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J Stenflo

Publications and source records attributed to J Stenflo.

At least 55 records · Page 3Linked to original sources
PubMed1994

Calcium-dependent interaction between gamma-carboxyglutamic acid-containing and N-terminal epidermal growth factor-like modules in factor X.

The N-terminal epidermal growth factor (EGF)-like module in factor X binds a single Ca2+ with low affinity (Kd = 2.2 mM). When it is linked to the gamma-carboxyglutamic acid (Gla)-containing module, however, the affinity increases approximately 20-fold (Kd = 120 microM), indicating an interaction between the two modules and making the site in the N-terminal EGF-like module essentially saturated at physiological Ca2+ concentrations. We have now used the thioredoxin system to probe Ca(2+)-induced conformational changes and interaction between modules in the light chain of factor X. Thioredoxin, in conjunction with thioredoxin reductase and NADPH, allows direct measurements of the rate and extent of disulfide bond reduction. Most disulfide bonds accessible to the reducing agent were found to be located in the light chain of the protein. Moreover, those disulfide bonds that were resistant to reduction by thioredoxin in the presence of Ca2+, but were readily reduced in the absence of the metal ion, were located in the N-terminal EGF-like module and in the Gla module, whereas disulfide bonds in the C-terminal EGF-like module appeared to be equally accessible whether Ca2+ was present or not. Comparison of the rate of disulfide bond reduction in the isolated modules with that in mixtures of modules indicated that a Ca(2+)-dependent interaction occurred between the Gla and the N-terminal EGF-like module. This interaction was mediated by the C-terminal alpha-helical part of the Gla module. The affinity between the modules was low and could not be determined accurately owing to competing equilibria, presumably Ca(2+)-dependent aggregation of the isolated Gla module. By comparing the rates of disulfide bond reduction in Gla module-containing fragments before and after decarboxylation of Gla, we could demonstrate that Ca2+ binding to sites in the Gla module as well as to the single site in the EGF-like module contribute to the interaction between the two modules.

1-Carboxyglutamic Acid↗
PubMed1994

Baculovirus-mediated expression of the epidermal growth factor-like modules of human factor IX fused to the factor XIIIa transamidation site in fibronectin. Evidence for a direct interaction between the NH2-terminal epidermal growth factor-like module of factor IXa beta and factor X.

Factor IX is a vitamin K-dependent procoagulant zymogen of a serine protease. In the presence of Ca2+ the active form of factor IX (factor IXa beta) forms a complex with factor VIIIa on suitable phospholipid surfaces such as aggregated platelets. This macromolecular complex rapidly activates factor X. We have previously provided data that suggest an interaction between the NH2-terminal epidermal growth factor (EGF)-like module of factor IXa beta and the substrate factor X. In an alternative approach to study this protein-protein interaction, we have expressed three recombinant baculovirus constructs encoding the EGF-like modules of human factor IX and a truncated form of fibronectin in a system based on the infection of insect cells (Spodoptera frugiperda 21). This strategy allows a simple one-step purification of the recombinant proteins on a gelatin-Sepharose column, followed by removal of the gelatin-binding part derived from fibronectin by proteolytic cleavage. The fusion proteins were isolated at yields of 20-50 micrograms/ml culture medium. The recombinant EGF-like modules contained 0.2-0.4 mol of erythro-beta-hydroxyaspartic acid/mol of protein, i.e. similar to the amount found in factor IX from human plasma, and appeared to be glycosylated at Ser-53. The NH2-terminal EGF-like module, which contained a transamidation acceptor site derived from fibronectin, was cross-linked by factor XIIIa in solution to intact and Gla-domainless factor X. There was no evidence of cross-linking to activated factor X or to factor X fragments containing only the gamma-carboxyglutamic acid module and the two EGF-like modules. The cross-linking results suggest a specific interaction between the NH2-terminal EGF-like module of factor IXa beta and the heavy chain of unactivated factor X. This interaction, albeit weak as judged by competition experiments, may be important for the targeting of factor X to the factor IXa beta-factor VIIIa complex on biological membranes and for the subsequent dissociation of factor Xa from the complex after activation.

Amino Acid Sequence↗
PubMed1994

The gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IXa beta. Effects on the serine protease module and factor X activation.

