CHARACTERIZATION OF A NEUTROPHIL CPYMOTRYPSIN-LIKE ENZYME THAT ACTIVATES PLATELETS

1987 ◽  
Author(s):  
M A Selak ◽  
M Chignard ◽  
J B Smith
Keyword(s):  
Platelet Activation ◽  
Cell Types ◽  
Cross Reactivity ◽  
Serotonin Release ◽  
Free Calcium ◽  
Cation Exchange Resins ◽  
Specific Stimulation ◽  
Exchange Resins ◽  
Stimulation Of

Communication between neutrophils and platelets was previously investigated by measuring platelet aggregation, serotonin release and changes in cytosolic free calcium subsequent to specific stimulation of neutrophils by fMet-Leu-Phe (FMLP) in a suspension of both cell types. The addition of the chemotactic peptide was shown to elicit secondary platelet activation as a consequence of primary stimulation of neutrophils. Cell-free supernatants from FMLP-stimulated neutrophils were capable of inducing platelet activation thus demonstrating that a factor released bv neutrophils was responsible for the observed platelet responses. After eliminating classical platelet agonists as the acitive agent, it was shown that an enzyme termed neutroohilin induced platelet calcium mobilization, secretion and aggregation. The current studies were conducted to characterize the mediator released bv neutrophils. Neutrophilin bound bo cation exchange resins but failed to bind to anion exchangers. The biological activity associated with neutroohilin was unaffected by leupeptin, only very weakly diminished by N-bosyl-Lvs-chloromethvl ketone and was strongly inhibited by N-tosvl-Phe-chloromethvl ketone, aloha-l-antitrvpsin, soybean trypsin inhibitor and Z-Glv-Leu-Phe-chloromethvl ketone. Neutroohilin was released from stimulated neutrophils only after cytochalasin B treatment, as was beta-glucuronidase, suggesting that both enzymes are located in azurophilic granules. Neutroohilin-induced platelet activation was inhibited bv antiserum to human catheosin G in a dose-deoendent manner but was unaffected by antiserum to human elastase or alpha-fetoprotein. The inhibitor sensitivity, immunological cross-reactivity, ionic properties and probable subcellular localization indicate that neutrophilin is a cationic chymotrvosin-like enzyme related, if not identical to, catheosin G. Neutroohilin-induced platelet activation could explain different pathological events in which platelets and neutroohils are known to be involved.

Synthesis and characterization of 19F NMR chelators for measurement of cytosolic free Ca

1987 ◽  
Vol 252 (4) ◽  
pp. C441-C449 ◽  
Author(s):  
L. A. Levy ◽  
E. Murphy ◽  
R. E. London
Keyword(s):  
Chemical Shifts ◽  
Cell Types ◽  
Free Calcium ◽  
Tetraacetic Acid ◽  
Nmr Studies ◽  
Cytosolic Free Calcium ◽  
Fluorine Nmr ◽  
Dissociation Constant Kd

Fluorine 19 nuclear magnetic resonance (NMR) studies of intracellular fluorinated calcium chelators provide a useful strategy for the determination of cytosolic free calcium levels in cells and perfused organs. However, the fluorinated chelator with the highest affinity for calcium ions which has been described to date. 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), exhibits a dissociation constant (Kd) value 5- to 10-fold greater than the intracellular calcium concentration levels in most cell types, thus limiting the ability of fluorine NMR to report these concentrations reliably. We have consequently designed and synthesized several fluorinated calcium chelators with higher affinity for calcium. The best of these, 2-(2-amino-4-methyl-5-fluorophenoxy)-methyl-8 aminoquinidine-N,N,N',N'-tetraacetic acid (quinMF), has a Kd value approximately 10 times lower than that of 5FBAPTA. Several of the newly synthesized indicators have different chemical shifts for the calcium complexed and uncomplexed chelators to allow the simultaneous use of two indicators. In addition to providing information about the level of cytosolic free calcium, chelators containing a quinoline ring exhibit considerable sensitivity to magnesium levels and hence have potential application for the determination of cytosolic-magnesium concentrations. Application of these chelators is illustrated by determination of the cytosolic-free calcium level in erythrocytes. Use of quinMF, the chelator with the lowest Kd value, gives a calcium value of 25-30 nM.

