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Biomedical subjects

Bruce Furie

Publications and source records attributed to Bruce Furie.

At least 37 records · Page 2Linked to original sources
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↗
PubMed2004

Role of platelet P-selectin and microparticle PSGL-1 in thrombus formation.

P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) are vascular adhesion molecules that play an important role in leukocyte-endothelial and leukocyte-platelet interaction during the inflammatory response. Their functions are now known to include a role in thrombus formation, specifically in relation to fibrin generation and propagation. Recent findings have demonstrated that leukocyte-derived microparticles, bearing both tissue factor and PSGL-1, circulate in the blood and accumulate in the developing platelet-rich thrombus following vessel wall injury, thus concentrating tissue factor at the site of vascular injury and initiating blood coagulation.

Animals↗
PubMed2004

The blood coagulation cascade.

PURPOSE OF REVIEW: The biochemistry of blood coagulation has been well defined over the past 50 years. Although much is known about the sequence of the proteolytic cascade and its regulation in the pathway to fibrin generation, many important questions remain unsolved about the mechanism of initiation and the structure of the protein complexes that form during blood coagulation. RECENT FINDINGS: This article summarizes some of the advances that have been made in this field from the last quarter of 2002 and during 2003. The papers, which vary in rigor and content, have been selected on the basis of their interest and possible contribution to knowledge in this field. Summaries are given of new findings on the source of factor V and the synthesis of factor VIII, the mechanism of tissue factor action in the initiation of blood coagulation, the structure and membrane-binding properties of the protein complexes formed, and regulation of the blood coagulation cascade. SUMMARY: Continued progress in this field offers opportunity for understanding the basis of thrombotic diseases and bleeding disorders, with the potential for defining novel targets for therapeutic applications. Some of the conclusions reviewed are conflicting, and further work will be necessary to place the results in the context of what has already been established. The structural biology of the coagulation proteins and understanding of hemostasis and thrombosis in a physiologic context have important implications for future work.

Animals↗
PubMed2004

Antibody cross-linking of human platelet P-selectin induces calcium entry by a mechanism dependent upon Fcgamma receptor IIA.

It is established that antibody-induced cross-linking of platelet surface receptors is able to activate platelets in a manner dependent upon FcgammaRIIA. This has not, however, previously been shown for the adhesion receptor P-selectin, and since there is evidence that P-selectin may couple to activation events, it was important to address whether antibody cross-linking of this receptor induced signalling events, and whether this was dependent on FcgammaRIIA. Here we show that although addition of soluble P-selectin ligand rPSGL-Ig alone is not able to induce calcium signalling, further addition of a full-length rabbit anti human IgG leads to a sustained rise in [Ca 2+ ]i. This was due to an increase in the frequency and amplitude of transient calcium spiking in single platelets. The response was dependent upon engagement of both P-selectin and FcgammaRIIA since blocking anti-bodies to either receptor inhibited the response. The calcium rise is mediated primarily by induction of a calcium entry mechanism involving the Na(+)-Ca(2+) exchanger operating in reverse mode, since it was blocked by inhibitors of Na(+)-Ca(2+) exchange, bepridil and 5 mM NiCl(2).

Antibodies↗
PubMed2004

Role of P-selectin and PSGL-1 in coagulation and thrombosis.

P-selectin and PSGL-1 are cell adhesion molecules, regulating the initial interactions between leukocytes and the blood vessel wall as well as between activated platelets and leukocytes. P-selectin is expressed on activated endothelial cells and platelets, while its major ligand PSGL-1 is expressed on leukocytes. Multiple studies have shown that these adhesion molecules are required for the normal recruitment of leukocytes during an inflammatory reaction. More recently, these adhesion molecules have been implicated in recruitment of leukocytes and leukocyte microparticles to thrombi. The P-selectin- and PSGL-1-dependent delivery of circulating microparticles to thrombi appears to be important for normal tissue factor accumulation and fibrin generation in thrombi.

Blood Coagulation↗
PubMed2003

Peritoneal macrophages express both P-selectin and PSGL-1.

