Biomedical subjects

B N Bouma

Publications and source records attributed to B N Bouma.

At least 19 recordsLinked to original sources

PubMed1992

Lysine-binding heterogeneity of Lp(a): consequences for fibrin binding and inhibition of plasminogen activation.

Lipoprotein(a) [Lp(a)] is recognized as an independent risk factor for atherosclerosis. Lp(a) consists of a LDL-like moiety with an additional glycoprotein, apo(a), linked to apolipoprotein B-100. Apo(a) has a high homology with plasminogen (Pg). In vivo, Pg is activated on a fibrin surface by tissue Pg activator (tPA). We prepared Lp(a) from plasma by sequential ultracentrifugation followed by lysine-sepharose affinity chromatography. We found that a changing (donor dependent) fraction of the Lp(a) did not bind to lysine-sepharose. This fraction, designated Lp(a)lys-, was further purified using gel filtration. Bound Lp(a) [Lp(a)lys+] was eluted with 0.2 M EACA. Apo(a) isoforms in both fractions were identical. In contrast Lp(a)lys+ inhibited Pg activation by tPA in vitro (IC50% 20 mg/l), whereas Lp(a)lys- did not. In addition Lp(a)lys- did not bind to CNBr-digested fibrinogen whereas Lp(a)lys+ did (Kd, app = 0.2 nM). Therefore we conclude that a changing donor dependent fraction of human plasma Lp(a) does not inhibit Pg activation in vitro and does not bind to CNBr-digested fibrinogen.

Binding Sites↗

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

Lupus anticoagulant activity is frequently dependent on the presence of beta 2-glycoprotein I.

Antiphospholipid antibodies (aPL) are defined by anticardiolipin antibody (aCL) ELISA and prolongation of phospholipid dependent coagulation assays (lupus anticoagulant; LAC). For the binding of aCL to cardiolipin a cofactor, beta 2-glycoprotein I (beta 2-GPI), is necessary. We have investigated whether the same cofactor is essential for LAC activity. Plasma from 6 LAC positive patients and 3 controls was depleted from beta 2-GPI by means of affinity chromatography. From the 6 LAC positive plasmas, 4 became LAC negative (tested with dRVVT) when beta 2-GPI was depleted and became positive again when purified beta 2-GPI (200 micrograms/ml) was added. A dose response curve showed that addition of 50 micrograms/ml beta 2-GPI to beta 2-GPI deficient patient plasma, led to a positive dRVVT. Depletion of, and addition of beta 2-GPI to plasma from controls had no effect on the dRVVT. Measurement of beta 2-GPI plasma levels in 19 LAC positive patients, 40 LAC negative patients and 15 controls showed no difference in beta 2-GPI levels. These results show that a combination of aPL and beta 2-GPI is essential not only for binding to cardiolipin, but also for LAC activity and imply that low beta 2-GPI levels (less than 50 micrograms/ml) can lead to false negative LAC tests. These observations may lead to new insights in the pathophysiological complications associated with aPL.

Adult↗

PubMed1992

Binding of vitronectin-thrombin-antithrombin III complex to human endothelial cells is mediated by the heparin binding site of vitronectin.

The interaction of vitronectin-thrombin-antithrombin III (VN.TAT) complex with endothelial cells (EC) was investigated. Binding was specific and time- and concentration-dependent. Kinetics revealed an apparent dissociation constant of 16 nM and 1.7 x 10(5) binding sites/endothelial cell. The binding determinant of the ternary complex was located on the VN moiety. Since the association of VN to TAT adds its specific properties to the VN.TAT complex, the involvement of the heparin binding domain and the cell attachment site of VN was investigated. Neither addition of RGD peptide nor blocking of the vitronectin receptor with a monoclonal antibody interfered with VN.TAT binding to EC. Addition of heparin, a VN-derived peptide comprising two heparin binding consensus sequences or a monoclonal antibody directed against the heparin binding domain on VN, completely inhibited VN.TAT binding to EC. These results indicate that the interaction is mediated through the heparin binding domain of VN. Digestion of heparan sulfate proteoglycans resulted in a decrease of VN.TAT binding to EC, indicating the involvement of heparin-like structures on the EC surface. Our findings point to an unrecognized mechanism by which VN may act as scavenger in order to enhance the clearance of end products of the clotting system via binding of the ternary VN.TAT complex to the luminal surface of EC.

