scholarly journals Cathepsin G binding to human platelets. Evidence for a specific receptor

1990 ◽  
Vol 266 (1) ◽  
pp. 55-62 ◽  
Author(s):  
M A Selak ◽  
J B Smith
Keyword(s):  
Binding Sites ◽  
Room Temperature ◽  
Cell Activation ◽  
Human Platelets ◽  
Cathepsin G ◽  
Native Enzyme ◽  
Hill Coefficient ◽  
Specific Receptor ◽  
Binding Studies ◽  
Binding Curves

We have shown previously that purified human neutrophil cathepsin G is a strong platelet agonist. We now demonstrate that cathepsin G exhibits saturable, reversible binding to human platelets which is characteristic of binding to a specific receptor. At room temperature, cathepsin G displayed apparent positive co-operativity of binding, as indicated by sigmoidal binding curves and a Hill coefficient greater than unity. By contrast, binding curves conducted with native enzyme at 0.5 degrees C displayed a much smaller degree of sigmoidicity, and binding studies performed with phenylmethanesulphonyl fluoride-treated enzyme at 22-25 degrees C exhibited hyperbolic binding curves. The concentrations of cathepsin G required to give half-saturation (S0.5) with inhibitor-treated enzyme or with native enzyme at either room temperature or 0.5 degrees C were all similar, suggesting that sigmoidal binding curves did not result from an alteration in the affinity of the binding sites for cathepsin G. However, platelets bound approximately twice as many molecules of native enzyme as molecules of phenylmethanesulphonyl fluoride-treated cathepsin G per cell. From these observations it can be inferred that the apparent positive co-operativity may in part reflect the exposure of binding sites due to the proteolytic activity of cathepsin G. However, this conclusion is not supported by experiments conducted with subsaturating cathepsin G concentrations, which demonstrated that ligand binding did not show an expected increase at longer time intervals. Measurement of Ca2+ mobilization and cathepsin G binding in the same platelet suspensions demonstrated that elevations in cytosolic free Ca2+ concentration had achieved near-maximal levels in the presence of 15 micrograms of cathepsin G/ml, whereas maximal binding was observed at approx. 35 micrograms/ml, indicating that only a fraction of the total binding sites need be occupied to elicit platelet activation. Pretreatment of platelets with forskolin or phorbol 12-myristate 13-acetate (PMA) decreased cathepsin G binding by approx. 60% and 40% respectively, indicating that the receptor may be desensitized or down-regulated by phosphorylation due to protein kinases. Since forskolin and PMA could diminish receptor availability by activating negative feedback mechanisms, inhibition of negative signal-transduction pathways could conversely play a role in the up-regulation of cathepsin G binding. In any event, these results show that cathepsin G is an agonist that must bind to platelets to initiate processes associated with cell activation, and suggest a role for cathepsin G in platelet function.

scholarly journals Internalization and down-regulation of the prostacyclin receptor in human platelets

1997 ◽  
Vol 325 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Serenella GIOVANAZZI ◽  
Maria R. ACCOMAZZO ◽  
Ornella LETARI ◽  
Daniela OLIVA ◽  
Simonetta NICOSIA
Keyword(s):  
Binding Sites ◽  
Organic Anion ◽  
Subcellular Fractionation ◽  
Human Platelets ◽  
Organic Anion Transporters ◽  
Camp Production ◽  
Binding Studies ◽  
Anion Transporters ◽  
Prostacyclin Receptor ◽  
High Affinity Binding Site

The internalization of [3H]iloprost, a prostacyclin analogue, was studied in human platelets by binding studies. After incubation with [3H]iloprost at 37 °C, addition of unlabelled ligand at either 37 °C or 4 °C caused dissociation of 74% and 52% of the bound ligand respectively, suggesting that a portion had been internalized. The percentage of [3H]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 °C or 4 °C was markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting agents (dinitrophenol and 2-deoxyglucose) completely inhibited internalization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated that, at 4 °C or in the absence of ligand, most of the receptor was present in membrane fractions (pellet at 27000 or 105000 g), whereas, when platelets were preincubated at 37 °C with iloprost, the receptor was found mainly in the cytosolic fraction. In platelets preincubated with iloprost at 4 °C, two classes of binding sites were present, whereas after preincubation at 37 °C only the lower-affinity sites were detected. After exposure to the agonist, iloprost-induced inhibition of platelet aggregation and activation of adenylate cyclase and cAMP production were significantly lower. Taken together, these data demonstrate that human platelets can internalize a high-affinity binding site for iloprost, presumably the prostacyclin receptor.

