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Biomedical subjects

Barbara C Furie

Publications and source records attributed to Barbara C Furie.

At least 19 recordsLinked to original sources
PubMed2006

Par4 is required for platelet thrombus propagation but not fibrin generation in a mouse model of thrombosis.

Thrombin, a central mediator of hemostasis and thrombosis, converts fibrinogen to fibrin and is a potent platelet activator. Activated platelets provide a surface for assembly of the tenase and prothrombinase complexes required for thrombin generation. The role of thrombin-induced platelet activation in platelet accumulation and its interplay with fibrin deposition during thrombus assembly has not been fully defined. We studied these processes during laser-induced thrombus formation by using real-time digital fluorescence microscopy in mice lacking protease-activated receptor-4 (Par4), which is necessary for thrombin responsiveness in mouse platelets. Juxtamural platelet accumulation immediately after laser injury was not different in wild-type and Par4(-/-) mice. However, subsequent growth of platelet thrombi was markedly diminished in Par4(-/-) mice. At the time of maximal thrombus size in wild type, platelet accumulation was more than 10-fold higher in wild type than in Par4(-/-) mice. P-selectin expression, a marker of platelet activation, was reduced and delayed in Par4(-/-) thrombi. In contrast to platelet activation and accumulation, the rate and amount of fibrin deposition, predominantly intramural and juxtamural in this model, were indistinguishable in Par4(-/-) and wild-type mice. These results suggest that platelet activation by thrombin is necessary for normal propagation of a platelet thrombus at a distance from the injured vessel wall and hence for normal thrombus growth. However, platelet activation by thrombin is unnecessary for initial and limited accumulation of platelets at or near the vessel wall, and this limited accumulation of platelets and/or platelet-independent mechanism(s) of thrombin generation are sufficient for normal fibrin deposition in this model.

Animals↗
PubMed2006

A single gamma-carboxyglutamic acid residue in a novel cysteine-rich secretory protein without propeptide.

Gamma-glutamyl carboxylase catalyzes the modification of specific glutamyl residues to gamma-carboxyglutamyl (Gla) residues in precursor proteins that possess the appropriate gamma-carboxylation recognition signal within the propeptide region. We describe the immunopurification and first biochemical characterization of an invertebrate high molecular weight Gla-containing protein with homologues in mammals. The protein, named GlaCrisp, was isolated from the venom of the marine cone snail Conus marmoreus. GlaCrisp gave intense signals in Western blot experiments employing the Gla-specific antibody M3B, and the presence of Gla was chemically confirmed by amino acid analysis after alkaline hydrolysis. Characterization of a full-length cDNA clone encoding GlaCrisp deduced a precursor containing an N-terminal signal peptide but, unlike other Gla-containing proteins, no apparent propeptide. The predicted mature protein of 265 amino acid residues showed considerable sequence similarity to the widely distributed cysteine-rich secretory protein family and closest similarity (65% identity) to the recently described substrate-specific protease Tex31. In addition, two cDNA clones encoding the precursors of two isoforms of GlaCrisp were identified. The predicted precursor isoforms differed at three amino acid positions (-6, 9, and 25). Analysis by Edman degradation and nanoelectrospray ionization mass spectrometry, before and after methyl esterfication, identified a Gla residue at amino acid position 9 in GlaCrisp. This is the first example of a Gla-containing protein without an obvious gamma-carboxylation recognition site. The results define a new class of Gla proteins and support the notion that gamma-carboxylation of glutamyl residues is phylogenetically older than blood coagulation and the vertebrate lineage.

1-Carboxyglutamic Acid↗
PubMed2006

Tissue factor pathway vs. collagen pathway for in vivo platelet activation.

The roles that the various platelet collagen receptors play in initial platelet adhesion and thrombus growth remain controversial. Here we summarize some of the pertinent data and discuss some recent studies of the initiation and propagation of platelet accumulation into thrombi and the initiation and propagation of thrombin generation. Mice lacking platelet surface glycoprotein VI (GPVI) form normal thrombi in the laser injury model but have a diminished thrombotic response to severe FeCl3 injury. We hypothesize that the paths to thrombus formation in these two models are different with interaction of GPVI and collagen predominant early after severe FeCl3-induced injury but platelet activation by thrombin predominant after laser-induced injury. Understanding of the response to insult in thrombosis models deepens our understanding of the process and provides a firm foundation for evaluation of anti-thrombotic therapy in these models.

Animals↗
PubMed2006

Cancer-associated thrombosis.

