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Research Interests: Chemistry, Medicine, Dogs, Humans, Animals, and 12 moreBlood, Haemostasis and Thrombosis, Blood Coagulation, Anticoagulants, Clinical Sciences, Hemophilia A, Disseminated Intravascular Coagulation, Hemophilia B, Factor Xa, Radioimmunoassay, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region... more
Glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) form a macromolecular complex on the activated platelet surface which contains the fibrinogen-binding site necessary for normal platelet aggregation. To identify the specific region of the fibrinogen molecule responsible for its interaction with the GPIIb-GPIIIa complex, purified fragment D1 (Mr = 100,000) and fragment E (Mr = 50,000) were prepared from plasmin digests of purified human fibrinogen. In addition, the polypeptide chain subunits A alpha, B beta, and gamma of fibrinogen were prepared. Using an enzyme-linked immunosorbent assay we have demonstrated that isolated fragment D1 in a solid phase system forms a complex with a mixture of GPIIb and GPIIIa. The binding of the GPIIb-GPIIIa mixture to fragment D1-coated plates reached saturation at 8 nM and to fibrinogen-coated plates at 24 nM. Isolated A alpha, B beta, and gamma chains were not reactive with added glycoproteins. Fragment E coated directly on plastic plates or immobilized on antibody-coated plastic plates did not form a complex with GPIIb-GPIIIa. Only fluid phase fibrinogen and fragment D1 but not fragment E were inhibitory toward formation of a complex between solid phase fibrinogen and GPIIb-GPIIIa. Isolated A alpha, B beta, and gamma chains at concentrations equivalent to fluid phase fibrinogen were inactive. Binding of fragment D1 but not fragment E to the GPIIb-GPIIIa complex was also demonstrated by rocket immunoelectrophoresis of the membrane glycoprotein mixture through a gel containing the individual fragments and subsequent autoradiography of the complex following exposure to 125I-anti-fibrinogen. These observations with isolated platelet membrane glycoproteins provide strong evidence that each of the D domains of the fibrinogen molecule interacts directly with the GPIIb-GPIIIa complex on the activated platelet surface, thus allowing formation of a tertiary molecular "bridge" across the surface of two adjacent activated platelets.
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... Page 12. 518 PROTEINS AT INTERFACES 10 CM Ε 3 4P CD N ~ >·» ο 0 JO 1 2 I Ab(E) (Hawiger) Ab(D) (Hawiger) Ab(Fg) (US Biol.) Ab(Fg) (Atlantic) ,^Γ-Π Ab(Fg) (Hawiger) AbdgG) (Sigma) Μ Ε Ρ Β Μ Ε Ρ Β Μ Ε Ρ Β Μ Ε Ρ Β Μ Ε Ρ Β Μ Ε Ρ Β... more
... Page 12. 518 PROTEINS AT INTERFACES 10 CM Ε 3 4P CD N ~ >·» ο 0 JO 1 2 I Ab(E) (Hawiger) Ab(D) (Hawiger) Ab(Fg) (US Biol.) Ab(Fg) (Atlantic) ,^Γ-Π Ab(Fg) (Hawiger) AbdgG) (Sigma) Μ Ε Ρ Β Μ Ε Ρ Β Μ Ε Ρ Β Μ Ε Ρ Β Μ Ε Ρ Β Μ Ε Ρ Β Methaerylate Polymers ...
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... 8. Fletcher, AP, Biederman, O., Moore, D., Alk-jaersig, N., Sherry, S. Abnormal plasminogen-plasmin system activity (fibrinolysis) in patients with hepatic cirrhosis: its cause and consequences. J. Clin. Invest. 43:681-695, 1964. 9.... more
... 8. Fletcher, AP, Biederman, O., Moore, D., Alk-jaersig, N., Sherry, S. Abnormal plasminogen-plasmin system activity (fibrinolysis) in patients with hepatic cirrhosis: its cause and consequences. J. Clin. Invest. 43:681-695, 1964. 9. Hawiger, J., Niewiarowski, S., Gurewich, V ...
