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1.
Transfus Med ; 20(5): 341-5, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20534030

RESUMO

OBJECTIVES: To contribute to the understanding of the biochemical changes associated with the RBC storage lesion. AIM: To investigate changes in O(2) equilibrium and on/off kinetic rates during routine cold storage. BACKGROUND: As RBCs are stored between 1 and 6°C numerous biochemical changes occur within the RBCs, including changes in the properties of the haemoglobin itself. This study serially analysed for the first time the O(2) equilibrium and on/off kinetic rates across the RBC membrane during routine storage. METHODS/MATERIALS: The oxygen binding (k(on) ) and offloading (k(off) ) constants were measured in fresh RBCs and then in AS-5-preserved RBCs at weekly intervals, along with oxygen equilibrium curves (OECs), 2,3-Diphosphoglycerate (2,3-DPG), p50 and the Hill number (n). RESULTS: The k(on) increased slightly as the 2,3-DPG and p50 decreased during storage, whereas the k(off) remained largely unchanged. The OECs demonstrated the expected increase in O(2) affinity, whereas the Hill number was unchanged during storage. CONCLUSION: In spite of the biochemical, structural and functional changes associated with the storage of RBCs, their in vitro interactions with oxygen were largely preserved through 42 days of storage.


Assuntos
Preservação de Sangue/normas , Eritrócitos/metabolismo , Oxigênio/metabolismo , 2,3-Difosfoglicerato/metabolismo , Preservação de Sangue/métodos , Transfusão de Eritrócitos/normas , Hemoglobinas/metabolismo , Humanos , Cinética , Oxigênio/análise , Temperatura , Fatores de Tempo
2.
Br J Haematol ; 123(5): 903-5, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14632782

RESUMO

A replication-incompetent adenovirus vector was administered to rhesus macaques at 1, 3 and 6 x 1012 particles/kg doses to investigate its toxicity. Platelet count decrements of 28%, 82% and 90%, respectively, were observed, with corresponding platelet half-lives of 69.0, 25.2 and 22.2 h (compared with 111 h in untreated animals). The platelet decline was equivalent for all three doses for 8 h, and platelet count recovery began as early as 8 h after infusion for low-dose recipients, or as late as 24 h for the medium and high dose recipients. These observations suggest that thrombocytopenia is a saturable, reversible consumptive process.


Assuntos
Infecções por Adenoviridae/complicações , Adenoviridae/genética , Plaquetas/virologia , Vetores Genéticos/administração & dosagem , Trombocitopenia/virologia , Animais , Engenharia Genética , Injeções Intravenosas , Macaca mulatta , Contagem de Plaquetas
3.
Transfus Med Rev ; 15(4): 268-81, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11668434

RESUMO

The cellular prion protein (PrPc) holds a central role in the pathophysiology of transmissible spongiform encephalopathies (TSE). The hallmark of these progressive neurodegenerative diseases is the accumulation of the protease-resistant, pathologic conformation of prion protein (PrPres) in the CNS. The conformational change is thought to be propagated by a template-like effect in which a normal prion protein (PrPc) interacts with its PrPres isoform and assumes the pathologic conformation. In its natural conformation, the prion protein is expressed on many different cell types, but its physiological function has yet to be clearly defined. PrPc expressed on blood cells or present in plasma may contribute to the transport of TSE infectivity found in blood of infected animal models. We examine the expression of PrPc on human and animal blood cells and its potential functional roles and discuss studies of transfusion-mediated transmission of TSE infectivity in animals.