Thrombosis is one of the major complications of malignant disease, but the underlying molecular and cellular basis remains elusive. A number of hypotheses have been put forth, including the expression of tissue factor by tumor cells, release of tissue factor during cell death following chemotherapy, intrinsic unique tumor procoagulants and microparticles. Exploration of a potential role of microparticles in cancer-associated thrombosis indicates that tissue factor microparticles are present in a spectrum of cancer patients known to have a high incidence of thromboembolic complications.

Cell Membrane↗
PubMed2006

Structure of human urokinase plasminogen activator in complex with its receptor.

The urokinase plasminogen activator binds to its cellular receptor with high affinity and initiates signaling cascades that are implicated in pathological processes including tumor growth, metastasis, and inflammation. We report the crystal structure at 1.9 angstroms of the urokinase receptor complexed with the urokinase amino-terminal fragment and an antibody against the receptor. The three domains of urokinase receptor form a concave shape with a central cone-shaped cavity where the urokinase fragment inserts. The structure provides insight into the flexibility of the urokinase receptor that enables its interaction with a wide variety of ligands and a basis for the design of urokinase-urokinase receptor antagonists.

Antibodies↗
PubMed2006

Glycoprotein VI-dependent and -independent pathways of thrombus formation in vivo.

The role of the collagen receptor glycoprotein VI (GPVI) in arteriolar thrombus formation was studied in FcRgamma-null mice (FcRgamma(-/-)) lacking platelet surface GPVI. Thrombi were induced with severe or mild FeCl(3) injury. Collagen exposure was significantly delayed and diminished in mild compared with severe FeCl(3) injury. Times to initial thrombus formation and vessel occlusion were delayed in FcRgamma(-/-) compared with wild-type mice after severe injury. Platelet accumulation in wild-type mice was decreased after mild compared with severe injury. However, there was little difference between platelet accumulation after severe or mild injury in FcRgamma(-/-). These data indicate a significant role for GPVI in FeCl(3)-induced thrombus formation. Pretreatment of wild-type mice with lepirudin further impaired mild FeCl(3)-induced thrombus formation, demonstrating a role for thrombin. Laser-induced thrombus formation in wild-type and FcRgamma(-/-) was comparable. Collagen exposure to circulating blood was undetectable after laser injury. Normalized for thrombus size, thrombus-associated tissue factor was 5-fold higher in laser-induced thrombi than in severe FeCl(3)-induced thrombi. Thus, platelet activation by thrombin appears to be more important after laser injury than platelet activation by GPVI-collagen. It may thus be important when considering targets for antithrombotic therapy to use multiple animal models with diverse pathways to thrombus formation.

Animals↗
PubMed2006

Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile.

The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.

1-Carboxyglutamic Acid↗
PubMed2005

Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development.

Circulating tissue factor accumulates in the developing thrombus and contributes to fibrin clot formation. To determine whether tissue factor derived from hematopoietic cells is delivered to the thrombus via tissue factor-bearing microparticles or circulating leukocytes expressing tissue factor on the plasma membrane, we compared the kinetics of tissue factor accumulation in the developing arteriolar thrombus with the time course of leukocyte-thrombus interaction and microparticle-thrombus interaction in the microcirculation of a living mouse using intravital high-speed widefield and confocal microscopy. Tissue factor rapidly accumulated in the developing thrombus, appearing immediately following vessel wall injury, reaching a first peak in approximately 100 s. In contrast, leukocyte-thrombus interaction was not observed until after 2-3 min following vessel wall injury. Maximal leukocyte rolling and firm leukocyte adherence on thrombi in wild-type mice were observed after approximately 8 min and were dependent on P-selectin and P-selectin glycoprotein ligand-1. This delay in P-selectin-dependent leukocyte rolling is a result of time-dependent platelet activation and P-selectin expression on the luminal surface of the thrombus. In contrast, microparticle accumulation in the developing arteriolar thrombus was rapid, and peak accumulation was within 60 s. The accumulation of hematopoietic cell-derived tissue factor in the developing thrombus correlates to the kinetics of microparticle accumulation and does not correlate temporally with leukocyte-thrombus interaction. These results indicate that tissue factor derived from hematopoietic cells is delivered by microparticles during the initial phase of thrombus development in vivo.

Animals↗
PubMed2005

Platelet PECAM-1 inhibits thrombus formation in vivo.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.

Animals↗
PubMed2005

PTP-1B is an essential positive regulator of platelet integrin signaling.