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Lipid A, the toxic principle of endotoxic lipopolysaccharide, and its precursor, Lipid X, interact with human platelets and modulate protein kinase C therein (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82,... more
Lipid A, the toxic principle of endotoxic lipopolysaccharide, and its precursor, Lipid X, interact with human platelets and modulate protein kinase C therein (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). We have now purified protein kinase C from human platelets and studied its interaction with endotoxic Lipids A and X. Protein kinase C-dependent phosphorylation of histone III-S was increased 15 times in the presence of Lipid A and 300 microM Ca2+. The Ca2+ requirement for such activation was lower when 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1,2-diolein were added. Lipid A also induced autophosphorylation of protein kinase C, and its activation was enhanced by phosphatidylserine without reducing the Ca2+ requirement. Kinetic analysis of protein kinase C activation induced by Lipid A, in regard to ATP as a substrate, demonstrated that Lipid A increased the rate of the reaction (Vmax) without modifying the affinity of the enzyme (Km) for the substrate. Lipid X inhibited the activation of the enzyme induced by Lipid A. Lipid X also inhibited protein kinase C activation by phosphatidylserine, 1,2-diolein, and PMA. However, 10 times more of Lipid X was required for 50% inhibition (IC50) when PMA was used as an activator of protein kinase C in the presence of phosphatidylserine than when Lipid A and 1,2-diolein were used. These results support the hypothesis that endotoxic Lipid A and Lipid X exert their biological effect in platelets through direct interactions with protein kinase C.
Research Interests: Biochemistry, Chemistry, Kinetics, Biology, Calcium, and 15 moreBiological Chemistry, Medicine, Biological Sciences, Humans, CHEMICAL SCIENCES, Glycolipids, Cell free System, Lipid A, Phosphatidylserine, Endotoxins, Isoelectric point, Blood platelets, Protein Kinase C, Medical and Health Sciences, and In Vitro Techniques
Research Interests: Chemistry, Bacteriology, Biology, Calcium, Medicine, and 15 moreBiological Sciences, Lysozyme, Copper, Staphylococcus aureus, Temperature, Gel Permeation Chromatography, Enzyme, Electrophoresis, Staphylococcus, Egg White, Cell Wall, Sodium Chloride, Lysis, Edetic Acid, and Medical and Health Sciences
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Excessive accumulation of collagen leads to fibrosis. Integrin α1β1 (Itgα1β1) prevents kidney fibrosis by reducing collagen production through inhibition of the EGF receptor (EGFR) that phosphorylates cytoplasmic and nuclear proteins. To... more
Excessive accumulation of collagen leads to fibrosis. Integrin α1β1 (Itgα1β1) prevents kidney fibrosis by reducing collagen production through inhibition of the EGF receptor (EGFR) that phosphorylates cytoplasmic and nuclear proteins. To elucidate how the Itgα1β1/EGFR axis controls collagen synthesis, we analyzed the levels of nuclear tyrosine phosphorylated proteins in WT and Itgα1-null kidney cells. We show that the phosphorylation of the RNA-DNA binding protein fused in sarcoma (FUS) is higher in Itgα1-null cells. FUS contains EGFR-targeted phosphorylation sites and, in Itgα1-null cells, activated EGFR promotes FUS phosphorylation and nuclear translocation. Nuclear FUS binds to the collagen IV promoter, commencing gene transcription that is reduced by inhibiting EGFR, down-regulating FUS, or expressing FUS mutated in the EGFR-targeted phosphorylation sites. Finally, a cell-penetrating peptide that inhibits FUS nuclear translocation reduces FUS nuclear content and collagen IV tran...
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Previously we showed that epidermal cells are able to use fibrinogen (FGN) as a migration substratum during wound closure. The goal of the present study was to determine the structural features of FGN that allow this migration. Pieces of... more
Previously we showed that epidermal cells are able to use fibrinogen (FGN) as a migration substratum during wound closure. The goal of the present study was to determine the structural features of FGN that allow this migration. Pieces of glass coated with native, fragmented, or other modified forms of FGN were implanted into full-thickness skin wounds of adult newts such that migrating epidermal cells would encounter the implant. In this system, a coating of FGN allowed considerably more migration than a coating of BSA. At high concentrations, heat-denatured FGN supported as much migration as the same amount of intact FGN. Fraction 1-9, a circulating form of FGN missing a 20-30K (K = 103Mr) carboxy-terminal segment of the Aα chain, was no less effective than intact FGN. Comparison of the isolated D1 and E fragments of FGN showed migration only on D1 but never to the extent seen on intact FGN containing the same amount of D1. Plasmin digestion of Di in the presence of EDTA, a process...
