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B Furie

Publications and source records attributed to B Furie.

At least 37 records · Page 2Linked to original sources
PubMed1999

Alpha-granule secretion from alpha-toxin permeabilized, MgATP-exposed platelets is induced independently by H+ and Ca2+.

In order to better understand granule release from platelets, we developed an alpha-toxin permeabilized platelet model to study alpha-granule secretion. Secretion of alpha-granules was analyzed by flow cytometry using P-selectin as a marker for alpha-granule release. P-selectin surface expression occurred when platelets were permeabilized in the presence of Ca2+. Responsiveness to Ca2+ was lost 30 min after permeabilization but could be reconstituted with MgATP. Alpha-toxin-permeabilized, MgATP-exposed platelets also degranulated within a pH range of 5.4-5.9 without exposure to and independent of Ca2+. ATP, GTP, CTP, UTP, and ITP supported Ca2+-induced alpha-granule secretion, while H+-induced alpha-granule secretion occurred only with ATP and GTP. Both Ca2+- and H+-induced alpha-granule secretion required ATP hydrolysis. Kinase inhibitors blocked both Ca2+- and H+-induced secretion. These data suggest that alpha-granule secretion in this permeabilized platelet system shares many characteristics with granule secretion studied in other permeabilized cell models. Furthermore, these results show that H+ can trigger alpha-granule release independent of Ca2+.

Adenosine Triphosphate↗
PubMed1999

The biology of P-selectin glycoprotein ligand-1: its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction.

Cell-cell interactions mediating leukocyte trafficking, thrombogenesis and inflammation are crucial for the host defense mechanism. The selectin family of integral membrane proteins includes E-selectin, L-selectin and P-selectin. Selectins mediate tethering and rolling of leukocytes to the vessel wall at the site of inflammation. The counter-receptor for P-selectin and possibly the other selectins is P-selectin glycoprotein ligand-1 (PSGL-1). This review focuses on the properties and biology of PSGL- 1.

Animals↗
PubMed1998

12-Hydroxyeicosatetraenoic acid upregulates P-selectin-induced tissue factor activity on monocytes.

12-Hydroxyeicosatetraenoic acid (12-HETE), a product of the platelet lipoxygenase pathway, amplifies tissue factor expression by P-selectin-stimulated monocytes in a time- and dose-dependent fashion. The same effect is observed when monocytes are incubated with Chinese hamster ovary cells transfected with the P-selectin cDNA. Both 5-HETE and leukotriene C4 are inactive in this system. Furthermore, the effect is not dependent on non-specific monocyte adhesion, since monocytes incubated with CHO cells expressing E-selectin do not express tissue factor, either in the presence or in the absence of 12-HETE. These results show that 12-HETE is a cofactor for the expression of tissue factor by monocytes.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1998

Interaction between soluble P-selectin and soluble P-selectin glycoprotein ligand 1: equilibrium binding analysis.

Leukocyte rolling in the vasculature is mediated by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Since cell-cell interaction mediated by P-selectin and PSGL-1 is cooperative and complex, we have developed a model system to examine the binding of P-selectin to PSGL-1 in a soluble system. Equilibrium binding analyses were performed with truncated forms of soluble human P-selectin and dimeric PSGL-1, both lacking the transmembrane domain and both produced in Chinese hamster ovary (CHO) cells. Soluble PSGL-1 (sPSGL-1), which contains no tryptophan residues and exhibits no intrinsic fluorescence, was harvested from CHO cells cotransfected with either fucosyltransferase III (sPSGL-1/Fuc-TIII) or fucosyltransferase VII (sPSGL-1/Fuc-TVII). Both fucosylation isoforms of sPSGL-1 bound to sP-selectin. The interaction of sP-selectin and sPSGL-1 was studied by monitoring changes in the intrinsic fluorescence of sP-selectin upon binding to sPSGL-1. Binding of sPSGL-1 to sP-selectin in the presence of calcium caused an increase in tryptophan fluorescence that could be reversed by the addition of ethylenediaminetetraacetic acid (EDTA). The fluorescence enhancement of sP-selectin by sPSGL-1 was used to generate binding isotherms, and these data were fitted to a bimolecular binding model. The binding constant, Kd, for the binding of sPSGL-1/Fuc-TIII and sPSGL-1/Fuc-TVII to sP-selectin was 3 +/- 2 nM and 80 +/- 44 nM, respectively. Monomeric sP-selectin bound to dimeric sPSGL-1 with a 2:1 stoichiometry. In a system in which both protein species are soluble and lack transmembrane domains, these results indicate high-affinity interaction between P-selectin and PSGL-1. Furthermore, the fucosylation pattern of PSGL-1 can affect its affinity for P-selectin. These binding constants can be used to explore models of cell adhesion in flow systems.

