to peak (around the 14th day) and lasted longer than the increases in the other acute reactive proteins.

Aged↗
PubMed1992

Combined inherited protein S and heparin co-factor II deficiency in a patient with upper limb thrombosis: a family study.

A 42-year-old Italian woman presenting with spontaneous deep vein thrombosis of the right arm, was found to have inherited a deficiency of both protein S (PS) and heparin co-factor II (HC II). The two defects seemed to segregate independently, since her son exhibited only a HC II deficiency while one of her sisters manifested only the PS defect. All affected patients appeared heterozygous for one or other or both deficiency states. The proposita and her sister exhibited a congenital PS deficiency consisting of normal or near normal levels of total PS antigen and C4b-binding protein (C4b-BP) but a moderate reduction both of free PS antigen and of PS functional activity. In addition, the proposita and her son had half normal levels of HC II antigen and activity. Except for the proposita, all were asymptomatic. Inherited deficiencies either of PS or of HC II have been associated with thrombotic manifestations. Since the proposita had an inherited combined defect of the two proteins, severe thrombotic events might be expected. However, this was not found to be the case. The role of HC II deficiency in the pathogenesis of thrombosis whether alone or combined remains to be fully investigated.

Adolescent↗
PubMed1992

Sex hormone-binding globulin, androgen-binding protein, and vitamin K-dependent protein S are homologous to laminin A, merosin, and Drosophila crumbs protein.

Androgen-binding protein (ABP) and sex hormone-binding globulin (SHBG) are extracellular steroid-binding proteins that are homologous to the COOH-terminal domain of vitamin K-dependent protein S, a protein important in blood clotting. We find that the sequences of ABP, SHBG, and protein S are also similar to two basement membrane proteins, laminin and merosin, and to an integral membrane protein, Drosophila crumbs protein. These latter three proteins have important roles in regulating differentiation and development. The sequence similarity corresponds to the G domain of laminin A chain, which binds heparin and type IV collagen. Analysis of a multiple alignment of these proteins reveals one well-conserved segment corresponding to the part of SHBG that binds to its membrane receptor and another corresponding to the part of protein S that binds to C4b-binding protein. The similarities suggest that ABP, SHBG, and protein S may also have functions related to that of laminin and merosin.

Amino Acid Sequence↗
PubMed1992

Study of the protein S system in HIV-infected patients: acquired protein S deficiency or unsuitable assays?

The plasma protein S (PS) system was studied in 120 HIV seropositive patients (CDC classification: group II: n = 35; group III: n = 6; groups IVA, IVC2 or IVE, n = 38; groups IVB, IVC1 or IVD, n = 41). Total PS antigen and C4b-binding protein (C4b-BP) values were not significantly different from control values. Free protein S levels assessed by an immunological method in the supernatant of PEG-precipitated plasma samples (PEG-fPS) were below the normal limit in 85 patients, lower values being found in patients with advanced HIV disease. No correlation was found between PEG-fPS levels and C4b-BP or total PS levels. At least 25 patients had a low functional PS value. Low functional levels of PS were found in each clinical group although there was no difference in the distribution of functional PS values among groups. Crossed immuno-electrophoresis showed an abnormal distribution of PS in some patients, but failed to confirm the marked decrease of free PS in patients with very low PEG-fPS. An impairment of the protein S system is observed in HIV-infected patients. However, the discrepancies found in some patients among the results of the various PS-related assays should lead to a cautious interpretation concerning the incidence of PS deficiency in HIV-infected patients.

Adult↗
PubMed1992

Free protein S deficiency in patients with chronic inflammatory bowel disease.

Free protein S, protein C, and C4b-binding protein (C4b-BP) were measured in randomly selected outpatients: 22 with Crohn's disease (CD) and 16 with ulcerative colitis (UC). Active disease was recorded in 10 patients with CD and 4 with UC. Fourteen patients (63.6%) with CD and 4 (25%) with UC had free protein S values below the normal range, with mean values of 62% and 78% of that found in healthy control subjects (p < 0.01). The C4b-BP level was 127% in patients with CD as compared with 89% in both healthy subjects and UC patients (p < 0.01). The protein C levels were similar in the three groups. The present results add to the factors already known favouring thromboembolic complications in inflammatory bowel disease and which might play a major role both for the pathogenesis and for the increased tendency to venous thromboembolism in these diseases.

Adult↗