Down-regulation of neutrophil functions by the ELR+CXC chemokine platelet basic protein

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 2965-2972 ◽  
Author(s):  
Jan E. Ehlert ◽  
Andreas Ludwig ◽  
Tobias A. Grimm ◽  
Buko Lindner ◽  
Hans-Dieter Flad ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Chemokine Receptors ◽  
Cell Activation ◽  
Basic Protein ◽  
Cathepsin G ◽  
Terminal Part ◽  
Down Regulation ◽  
Cxc Chemokine ◽  
Continuous Accumulation

Abstract The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR+ CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2– but not CXCR-1– dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

Down-regulation of neutrophil functions by the ELR+CXC chemokine platelet basic protein

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 2965-2972 ◽  
Author(s):  
Jan E. Ehlert ◽  
Andreas Ludwig ◽  
Tobias A. Grimm ◽  
Buko Lindner ◽  
Hans-Dieter Flad ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Chemokine Receptors ◽  
Cell Activation ◽  
Basic Protein ◽  
Cathepsin G ◽  
Terminal Part ◽  
Down Regulation ◽  
Cxc Chemokine ◽  
Continuous Accumulation

The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR+ CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2– but not CXCR-1– dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

scholarly journals Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets

1984 ◽  
Vol 223 (3) ◽  
pp. 901-909 ◽  
Author(s):  
E Kloprogge ◽  
J W N Akkerman
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Human Platelets ◽  
Equilibrium Analysis ◽  
Binding Studies ◽  
Prolonged Incubation ◽  
Fibrinogen Receptor ◽  
Receptor Aggregation ◽  
Specific Receptors ◽  
High Doses

The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82×10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87×10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.

Interaction Of Tritiated-Quinine With Human Platelets In The Presence Of A Quinine-Dependent Antibody

1981 ◽  
Author(s):  
D J Christie ◽  
R H Aster
Keyword(s):  
Binding Sites ◽  
Room Temperature ◽  
Hydrogen Exchange ◽  
Human Platelets ◽  
Drug Binding ◽  
Drug Induced ◽  
Catalytic Hydrogen ◽  
Normal Human ◽  
Membrane Bound ◽  
Drug Binding Sites

In drug-induced immune thrombocytopenia, it has not been established with certainty whether drug binds first to the platelet membrane or to antibody to promote platelet-antibody binding. The association of the drug, quinine, and a quinine dependent antibody (obtained from a patient with drug-induced thrombocytopenia) with human platelets was studied using a tritiated derivative of quinine prepared by catalytic hydrogen exchange. Tritiated quinine binding to platelets was measured in the presence of the antibody or normal human serum (NHS). Platelets were stirred with the appropriate reagents at room temperature for 20 minutes and washed. Bound tritiated quinine was measured by scintillation techniques.Two types of drug binding sites, antibody-dependent and antibody-independent, were demonstrated on platelets. In the presence of NHS, fewer than 20,000 molecules of drug became tightly bound to the membrane. In the presence of excess antibody, approximately 200,000 molecules of quinine became membrane-bound. Antibody did not bind to platelets in the absence of drug. These findings appear to provide the first demonstration of antibody-dependent binding of quinine to platelets and suggest that interaction of drug and antibody in the soluble phase precedes the binding of either constituent to the platelets (“innocent bystander mechanism”). The use of radioactive drugs appears to provide a useful tool for the investigation of the molecular basis of drug- induced immune disorders.

scholarly journals Platelet-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms

1986 ◽  
Vol 240 (2) ◽  
pp. 403-412 ◽  
Author(s):  
E Kloprogge ◽  
J W Akkerman
Keyword(s):  
Platelet Activation ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
A Minor

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [(3H]arachidonate mobilization and thromboxane synthesis) and secretion [(14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.