scholarly journals Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 547-555 ◽  
Author(s):  
S De Reys ◽  
MF Hoylaerts ◽  
M De Ley ◽  
J Vermylen ◽  
H Deckmyn
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Platelet Activation ◽  
Cell Types ◽  
Serotonin Release ◽  
Platelet Membrane ◽  
Cross Linking ◽  
Antiplatelet Antibody ◽  
Affinity Isolation ◽  
Isoelectric Ph

Abstract A monoclonal antiplatelet antibody (MA-13G8E1) is described that dose- dependently induces platelet aggregation and serotonin release in an Fc- independent fashion. Whereas platelets were equally aggregated by F(ab')2 fragments of this monoclonal antibody (MoAb), its Fab fragments, on the other hand, were inactive, indicating that divalent interaction is an essential requirement to induce platelet activation by MA-13G8E1. In addition, we could show that platelet epitope cross- linking by MA-13G8E1 occurred on the same platelet. MA-13G8E1 stimulated platelet phospholipase C (PLC) and induced activation of protein kinase C (PKC), both of which were almost unaffected by aspirin pretreatment. Furthermore, PLC activation appeared to be a direct antibody-mediated effect, since intracellular Ca2+ rises were not inhibited by EGTA, cytochalasin B, or aggregation-blocking MA-16N7C2 (antiglycoprotein [anti-GP]IIb/IIa). The MA-13G8E1 antigen is constitutively expressed on resting platelets of different species (7,100 +/- 800 molecules per human platelet), but not on other cell types tested. Both immunoprecipitation and affinity isolation by MA- 13G8E1 showed two low-molecular weight proteins (45 and 36 kD), having slightly acidic isoelectric pH levels (4.5 to 5.5) and forming multimolecular complexes. In conclusion, we found an MoAb that is able to induce platelet activation in an Fc-independent fashion. The mechanism involves cross-linking of a hitherto undescribed platelet membrane protein, leading to PLC and PKC stimulation.

Further Characterization of Human Platelet Activation in the Absence of Aggregation: Phosphorylations of Specific Proteins and Relationship with Platelet Secretion

1984 ◽  
Vol 51 (02) ◽  
pp. 198-203 ◽  
Author(s):  
Danièle Nunez ◽  
Sylviane Levy-Toledano
Keyword(s):  
Platelet Activation ◽  
Serotonin Release ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Release Reaction ◽  
Specific Proteins ◽  
Relationship Of ◽  
The Relationship ◽  
Platelet Secretion

SummaryPlatelet aggregation and secretion have been described to be associated with phosphorylation reactions. Thrombasthénie and EDTA-treated control platelets undergo a normal serotonin release in the absence of aggregation. We now studied the phosphorylation of specific proteins associated with platelet secretion. In the presence of ionophore, significant increases occurred in the phosphorylation of two polypeptides of 43,000 and 20,000 molecular weight (P43 and P20) in a concentration dependent manner, and this was accompanied by an increase in the 14C-5HT release. The 32P-labelling of P43 and P20 reaches a peak within 1 min of platelet activation and is followed by a rapid dephosphorylation over the next 2-10 min. While the P20 is identified as the myosin light chain, the identity and the function of the P43 remain unknown.Isoelectric focusing separates 4 proteins from P43 during two dimensional electrophoresis, but only one of them is phosphorylated by A 23187. Chlorpromazine (CPZ) and trifluoperazine (TFP) inhibit the P43 and P20 phosphorylation as well as the 14C- 5HT release in a dose dependent manner. The inhibitory action of the drugs is more pronounced for P43 than for P20, especially when the reactions are carried out at 20° C instead of at 37° C, while the release reaction is still inhibited under these conditions. These results allow different hypotheses for the relationship of phosphorylation-secretion and indicate the importance of one of these proteins (P43) for the release reaction.