Blood coagulation factors IX and X are two serine proteases with a similar modular structure. The non-catalytic part of each protein consists of a gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. We have now found that the NH2-terminal EGF-like module of both factors IX and X inhibits factor Xa formation in a Gla-independent manner, both in the presence and absence of phospholipid and the cofactor, factor VIIIa. In contrast, the COOH-terminal EGF-like module has no such effect. Our data indicate that the NH2-terminal EGF-like module of factor IXa beta interacts either with the corresponding module or with the serine protease module in the substrate, factor X, without affecting the hydrolysis of low molecular weight substrates. Using antibodies as structural probes, we found that Ca2+ binding to the Gla module of factor IXa beta induces a conformational transition in the serine protease module. No evidence was found for a direct interaction between the Gla module and factor VIIIa. We therefore propose that the Gla module in factor IXa beta is indirectly involved in the cofactor interaction, in that Ca2+ binding to sites in this module induces a conformation in the serine protease module that is commensurate with factor VIIIa interaction. In addition, the immunochemical approach revealed a Gla-independent Ca2+ binding site in the serine protease module (apparent Kd of approximately 120 microM) that also might influence its conformation. Antibodies against the EGF-like modules of factor IX were used to probe Ca2+ binding to these modules in intact and in Gla-domainless factor IXa beta. The data indicate a Ca2+ binding site with an apparent Kd of approximately 50 microM in the NH2-terminal EGF-like module of both factor IX species.

1-Carboxyglutamic Acid↗
PubMed1993

Calcium affinity of the NH2-terminal epidermal growth factor-like module of factor X. Effect of the gamma-carboxyglutamic acid-containing module.

The NH2-terminal epidermal growth factor (EGF)-like module of vitamin K-dependent coagulation factors IX and X and protein C each has one calcium binding site. This module (residues 45-86) from factor X has been isolated previously and found to bind calcium with a Kd of 2.2 mM at physiological pH and ionic strength. We have now demonstrated that it binds calcium with a Kd of 120 microM in a fragment that consists of the Gla module and the NH2-terminal EGF-like module. The presence of the Gla module (residues 1-44) increases the calcium affinity of the site in the EGF-like module approximately 20-fold, thus making it essentially saturated in vivo. Decarboxylation of the Gla residues to Glu has no significant effect on the calcium affinity of the EGF-like module. A proteolytic fragment of factor X (residues 29-86) and a synthetic peptide (residues 34-86), folded to a native conformation, were used to demonstrate that the contribution of the Gla module to the calcium affinity of the site in the EGF-like module is mediated by its 17 COOH-terminal residues, 12 of which form an alpha-helix in the intact Gla module. In the NMR structure of the NH2-terminal EGF-like module in factor X, five calcium ligating groups have been identified (Selander-Sunnerhagen, M., Ullner, M., Persson, E., Teleman, O., Stenflo, J., and Drakenberg, T. (1992) J. Biol. Chem. 267, 19642-19649). As calcium usually requires seven to eight oxygen ligands, there is reason to believe that the Gla module contributes ligands, or negative charge, to increase the calcium affinity. Our findings suggest that the calcium affinity of EGF-like modules in other proteins may also be influenced by neighboring modules.

1-Carboxyglutamic Acid↗
PubMed1993

The effect of aspartate hydroxylation on calcium binding to epidermal growth factor-like modules in coagulation factors IX and X.

Hydroxylation of aspartic acid to erythro-beta-aspartic acid (Hya) occurs in epidermal growth factor (EGF)-like modules in numerous extracellular proteins with diverse functions. Several EGF-like modules with the consensus sequence for hydroxylation bind Ca2+, and it has therefore been suggested that the hydroxyl group is essential for Ca2+ binding. To determine directly the influence of beta-hydroxylation on calcium binding in the EGF-like modules from coagulation factors IX and X, we have now measured calcium binding to both the fully beta-hydroxylated and the non-beta-hydroxylated modules of the two proteins. At low ionic strength, the Hya-containing module of factor X has a slightly higher Ca2+ affinity, but at physiological salt concentrations this difference is no longer significant for either factor IX or X. Analysis of the 1H NMR chemical shift differences between the hydroxylated and nonhydroxylated factor X modules show that hydroxylation has no effect on the domain fold. Furthermore, measurements on factor IX show that hydroxylation has no effect on the Ca2+/Mg2+ specificity of the ion binding site. We conclude that the hydroxyl group is not a direct ligand for the calcium ion in these EGF-like modules, nor is it essential for high-affinity Ca2+ binding.