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Lipid A, the toxic principle of endotoxic lipopolysaccharide, and its precursor, Lipid X, interact with human platelets and modulate protein kinase C therein (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82,... more
Lipid A, the toxic principle of endotoxic lipopolysaccharide, and its precursor, Lipid X, interact with human platelets and modulate protein kinase C therein (Grabarek, J., Timmons, S., and Hawiger, J. (1988) J. Clin. Invest. 82, 964-971). We have now purified protein kinase C from human platelets and studied its interaction with endotoxic Lipids A and X. Protein kinase C-dependent phosphorylation of histone III-S was increased 15 times in the presence of Lipid A and 300 microM Ca2+. The Ca2+ requirement for such activation was lower when 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1,2-diolein were added. Lipid A also induced autophosphorylation of protein kinase C, and its activation was enhanced by phosphatidylserine without reducing the Ca2+ requirement. Kinetic analysis of protein kinase C activation induced by Lipid A, in regard to ATP as a substrate, demonstrated that Lipid A increased the rate of the reaction (Vmax) without modifying the affinity of the enzyme (Km) for the substrate. Lipid X inhibited the activation of the enzyme induced by Lipid A. Lipid X also inhibited protein kinase C activation by phosphatidylserine, 1,2-diolein, and PMA. However, 10 times more of Lipid X was required for 50% inhibition (IC50) when PMA was used as an activator of protein kinase C in the presence of phosphatidylserine than when Lipid A and 1,2-diolein were used. These results support the hypothesis that endotoxic Lipid A and Lipid X exert their biological effect in platelets through direct interactions with protein kinase C.
Research Interests: Biochemistry, Chemistry, Kinetics, Biology, Calcium, and 15 moreBiological Chemistry, Medicine, Biological Sciences, Humans, CHEMICAL SCIENCES, Glycolipids, Cell free System, Lipid A, Phosphatidylserine, Endotoxins, Isoelectric point, Blood platelets, Protein Kinase C, Medical and Health Sciences, and In Vitro Techniques
Research Interests: Biochemistry, Chemistry, Polymers, Medicine, Fibrinogen, and 15 moreThrombin, Humans, Blood, Peptides, Clinical Sciences, Hydrogen Bonding, Staphylococcus, Genetic variation, Hydrogen-Ion Concentration, Serine Protease, Binding Site, binding sites, plasmin, Cardiovascular medicine and haematology, and Paediatrics and reproductive medicine
Immune injury to human platelets by drugs, bacteria, and viruses which form antigenantibody complexes is mediated by the IgG Fc receptor on human platelets and results in thrombocytopenia. We studied whether immune injury to human... more
Immune injury to human platelets by drugs, bacteria, and viruses which form antigenantibody complexes is mediated by the IgG Fc receptor on human platelets and results in thrombocytopenia. We studied whether immune injury to human platelets mediated by the IgG Fc receptor can be prevented by prostacyclin (Prostagland in 12, PGI2), a novel prost glandin generated by the blood vessel wall. Immune injury to human platelets in whole plasma was elicited by Protein A-bearing staphylococci. Protein A induces binding of I to the human platelet Fc receptor, which results in platelet aggregation and 3H-seroton release in whole plasma. Excess of isolated Fc fragment inhibits aggregation and serotonin release in this model of immune injury. Synthetic PGI2 protected human platelets from this IgG Fc fragment-mediated immune injury in whole plasma. Inhibition was prompt (1 to 5 min) and dose dependent, reaching maximum at 10-6M of PGI2. Removal of plasma proteins and use of IgG-coated cells did no...