Assuntos
Células Sanguíneas/química , Expressão Gênica , Doenças Priônicas/etiologia , Príons/sangue , Príons/genética , Animais , Células-Tronco Hematopoéticas/química , Humanos , Camundongos , Camundongos Knockout , Doenças Priônicas/sangue , Doenças Priônicas/prevenção & controle , Doenças Priônicas/transmissão , Príons/fisiologia , Reação Transfusional
4.
Br J Haematol ; 112(4): 1048-54, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11298605

RESUMO

Several lines of evidence point to the potential role of nitric oxide (NO) in the pathophysiology, as well as in the therapy, of sickle cell disease (SCD). In this study, we compared the effects of NO on platelets from normal individuals and from patients with SCD. Three NO donors were used to deliver NO to platelets: sodium 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitrosocysteine (CysNO) and sodium trioxdintrate (OXINO or Angeli's salt). ADP-induced platelet aggregation, CD62P expression, PAC-1 binding and calcium elevation were evaluated in paired studies of normal and SCD subjects. DEANO significantly reduced aggregation in SCD platelets compared with normal platelets. DEANO similarly reduced the extent of CD62P expression in SCD platelets. All NO donors reduced PAC-1 binding, but there were no significant differences between platelets from normal or SCD subjects. Calcium elevation, as induced by ADP, was not altered by the presence of NO donors. However, when platelets were stimulated with thrombin, there was an increased initial response of SCD platelets compared with normal platelets. Taken together, these data suggest that the mode of NO delivery to platelets may produce various physiological responses and the optimization of NO delivery may contribute to reducing platelet aggregation in sickle cell disease.


Assuntos
Cisteína/análogos & derivados , Doadores de Óxido Nítrico/farmacologia , Agregação Plaquetária/efeitos dos fármacos , S-Nitrosotióis , Traço Falciforme/sangue , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Plaquetas/metabolismo , Cálcio/metabolismo , Estudos de Casos e Controles , Cisteína/farmacologia , Dietilaminas/farmacologia , Fosfatase 2 de Especificidade Dupla , Hemostáticos/farmacologia , Humanos , Nitritos/farmacologia , Óxidos de Nitrogênio , Compostos Nitrosos/farmacologia , Selectina-P/análise , Proteína Fosfatase 2 , Proteínas Tirosina Fosfatases/metabolismo , Trombina/farmacologia
6.
Br J Haematol ; 110(2): 472-80, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10971411

RESUMO

The host prion protein, PrPc, and its conformationally changed isoform, PrPsc, play an essential role in the transmissible spongiform encephalopathy (TSE) infections. The prion hypothesis postulates that PrPsc is the TSE infectious agent and that it serves as a template to convert host PrPc to additional PrPsc. Blood of experimentally TSE-infected rodents has been shown to contain TSE infectivity. If blood-borne TSE infectivity requires association with PrPc, differences in the distribution of PrPc in blood could affect the amount and distribution of blood-borne infectivity in different hosts. We have compared the distribution of PrPc on the peripheral blood cells of humans, hamsters and mice using quantitative flow cytometry. Human lymphocytes, monocytes and platelets displayed much greater quantities of PrPc than corresponding mouse cells. Mouse platelets did not express any detectable PrPc. A similar low level of PrPc was found on both human and mouse red blood cells. None of the hamster peripheral blood cells displayed detectable amounts of PrPc. If PrPc contributes to the propagation or transport of TSE infectivity in blood, the species differences in PrPc distribution reported here need to be considered when extrapolating the results of rodent TSE transmission studies with blood and blood components to humans.


Assuntos
Príons/sangue , Animais , Anticorpos Monoclonais , Plaquetas/imunologia , Cricetinae , Eritrócitos/imunologia , Feminino , Citometria de Fluxo , Humanos , Linfócitos/imunologia , Masculino , Camundongos , Monócitos/imunologia , Príons/imunologia
8.
Am J Clin Pathol ; 111(5): 693-9, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10230361

RESUMO

Conventional platelet storage in a blood bank is up to 5 days at room temperature in plasma. We investigated the optimal medium for assessing the quality of stored platelets by comparing in vitro test responses after resuspension in autologous plasma prepared from platelet-rich plasma after 5 days of storage at room temperature, autologous plasma stored cell-free for 5 days at room temperature, or autologous plasma stored cell-free for 5 days at -20 degrees C. Five-day-old platelets were prepared from aliquots of the same unit and resuspended in I of the 3 plasma preparations. The platelet-plasma mixtures were monitored for changes in pH, mean platelet volume, hypotonic shock response, P-selection expression, and aggregation. There were statistically significant differences between platelets resuspended in original plasma and platelets resuspended in either plasma stored cell-free at room temperature or frozen, with regard to hypotonic shock response, agonist-induced aggregation, and P-selectin expression. Plasma stored with platelets for 5 days yielded inferior platelet function test responses when compared with plasma stored cell-free at room temperature or frozen. Therefore, for direct comparison of platelet responses following novel storage methods, the resuspending plasma should be stored under the same conditions as the control platelet unit.