Alkylation↗
PubMed1997

Role of gamma-carboxyglutamic acid in the calcium-induced structural transition of conantokin G, a conotoxin from the marine snail Conus geographus.

Conantokin G is a gamma-carboxyglutamic acid- (Gla-) containing conotoxin isolated from the venom of the marine cone snail Conus geographus. This 17-residue polypeptide, which contains five gamma-carboxyglutamic acid residues, is a N-methyl-d-aspartate- (NMDA-) type glutamate receptor antagonist. To investigate the role of gamma-carboxyglutamic acid in the calcium-induced structural transition of conantokin G, we determined the three-dimensional structure of the conantokin G/Ca2+ complex by two-dimensional 1H NMR spectroscopy and compared it to the high-resolution structure of conantokin G in the absence of metal ions [Rigby et al. (1997) Biochemistry 36, 6906]. Complete resonance assignments were made by two dimensional 1H NMR spectroscopy at pH 5.6 in the presence of saturating amounts of Ca2+. Distance geometry and simulated annealing methods were used to derive 23 convergent structures from a set of 302 interproton distance restraints and two torsion angle measurements. A high-resolution structure, with the backbone root mean square deviation to the geometric average of the 23 structures of 0.6 +/- 0.1 A, contains a linear alpha-helix from Gla 3 to Lys 15. Gla residues 3, 7, 10, and 14 are aligned in a linear array on one face of the helix. A genetic algorithm was applied to determine the calcium positions in conantokin G, and the conantokin G/Ca2+ complex refined by molecular simulation. Upon binding of Ca2+ to gamma-carboxyglutamic acid, conantokin G undergoes a conformational transition from a distorted curvilinear 310 helix to a linear alpha-helix. Occupancy of the metal binding sites, defined by gamma-carboxyglutamic acids, results in formation of a calcium-carboxylate network that linearizes the helix and exposes the hydrophobic amino acids on the opposite face of the helix.

1-Carboxyglutamic Acid↗
PubMed1997

The gamma-carboxylation recognition site is sufficient to direct vitamin K-dependent carboxylation on an adjacent glutamate-rich region of thrombin in a propeptide-thrombin chimera.

The propeptides of the vitamin K-dependent proteins contain a gamma-carboxylation recognition site that is required for gamma-glutamyl carboxylation. To determine whether the propeptide is sufficient to direct carboxylation, two mutant prothrombin species were expressed and characterized with regard to posttranslational gamma-carboxylation. A double point mutant, in which serine substituted for cysteines 17 and 22 disrupted a conserved loop formed by a disulfide bond, was fully carboxylated when expressed in Chinese hamster ovary cells. A propeptide/thrombin chimeric protein, constructed by deleting the Gla, aromatic amino acid stack, and kringle domains of prothrombin, has the signal peptide and propeptide juxtaposed to a glutamate-rich COOH-terminal region of prothrombin, residues 249-530. Of the 8 glutamic acid residues contained within the first 40 residues of the NH2 terminus adjacent to the propeptide, at least seven were fully carboxylated as demonstrated by direct gamma-carboxyglutamic acid analysis of the alkaline hydrolysate and by NH2-terminal sequence analysis. These results indicate that the gamma-carboxylation recognition site within the prothrombin propeptide in a prothrombin propeptide-thrombin chimeric protein is sufficient to direct gamma-carboxylase-catalyzed carboxylation of adjacent glutamic acid residues in a glutamate-rich region of thrombin that is not normally gamma-carboxylated. Furthermore, the disulfide loop in the Gla domain of prothrombin is not required for complete carboxylation.