Amino Acid Sequence↗
PubMed1993

Effects of Ca2+ binding on the protease module of factor Xa and its interaction with factor Va. Evidence for two Gla-independent Ca(2+)-binding sites in factor Xa.

The assembly of macromolecular complexes containing factors Xa and Va on suitable phospholipid surfaces is crucial for rapid activation of prothrombin. We have used quantitative affinity chromatography to characterize the interaction between factor Va and intact factor Xa on the one hand and between factor Va and factor Xa lacking the gamma-carboxyglutamic acid (Gla)-containing module on the other. The dissociation constants were found to be 1.0 +/- 0.1 and 9.5 +/- 1.8 microM, respectively. There was good agreement between these dissociation constants and the concentrations of active site-inhibited factor Xa and Gla-domainless factor Xa that caused half-maximal inhibition of prothrombin activation. To investigate whether the noncatalytic modules of factor Xa interacted directly with factor Va, intact modules were isolated from proteolytic digests of factor X and used as inhibitors of prothrombin activation. The inhibitory effect observed with the isolated Gla module in the absence of phospholipid was due to inhibition of the amidolytic activity of factor Xa rather than to an interaction with factor Va. The epidermal growth factor-like modules did not inhibit prothrombin activation. Using antibodies specific for calcium-dependent epitopes in the serine protease module of factor Xa we demonstrated that Ca2+ binding to the Gla module alters the conformation of the catalytic module. Half-maximal binding was observed at approximately 0.8 mM Ca2+. Evidence was also obtained for the presence of two Gla-independent Ca(2+)-binding sites in factor Xa. One of these sites, located in the NH2-terminal epidermal growth factor-like module, was half-saturated at approximately 60 microM Ca2+ in intact factor Xa and at approximately 1.2 mM Ca2+ in Gla-domainless factor Xa. This site appeared not to influence the conformation of the protease module. The second site, which was half-saturated at approximately 0.16 mM Ca2+, appeared to reside in the serine protease module and to alter its conformation as judged by binding of antibodies specific for calcium-dependent epitopes.

1-Carboxyglutamic Acid↗
PubMed1992

Comparison of the Ca2+ binding properties of the gamma-carboxyglutamic acid-containing module of protein Z in the intact protein and in N-terminal fragments.

Protein Z is a vitamin K-dependent plasma protein of unknown function. Its modular structure is identical with those of factors VII, IX, X, and protein C. These proteins have an N-terminal gamma-carboxyglutamic acid (Gla)-containing module which binds six to ten Ca2+. In factors IX, X, and protein C, the adjacent epidermal growth factor (EGF)-like module binds one Ca2+ whereas the EGF-like module in protein Z does not. We have compared the Ca2+ binding properties of a fragment of protein Z comprising the Gla and N-terminal EGF-like modules (pZ-GlaEGFN) with those of intact protein Z and the isolated Gla module by measuring the Ca(2+)-induced quenching of the intrinsic protein fluorescence. The similar Ca2+ affinities of pZ-GlaEGFN and protein Z indicate that pZ-GlaEGFN has a native conformation and normal Ca2+ binding properties. A comparison of the Ca2+ binding to pZ-GlaEGFN with those to the corresponding fragments of factors IX, X, and protein C indicate that Ca2+ binding to the N-terminal EGF-like modules in the latter proteins does not influence the folding and Ca2+ binding properties of their Gla modules. Furthermore, the Ca(2+)-induced fluorescence enhancements of GlaEGF fragments from factors IX, X, and protein C appear to be caused by Ca2+ binding to the site in the EGF-like modules since it is not observed for pZ-GlaEGFN.

1-Carboxyglutamic Acid↗
PubMed1992

Modulation of fibrinolysis by thrombospondin.