Amino Acid Sequence↗

PubMed1992

The contact activation proteins: a structure/function overview.

In recent years, extensive knowledge has been obtained on the structure/function relationships of blood coagulation proteins. In this overview, we present recent developments on the structure/function relationships of the contact activation proteins: factor XII, high molecular weight kininogen, prekallikrein, and factor XI, with the emphasis on the localization of domains on these proteins that are involved in the interaction with activators, substrates and cofactors.

Amino Acid Sequence↗

PubMed1991

In vitro studies of antiphospholipid antibodies and its cofactor, beta 2-glycoprotein I, show negligible effects on endothelial cell mediated protein C activation.

The effect of sera and purified IgG isolated from plasma of 46 patients with systemic lupus erythematosus (SLE) and 9 healthy donors on the endothelial cell (EC) mediated protein C activation was investigated. Out of the 46 SLE sera used, 19 were antiphospholipid antibodies (aPL) positive. From 12 patients IgG was isolated, of which 6 contained aPL. EC were first incubated with IgG (7 mg/ml) or serum (1:1 diluted) for 1 h and then tested for their ability to promote protein C activation by thrombin, with the cells either in a monolayer or in a suspension. The normal range (mean of control values +/- 2 SD) of protein C activation was 80-120%. In contrast to others, we could not detect an inhibition of protein C activation by any of the patient IgG's or sera. The recently described cofactor for binding of antiphospholipid antibodies to phospholipids, beta 2-glycoprotein I, was purified and added to the purified IgG's. A combination of these two components did not inhibit the EC mediated protein C activation by thrombin. This study suggests that the inhibition of the protein C activation, mediated by EC, is not a general mechanism by which aPL related thrombosis can be explained.

Adolescent↗

PubMed1991

Formation of meizothrombin as intermediate in factor Xa-catalyzed prothrombin activation on endothelial cells. The influence of thrombin on the reaction mechanism.

Incubation of prothrombin on cultured human umbilical vein endothelial cells with factor Xa and calcium ions induced the activation of prothrombin. The mechanism of prothrombin activation was analyzed on sodium dodecyl sulfate gels using immuno- and amido-blotting techniques. It was demonstrated that meizothrombin was formed as an intermediate in prothrombin activation on the endothelial cell surface. In addition, considerable amounts of meizothrombin des-fragment-1 accumulated during prothrombin activation and were not further converted to thrombin. Although preincubation of the endothelial cells with thrombin did not influence the formation of meizothrombin, addition of hirudin to the prothrombin activation mixture inhibited the formation of meizothrombin and meizothrombin des-fragment-1 almost completely. This indicated that the activity of endogenously formed thrombin influenced the formation of meizothrombin via a feedback mechanism. The increased formation of meizothrombin and accumulation of meizothrombin des-fragment-1 in a latter phase of prothrombin activation points to a regulatory mechanism in hemostasis which subdues the formation of the procoagulant alpha-thrombin.

Calcium↗

PubMed1991

Structure-function studies on human C4b-binding protein using monoclonal antibodies.

Human C4b-binding protein (C4BP) is a multimeric regulatory complement component interacting with vitamin K-dependent protein S and complement C4b. Using hybridoma technology, a panel of monoclonal antibodies (mAb) specific for intact human C4BP and its 160-kDa chymotryptic central core fragment were prepared to study the structure-function relationships of C4BP. By Western blot analysis and competition experiments, four distinct groups of mAb were identified and mapped on the C4BP molecule. By rotary shadowing, spider-like images of C4BP-antibody complexes were obtained and immunoelectron microscopy provided some information on the stoichiometry of the antibody-C4BP interaction. Certain antibodies interacted with C4BP molecules only at a ratio of 1:1. Others formed complexes of two or more antibodies bound to homologous sites on the C4BP molecule. Using an enzyme-linked immunosorbent sandwich assay for the measurement of the complex formation between protein S and C4BP, mAb against the central core and the disulfide-linked beta chain of C4BP were identified that inhibited the binding of protein S to C4BP. In a binding assay using 125I-labeled C4BP and solid-phase C4b, the inhibitory effect of one group of anti-C4BP mAb on the binding of C4BP to C4b was demonstrated.

Antibodies, Monoclonal↗

PubMed1990

Proteolysis of human apolipoprotein B: effect on quantitative immunoturbidimetry.