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Fibrinogen is a plasma factor required for aggregation of human platelets by ADP. The mechanism of platelet-ADP-fibrinogen interaction was studied by measuring the equilibrium binding of 125I-fibrinogen to human platelets separated from... more
Fibrinogen is a plasma factor required for aggregation of human platelets by ADP. The mechanism of platelet-ADP-fibrinogen interaction was studied by measuring the equilibrium binding of 125I-fibrinogen to human platelets separated from plasma proteins. Binding of 125I-fibrinogen to platelets not stimulated with ADP was low and unaffected by an excess of unlabel led fibrinogen. However, when platelets were stimulated with 4μM of ADP, there was an eightfold increase In the number of available binding sites for human fibrinogen, with affinity constant of 1.9 x 109M-1. This striking increase in fibrinogen receptor sites on human platelets was specific for ADP as contrasted to ATP, AMP, and adenosine. Prostacyclin (Prostaglandin I2, PGI2), a novel prostaglandin produced by the blood vessel wall, completely blocked this ADP-induced increase in fibrinogen receptor sites on human platelets. The effect of PGI2 was prompt and concentration dependent, reaching maximum at 10-9M. 6-keto PGF2 a ...
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Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization... more
Reactivity of platelets with an artificial surface exposed to whole blood is correlated with the concentration of adsorbed fibrinogen detectable by antifibrinogen antibodies. To examine the effect on platelets of the organization (distribution, orientation, conformation) of fibrinogen adsorbed on a hydrophobic surface, we studied the binding of polyclonal and monoclonal antifibrinogen antibodies to polyalkyl methacrylate polymers previously exposed to purified fibrinogen solution or diluted plasma and compared the results with platelet retention in methacrylate bead columns. There was an increase in platelet retention following diluted plasma pretreatment, which was eliminated by a polyclonal antibody against fibrinogen or against a gamma-(395-411) peptide from fibrinogen and was reduced by monoclonal antibodies (4A5, 4-2) against other COOH-terminal gamma-chain epitopes. Monoclonal antibody 10E5 against the fibrinogen receptor GpIIb/IIIa totally inhibited platelet retention in the ...
Research Interests: Physiology, Chemistry, Kinetics, Medicine, Adsorption, and 14 moreAntibodies, Platelet, Fibrinogen, Humans, Monoclonal Antibodies, Medical Physiology, Platelet activation mechanism, Polyclonal Antibodies, Monoclonal Antibody, Protein Binding, Biochemistry and cell biology, Blood platelets, epitope, and In Vitro Techniques
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Publisher Summary This chapter discusses platelet secretory pathways. Secretion is initiated in response to agonists such as thrombin, collagen, epinephrine, and adenosine diphosphate (ADP) acting on specific membrane receptors. At least... more
Publisher Summary This chapter discusses platelet secretory pathways. Secretion is initiated in response to agonists such as thrombin, collagen, epinephrine, and adenosine diphosphate (ADP) acting on specific membrane receptors. At least three stimulatory pathways in human platelets can be initiated all at once, or individually, depending on the nature and potency of the agonist-evoked stimulus. The first pathway involves archidonic acid, which is transformed by the enzyme cyclooxygenase into biologically active endoperoxides. Endoperoxides are converted enzymatically into thromboxane that triggers the secretory response through a poorly understood mechanism. The second stimulatory pathway is controlled by diacylglycerol, which is another phospholipid-derived messenger molecule. The third pathway involves the Ca 2+ /calmodulin complex, which activates myosin light-chain kinase. The ensuing phosphorylation of myosin light chain is associated with the early shape change of platelets and precedes their secretory response.