Assuntos
Plaquetas/fisiologia , Preservação de Sangue/métodos , Plasma , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células Cultivadas , Humanos , Concentração de Íons de Hidrogênio , Soluções Hipotônicas/farmacologia , Selectina-P/metabolismo , Agregação Plaquetária/fisiologia , Contagem de Plaquetas
9.
Br J Haematol ; 103(1): 276-82, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9792322

RESUMO

The surface expression of prion protein (PrP(C)) on human platelets, as detected by flow cytometry with the monoclonal antibody 3F4, increased more than two-fold (4300 v 1800 molecules/platelet) after full activation. Maximal surface expression of PrP(C) occurred within 3 min of platelet activation and declined to approximately half of maximal levels by 2 h at 37 degrees C. In comparison, PrP(C) on the surface of platelets, activated at 22 degrees C took 10 min to reach maximum but then remained constant for 2 h. In sonicated resting platelets, PrP(C) and P-selectin remained in intact granules after subcellular fractionation. Both glycoproteins were found in the ruptured membranes of activated platelets, suggesting that the PrP(C) was translocated from internal granules to the plasma membrane during activation, as is P-selectin. Platelet PrP(C) was not removed from the surface of platelets by phosphatidylinositol-specific phospholipase C (PIPLC) treatment but was degraded by proteinase K. Platelets may serve as a useful model for following the cellular processing of PrP(C).


Assuntos
Plaquetas/enzimologia , Fosfatidilcolinas/metabolismo , Proteínas PrPC/metabolismo , Fosfolipases Tipo C/metabolismo , Western Blotting , Humanos , Ativação Plaquetária , RNA Mensageiro/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-9507752

RESUMO

Chemically modified hemoglobins are potential oxygen-carrying blood substitutes, but their in vivo administration has been associated with a variety of unexpected side events, including increased platelet reactivity. We studied the effects of hemoglobin A0 (HbA0) and alpha-crosslinked hemoglobin (alpha-DBBF) on platelets in vitro. Neither hemoglobin A0 nor alpha-DBBF activated platelets when added alone, but both proteins potentiated submaximal agonist-induced platelet aggregation without increasing other markers of platelet activation such as serotonin secretion. Only agonists that are known to cause release of platelet arachidonic acid (AA) were potentiated while aggregation induced by ADP, which does not release AA, was not potentiated. Blockade of the thromboxane receptor with SQ-29,548 prevented the HbA0-induced and the alpha-DBBF-induced potentiation suggesting that the AA/thromboxane signaling pathway mediates the interaction of platelets with hemoglobin.


Assuntos
Aspirina/análogos & derivados , Plaquetas/química , Hemoglobina A/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Receptores de Tromboxanos/sangue , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/agonistas , Ácido Araquidônico/agonistas , Ácido Araquidônico/metabolismo , Aspirina/farmacologia , Substitutos Sanguíneos/farmacologia , Colágeno/agonistas , Colágeno/efeitos dos fármacos , Sinergismo Farmacológico , Heme/farmacologia , Humanos , Proteínas de Membrana/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/efeitos dos fármacos , Receptores de Prostaglandina/efeitos dos fármacos , Receptores de Tromboxano A2 e Prostaglandina H2 , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Trombina/efeitos dos fármacos , Trombina/metabolismo
12.
Free Radic Biol Med ; 22(6): 1055-63, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9034245