Thrombin stimulation of human platelets:Phosphoinositides as the only source of the diacylglycerol moiety in phosphatidic acid

1987 ◽  
Author(s):  
O-B Tysnes ◽  
AJ M Verhoeven ◽  
H Holmsen
Keyword(s):  
Phosphatidic Acid ◽  
Platelet Activation ◽  
Cell Types ◽  
Human Platelets ◽  
Stimulus Response ◽  
Low Concentrations ◽  
Higher Doses ◽  
Stimulation Of

Recent reports on various cell types including platelets have concluded in the heterogenous production of phosphatidic acid (PA) during the stimulus-response coupling. Human platelets were pulse-labelled simultaneously with [32P] P. and [32H]glycerol. Extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. When the cells were stimulated with low concentrations of thrombin, the production of [32P] PA was evident without measurable production of [3H] PA. At higher doses of the agonist,[3H] PA was formed, but distinctly later than [32P] PA.This suggested a heterogenous production of PA upon thrombin stimulation of platelets. The specific H-radioactivity of PA in unstimulated cells was about 50% of that of the phosphoinositides. Upon l80 sec of stimulation with 0.5 U/ml of thrombin, the specific [3H] PA radioactivity increased to the level of the phosphoinositides which remained constant during platelet activation. Since other phospholipids incorporate [3H] glycerol much slower than the phosphoinositides, these latter remain the only possible source of the diacylglycerol moiety of PA. The specific 32P-radioactivity of PA in unstimulated cells was only 4% of that of α-ATP and similar to the specific 32P-radioactivity of phosphatidylinositol in unstimulated platelets. After Jyijin of stimulation with 0.5 U/ml of thrombin, specific [32P] PA was similar to that of γ-ATP. The descrepancy in [32P] Pi. and [3H] glycerol incorporation into PA upon thrombin stimulation of platelets is therefore mainly due to a thrombin-induced shift inspecific 32P-radioactivity in PA.

scholarly journals Desensitization and antagonism of vasopressin-induced phosphoinositide metabolism and elevation of cytosolic free calcium concentration in human platelets

1986 ◽  
Vol 234 (1) ◽  
pp. 67-73 ◽  
Author(s):  
W K Pollock ◽  
D E MacIntyre
Keyword(s):  
Platelet Activation ◽  
Human Platelet ◽  
Receptor Occupancy ◽  
Human Platelets ◽  
Free Calcium ◽  
Phosphoinositide Metabolism ◽  
Subsequent Challenge ◽  
Free Calcium Concentration ◽  
20 Nm ◽  
Stimulation Of

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.

scholarly journals Fc-independent cross-linking of a novel platelet membrane protein by a monoclonal antibody causes platelet activation

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 547-555
Author(s):  
S De Reys ◽  
MF Hoylaerts ◽  
M De Ley ◽  
J Vermylen ◽  
H Deckmyn
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Platelet Activation ◽  
Cell Types ◽  
Serotonin Release ◽  
Platelet Membrane ◽  
Cross Linking ◽  
Antiplatelet Antibody ◽  
Affinity Isolation ◽  
Isoelectric Ph

A monoclonal antiplatelet antibody (MA-13G8E1) is described that dose- dependently induces platelet aggregation and serotonin release in an Fc- independent fashion. Whereas platelets were equally aggregated by F(ab')2 fragments of this monoclonal antibody (MoAb), its Fab fragments, on the other hand, were inactive, indicating that divalent interaction is an essential requirement to induce platelet activation by MA-13G8E1. In addition, we could show that platelet epitope cross- linking by MA-13G8E1 occurred on the same platelet. MA-13G8E1 stimulated platelet phospholipase C (PLC) and induced activation of protein kinase C (PKC), both of which were almost unaffected by aspirin pretreatment. Furthermore, PLC activation appeared to be a direct antibody-mediated effect, since intracellular Ca2+ rises were not inhibited by EGTA, cytochalasin B, or aggregation-blocking MA-16N7C2 (antiglycoprotein [anti-GP]IIb/IIa). The MA-13G8E1 antigen is constitutively expressed on resting platelets of different species (7,100 +/- 800 molecules per human platelet), but not on other cell types tested. Both immunoprecipitation and affinity isolation by MA- 13G8E1 showed two low-molecular weight proteins (45 and 36 kD), having slightly acidic isoelectric pH levels (4.5 to 5.5) and forming multimolecular complexes. In conclusion, we found an MoAb that is able to induce platelet activation in an Fc-independent fashion. The mechanism involves cross-linking of a hitherto undescribed platelet membrane protein, leading to PLC and PKC stimulation.

scholarly journals Isolation and characterization of a factor from calf serum that promotes the pigmentation of embryonic and transformed melanocytes.