Thrombospondin is a large, trimeric glycoprotein secreted by activated platelets and growing cells. Thrombospondin copolymerizes with fibrin during blood coagulation and deposits in extracellular matrix. We found that thrombospondin is a slow (rate constant approximately 6.3 x 10(3) M-1 sec-1), tight-binding (Kd < 10(-9) M) inhibitor of plasmin as determined by loss of amidolytic activity, loss of ability to degrade fibrinogen, and decreased lysis zones in fibrin plate assays (Biochemistry 31: 265-269, 1992). Thrombospondin also slowly inhibits urokinase plasminogen activator. The lysis zone when urokinase is put on fibrin plates made from whole plasma is less if thrombospondin is present. The stoichiometry of inhibition is approximately one mole plasmin:one mole thrombospondin trimer, a somewhat surprising result considering the trimeric nature of thrombospondin. These results indicate that thrombospondin is an important regulator of fibrinolysis and degradation of extracellular matrix, particularly when these processes are initiated by urokinase and even when other inhibitors of fibrinolysis are present.

Animals↗
PubMed1992

How an epidermal growth factor (EGF)-like domain binds calcium. High resolution NMR structure of the calcium form of the NH2-terminal EGF-like domain in coagulation factor X.

Domains homologous to the epidermal growth factor (EGF) are important building blocks for extracellular proteins. Proteins containing these domains have been shown to function in such diverse biological processes as blood coagulation, complement activation, and the developmental determination of embryonic cell fates. Many of these proteins require calcium for their biological function. In the case of coagulation factors IX and X and anticoagulants proteins C and S, calcium has been found to bind to the EGF-like domains. We have now determined the three-dimensional structure of the calcium-bound form of the NH2-terminal EGF-like domain in coagulation factor X by two-dimensional NMR and simulated folding. Ligands to the calcium ion are the two backbone carbonyls in Gly-47 and Gly-64, as well as the side chains in Gln-49, erythro-beta-hydroxyaspartic acid (Hya) 63, and possibly Asp-46. The conserved Asp-48 is not a ligand in our present structures. The remaining ligands are assumed to be solvent molecules or, in the intact protein, ligands from neighboring domains. Other proteins interacting in a calcium-dependent manner may also contribute ligands. A comparison with the calcium-free form shows that calcium binding induces strictly local structural changes in the domain. Residues corresponding to the side chain ligands in factor X are conserved in many other proteins, such as the integral membrane protein TAN-1 of human lymphocytes and its developmentally important homolog, Notch, in Drosophila. Calcium binding to EGF-like domains may be crucial for numerous protein-protein interactions involving EGF-like domains in coagulation factors, plasma proteins, and membrane proteins. Therefore, there is reason to believe that this novel calcium site plays an important role in the biochemistry of extracellular proteins.

Amino Acid Sequence↗
PubMed1992

Three-dimensional structure of the apo form of the N-terminal EGF-like module of blood coagulation factor X as determined by NMR spectroscopy and simulated folding.

The three-dimensional structure of a 42-residue fragment containing the N-terminal EGF-like module of blood coagulation factor X was determined by means of 2D NMR spectroscopy and computer simulation. The spectroscopic data consisted of 370 NOE distances and 27 dihedral angle constraints. These were used to generate peptide conformations by molecular dynamics simulation. The simulations used a novel functional form for the constraint potentials and were performed with two time steps to ensure rapid execution. Apart from preliminary runs to aid assignment of NOEs, 60 runs resulted in 13 accepted structures, which have two antiparallel beta sheets, no alpha helices, and five tight turns. There is no hydrophobic cluster. The root mean square deviation for the backbone of the 13 conformations is 0.65 +/- 0.11 A against their mean conformation. About half of the side chains have well-defined structure. The overall conformation is similar to that of murine EGF.

Amino Acid Sequence↗
PubMed1992

Effects of gamma-carboxyglutamic acid and epidermal growth factor-like modules of factor IX on factor X activation. Studies using proteolytic fragments of bovine factor IX.