RESUMO

Peroxynitrite (ONOO-) induces nitration of tyrosine residues and inhibits tyrosine phosphorylation in cell free systems. We investigated the effect of peroxynitrite on protein tyrosine nitration and phosphorylation in resting or thrombin-activated platelets. Peroxynitrite (150 microM) rapidly induced tyrosine nitration of 187, 164, 113, 89, and 61 kDa proteins in gel-filtered platelets which persisted up to 4.5 h. Repeated exposure of platelets to peroxynitrite produced increasing levels of nitration. Peroxynitrite also rapidly increased tyrosine phosphorylation of 120, 117, 95, 80-85, and 70 kDa platelet proteins, but this decreased by 5 min. The same pattern of tyrosine phosphorylation, but with higher intensity, was induced by thrombin in control platelets. Pretreatment of platelets with peroxynitrite decreased thrombin-induced tyrosine phosphorylation at 0.05 and 1 U/ml thrombin but not at 2 U/ml thrombin. Platelet activation responses such as P-selectin expression, serotonin secretion, and aggregation were also decreased by peroxynitrite treatment at low thrombin concentrations. Peroxynitrite exposure and tyrosine nitration decreased platelet sensitivity to thrombin but did not absolutely prevent tyrosine phosphorylation and other platelet responses.


Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Nitratos/sangue , Nitratos/farmacologia , Fosfotirosina/sangue , Tirosina/sangue , Plaquetas/fisiologia , Proteínas Sanguíneas/metabolismo , Humanos , Selectina-P/sangue , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Serotonina/sangue , Trombina/farmacologia
13.
Biochim Biophys Acta ; 1314(1-2): 1-12, 1996 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-8972712

RESUMO

Prostaglandin E1(PGE1) inhibits tyrosine phosphorylation induced by low thrombin concentration (0.05 U/ml), but this is overcome by a high thrombin (2.0 U/ml) concentration. Thromboxane A2 and ADP are endogenous platelet agonists released during platelet activation which potentiate platelet responses. We investigated how these endogenous agonists influenced the effects of PGE1 on thrombin (2.0 U/ml)-induced tyrosine phosphorylation by removing released ADP with apyrase (2.0 U/ml) and by inhibiting thromboxane A2 synthesis with indomethacin (1 microM). Adding PGE1 (1 microM) before thrombin in apyrase/indomethacin(A/I)-treated platelets selectively prevented thrombin-induced tyrosine phosphorylation of a 117 kDa protein while other substrates were not affected. This selective effect was evident only in the presence of apyrase and was not dependent on indomethacin. Addition of PGE1 to A/I-treated platelets after thrombin also caused selective tyrosine dephosphorylation of the 117 kDa protein. Conditions which prevented thrombin-induced 117 kDa protein tyrosine phosphorylation also decreased fibrinogen binding to platelets. The 117 kDa protein was identified as the focal adhesion kinase (FAK) by immunoprecipitation with a monoclonal antibody to FAK and by absence of its tyrosine phosphorylation in the presence of RGDS peptide which inhibits fibrinogen binding and platelet aggregation. Thus, released endogenous ADP selectively prevents PGE1-mediated tyrosine dephosphorylation of platelet FAK most likely by stabilizing fibrinogen binding to platelets.


Assuntos
Difosfato de Adenosina/fisiologia , Alprostadil/fisiologia , Plaquetas/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Trombina/farmacologia , Tromboxano A2/fisiologia , Tirosina/metabolismo , Plaquetas/metabolismo , Cálcio/metabolismo , AMP Cíclico/fisiologia , Citosol/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Fosforilação , Ativação Plaquetária/efeitos dos fármacos
14.
J Biol Chem ; 271(32): 19524-9, 1996 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-8702645