Apolipoprotein (Apo) B100 is the obligatory structural protein of low and very low density lipoproteins. We studied the appearance of a kallikrein-induced proteolytic fragment of Apo B100, identified by sodium dodecyl sulphate (SDS)-polyacrylamide electrophoresis as Apo B74, in plasma samples obtained from 7 men and 7 women. We also studied the effect of Apo B74 on total Apo B immunoreactivity by immunoturbidimetry. Apo B74 was found in plasma from 4 of the 7 women when the samples had been stored at 4 degrees C in glass or plastic tubes, but in some of the samples from the men. These 4 women used oral estrogens. The plasma samples which contained Apo B74 showed evidence of kallikrein activation. Presence of Apo B74 resulted in an underestimation of total Apo B immunoreactivity by 13% (P less than 0.05).

Adult↗

PubMed1990

Identification of different forms of human C4b-binding protein lacking beta-chain and protein S binding ability.

Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical alpha-chains (70 kDa) and one beta-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the beta-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silver-staining and with Western-blotting using monoclonal antibodies specific for the beta-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 x 10(8) and 3.21 x 10(8) M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the beta-chain and was capable of protein S binding (KA: 4.35 x 10(8) M-1). The two other forms of C4BP lacked the beta-chain and were unable to bind protein S.

Binding Sites↗

PubMed1990

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.

Amino Acid Sequence↗

PubMed1990

The effect of von Willebrand factor on activation of factor VIII by factor Xa.

Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 U/ml) with 0.1 microgram/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 micrograms/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 micrograms/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12, 20/21, 23, 27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa.

Factor VIII↗

PubMed1990

The binding site of human C4b-binding protein on complement C4 is localized in the alpha'-chain.

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. We now demonstrate that C4b contains a binding site for C4BP, which is localized on the alpha'-chain of C4b. SDS-PAGE of C C4 and C4b both under reducing and nonreducing conditions was followed by a radiolabeled C4BP ligand blotting procedure. It was demonstrated that the C4BP binding site on C4b is localized on the alpha'-chain. In addition, we found C4BP binding to the alpha-chain of C4, which suggests that the binding site for C4BP becomes available after reduction of the C4 molecule. Direct binding of C4BP to the alpha- and alpha'-chains of C4 and C4b was demonstrated in a radio-labeled C4BP binding assay with the reduced and alkylated isolated chains. mAb against the alpha'-chain of C4b were prepared, characterized, and evaluated for their ability to block the binding of 125I-C4BP to C4b. Two mAb specific for the alpha'-chain of C4b were found that completely abolished C4BP binding to intact C4b. Other mAb recognizing both the alpha- and alpha'-chain of C4 and C4b demonstrated only minor inhibitory effect on the binding of C4BP to C4b. In conclusion, we have localized the C4BP binding site on the alpha'-chain of C4b and have demonstrated that this binding can be inhibited by mAb specific for the alpha'-chain.

Antibodies, Monoclonal↗

PubMed1990

Studies on components of the contact phase system in patients with advanced gastrointestinal cancer.

The authors have studied components of the contact system in plasma obtained from patients with advanced gastrointestinal cancer. Plasma samples from 118 healthy blood donors served as controls. Plasma prekallikrein (PKK) values, evaluated by chromogenic peptide substrate technique, were significantly decreased in patients with cancer compared with healthy blood donors. High molecular weight kininogen (HMwK) and Hageman factor (FXII) values, assayed by immunochemical techniques, were also decreased in the patients with cancer. The changes of contact factors were most pronounced in patients with liver metastasis. The most striking observation in our study, however, was the elevated inhibitor values in patients with cancer. Alpha-2-macroglobulin (alpha 2-M) and C1 inhibitor (C1INH) values, determined both by functional and immunochemical techniques, were markedly increased in patients with cancer. In conclusion, this study shows that patients with intestinal cancer have reduced values of contact factors and markedly elevated inhibitor values which indicate that development of malignant tumors in the gastrointestinal tract is associated with changes in the contact system of plasma.

Aged↗

PubMed1990

Methylprednisolone induces activation of the contact system in a dose-dependent manner. An in vitro study.