Macrophages, phagocytic cells involved in an early phase of host defense, are known to express the P-selectin ligand, PSGL-1. Heretofore, P-selectin has only been found on platelets and endothelial cells. Here, we demonstrate that peritoneal macrophages isolated by peritoneal lavage of unchallenged mice express P-selectin on the plasma membrane. The peritoneal macrophages synthesize P-selectin, as indicated by metabolic labeling experiments. P-Selectin is constitutively expressed on the extracellular surface of macrophages but is only partially colocalized with PSGL-1. P-Selectin is rapidly translocated from the macrophage plasma membrane to intracellular vesicles and to lysosomes. Peritoneal macrophages assemble into cell strings under flow conditions based upon macrophage-macrophage interactions mediated by P-selectin and PSGL-1. This is the first description of a leukocyte shown to express both P-selectin and PSGL-1.

Animals↗
PubMed2003

Initial accumulation of platelets during arterial thrombus formation in vivo is inhibited by elevation of basal cAMP levels.

Platelet accumulation at sites of vascular injury is the primary event in arterial thrombosis. Initial platelet accrual into thrombi is mediated by interactions of platelet adhesion receptors with ligands on the injured endothelium or in the sub-endothelial matrix. The role of intracellular signals in initial platelet accumulation at sites of endothelial injury, however, is the subject of debate. We have used a newly discovered inhibitor of phosphodiesterase 3A (PDE3A) and the well-characterized PDE3A inhibitor, cilostazol, to modulate 3',5'-cyclic adenosine monophosphate (cAMP) levels in an in vivo model that enables the kinetic analysis of platelet accumulation. These studies demonstrate that elevation of basal cAMP levels results in an overall decline in platelet accumulation at the site of vascular injury. In particular, the initial rate of accumulation of platelets is inhibited by elevation of cAMP. Analysis of the kinetics of individual platelets at injury sites using intravital microscopy demonstrates that cAMP directs the rate at which platelets attach to and detach from thrombi. These studies demonstrate that cAMP in circulating platelets controls attachment to and detachment from sites of arteriolar injury. Thus, the status of the intracellular signaling machinery prior to engagement of platelet receptors influences the rate of platelet accumulation during thrombus formation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins.

In a calcium-dependent interaction critical for blood coagulation, vitamin K-dependent blood coagulation proteins bind cell membranes containing phosphatidylserine via gamma-carboxyglutamic acid-rich (Gla) domains. Gla domain-mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologic time scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovine prothrombin Gla domain. The serine head group binds Gla domain-bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gla domains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are being identified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in other physiologic processes.

1-Carboxyglutamic Acid↗
PubMed2003

Novel role of vitamin k in preventing oxidative injury to developing oligodendrocytes and neurons.

Oxidative stress is believed to be the cause of cell death in multiple disorders of the brain, including perinatal hypoxia/ischemia. Glutamate, cystine deprivation, homocysteic acid, and the glutathione synthesis inhibitor buthionine sulfoximine all cause oxidative injury to immature neurons and oligodendrocytes by depleting intracellular glutathione. Although vitamin K is not a classical antioxidant, we report here the novel finding that vitamin K1 and K2 (menaquinone-4) potently inhibit glutathione depletion-mediated oxidative cell death in primary cultures of oligodendrocyte precursors and immature fetal cortical neurons with EC50 values of 30 nm and 2 nm, respectively. The mechanism by which vitamin K blocks oxidative injury is independent of its only known biological function as a cofactor for gamma-glutamylcarboxylase, an enzyme responsible for posttranslational modification of specific proteins. Neither oligodendrocytes nor neurons possess significant vitamin K-dependent carboxylase or epoxidase activity. Furthermore, the vitamin K antagonists warfarin and dicoumarol and the direct carboxylase inhibitor 2-chloro-vitamin K1 have no effect on the protective function of vitamin K against oxidative injury. Vitamin K does not prevent the depletion of intracellular glutathione caused by cystine deprivation but completely blocks free radical accumulation and cell death. The protective and potent efficacy of this naturally occurring vitamin, with no established clinical side effects, suggests a potential therapeutic application in preventing oxidative damage to undifferentiated oligodendrocytes in perinatal hypoxic/ischemic brain injury.

Animals↗
PubMed2003

The Gla domain of factor IXa binds to factor VIIIa in the tenase complex.

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.

Amino Acid Sequence↗
PubMed2003

Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin.