RESUMO

Tyrosine phosphorylation is a potential mechanism for mediating store-operated calcium (SOC) influx in platelets and other nonexcitable cells. Thapsigargin induces calcium-dependent tyrosine phosphorylation and SOC influx in platelets. We prevented thapsigargin-induced tyrosine phosphorylation by buffering cytosolic calcium rise with the calcium chelator 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetomethoxyester (BAPTA-AM). Calcium influx, induced by thapsigargin and measured by 45Ca2+ accumulation, persisted in BAPTA-loaded platelets in the absence of tyrosine phosphorylation. This calcium influx was blocked by the SOC influx inhibitor SKF-96365. Tyrosine kinase inhibitors have been used to demonstrate a role for tyrosine phosphorylation in SOC influx. We compared the effects of four tyrosine kinase inhibitors genistein, methyl-2, 5-dihydroxycinnamate (erbstatin analog), tyrphostin A47, and lavendustin A, on thapsigargin-induced tyrosine phosphorylation in control platelets and on thapsigargin-induced SOC influx into BAPTA-loaded platelets in absence of tyrosine phosphorylation. Tyrphostin A47 prevented all measurable tyrosine phosphorylation in control platelets, but did not decrease calcium influx into BAPTA-loaded platelets. Genistein and the erbstatin analog were poor inhibitors of tyrosine phosphorylation but decreased SOC influx into BAPTA-loaded platelets to 55.8 +/- 3% and 51.9 +/- 7.5% of control, respectively. Lavendustin A did not decrease tyrosine phosphorylation or calcium influx. Thus, thapsigargin-induced SOC influx can occur without detectable tyrosine phosphorylation and the inhibition of SOC influx by tyrosine kinase inhibitors does not correlate with their ability to prevent tyrosine phosphorylation.


Assuntos
Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Terpenos/farmacologia , Tirosina/metabolismo , Plaquetas/metabolismo , Cálcio/química , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Quelantes , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Transporte de Íons , Fosforilação , Proteínas Tirosina Quinases/antagonistas & inibidores , Tapsigargina
15.
Biochem J ; 295 ( Pt 2): 525-9, 1993 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-8240252

RESUMO

Cytochrome P-450 has been suggested as a mediator of the signal between depleted platelet calcium stores and an increase in plasma membrane permeability to calcium which follows depletion of the stores. This hypothesis is based on the observations that inhibitors of cytochrome P-450, such as the imidazole antifungal agents, also inhibit influx of a calcium surrogate (manganese) into calcium-depleted platelets. We tested the effects of econazole and of a cytochrome P-450 inhibitor, carbon monoxide (CO), on thapsigargin (TG)-induced platelet 45Ca2+ influx. TG specifically depletes internal calcium stores and activates store-regulated calcium influx. Econazole blocked 45Ca2+ influx when it was added before TG (IC50 11 microM). Econazole at a concentration (20 microM) that inhibited 83% of TG-induced calcium influx was not inhibitory to TG-induced calcium efflux from 45Ca(2+)-loaded platelets, and did not affect calcium fluxes in resting platelets. This econazole concentration was also inhibitory to calcium influx even when it was added after the stores had been calcium-depleted by EGTA and TG for 15 min and the signal to increase calcium influx had already been generated. Inhibition of cytochrome P-450 with CO bubbled through platelet suspensions did not change calcium influx in resting cells and potentiated TG-induced calcium influx (160% of control calcium accumulation at 20 min). This effect appeared to be concentration-dependent, such that a 5 min exposure to CO produced a greater influx potentiation than a 3 min exposure. These observations indicate that (1) cytochrome P-450 does not mediate store-regulated calcium influx, and (2) econazole probably inhibits store-regulated calcium influx by an alternative mechanism, such as interaction with plasma membrane calcium channels.


Assuntos
Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Inibidores das Enzimas do Citocromo P-450 , Econazol/farmacologia , Terpenos/antagonistas & inibidores , Transporte Biológico , Plaquetas/enzimologia , Plaquetas/metabolismo , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Monóxido de Carbono/farmacologia , Humanos , Técnicas In Vitro , Tapsigargina
16.
Biochem J ; 294 ( Pt 3): 675-80, 1993 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-7691054