The effect of methylprednisolone sodium succinate (MP) on the contact system of plasma was studied in human citrated pool plasma. Contact activation was demonstrated by the presence of plasma kallikrein (KK) activity and activated Hageman factor (FXIIa) and/or KK in complex with C1 inhibitor (C1inh), detected by chromogenic peptide substrates or radioimmunoassays, using monoclonal antibodies directed to neodeterminants exposed on complexed C1inh, respectively. When plasma and different doses of MP were incubated for a period of 24 hours, the highest dose of MP (10 mg/ml) gave rapid and marked increases in KK activities and concentrations of C1inh complexes. MP at 5 mg/ml plasma also induced activation of the contact system, although this activation was less pronounced. Even the lower dose of MP (1 mg/ml), which is equivalent to doses used in humans, increased plasma concentrations of KK-C1inh complexes. In conclusion, this in vitro study shows that MP in a dose-dependent way activates the contact system of plasma.

Complement C1 Inactivator Proteins↗

PubMed1990

The localization of heparin-binding fragments on human C4b-binding protein.

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP interacts with C C4b on a domain located in a 48-kDa chymotryptic fragment. We now demonstrate that C4BP contains heparin-binding fragments, which are located within the C4b binding domain. We have used an assay using heparin coupled to Sepharose CL-6B to show that 125I-C4BP binds to heparin in a time-dependent, saturable, and reversible manner. Binding could be inhibited by purified 48-kDa fragments and direct binding on the 48-kDa fragments to heparin-Sepharose was demonstrated by SDS-PAGE. mAb against native C4BP and the isolated 160-kDa central core fragment were evaluated for their ability to block the binding of 125I-C4BP to heparin and C4b. The relative efficacy of mAb against intact C4BP in blocking C4BP binding to heparin-Sepharose was similar to that for blocking 125I-C4BP binding to C4b. In addition, heparin blocked the binding of 125I-C4BP to C4b and vice versa. It is therefore likely that the heparin-binding fragments are localized on or close to the C4b-binding site of C4BP.

Antibodies, Monoclonal↗

PubMed1990

Elucidating the structural chemistry of glycosaminoglycan recognition by protein C inhibitor.

Glycosaminoglycans (GAGs) including heparin accelerate the inhibition of serine proteases by serine protease inhibitors (serpins), an essential process in regulating blood coagulation. to analyze the molecular basis for GAG recognition by the plasma serpin protein C inhibitor (PCI; also known as plasminogen activator inhibitor 3), we have constructed a complete, energy-minimized, three-dimensional model of PCI by using the structure of homologous alpha 1-antitrypsin as a template. Sequence analysis, hydrogen-bonding environment, and shape complementarity suggested that the N-terminal residues of PCI, which are not homologous to those of alpha 1-antitrypsin, form an amphipathic alpha-helix, here designated A+ since it precedes the alpha 1-antitrypsin A helix. Electrostatic calculations revealed a single, highly positive surface region arising from both the A+ and H helices, suggesting that this two-helix motif is required for GAG binding by PCI. The dominant role of electrostatic interactions in PCI-heparin binding was confirmed by the strong ionic strength dependence of heparin stimulation. The involvement of the A+ helix in heparin binding was verified by demonstrating that an anti-PCI antibody that specifically binds the A+ peptide blocks heparin binding.

Amino Acid Sequence↗

PubMed1989

Purified human plasma kallikrein does not stimulate but primes neutrophils for superoxide production.

In patients with septicemia and septic shock the contact phase of blood coagulation is activated. It has been suggested that polymorphonuclear leukocytes (PMN) are directly activated by purified plasma kallikrein. This has been recently questioned because granulocytic elastase release induced by recalcification of normal and prekallikrein-deficient plasma was similar. We studied the interaction of different preparations of purified human plasma kallikrein with PMN. Cytosolic calcium shifts were measured with the quin2 method, PMN aggregation was assayed in an aggregometer, and superoxide production was quantitated as superoxide dismutase inhibitable cytochrome c reduction in a continuous assay. No increase of cytosolic free calcium was found during at least 5 min after adding 10 micrograms/ml plasma kallikrein to PMN. Similarly, highly purified plasma kallikrein from two different sources did not induce PMN aggregation at all, nor did it stimulate superoxide production. However, sequential exposure of PMN to plasma kallikrein and formylpeptide increased the superoxide production compared to stimulation with formylpeptide alone. This phenomenon which is called priming was observed at plasma kallikrein concentrations greater than or equal to 7 micrograms/ml. The active site of the molecule was required for the priming, because plasma prekallikrein, active site-inactivated plasma kallikrein, and soybean trypsin inhibitor treated kallikrein did not prime PMN. This indicates that the contact activation system may play a role in host defence against bacterial infection.

Calcium↗