Using a laser-induced endothelial injury model, we examined thrombus formation in the microcirculation of wild-type and genetically altered mice by real-time in vivo microscopy to analyze this complex physiologic process in a system that includes the vessel wall, the presence of flowing blood, and the absence of anticoagulants. We observe P-selectin expression, tissue factor accumulation, and fibrin generation after platelet localization in the developing thrombus in arterioles of wild-type mice. However, mice lacking P-selectin glycoprotein ligand 1 (PSGL-1) or P-selectin, or wild-type mice infused with blocking P-selectin antibodies, developed platelet thrombi containing minimal tissue factor and fibrin. To explore the delivery of tissue factor into a developing thrombus, we identified monocyte-derived microparticles in human platelet-poor plasma that express tissue factor, PSGL-1, and CD14. Fluorescently labeled mouse microparticles infused into a recipient mouse localized within the developing thrombus, indicating that one pathway for the initiation of blood coagulation in vivo involves the accumulation of tissue factor- and PSGL-1-containing microparticles in the platelet thrombus expressing P-selectin. These monocyte-derived microparticles bind to activated platelets in an interaction mediated by platelet P-selectin and microparticle PSGL-1. We propose that PSGL-1 plays a role in blood coagulation in addition to its known role in leukocyte trafficking.

Animals↗
PubMed2003

PSGL-1 participates in E-selectin-mediated progenitor homing to bone marrow: evidence for cooperation between E-selectin ligands and alpha4 integrin.

The nature and exact function of selectin ligands involved in hematopoietic progenitor cell (HPC) homing to the bone marrow (BM) are unclear. Using murine progenitor homing assays in lethally irradiated recipients, we found that the P-selectin glycoprotein ligand-1 (PSGL-1) plays a partial role in HPC homing to the BM (a reduction of about 35% when the P-selectin binding region is blocked). Blockade of both PSGL-1 and alpha4 integrin did not further enhance the effect of anti-alpha4 integrin (a reduction of about 55%). We suspected that E-selectin ligands might contribute to the remaining homing activity. To test this hypothesis, HPC homing assays were carried out in E-selectin-deficient recipients and revealed a profound alteration in HPC homing when E-selectin and alpha4 integrin were inactivated (> 90% reduction). Competitive assays to test homing of long-term repopulating stem cells revealed a drastic reduction (> 99%) of the homed stem cell activity when both alpha4 integrin and E-selectin functions were absent. Further homing studies with PSGL-1-deficient HPCs pretreated with anti-alpha4 integrin antibody revealed that PSGL-1 contributes to approximately 60% of E-selectin ligand-mediated homing activity. Our results thus underscore a major difference between mature myeloid cells and immature stem/progenitor cells in that E-selectin ligands cooperate with alpha4 integrin rather than P-selectin ligands.

Animals↗
PubMed2002

P-, E-, and L-selectin mediate migration of activated CD8+ T lymphocytes into inflamed skin.

P- and E-selectin mediate CD4+ Th1 cell migration into the inflamed skin in a murine contact hypersensitivity model. In this model, not only CD4+ T cells but also CD8+ T cells infiltrate the inflamed skin, and the role of CD8+ type 1 cytotoxic T (Tc1) cells as effector cells has been demonstrated. Here we show that in mice deficient in both P- and E-selectin, the infiltration of CD8+ T cells in the inflamed skin is reduced, suggesting the role of these selectins in CD8+ T cell migration. We directly studied the role of selectins using in vitro-generated Tc1 cells. These cells are able to migrate into the inflamed skin of wild-type mice. This migration is partially mediated by P- and E-selectin, as shown by the reduced Tc1 cell migration into the inflamed skin of mice deficient in both P- and E-selectin or wild-type mice treated with the combination of anti-P-selectin and anti-E-selectin Abs. During P- and E-selectin-mediated migration of Tc1 cells, P-selectin glycoprotein ligand-1 appears to be the sole ligand for P-selectin and one of the ligands for E-selectin. P- and E-selectin-independent migration of Tc1 cells into the inflamed skin was predominantly mediated by L-selectin. These observations indicate that all three selectins can mediate Tc1 cell migration into the inflamed skin.

Animals↗
PubMed2002

Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.

We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. We observed platelet deposition, tissue factor accumulation and fibrin generation after laser-induced endothelial injury in a single developing thrombus. The initiation of blood coagulation in vivo entailed the initial accumulation of tissue factor on the upstream and thrombus-vessel wall interface of the developing thrombus. Subsequently tissue factor was associated with the interior of the thrombus. Tissue factor was biologically active, and was associated with fibrin generation within the thrombus.

Animals↗