RESUMO

When intracellular Ca2+ pools are released during platelet stimulation by thrombin, elevation of platelet cytosolic Ca2+ concentration induces tyrosine phosphorylation of a 130 kDa protein, and refilling the pools mediates dephosphorylation of this protein [Vostal, Jackson and Shulman (1991) J. Biol. Chem. 266, 16911-16916]. In the present work the 130 kDa protein was identified as vinculin by the following criteria. (1) It is detected on protein immunoblots of thrombin-activated platelets by both monoclonal anti-phosphotyrosine and anti-vinculin antibodies. (2) Removal of N-linked sugars with peptide-N-glycosidase or reduction did not change the molecular mass of vinculin or of the 130 kDa protein on SDS/PAGE. (3) The 130 kDa tyrosine-phosphorylated protein associates with Triton-soluble fraction of platelets as does vinculin. (4) The 130 kDa protein immunoprecipitated by anti-vinculin monoclonal antibody reacts with anti-phosphotyrosine antibody; when immunoprecipitated by anti-phosphotyrosine antibody it reacts with anti-vinculin antibody. (5) The 130 kDa tyrosine-phosphorylated protein and vinculin focus isoelectrically at pI 5.4-5.8. Our finding that vinculin is a major platelet protein that undergoes Ca(2+)-dependent tyrosine phosphorylation during platelet activation may provide clues to the function of this protein.


Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Ativação Plaquetária , Vinculina/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Glicosídeo Hidrolases/metabolismo , Humanos , Técnicas In Vitro , Fosfoproteínas/metabolismo , Fosfotirosina , Molécula-1 de Adesão Celular Endotelial a Plaquetas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
17.
J Biol Chem ; 266(25): 16911-6, 1991 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-1832159

RESUMO

Depletion of intracellular calcium stores appears to increase plasma membrane permeability for calcium by an as yet obscure mechanism. We found that the Ca2+ ionophore, A23187, and thrombin elevate cytosolic calcium ([Ca2+]i) equally and cause tyrosine phosphorylation of a 130-kDa protein and to a lesser extent 80- and 60-kDa proteins. Chelation of [Ca2+]i by 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid/acetomethoxy ester decreased thrombin-induced tyrosine phosphorylation responses. These results suggested that [Ca2+]i elevation promotes tyrosine phosphorylation. Tyrosine phosphorylation persisted in the presence or absence of extracellular calcium after thrombin stimulation but subsided rapidly after A23187 addition if extracellular calcium was present. When Ca2+/ATPase activity, which is apparently required to maintain calcium stores, is inhibited by low temperature, tyrosine phosphorylation of the 130-kDa protein occurs. Rewarming platelets reverses tyrosine phosphorylation only if extracellular calcium is present. Thapsigargin, a calcium ATPase inhibitor, also induces tyrosine phosphorylation of the 130-kDa protein and prevents dephosphorylation of this protein when added prior to rewarming. These observations suggest that homeostatic levels of calcium in storage compartments favor tyrosine dephosphorylation of specific proteins. Thus the levels of [Ca2+]i and stored calcium appear to control tyrosine phosphorylation antagonistically. Tyrosine phosphorylation may play a role in regulating calcium channel function.


Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Tirosina/metabolismo , Western Blotting , Calcimicina/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Fura-2 , Humanos , Cinética , Fosforilação , Temperatura , Terpenos/farmacologia , Tapsigargina , Trombina/farmacologia
18.
Thromb Res ; 63(3): 299-309, 1991 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-1957274

RESUMO

The hepatic asialoglycoprotein receptor (AGPR) system can efficiently internalize and degrade circulating glycoproteins which lack terminal sialic acids on their carbohydrate chains. Since pro-thrombin is a glycosylated plasma protein, possible involvement of AGPR system in its clearance from circulation was evaluated. The half lives of bovine 125I-prothrombin and 125I-asialoprothrombin, injected intravenously into rats, were 192 and 1.8 minutes, respectively. Asialoprothrombin appeared to be cleared by the hepatic AGPRs since 33% of it accumulated in the liver at 30 minutes and its clearance was competitively blocked by simultaneous administration of increasing amounts of asialofetuin. Only 5% of prothrombin accumulated in the liver at 3 hours and injections asialofetuin in amounts capable of saturating the AGPR for the duration of four asialoprothrombin half lives had no effect on the disappearance of prothrombin. Our observations indicate that, although asialoprothrombin is readily cleared from plasma by the AGPR system, prothrombin is not. Thus these receptors do not appear to be involved in physiological processes that control prothrombin half life.