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Isolation of Platelet Membranes, Subcellular Organelles, and Cytoskeleton: J. Hawiger, Introduction to Platelet Structural and Functional Organization. Platelet Membranes and Granules: N. Crawford, K.S. Authi, and N. Hack, Isolation and... more
Isolation of Platelet Membranes, Subcellular Organelles, and Cytoskeleton: J. Hawiger, Introduction to Platelet Structural and Functional Organization. Platelet Membranes and Granules: N. Crawford, K.S. Authi, and N. Hack, Isolation and Characterization of Platelet Membranes Prepared by Free Flow Electrophoresis. M.J. Broekman, Homogenization by Nitrogen Cavitation Technique Applied to Platelet Subcellular Fractionation. J.T. Harmon, N.J. Greco, and G.A. Jamieson, Isolation of Human Platelet Plasma Membranes by Glycerol Lysis. M.H. Fukami, Isolation of Dense Granules from Human Platelets. Platelet Cytoskeleton: J.E.B. Fox, C.C. Reynolds, and J.K. Boyles, Studying the Platelet Cytoskeleton in Triton X-100 Lysates. S. Rosenberg-Schaier, Purification and Characterization of Platelet Actin, Actin-Binding Proteins, and ~ga-Actin. J.L. Daniel and J.R. Sellers, Purification and Characterization of Platelet Myosin. J. Bryan, Isolation and Characterization of Platelet Gelsolin. N.C. Collier and K. Wang, Purification and Properties of Human Platelet P235: Talin. J.G. White, Ultrastructural Analysis of Platelet Contractile Apparatus. Platelet Receptors: Assays and Purification: J. Hawiger, Repertoire of Platelet Receptors. Receptors for Platelet Agonists: D.E. Macfarlane, 2-Methylthioadenosine [*gb-32P]Diphosphate: Synthesis and Use as Probe of Platelet ADP-Receptors. W.R. Figures, L.M. Scearce, R.F. Colman, and R.W. Colman, Interaction of Nucleotide Affinity Analog 5'p-Fluorosulfonylbenzoyladenosine with Platelet ADP Receptor: Aggregin. E.R. Simons, T.A. Davies, S.M. Greenberg-Sepersky, and N.E. Larsen, Thrombin Receptors on Human Platelets. D.R. Phillips and M.C. Berndt, Platelet Membrane Glycoprotein V Purification. S.E. Domino, M.G. Repaske, C.A. Bonner, M.E. Kennedy, S. Brandon, and L.E. Limbird, Identification, Solubilization, and Affinity Chromatography of Human Platelet ~ga2-Adrenergic Receptors. J.R. Peters, D.P. Geaney, and D.G. Grahame-Smith, 5-[3H]Hydroxytryptamine and [3H]Lysergic Acid Diethylamide Binding to Human Platelets. G. Rudnick and C.J. Humphreys, Platelet Serotonin Transporter. F.H. Valone, Quantitation of Binding of Platelet Activating Factor 1-0-Alkyl-2-acetyl-2-sn-glycero-3-phosphorylcholine to Intact Platelets and Platelet Membranes. Receptors for Adhesive Proteins: J. Hawiger and S. Timmons, Binding of Fibrinogen and von Willebrand Factor to Platelet Glycoprotein Iib-IIIa Complex. D.R. Phillips, L. Fitzgerald, L. Parise, and B. Steiner, Platelet Membrane Glycoprotein IIb-IIIa Complex: Purification, Characterization, and Reconstitution into Phospholipid Vesicles. Z.M. Ruggeri, T.S. Zimmerman, S. Russell, R. Bader, and L. DeMarco, Von Willebrand Factor Binding to Platelet Glycoprotein Ib Complex. A.N. Wicki, J.M. Clemetson, B. Steiner, W. Schnippering, and K.J. Clemetson, Isolation and Characterization of Glycoprotein Ib. J. Loscalzo and R.I. Handin, Platelet Glycocalicin. L.E. Scudder, E.L. Kalomiris, and B.S. Coller, Preparation and Functional Characterization of Monoclonal Antibodies against Glycoprotein Ib. J. Forsyth, E.F. Plow, and M.H. Ginsberg, Fibronectin Binding to Platelets. Receptors for Clotting Factors and Other Ligands: M.E. Nesheim, R.P. Tracy, P.B. Tracy, D.S. Boskovic, and K.G. Mann, Mathematical Simulation of Prothrombinase. P.B. Tracy, M.E. Nesheim, and K.G. Mann, Platelet Factor Xa Receptor. D. Sinha and P.N. Walsh, Binding of Coagulation Factor XIa to a Receptor on Human Platelets. J.S. Greengard and J.H. Griffin, High Molecular Weight Kininogen Receptor. E. Koller and F. Koller, Binding Characteristics of Homologous Plasma Lipoproteins to Human Platelets. A.S. Hajek and J.H. Joist, Platelet Insulin Receptor. General Approaches to Receptor Analysis: J.V. Staros, N.J. Kotite, and L.W. Cunningham, Membrane-Impermeant Cross-Linking Reagents for Structural and Functional Analyses of Platelet Membrane Glycoproteins. D.R. Phillips, Surface Labeling of Platelet Membrane Glycoproteins. B. Adelman, P. Carlson, and R.I. Handin, Evaluation of Platelet Surface Antigens by Fluorescence Flow Cytometry. D.S. Beardsley, Identification of Platelet Membrane Target Antigens for Human Antibodies by Immunoblotting. S. Karpatkin, S. Shulman, and L. Howard, Crossed Immunoelectrophoresis of Human Platelet Membranes. R.M. Albrecht, O.E. Olorundare, S.R. Simmons, J.C. Loftus, and D.F. Mosher, Use of Correlative Microscopy with Colloidal Gold Labeling to Demonstrate Platelet Receptor Distribution and Movement.