Outside-in integrin alphaIIbbeta3 signaling is required for normal platelet thrombus formation and is triggered by c-Src activation through an unknown mechanism. In this study, we demonstrate an essential role for protein-tyrosine phosphatase (PTP)-1B in this process. In resting platelets, c-Src forms a complex with alphaIIbbeta3 and Csk, which phosphorylates c-Src tyrosine 529 to maintain c-Src autoinhibition. Fibrinogen binding to alphaIIbbeta3 triggers PTP-1B recruitment to the alphaIIbbeta3-c-Src-Csk complex in a manner that is dependent on c-Src and specific tyrosine (tyrosine 152 and 153) and proline (proline 309 and 310) residues in PTP-1B. Studies of PTP-1B-deficient mouse platelets indicate that PTP-1B is required for fibrinogen-dependent Csk dissociation from alphaIIbbeta3, dephosphorylation of c-Src tyrosine 529, and c-Src activation. Furthermore, PTP-1B-deficient platelets are defective in outside-in alphaIIbbeta3 signaling in vitro as manifested by poor spreading on fibrinogen and decreased clot retraction, and they exhibit ineffective Ca2+ signaling and thrombus formation in vivo. Thus, PTP-1B is an essential positive regulator of the initiation of outside-in alphaIIbbeta3 signaling in platelets.

Animals↗
PubMed2005

Precursors of novel Gla-containing conotoxins contain a carboxy-terminal recognition site that directs gamma-carboxylation.

Vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamyl residues to gamma-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the gamma-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel gamma-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs gamma-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue "postpeptide" from the Gla-TxXI precursor reduced the K(m) for the reaction of the Conus gamma-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it was positioned at the N- or C-terminal end of the mature toxin. Comparison of the postpeptides to propeptides from other conotoxins suggested some common elements, and amino acid substitutions of these residues perturbed gamma-carboxylation of the Gla-TxXI peptide. The demonstration of a functional and transferable C-terminal postpeptide in these conotoxins indicates the presence of the gamma-carboxylation recognition site within the postpeptide and defines a novel precursor structure for vitamin K-dependent polypeptides. It also provides the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.

1-Carboxyglutamic Acid↗
PubMed2005

Thrombus formation in vivo.

To examine thrombus formation in a living mouse, new technologies involving intravital videomicroscopy have been applied to the analysis of vascular windows to directly visualize arterioles and venules. After vessel wall injury in the microcirculation, thrombus development can be imaged in real time. These systems have been used to explore the role of platelets, blood coagulation proteins, endothelium, and the vessel wall during thrombus formation. The study of biochemistry and cell biology in a living animal offers new understanding of physiology and pathology in complex biologic systems.

Animals↗
PubMed2004

Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation during thrombus propagation.

Tissue factor (TF) is expressed on nonvascular cells and cells within the vessel wall and circulates in blood associated with microparticles. Although blood-borne TF accumulates into the developing thrombus during thrombus formation, the contribution of blood-borne TF and vessel wall TF to thrombin generation in vivo following vessel injury is unknown. To determine the source and role of blood-borne microparticle TF, we studied arterial thrombus formation in a living mouse using intravital microscopy. Platelet, TF, and fibrin accumulation in the developing thrombus was compared in wild-type and low TF mice. Compared to wild-type mice, low TF mice formed very small platelet thrombi lacking TF or fibrin. Wild-type and low TF mice received transplants of bone marrow from wild-type and low TF mice. Arterial thrombi in low TF bone marrow/wild-type chimeric mice had decreased size and decreased TF and fibrin levels. Arterial thrombi in wild-type bone marrow/low TF chimeric mice showed decreased platelet thrombus size but normal TF and fibrin levels. This demonstrates that blood-borne TF associated with hematopoietic cell-derived microparticles contributes to thrombus propagation.

Animals↗
PubMed2004

Isolation and characterization of three novel Gla-containing Conus marmoreus venom peptides, one with a novel cysteine pattern.

One defining characteristic of Conus venom peptides is the high frequency of posttranslational modifications found. We report the discovery and initial characterization of three novel gamma-carboxyglutamic acid (Gla)-containing conotoxins, Gla-MrII, Gla-MrIII, and Gla-MrIV, isolated from the venom of the mollusc-hunting cone snail Conus marmoreus. Peptide Gla-MrII, a 50 amino acid residue peptide, carries eight cysteine residues arranged in a novel cysteine pattern, and five gamma-carboxyglutamic acid residues. The monoisotopic molecular mass was determined by electrospray ionization mass spectrometry to 5860.23 Da, consistent with the peptide having the cysteine residues disulphide-bonded and having a free acid C-terminus. Peptides Gla-MrIII and Gla-MrIV each contain two gamma-carboxyglutamic acid residues and share little sequence similarity to previously identified conotoxins. Both peptides contain four cysteine residues that are positioned in the linear sequence in a manner reminiscent of conotoxins belonging to cysteine scaffold superfamily T (scaffold T-1). Determination of the monoisotopic molecular masses revealed that Gla-MrIII is amidated at its C-terminus while Gla-MrIV has a free C-terminal acid.