Assuntos
Fígado/metabolismo , Protrombina/farmacocinética , Receptores Imunológicos/fisiologia , Animais , Receptor de Asialoglicoproteína , Bovinos , Meia-Vida , Humanos , Masculino , Taxa de Depuração Metabólica , Protrombina/metabolismo , Ratos , Ratos Endogâmicos
19.
Blood ; 75(11): 2194-203, 1990 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-2189507

RESUMO

We have found that Western blots (WBs) of whole platelets exposed to normal autologous or homologous sera commonly have bands at 90 to 95 (95) Kd, and less often at 100 to 110, 80 to 85, 60 to 75, and 50 to 60 Kd when developed with antiglobulins. The percentages of normal sera producing a 95-Kd band with anti-immunoglobulin G (IgG), -IgA, and -IgM are 85, 50, and 30, respectively. Antiglobulin reagents alone also produce background bands on WBs that we have shown correspond to levels of platelet-associated Igs (PAIgs) or their derivatives. Titers of 95 Kd-reactive IgG in normal sera range from 10 to 1,280 (85% less than or equal to 50), and the reaction appears to be partially F(ab')2-mediated. The 95-Kd protein is internal and differs in many respects from surface glycoproteins IIIa, IV, and V of similar apparent molecular weight. In thrombocytopenic patients there was no correlation between severity of thrombocytopenia or PAIgG of platelet eluates and corresponding serum titers of 95 Kd-reactive IgG. Some WB reactions previously reported as evidence of autoimmunity may represent normal variations in reactions of Igs with internal platelet proteins. These reactions may be immunospecific or analogous to nonspecific, partially F(ab')2-dependent binding of Igs by certain bacterial proteins.


Assuntos
Proteínas Sanguíneas/imunologia , Imunoglobulinas/imunologia , Glicoproteínas da Membrana de Plaquetas/imunologia , Plaquetas/imunologia , Plaquetas/metabolismo , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Western Blotting , Feminino , Imunofluorescência , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/imunologia , Imunoglobulinas/metabolismo , Masculino , Peso Molecular , Glicoproteínas da Membrana de Plaquetas/análise , Glicoproteínas da Membrana de Plaquetas/metabolismo
20.
Biochem Biophys Res Commun ; 165(1): 27-36, 1989 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-2531581

RESUMO

The active site for uteroglobin inhibition of phospholipase A2 has been localized to a nonapeptide (P1) which is partially homologous to a nonapeptide (P2) in lipocortin, which also inhibits phospholipase A2. P1 and P2 share an identical tetrapeptide (P4) which is required for inhibition, although P4 alone does not inhibit this enzyme. We found the mechanism of inhibition of platelet aggregation and secretion by the nonapeptides and P4 varied depending on whether platelets were thrombin- or ADP-activated. All three peptides decrease thrombin esterolytic activity and thereby inhibit thrombin-induced platelet activation. P1 decreases ADP-induced aggregation and serotonin secretion by inhibiting phospholipase A2 whereas P4 decreases only aggregation by blocking fibrinogen binding to activated platelets. The P4 sequence in P1 may affect the interaction of P1 with platelets since the presence of P4 potentiates P1 inhibition of platelet activation.


Assuntos
Plaquetas/fisiologia , Proteínas de Ligação ao Cálcio/farmacologia , Glicoproteínas/farmacologia , Oligopeptídeos/farmacologia , Fosfolipases/antagonistas & inibidores , Inibidores da Agregação Plaquetária/farmacologia , Uteroglobina/farmacologia , Difosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Anexinas , Ácido Araquidônico , Ácidos Araquidônicos/farmacologia , Plaquetas/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/síntese química , Humanos , Cinética , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Uteroglobina/síntese química
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