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ABSTRACT
Research Interests: Cardiology, Biology, Enzyme Inhibitors, Medicine, Dogs, and 14 moreInternal Medicine, Platelet aggregation, Animals, Oxidoreductases, Clinical Sciences, Myocardial Infarction, Coronary heart disease, Occlusion, Circulation, Coronary Artery Disease, Imidazoles, Ventricular Fibrillation, Blood platelets, and Cardiovascular medicine and haematology
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Hyperlipidemia, the hallmark of Metabolic Syndrome that afflicts millions of people worldwide, exacerbates life-threatening infections. We present a new evidence for the mechanism of hyperlipidemic hypersensitivity to microbial... more
Hyperlipidemia, the hallmark of Metabolic Syndrome that afflicts millions of people worldwide, exacerbates life-threatening infections. We present a new evidence for the mechanism of hyperlipidemic hypersensitivity to microbial inflammation caused by pathogen-derived inducer, LPS. We demonstrate that hyperlipidemic animals succumbed to a non-lethal dose of LPS whereas normolipidemic controls survived. Strikingly, survival of hyperlipidemic animals was restored when the nuclear import of stress-responsive transcription factors (SRTFs), Sterol Regulatory Element-Binding Proteins (SREBPs), and Carbohydrate-Responsive Element-Binding Proteins (ChREBPs) was impeded by targeting the nuclear transport checkpoint with cell-penetrating, biselective nuclear transport modifier (NTM) peptide. Furthermore, the burst of proinflammatory cytokines and chemokines, microvascular endothelial injury in the liver, lungs, heart, and kidneys, and trafficking of inflammatory cells were also suppressed. To ...
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... JACEK HAWIGER, ALEKSANDRA HAWIGER, AND M. GLENN KOENIG~ George Hunter Laboratory, Department of Medicine, Vanderbilt University School o j Medicine NarhviUe, Tennessee 37203 ... Newman + + + + + Newman D,C - Smith diffuse + - - - -... more
... JACEK HAWIGER, ALEKSANDRA HAWIGER, AND M. GLENN KOENIG~ George Hunter Laboratory, Department of Medicine, Vanderbilt University School o j Medicine NarhviUe, Tennessee 37203 ... Newman + + + + + Newman D,C - Smith diffuse + - - - - Zak ...