Neutrophil Proteases Can Inactivate Human PAR3 and Abolish the Co-receptor Function of PAR3 on Murine Platelets

2001 ◽  
Vol 85 (03) ◽  
pp. 533-538 ◽  
Author(s):  
Albana Cumashi ◽  
Helenia Ansuini ◽  
Nicola Celli ◽  
Antonio De Blasi ◽  
Peter O’Brien ◽  
...  
Keyword(s):  
Binding Sites ◽  
Family Members ◽  
Human Platelets ◽  
Cathepsin G ◽  
Model Systems ◽  
Cos Cells ◽  
N Terminus ◽  
Tethered Ligand ◽  
Species Specific ◽  
Receptor Family

SummaryThree members of the protease-activated receptor family, PAR1, PAR3 and PAR4, are activated when thrombin cleaves the receptor N-terminus, exposing a tethered ligand. Proteases other than thrombin can also cleave PAR family members and, depending upon whether this exposes or removes the tethered ligand, either activate or disable the receptor. For example, on human platelets PAR1 is disabled by cathepsin G, although aggregation still occurs because cathepsin G can activate PAR4. The present studies examine the interaction of cathepsin G and a second neutrophil protease, elastase, with PAR3 using two model systems: COS-7 cells transfected with human PAR3 and mouse platelets, which express PAR3 and PAR4, but not PAR1. In contrast to human platelets, cathepsin G did not aggregate murine platelets, and prevented their activation only at low thrombin concentrations. Elastase had no effect on thrombin responses in mouse platelets, but when added to COS cells expressing human PAR3, both cathepsin G and elastase prevented activation of phospholipase C by thrombin. Notably, this inhibition occurred without loss of the binding sites for two monoclonal antibodies that flank the tethered ligand on human PAR3. We therefore conclude that 1) exposure to cathepsin G disables signaling through human PAR3, and prevents murine PAR3 from serving its normal role, which is to facilitate PAR4 cleavage at low thrombin concentrations, 2) elastase disables human, but not murine, PAR3, 3) in contrast to human PAR4, mouse PAR4 will not support platelet aggregation in response to cathepsin G, and 4) the inactivation of human PAR3 by cathepsin G and elastase involves a mechanism other than amputation of the tethered ligand domain. These results extend the range of possible interactions between PAR family members and proteases, and provide further support for species-specific differences in the interaction of these receptors with proteases other than thrombin.

scholarly journals Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide–adenine dinucleotide-binding studies

1978 ◽  
Vol 169 (3) ◽  
pp. 577-588 ◽  
Author(s):  
Alberto Lodola ◽  
S. Peter Spragg ◽  
J. John Holbrook
Keyword(s):  
Malate Dehydrogenase ◽  
Binding Sites ◽  
High Yield ◽  
British Library ◽  
Enzyme Molecule ◽  
Binding Studies ◽  
Fructose 1,6 Bisphosphate ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Binding Curves

1. Two methods of preparing pig heart soluble malate dehydrogenase are described. A slow method yields an enzyme composed of three electrophoretically separable subforms. The more rapid method reproducibly gives a high yield of an enzyme that consists predominantly of the least acid subform. 2. The A1%1cm of the protein was redetermined as 15 at 280nm. By using this value the enzyme molecule was found to contain two independent and indistinguishable NADH-binding sites in titrations with NADH. 3. No evidence was found for the dissociation of the enzyme in the concentration range 0.02–7.2μm. 4. l-Malate (0.1m) tightened the binding of NADH to both pig and ox heart enzyme (2-fold), but, in contrast with the report by Mueggler, Dahlquist & Wolfe [(1975) Biochemistry14, 3490–3497], did not cause co-operative interactions between the binding sites. 5. Fructose 1,6-bisphosphate had no effect on the binding of NADH to the pig heart enzyme, but with the ox heart enzyme the NADH is slowly oxidized. This slow oxidation explains the ‘sigmoidal’ binding curves obtained when NADH was added to ox heart soluble malate dehydrogenase in the presence of fructose 1,6-bisphosphate [Cassman (1973) Biochem. Biophys. Res. Commun.53, 666–672] without the postulate of site–site interactions. 6. It is concluded that neither l-malate nor fructose 1,6-bisphosphate could in vivo modulate the activity of soluble malate dehydrogenase and alter the rates of transport of NADH between the cytosol and the mitochondrion. 7. Details of the preparation of soluble malate dehydrogenase have been deposited as Supplementary Publication SUP 50080 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1978) 169, 5.