Amino Acid Sequence↗
PubMed1997

Propeptide and glutamate-containing substrates bound to the vitamin K-dependent carboxylase convert its vitamin K epoxidase function from an inactive to an active state.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational conversion of glutamic acid to gamma-carboxyglutamic acid in precursor proteins containing the gamma-carboxylation recognition site (gamma-CRS). During this reaction, glutamic acid is converted to gamma-carboxyglutamic acid while vitamin KH2 is converted to vitamin K 2,3-epoxide. Recombinant bovine carboxylase was purified free of gamma-CRS-containing propeptide and endogenous substrate in a single-step immunoaffinity procedure. We show that in the absence of gamma-CRS-containing propeptide and/or glutamate-containing substrate, carboxylase has little or no epoxidase activity. Epoxidase activity is induced by Phe-Leu-Glu-Glu-Leu (FLEEL) (9.2 pmol per min per pmol of enzyme), propeptide, residues -18 to -1 of proFactor IX (3.4 pmol per min per pmol of enzyme), FLEEL and propeptide (100 pmol per min per pmol of enzyme), and proPT28 (HVFLAPQQARSLLQRVRRANTFLEEVRK, residues -18 to +10 of human acarboxy-proprothrombin), (5.3 pmol per min per pmol of enzyme). These results indicate that in the absence of propeptide or glutamate-containing substrate, oxygenation of vitamin K by the carboxylase does not occur. Upon addition of propeptide or glutamate-containing substrate, the enzyme is converted to an active epoxidase. This regulatory mechanism prevents the generation of a highly reactive vitamin K intermediate in the absence of a substrate for carboxylation.

1-Carboxyglutamic Acid↗
PubMed1997

Three-dimensional structure of a gamma-carboxyglutamic acid-containing conotoxin, conantokin G, from the marine snail Conus geographus: the metal-free conformer.

Conantokin G is a gamma-carboxyglutamic acid-containing conotoxin from the venom of the marine cone snail Conus geographus. The 17-residue peptide, which contains five gamma-carboxyglutamic acid (Gla) residues and an amidated C-terminal asparagine amide, was synthesized chemically in a form identical to the natural conantokin G. To gain insight into the role of gamma-carboxyglutamic acid in the structure of this peptide, we determined the three-dimensional structure of conantokin G by 1H NMR and compared its structure to other conotoxins and to the gamma-carboxyglutamic acid-containing regions of the vitamin K-dependent blood-clotting proteins. Complete resonance assignments were made by two-dimensional 1H NMR spectroscopy in the absence of metal ions. NOE cross-peaks d(alphaN), d(NN), and d(betaN) provided interproton distance information, and vicinal spin-spin coupling constants 3J(HN alpha) were used to calculate phi torsion angles. Distance geometry and simulated annealing methods were used to derive 20 convergent structures from a set of 227 interproton distance restraints and 13 torsion angle measurements. The backbone rmsd to the geometric average for 20 final structures is 0.8 +/- 0.1 A. Conantokin G consists of a structured region commencing at Gla 3 and extending through arginine 13. This structure includes a partial loop centered around Gla 3 and Gla 4, a distorted type I turn between glutamine 6 and glutamine 9, and two type I turns involving Gla 10, leucine 11, and isoleucine 12 and arginine 13. Together, these two turns define approximately 1.6 turns of a distorted 3(10) helix. The observed structure possesses structural elements similar to those seen in the disulfide-linked conotoxins.

1-Carboxyglutamic Acid↗
PubMed1997

The propeptide of the vitamin K-dependent carboxylase substrate accelerates formation of the gamma-glutamyl carbanion intermediate.

Vitamin K-dependent carboxylase catalyzes the post-translational gamma-carboxylation of 9-12 glutamyl residues of several blood coagulation proteins. Carboxylase purified from Chinese hamster ovary (CHO) cells as a recombinant FLAG-carboxylase fusion protein [Sugiura, I., et al. (1996) J. Biol. Chem. 271, 17837-17844] was utilized with pentapeptide substrate FL[3H-R,S]EAL with high specific radioactivity to probe the timing of glutamyl Cgamma-3H cleavage relative to Cgamma-COO- bond formation by 14CO2 incorporation rates. Studies were conducted over a range of NaH14CO3 concentrations to assess uncoupling of gamma-glutamyl carbanion formation and over a range of concentrations of ProPT18, the 18-residue peptide corresponding to the -18 to -1 propeptide region of prothrombin known to affect the catalytic efficiency of carboxylase. At saturation, ProPT18 accelerates Cgamma-3H cleavage 11-13-fold and Cgamma-14CO2- formation 6-7-fold, converting a Cgamma-3H cleavage/Cgamma-14CO2- formation ratio of 1.2-1.4 in the absence of ProPT18 to 2.3-2.8 in its presence, a relative increase in and uncoupling of Cgamma-3H cleavage from C-C bond formation. When the HCO3- concentration was varied, the V/K3H+/V/K14CO2 ratios rose as HCO3- fractional saturation dropped to a ratio of 9.3-10.8/l at low bicarbonate, indicating an uncoupling of nine out of ten gamma-glutamyl carbanion formations from carboxylative capture, consistent with prior reports on microsomal enzyme [Larson, A. E., et al. (1981) J. Biol. Chem. 256, 11032-11035]. These results with pentapeptide substrate FLEAL validate reversible gamma-glutamyl carbanion formation by pure carboxylase and indicate the ProPT18 increase in catalytic efficiency is in selective lowering of an energy barrier preceding the gamma-glutamyl carbanion intermediate.