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<p>(A) Wild type C57BL/6 mice were challenged i.p. with a lethal dose of LPS (800 µg) and treated with i.p. injections of NTM (cSN50.1 peptide) or diluent (saline) following a prophylactic protocol as in <a... more
<p>(A) Wild type C57BL/6 mice were challenged i.p. with a lethal dose of LPS (800 µg) and treated with i.p. injections of NTM (cSN50.1 peptide) or diluent (saline) following a prophylactic protocol as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110183#pone-0110183-g004" target="_blank">Figure 4A</a>. Blood was collected at baseline and 2 or 6 h after LPS challenge and a multiplex assay was used to measure 32 analytes in plasma. Twenty-four analytes were significantly altered by NTM treatment, as determined by repeated measures two-way analysis of variance with Sidak’s post-test. Twenty-three were reduced, while anti-inflammatory IL-10 was increased by NTM treatment. Results are shown as the % inhibition or increase by NTM compared to saline control set to 100% at the time point demonstrating maximal expression for that analyte. <i>n</i> = 10 animals/group. (B) Comparison of prophylactic and therapeutic NTM treatment protocols on selected plasma cytokine and chemokine levels in the high-dose LPS model of endotoxic shock. Data are presented as mean ± standard error, <i>n</i> = 5 −10 animals/group.</p
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There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration... more
There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against…
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The activation of factor X by factor VII/VIIa-tissue factor or factor IXa plays a pivotal role in the hemostatic mechanism. This reaction results in the liberation of a peptide from the zymogen for which we have developed a sensitive and... more
The activation of factor X by factor VII/VIIa-tissue factor or factor IXa plays a pivotal role in the hemostatic mechanism. This reaction results in the liberation of a peptide from the zymogen for which we have developed a sensitive and specific radioimmunoassay (RIA), The native peptide was purified from activated human factor X by hydroxylapatite chromatography and reverse-phase high pressure liquid chromatography (HPLC). Gel filtration experiments demonstrated that the peptide was not physically associated with the enzyme. A 15 amino acid peptide with the COOH-terminal sequence of the activation fragment was synthesized using the solid-phase method of Merrifield. Antisera were raised in rabbits to the synthetic analogue coupled to bovine serum albumin with glutaraldehyde. The antibody population obtained was used to construct a double antibody RIA and was able to measure as little as 0.02 nM of this component. The antibody reactivity toward the factor X zymogen was negligible (l...
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Human von Willebrand factor (vWF) and fibrinogen are adhesive plasma glycoproteins essential for formation of a platelet hemostatic plug. We investigated the role of ADP and fibrinogen in binding of vWF to platelets in vitro. Binding of... more
Human von Willebrand factor (vWF) and fibrinogen are adhesive plasma glycoproteins essential for formation of a platelet hemostatic plug. We investigated the role of ADP and fibrinogen in binding of vWF to platelets in vitro. Binding of 125I-labeled vWF to human platelets separated from plasma proteins and treated with ADP was specific, and time and concentration dependent, reaching equilibrium at 20 min and approaching saturation at 12 micrograms/ml. The binding was inhibited by EDTA and by prostaglandin I2, a known activator of platelet adenylate cyclase. A purine nucleotide affinity analog, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies the ADP binding sites on the human platelet membrane, prevented binding of vWF induced with ADP, as well as with human thrombin and with ionophore A23187, agents known to cause platelet ADP secretion. By comparison, FSBA did not inhibit binding of vWF induced by ristocetin, indicating that the ristocetin mechanism is no...
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We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., &amp; Hawiger, J.... more
We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., &amp; Hawiger, J. (1984) Biochemistry 23, 1767]. To study the correlation between the structure of this segment of the gamma chain and its reactivity toward receptors on ADP-activated human platelets, we designed a series of analogues containing replacements at 9 out of 12 positions. A double substitution of the normal His400-His401 sequence by Ala-Ala reduced the inhibitory potency of the dodecapeptide 3-fold. When Lys406 was replaced by Arg, the inhibitory potency of the dodecapeptide decreased 15 times. On the other hand, substitution of Ala408 with Arg increased the inhibitory potency of the dodecapeptide 6-fold. A drastic decrease in the reactivity of the dodecapeptide toward platelet receptors was observed when Val411 was replaced by leucine or cysteine or tyrosine. A 3-fold decrease in reactivity was noted when Val411 was substituted with phenylalanine. Amidation of the carboxy-terminal Val411 also produced a significant