1985 ◽  
Vol 100 (5) ◽  
pp. 1493-1498 ◽  
Author(s):  
J A Jerdan ◽  
H H Varner ◽  
J H Greenberg ◽  
V J Horn ◽  
G R Martin
Keyword(s):  
Molecular Sieve ◽  
Cultured Cells ◽  
Calf Serum ◽  
Submaxillary Gland ◽  
Cross Reactivity ◽  
Serum Enzyme ◽  
Melanin Biosynthesis ◽  
Isolation And Characterization ◽  
Stimulation Of

A protein (Mr = 63,000) from calf serum that promotes the pigmentation of cultured chick neural crest and mouse melanoma cells has been partially isolated and characterized in this study. The stimulation of melanin synthesis in cultured cells was used to follow its activity during purification. The pigment-promoting factor was isolated by sequential column chromatography on dye-agarose matrices followed by hydroxyapatite and high pressure molecular sieve chromatography. The factor was found to stimulate melanin biosynthesis at 2-4 micrograms/ml and was specific for melanin-producing cells and their precursors. Antibodies raised in rabbits against the factor inhibited its pigment-promoting activity as well as that of whole calf serum. Enzyme-linked immunoadsorbent assays demonstrated that calf and bovine sera contain molecules that cross-react with the pigment-promoting factor. Horse, human, rat, and chicken sera, which lack the biological activity, also lacked immunological cross-reactivity. Extracts of certain tissues, particularly the submaxillary gland, were observed to be rich sources of pigment-promoting activity.

Novel Single Agents with Combined Anti-Coagulant and Anti-Platelet Activities: Potential Treatment Option for the Management of HIT.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1884-1884
Author(s):  
Jeanine M. Walenga ◽  
Margaret Prechel ◽  
Walter P. Jeske ◽  
Meredith K. McDonald ◽  
James M. Daikur ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Treatment Options ◽  
Clinical Trial Data ◽  
Therapeutic Agents ◽  
Cross Reactivity ◽  
Serotonin Release ◽  
Thrombin Inhibitor ◽  
The Novel ◽  
Anti Platelet Drug ◽  
Fxa Inhibitor