Factor IX is a vitamin K-dependent zymogen of a serine protease. The NH2-terminal half of the molecule consists of a Ca(2+)-binding gamma-carboxyglutamic acid (Gla)-containing module and two modules homologous to the epidermal growth factor (EGF) precursor. To elucidate the role of these non-catalytic modules of factor IXa beta in factor X activation, we have isolated and characterized fragments of bovine factor IX, containing one or both of the EGF-like modules as well as these modules linked to the Gla module. The fragments were used as inhibitors of factor IXa beta-mediated factor X activation in a plasma clotting system and in systems with purified components of the Xase complex. Fragments consisting of either the two EGF-like modules of factor IX linked together or the NH2-terminal EGF-like module alone were found to inhibit factor Xa generation both in the presence and absence of the cofactor, factor VIIIa. Moreover, a fragment consisting of the corresponding modules of factor X had a similar effect. We therefore propose that factor IXa beta and factor X interact directly through their EGF-like modules on or in the vicinity of a phospholipid surface. We have also found that the isolated Gla module of factor IX inhibits the formation of factor Xa both in the presence and absence of phospholipid but not in the absence of factor VIIIa. Our results are compatible with a model of the Xase complex, in which both the serine protease part and the Gla module of factor IXa beta interact with factor VIIIa.

1-Carboxyglutamic Acid↗
PubMed1992

Identification of structural domains in protein C involved in its interaction with thrombin-thrombomodulin on the surface of endothelial cells.

The structural domains of protein C involved in its interaction with thrombin-thrombomodulin on the endothelial cell surface have been investigated using isolated intact domains of bovine protein C produced from controlled proteolytic digests of the protein. The fragments investigated include the gamma-carboxyglutamic acid (Gla)-rich module, the two epidermal growth factor (EGF)-like modules, and a fragment consisting of the Gla and the two EGF-like modules. The effects of these fragments on the catalytic efficiency (Km and Vmax) of activation of protein C by the endothelial cell surface thrombin-thrombomodulin complex (IIa-TM) have been evaluated in vitro using a stirred microcarrier cell culture of bovine aortic endothelial cells and purified proteins. Neither the Gla nor the two EGF-like modules alone had any discernible effect on protein C activation. The intact Gla-EGF fragment, however, inhibited protein C activation. The results are consistent with a rapid equilibrium competitive inhibition model, in which the Gla-EGF fragment competes with protein C for binding to IIa-TM, and indicate that the Gla-EGF fragment alone accounts for most of the binding energy of intact protein C for IIa-TM. In addition, a requirement for the Gla residues of protein C for binding is implied by the observation that heat-decarboxylated Gla-EGF fragment was not an inhibitor of protein C activation. In addition, chloromethyl ketone-inactivated activated protein C was found to bind to IIa-TM with the same affinity as protein C, suggesting that the changes which occur in protein C upon activation do not affect that part of the protein responsible for binding to IIa-TM, that is the Gla-EGF region. The Gla-EGF region from factor X also weakly inhibited the IIa-TM activation of protein C.

1-Carboxyglutamic Acid↗
PubMed1992

Thrombospondin is a slow tight-binding inhibitor of plasmin.

Thrombospondin is a multifunctional glycoprotein of platelet alpha-granules and a variety of growing cells. We demonstrate that thrombospondin is a slow tight-binding inhibitor of plasmin as determined by loss of amidolytic activity, loss of ability to cleave fibrinogen, and decreased lysis zones in fibrin plate assays. Stoichiometric titrations indicate that approximately 1 mol of plasmin interacts with 1 mol of thrombospondin, an unexpected result considering the trimeric nature of thrombospondin. Plasmin in a complex with streptokinase or bound to epsilon-aminocaproic acid is protected from inhibition by thrombospondin, thereby implicating the lysine-binding kringle domains of plasmin in the inhibition process. Thrombospondin also inhibits urokinase plasminogen activator, but more slowly than plasmin, stimulates the amidolytic activity of tissue plasminogen activator, and has no effect on the amidolytic activity of alpha-thrombin or factor Xa. These results, therefore, identify thrombospondin as a new type of serine proteinase inhibitor and potentially important regulator of fibrinolysis.

Blood Platelets↗
PubMed1992

Protein C inhibitor in human body fluids. Seminal plasma is rich in inhibitor antigen deriving from cells throughout the male reproductive system.