decrease in dodecapeptide reactivity. With seven residues (His400, His401, Leu402, Lys406, Gln407, Asp410, and Val411) preserved, substitution of the intervening five amino acids with nonpolar leucine or polar serine, increasing or decreasing the hydrophobicity of the dodecapeptide, reduced more than 16-fold its inhibitory potency. Rabbit antibody Fab fragments directed against the human fibrinogen gamma-chain peptide encompassing residues 385-411 inhibited 50% of 125I-fibrinogen binding at a 2:1 stoichiometry with regard to 125I-fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Research Interests: Biochemistry, Chemistry, Kinetics, Medicine, Fibrinogen, and 14 moreHumans, Beta decay, Peptides, Blood Coagulation, Dose Response Relationship, Chemical Reaction, Amino Acid Profile, Amino Acid Sequence, Glutamic Acid, Blood cells, Biochemistry and cell biology, Molecular Structure, Molecular Sequence Data, and Medical biochemistry and metabolomics
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Human platelet factor 4 antigen (PF4 antigen) was measured in platelets and in plasma by means of single radial immunodiffusion. Anti-PF4 antibody obtained in rabbits by injecting highly purified human PF4 was monospecific in double... more
Human platelet factor 4 antigen (PF4 antigen) was measured in platelets and in plasma by means of single radial immunodiffusion. Anti-PF4 antibody obtained in rabbits by injecting highly purified human PF4 was monospecific in double immunodiffusion and in quantitative &quot;rocket&quot; immunoelectrophoresis. A high degree of correlation was observed between the precipitation zones in the radial immunodiffusion method and the amount of purified PF4 (in the range of 0.6 to 50.0 mug per milliliter) or the number of platelets in plasma (in the range of 5 x 10(6) to 1.6 x 10(8) platelets per milliliter applied. The sensitivity of the method was 30 to 125 times higher as compared with clotting assay (antiheparin activity) and the standard error of the method was 2.3 per cent. The method was specific for the antigen present in platelets since human leukocytes and erythrocytes gave negative results. Release of PF4 antigen from washed platelets challenged with thrombin, collagen, ADP, and antigen-antibody complexes was measured by the radial immunodiffusion assay. It usually paralleled the release of 3H-serotonin but PF4 antigen was a more sensitive marker for platelet release reaction. Release of PF4 antigen was usually 2 to 4 times higher than release of the antiheparin activity as measured by clotting assay when both were compared as percentage of total content in platelets. The level of PF4 antigen was determined in platelet-rich plasma (PRP) and platelet-free plasma (PFP) obtained from 12 healthy volunteers. While the mean level of extraplatelet pool of PF4 antigen in PFP was 0.72 +/- 0.92 mug per milliliter, PRP contained 80 +/- 22 mug of PF4 antigen per 10(9) platelets. Addition of thrombin (1 U. per milliliter) liberated all of the PF4 antigen (78 +/- 24 mug) present in PRP but ADP (50 muM) released only 31 +/- 22 mug of PF4 antigen per 10(9) platelets. The presence of heparin did not interfere with the assay of intraplatelet or extraplatelet PF4 by single radial immunodiffusion. The method described represents a simple, sensitive, quantitative, and specific assay for human PF4 antigen possessing antiheparin activity.
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Invariant natural killer T (iNKT) cell-derived cytokines have important functions in inflammation, host defense, and immunoregulation. Yet, when and how iNKT cells undergo effector differentiation, which endows them with the capacity to... more
Invariant natural killer T (iNKT) cell-derived cytokines have important functions in inflammation, host defense, and immunoregulation. Yet, when and how iNKT cells undergo effector differentiation, which endows them with the capacity to rapidly secrete cytokines upon activation, remains unknown. We discovered that granulocyte-macrophage colony-stimulating factor (Csf-2)-deficient mice developed iNKT cells that failed to respond to the model antigen alpha-galactosylceramide because of an intrinsic defect in the fusion of secretory vesicles with the plasma membrane. Exogenous Csf-2 corrected the functional defect only when supplied during the development of thymic, but not mature, splenic Csf-2-deficient iNKT cells. Thus, we ascribe a unique function to Csf-2, which regulates iNKT cell effector differentiation during development by a mechanism that renders them competent for cytokine secretion.
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ABSTRACT
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Research Interests:
Research Interests:
Stimulation of macrophages with lipopolysaccharide (LPS) leads to the production of cytokines that elicit massive liver apoptosis. We investigated the in vivo role of stress-responsive transcription factors (SRTFs) in this process... more
Stimulation of macrophages with lipopolysaccharide (LPS) leads to the production of cytokines that elicit massive liver apoptosis. We investigated the in vivo role of stress-responsive transcription factors (SRTFs) in this process focusing on the precipitating events that are sensitive to a ...