scholarly journals Monoclonal antibody studies of creatine kinase. Antibody-binding sites in the N-terminal region of creatine kinase and effects of antibody on enzyme refolding

1987 ◽  
Vol 248 (1) ◽  
pp. 53-59 ◽  
Author(s):  
G E Morris ◽  
L C Frost ◽  
P A Newport ◽  
N Hudson
Keyword(s):  
Creatine Kinase ◽  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Binding Sites ◽  
Terminal Region ◽  
Native Enzyme ◽  
Single Amino Acid ◽  
Binding Studies ◽  
Simple Method ◽  
Variable Regions

(1) The binding sites of two monoclonal antibodies, CK-2A7 and CK-5H5, have been located to a 60-amino-acid sequence in the N-terminal region of creatine kinase (CK) by the use of chemical cleavage with formic acid (which cleaves proteins at Asp-Pro bonds) and cyanogen bromide (which cleaves at Met residues). (2) A simple method for preparing chemically-cleaved fragments of proteins for electrophoresis and Western blotting is described. (3) Binding studies with CK preparations from different animal species show that single amino acid changes at residues 39 or 82 prevent binding of CK-2A7 and CK-5H5 respectively. We suggest that Lys-39 and Glu-82 form parts of the binding sites on CK for the two monoclonal antibodies. The two sites lie in variable regions at each end of a highly-conserved sequence (residues 46 to 79) and are inaccessible to antibody in the native enzyme. (4) One of the antibodies, CK-2A7, inhibits the refolding of CK to native enzyme after denaturation by urea.

Detection Of High Affinity Fibrinogen Binding Sites On Human Platelets Stimulated By ADP: Comparison Of Two Methods Of Platelet Separation

1981 ◽  
Author(s):  
Stefan Niewiarowski ◽  
Thomas A Morinelli ◽  
Elizabeth Kornecki
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Gel Filtration ◽  
Human Platelets ◽  
Binding Studies ◽  
Test Analysis ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
Specific Receptors

Binding of fibrinogen to specific receptors on human platelets exposed by ADP results in platelet aggregation. There are controversial data regarding classes and number of fibrinogen receptors, the values range from one to two classes and 1,000-80,000 receptors per platelet as reported in the literature. We have studied the interaction of fibrinogen with a) platelets washed by differential centrifugation according to Mustard and colleagues (washed platelets - WP) and with b) gel-filtered platelets (GFP). Platelet aggregation was studied with 100 μM ADP and with various concentration of fibrinogen. Maximal velocities of aggregation for WP and GFP were 81 and 47 units per min, respectively, and the Km values for fibrinogen calculated from the rate of aggregation were 0.9 × 10-7M for WP and 5.8 × 10-7M for GFP. The level of platelet fibrinogen released into the suspension from WP and GFP amounted to 2.4 μg and 15.0 μg per 10 9 platelets/ml, respectively, as measured by the staphylococcal clumping test. Analysis of 125I-fibrinogen binding data by the method of Scatchard and Feldman revealed 1,300 high affinity receptors (KD 3.2 × 10-8M) and 80,000 low affinity receptors (KD 5.6 × 10-5M) for WP. The binding of 125I-fibrinogen to GFP was greatly diminished. The number of fibrinogen receptors exposed by ADP on GFP and their binding affinity are under investigation in our laboratory. In conclusion, GFP were less sensitive to fibrinogen than were WP as shown in the aggregation and 125I-fibrinogen binding studies. It appears that the method of platelet separation is critical for the assessment of fibrinogen binding. Platelet activation and release of intact platelet fibrinogen during gel-filtration may interfere with the detection of high affinity fibrinogen binding sites.

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