1-Carboxyglutamic Acid↗
PubMed1997

Refinement of the NMR solution structure of the gamma-carboxyglutamic acid domain of coagulation factor IX using molecular dynamics simulation with initial Ca2+ positions determined by a genetic algorithm.

A genetic algorithm (GA) successfully identified the calcium positions in the crystal structure of bovine prothrombin fragment 1 bound with calcium ions (bf1/Ca). The same protocol was then used to determine the calcium positions in a closely related fragment, the Gla domain of coagulation factor IX, the structure of which had previously been determined by NMR spectroscopy in the presence of calcium ions. The most frequently occurring low-energy structure found by GA was used as the starting structure for a molecular dynamics refinement. The molecular dynamics simulation was performed using explicit water and the Particle-Mesh Ewald method to accommodate the long-range electrostatic forces. While the overall conformation of the NMR structure was preserved, significant refinement is apparent when comparing the simulation average structure with its NMR precursor in terms of the N-terminal (Tyr1-N) network, the total number of hydrogen bonds, the calcium ion coordinations, and the compactness of the structure. It is likely that the placement of calcium ions in the protein is critical for refinement. The calcium ions apparently induce structural changes during the course of the simulation that result in a more compact structure.

1-Carboxyglutamic Acid↗
PubMed1997

gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function.

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.

1-Carboxyglutamic Acid↗
PubMed1997

Leukocyte crosstalk at the vascular wall.

P-selectin is an adhesion molecule on platelets and endothelial cells that mediates the interaction of these cells with leukocytes. In addition to its docking function in cell-cell recognition, P-selectin is involved in cell signalling and cell communication. Upon platelet activation, P-selectin undergoes rapid phosphorylation and dephosphorylation. The biological role of these phosphorylation events within platelets is unknown. P-selectin, upon contact with its receptor on monocytes, initiates the de novo biosynthesis of tissue factor and other cytokines. Tissue factor upregulation on monocytes is mediated by PSGL-1 and is independent of CD14, the LPS receptor.

Animals↗
PubMed1997

Structure and mechanism of action of the vitamin K-dependent gamma-glutamyl carboxylase: recent advances from mutagenesis studies.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamic acid to gamma-carboxyglutamic acid in substrate proteins. The enzyme has recently been purified and the cDNA encoding the enzyme has been cloned. The availability of recombinant enzyme provides the opportunity to probe the mechanism of this unique enzyme. The binding sites for the gamma-carboxylation recognition site containing propeptide and carboxylatable glutamate residues of a vitamin K-dependent substrate protein have been localized to the amino-terminal 250 residues of the enzyme. Regions of the carboxy-terminal of the enzyme are important for conversion of vitamin K hydroquinone to vitamin K epoxide, a reaction that occurs concomitantly with carboxylation and is catalyzed by the vitamin K-dependent carboxylase. Using pure recombinant vitamin K-dependent carboxylase it has been demonstrated that catalysis of vitamin K oxygenation by the enzyme is regulated by the availability of carboxylatable substrate.

Animals↗
PubMed1997

Platelet-leukocyte-endothelial cell interaction on the blood vessel wall.

Leukocytes, platelets, and endothelial cells interact at sites of vascular injury and inflammation through adhesion receptors on the cell surface. On binding of ligand to receptor, these receptors initiate intracellular signaling that leads to the modulation of several biological properties of the cells involved. These finely regulated processes involve several classes of cell adhesion molecules: integrins, immunoglobulin-like proteins, selectins, and mucin-like proteins as well as an array of soluble mediators. Interaction of these cell adhesion molecules serves to recruit circulating cells to the blood vessel endothelium or to accumulated platelets on the vessel wall and to foster cell-cell communication. The importance of these interactions to inflammation, blood coagulation, and the immune response is outlined.