1-Carboxyglutamic Acid↗
PubMed2004

The first gamma-carboxyglutamic acid-containing contryphan. A selective L-type calcium ion channel blocker isolated from the venom of Conus marmoreus.

Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to gamma-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a K(D) of 0.63 mM. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.

1-Carboxyglutamic Acid↗
PubMed2004

The metal-free and calcium-bound structures of a gamma-carboxyglutamic acid-containing contryphan from Conus marmoreus, glacontryphan-M.

Glacontryphan-M, a novel calcium-dependent inhibitor of L-type voltage-gated Ca(2+) channels expressed in mouse pancreatic beta-cells, was recently isolated from the venom of the cone snail Conus marmoreus (Hansson, K., Ma, X., Eliasson, L., Czerwiec, E., Furie, B., Furie, B. C., Rorsman, P., and Stenflo, J. (2004) J. Biol. Chem. 278, 32453-32463). The conserved disulfide-bonded loop of the contryphan family of conotoxins including a D-Trp is present; however, unique to glacontryphan-M is a histidine within the intercysteine-loop and two gamma-carboxyglutamic acid (Gla) residues, formed by post-translational modification of glutamic acid. The two calcium-binding Gla residues are located in a four residue N-terminal extension of this contryphan. To better understand the structural and functional significance of these residues, we have determined the structure of glacontryphan-M using two-dimensional (1)H NMR spectroscopy in the absence and presence of calcium. Comparisons of the glacontryphan-M structures reveal that calcium binding induces structural perturbations within the Gla-containing N terminus and the Cys(11)-Cys(5)-Pro(6) region of the intercysteine loop. The backbone of N-terminal residues perturbed by calcium, Gla(2) and Ser(3), moves away from the His(8) and Trp(10) aromatic rings and the alignment of the D-Trp(7) and His(8) aromatic rings with respect to the Trp(10) rings is altered. The blockage of L-type voltage-gated Ca(2+) channel currents by glacontryphan-M requires calcium binding to N-terminal Gla residues, where presumably histidine and tryptophan may be accessible for interaction with the channel. The backbone C alpha conformation of the intercysteine loop of calcium-bound glacontryphan-M superimposes on known structures of contryphan-R and Vn (0.83 and 0.66 A, respectively). Taken together these data identify that glacontryphan-M possesses the canonical contryphan intercysteine loop structure, yet possesses critical determinants necessary for a calcium-induced functionally required conformation.

1-Carboxyglutamic Acid↗
PubMed2004

Crystal structure of the calcium-stabilized human factor IX Gla domain bound to a conformation-specific anti-factor IX antibody.

The binding of Factor IX to membranes during blood coagulation is mediated by the N-terminal gamma-carboxyglutamic acid-rich (Gla) domain, a membrane-anchoring domain found on vitamin K-dependent blood coagulation and regulatory proteins. Conformation-specific anti-Factor IX antibodies are directed at the calcium-stabilized Gla domain and interfere with Factor IX-membrane interaction. One such antibody, 10C12, recognizes the calcium-stabilized form of the Gla domain of Factor IX. We prepared the fully carboxylated Gla domain of Factor IX by solid phase peptide synthesis and crystallized Factor IX-(1-47) in complex with Fab fragments of the 10C12 antibody. The overall structure of the Gla domain in the Factor IX-(1-47)-antibody complex at 2.2 A is similar to the structure of the Factor IX Gla domain in the presence of calcium ions as determined by NMR spectroscopy (Freedman, S. J., Furie, B. C., Furie, B., and Baleja, J. D. (1995) Biochemistry 34, 12126-12137) and by x-ray crystallography (Shikamoto, Y., Morita, T., Fujimoto, Z., and Mizuno, H. (2003) J. Biol. Chem. 278, 24090-24094). The complex structure shows that the complementarity determining region loops of the 10C12 antibody form a hydrophobic pocket to accommodate the hydrophobic patch of the Gla domain consisting of Leu-6, Phe-9, and Val-10. Polar interactions also play an important role in the antibody-antigen recognition. Furthermore, the calcium coordination network of the Factor IX Gla domain is different than in Gla domain structures of other vitamin K-dependent proteins. We conclude that this antibody is directed at the membrane binding site in the omega loop of Factor IX and blocks Factor IX function by inhibiting its interaction with membranes.

Amino Acid Sequence↗