Abstract A recently introduced series of antithrombotic agents brings the novel characteristic of dual anti-coagulant and anti-platelet actions in one molecule. These low molecular weight, synthetic, serine protease inhibitors, depending on structural modifications, have variable ratios of both anti-thrombin and anti-platelet activities. Studies have shown that these agents produce stronger antithrombotic actions relative to single targeted therapeutic agents (O. Iqbal #P0521 and D. Hoppensteadt #OR335, ISTH meeting Sydney, Australia August 2005). Heparin-induced thrombocytopenia (HIT) is an adverse effect of heparin in which both thrombin generation and platelet activation augment hypercoagulable and inflammatory states leading to a high probability of developing severe thrombosis. Current guidelines for patients who have HIT recommend the use of a direct thrombin inhibitor (DTI) to prevent or treat associated thrombosis. Clinical trial data, as well as practice outcomes, show that DTI treatment alone is not sufficient to overcome the pathology and resultant thrombosis in all HIT patients. Thus, more optimal treatment options are needed. A focus of treatment on inhibiting both platelet and coagulation activation is logical based on the pathophysiology of HIT. This study was undertaken to determine if the novel CanAm series of agents may have a role in the management of patients with HIT. Eight agents (MC45301, MC45308, CA207, CA216, CA234, CA247, CA250, CA254) with varying ratios of anticoagulant/anti-platelet activities were studied using the 14C-serotonin release assay (SRA) and flow cytometry for the detection of platelet P-selectin expression and platelet microparticle generation. The DTI argatroban, the FXa inhibitor fondaparinux, and the platelet GPIIb/IIIa receptor antagonist eptifibatide were included for comparison. Both cross-reactivity to HIT antibodies and amelioration of HIT antibody-induced platelet activation were assessed. In the absence of heparin, at 1-100 μg/ml none of the CanAm agents caused platelet activation in the presence of serum from patients with HIT (n=12) ruling out cross-reactivity with HIT antibodies. None of the comparator drugs showed cross reactivity with HIT antibodies. In the presence of heparin (0.1 U/ml) and serum from patients with HIT (n=12), the CanAm agents were able to inhibit all platelet activation responses at concentrations of 10–100 μg/ml. In comparison, the DTI and FXa inhibitor were not able to inhibit the HIT antibody/heparin induced platelet activation with any HIT serum. The GPIIb/IIIa inhibitor, however, showed a concentration-dependent inhibition of the platelet activities with complete blockade at 1 μg/ml, suggesting the importance of platelet activation inhibition for the treatment of HIT. Thus, compared to mono-therapeutic agents such as DTIs and fondaparinux, the dual-acting CanAm agents not only lack cross reactivity with HIT antibodies, but they have the added ability to block the antibody-induced platelet activation that occurs during an acute episode of HIT. A dual-acting anticoagulant/anti-platelet drug may, therefore, be of more value than single targeted anti-thrombin drugs for the management of HIT and associated thrombosis.

Abstract 523: Expression, Activation and Function of Integrin α M β 2 on Neutrophil-derived Microparticles

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Neil M Woody ◽  
Dorota Szpak ◽  
Edward F Plow ◽  
Elzbieta Pluskota
Keyword(s):  
Cardiovascular Diseases ◽  
Platelet Activation ◽  
Cell Types ◽  
Time Dependent ◽  
Human Neutrophils ◽  
Active Conformation ◽  
Adhesion Receptor ◽  
And Function ◽  
Stimulation Of

The levels of circulating leukocyte-derived microparticles (MPs) are elevated in thrombosis and most cardiovascular diseases, and it has been suggested that MPs are not only markers of disease but also active contributors to pathogenesis via their interaction with various cell types. Such interactions will depend on the presence and activity of molecules expressed on the MP surface. In our study we have considered the presence and activation state of a major adhesion receptor on the surface of leukocytes, integrin α M β 2 (CD11b/18, Mac-1) on MPs derived from human neutrophils (PMN). MPs were obtained from resting or agonist-stimulated PMNs, and the presence of α M β 2 was assessed by FACS. α M β 2 expression was significantly enhanced on MPs derived from stimulated PMN (e.g., resting MPs: MFI = 3.5 ± 0.7 vs PMA-MPs: MFI = 20 ± 4, and fLMP-MPs: MFI = 24 ± 5). PSGL-1 was expressed by MPs but levels were not affected by stimulation. Of note, α M β 2 expressed on MPs obtained from stimulated PMNs was in active conformation as assessed using mAb (CBRM1/5) to activation-dependent epitope of this integrin. Recognition of a protein ligand, fibrinogen, by activated α M β 2 on the MPs was also demonstrable. With evidence of a functionally active integrin, we sought to evaluate its role in mediating MP interaction with platelets. MP binding to isolated, resting platelets was dose and time dependent. However, MPs expressing activated α M β 2 bound to more platelets, and only MPs obtained from stimulated PMNs induced platelet activation as assessed P-selectin expression (resting-MPs: MFI = 25.1 ± 6.5 vs PMA-MPS: MFI = 293 ± 60, n = 4). MP binding and platelet activation were reduced by ~40–50% by function-blocking mAbs to α M β 2 and GPIbα, implicated as a major counter-receptor for α M β 2 . Complete abrogation of these events was achieved by the combination of blocking reagents to both α M β 2 /GPIbα and to PSGL-1/P-selectin. In conclusion, MPs released by stimulated PMNs express activated α M β 2 . This integrin cooperates with PSGL-1 to mediate interaction with and activation of platelets.

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