Blood Platelets↗
PubMed1997

Interactions of neutrophils and coagulation proteins.

Some of the interactions between coagulation factors and neutrophils are described. Proteins of the contact system, FXa, thrombin, and fibrinogen all bind to various sites on the neutrophil. This binding has a dual purpose: the assembly of coagulation complexes such as the prothrombinase complex and the contact system on the neutrophil membrane and influencing the various neutrophil functions including chemotaxis, aggregation, degranulation, and transendothelial migration. In addition, neutrophil elastase degrades many coagulation proteins, thus modulating both the thrombotic and the fibrinolytic systems. These interactions should be viewed in a wider context as part of the links between the coagulation and inflammation pathways.

Blood Coagulation Factors↗
PubMed1996

Structure and function of the epidermal growth factor domain of P-selectin.

P-selectin is a multidomain adhesion protein on the surface of activated platelets and endothelial cells that functions in the recruitment of leukocytes to the site of inflammation. The amino-terminal lectin and EGF domains constitute the ligand recognition unit. We have produced a synthetic 40-residue P-selectin EGF domain (P-sel:EGF) to examine the structure and function of this domain independent of P-selectin. The peptide was folded in vitro and exhibited the same disulfide bonding pattern as other EGF-like domains. P-sel:EGF did not inhibit P-selectin-mediated cellular adhesion assays, indicating that the lectin domain is also required. We undertook the study of the P-selectin EGF by 1H NMR to determine its structure independent of the lectin domain and to compare its structure to that of E-selectin determined crystallographically [Graves et al. (1994) Nature 367, 532]. Although the binding of P-selectin to its carbohydrate ligand is calcium dependent, and some EGF domains have calcium binding sites, addition of calcium had no effect on the NMR spectrum or on the pH-induced changes. Nearly complete resonance assignments were made from 2D 1H NMR spectra at pH 6.0. Two sections of antiparallel beta-sheet were identified on the basis of the pattern of long-range NOEs, 3JHN alpha coupling constants, and slowly exchanging amides. The solution structure of the peptide backbone was determined using distance geometry and simulated annealing calculations. The backbone RMSD to the geometric average for 19 final structures is 0.64 +/- 0.17 A. The resulting fold closely resembles that of other EGF-like peptides, including the E-selectin EGF domain (RMSD approximately 1.08 A). However, compared to the E-selectin EGF structure which also contains the lectin domain, some residues from 1-11 are less ordered, and novel contacts occur between the amino terminus and the core beta-sheet. Despite marked structural homology of the selectin polypeptide backbones, the selectin EGF surfaces show unique distributions of charged residues, a feature that likely correlates to the functional differences.

Amino Acid Sequence↗
PubMed1996

Biosynthesis of prothrombin: intracellular localization of the vitamin K-dependent carboxylase and the sites of gamma-carboxylation.

Prothrombin is a vitamin K-dependent blood coagulation protein that undergoes posttranslational gamma-carboxylation and propeptide cleavage during biosynthesis. The propeptide contains the gamma-carboxylation recognition site that directs gamma-carboxylation. To identify the intracellular sites of carboxylation and propeptide cleavage, we monitored the synthesis of prothrombin in Chinese hamster ovary cells stably transfected with the prothrombin cDNA by immunofluorescent staining. The vitamin K-dependent carboxylase was located in the endoplasmic reticulum and Golgi complex. Antibodies specific to prothrombin processing intermediates were used for immunocytolocalization. Anti-des-gamma-carboxyprothrombin antibodies stained only the endoplasmic reticulum whereas antiproprothrombin antibodies (specific for the propeptide) and antiprothrombin:Mg(II) antibodies (which bind the carboxylated forms of proprothrombin and prothrombin) stained both the endoplasmic reticulum and the Golgi complex. Antiprothrombin:Ca(II)-specific antibodies (which bind only to the carboxylated form of prothrombin lacking the propeptide) stained only the Golgi complex and secretory vesicles, and colocalized with antimannosidase II and anti-p200 in the juxtanuclear Golgi complex. These results indicate that uncarboxylated proprothrombin undergoes complete gamma-carboxylation in the endoplasmic reticulum and that gamma-carboxylation precedes propeptide cleavage during prothrombin biosynthesis.

Acylation↗