An assay was developed for the measurement of human protein C inhibitor antigen (PCI) in blood plasma and other biological fluids. Both native PCI, modified inhibitor, and complexes of inhibitor with activated protein C or plasma kallikrein could be measured with the assay. Inhibitor antigen concentrations were found to be very high in seminal plasma (greater than 200 mg/liter), more than 40 times the concentration of PCI found in blood plasma. The inhibitor in seminal plasma was unable to form complexes with activated protein C. Gel filtration and immunoblotting findings indicated that the inhibitor in seminal plasma is present in a high molecular mass complex or cleaved to its modified form. As PCI antigen was absent from seminal plasma of patients with dysfunctional seminal vesicles, the seminal vesicle glands would appear to be the major source of seminal plasma PCI, a conclusion supported by immunohistochemical demonstration of the presence of PCI epitopes in the secretory epithelium of the seminal vesicles. Specific PCI immunoreactivity was also shown to be present in the testes, the epididymis glands, and the prostate, suggesting the inhibitor to have a complex or multiple function in the male reproductive system. Conclusive evidence of a local synthesis of PCI in the four male sex glands was provided by Northern blot analysis of RNA from these organs.

Adolescent↗
PubMed1991

Interaction of human protein Z with thrombin: evaluation of the species difference in the interaction between bovine and human protein Z and thrombin.

Protein Z is a vitamin K-dependent protein of unknown function present in normal human and bovine plasma. Binding and kinetic studies showed that bovine protein Z interacts with bovine thrombin with a dissociation constant of 0.11 microM in a Ca(2+)-independent fashion and that thrombin becomes associated with phospholipid vesicles in the presence of protein Z but not in its absence (Hogg, P. J. and Stenflo, J. (1991) J. Biol. Chem., in press). In the present study the interaction of human protein Z with human thrombin and the influence of human protein Z on the association of thrombin with phospholipid vesicles was evaluated. In contrast to bovine protein Z, human protein Z bound human DIP-thrombin with a 20-fold weaker affinity at 1.5 mM Ca2+ and in a Ca(2+)-dependent fashion. Human protein Z was also less effective than bovine protein Z in promoting the association of thrombin with phospholipid vesicles. Also, bovine protein Z cleaved by thrombin at Arg-365 bound DIP-thrombin with a 10-fold weaker affinity than did native bovine protein Z. The data suggest that the species difference in the interaction between protein Z and thrombin can be explained by a difference in the COOH-terminal region of bovine protein Z versus human protein Z.

Animals↗
PubMed1991

Interaction of vitamin K-dependent protein Z with thrombin. Consequences for the amidolytic activity of thrombin and the interaction of thrombin with phospholipid vesicles.

Protein Z is a vitamin K-dependent protein of unknown function present in normal bovine plasma at a concentration of approximately 0.1 microM. Quantitative affinity chromatographic studies using diisopropylphosphoryl (DIP)-thrombin-Affi-Gel 10 as the affinity matrix and free DIP-thrombin as the competitor demonstrated that protein Z interacts with DIP-thrombin with a dissociation constant of 0.15 +/- 0.05 microM. Binding was independent of Ca2+. Protein C and factor IX, other vitamin K-dependent clotting proteins with the same domain structure as that of protein Z, did not interact with immobilized DIP-thrombin under these conditions; and factor X interacted with an affinity 20-fold lower than that for protein Z. The Michaelis constant, Km, for hydrolysis of pyro-Glu-Pro-Arg-p-nitroanilide by thrombin was increased 1.8-fold, from 130 to 230 microM, as a result of the binding of protein Z and the Km for H-Val-Leu-Arg-p-nitroanilide 1.4-fold, from 390 to 560 microM. From these kinetic studies, a dissociation constant of 0.11 +/- 0.04 microM was calculated for the binding of protein Z to alpha-thrombin. Protein Z bound to large phospholipid vesicles (25% phosphatidylserine, 75% phosphatidylcholine) with a dissociation constant of 0.39 +/- 0.16 microM at a phospholipid to protein ratio of 82 mol of phospholipid/mol of protein Z at saturation. In the presence of protein Z thrombin associated with phospholipid vesicles, whereas thrombin did not interact with phospholipid vesicles in the absence of protein Z. These studies, therefore, demonstrate a physiologically relevant interaction between protein Z and thrombin. They also suggest a mechanism whereby thrombin is localized to an injury site by virtue of its interaction with protein Z bound to phospholipid surfaces.

Amidohydrolases↗