RESUMO
BACKGROUND: Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK). OBJECTIVE: The present study was carried out to elucidate the mechanisms regulating MLC (Ser)19 and (Thr)18 phosphorylation and the functional consequence of each phosphorylation event in platelets. RESULTS: Induction of 2MeSADP-induced shape change occurs within 5s along with robust phosphorylation of MLC (Ser)19 with minimal phosphorylation of MLC (Thr)18. Selective activation of G(12/13) produces both slow shape change and comparably slow MLC (Thr)18 and (Ser)19 phosphorylation. Stimulation with agonists that trigger ATP secretion caused rapid MLC (Ser)19 phosphorylation while MLC (Thr)18 phosphorylation was coincident with secretion. Platelets treated with p160(ROCK) inhibitor Y-27632 exhibited a partial inhibition in secretion and had a substantial inhibition in MLC (Thr)18 phosphorylation without effecting MLC (Ser)19 phosphorylation. These data suggest that phosphorylation of MLC (Ser)19 is downstream of Gq/Ca(2+) -dependent mechanisms and sufficient for shape change, whereas MLC (Thr)18 phosphorylation is substantially downstream of G(12/13) -regulated Rho kinase pathways and necessary, probably in concert with MLC (Ser)19 phosphorylation, for full contractile activity leading to dense granule secretion. Overall, we suggest that the amplitude of the platelet contractile response is differentially regulated by a least two different signaling pathways, which lead to different phosphorylation patterns of the myosin light chain, and this mechanism results in a graded response rather than a simple on/off switch.
Assuntos
Plaquetas/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Serina/química , Treonina/química , Difosfato de Adenosina/química , Cálcio/química , Cálcio/metabolismo , Forma Celular , Humanos , Cinética , Fosforilação , Agregação Plaquetária , Transdução de Sinais , Quinases Associadas a rho/metabolismoRESUMO
BACKGROUND: It has been reported that the association of glycoprotein VI (GPVI) with lipid rafts regulates GPVI signaling in platelets. OBJECTIVE: Secreted adenosine 5'-diphosphate (ADP) potentiates GPVI-induced platelet aggregation at particular agonist concentrations. We have investigated whether the decrease in GPVI signaling, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. METHODS: We disrupted platelet lipid rafts with methyl-beta-cyclodextrin and measured signaling events downstream of GPVI activation. RESULTS: Lipid raft disruption decreases aggregation induced by low concentrations of convulxin, but this decrease is almost eliminated in the presence of ADP antagonists. Signaling indicators, such as protein phosphorylation and calcium mobilization, were not affected by raft disruption in collagen or convulxin stimulated platelets. Interestingly, however, raft disruption directly reduced GPVI signaling induced by collagen-related peptide. CONCLUSIONS: Lipid rafts do not directly contribute to signaling by the physiologic agonist collagen. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates GPVI signaling.
Assuntos
Microdomínios da Membrana/fisiologia , Glicoproteínas da Membrana de Plaquetas/agonistas , Difosfato de Adenosina/antagonistas & inibidores , Plaquetas , Colágeno/farmacologia , Retroalimentação Fisiológica , Humanos , Agregação Plaquetária , Transdução de SinaisRESUMO
Trifluoperazine (TFP) in concentrations up to 10-15 microM increased the formation of phosphatidic acid (PA) in platelets treated with 0.5 U/ml of thrombin, while higher concentrations of TFP inhibited formation of PA. Liberation of arachidonate (AA) from platelet phospholipids was progressively inhibited as the concentration of TFP increased. At thrombin doses lower than 0.1 U/ml TFP, (less than or equal to 25 microM) enhanced PA formation with either no effect of AA liberation (6 donors) or with much greater enhancement of PA formation than the decrease in liberation of AA (3 donors). The enhancement of PA formation by TFP did therefore not seem to be due to inhibition of phospholipase A2 (PLA2) by the phenothiazine, which has been suggested. We show further that TFP inhibits PA phosphohydrolase in platelet lysates, although with complex kinetics. It is therefore concluded that the enhancement of thrombin-induced PA production by TFP is not caused by inhibition of PLA2 but could be due to TFP-induced inhibition of PA phosphohydrolase.
Assuntos
Plaquetas/efeitos dos fármacos , Ácidos Fosfatídicos/sangue , Trifluoperazina/farmacologia , Ácido Araquidônico , Ácidos Araquidônicos/sangue , Plaquetas/metabolismo , Humanos , Hidrólise , Técnicas In Vitro , Cinética , Fosfatidato Fosfatase/antagonistas & inibidores , Fosfatidato Fosfatase/sangue , Trombina/farmacologiaAssuntos
Plaquetas/enzimologia , Glicosídeo Hidrolases/sangue , Lisossomos/enzimologia , Colorimetria/métodos , Glicosídeo Hidrolases/metabolismo , Humanos , Indicadores e Reagentes , Valores de Referência , Espectrofotometria/métodos , beta-Galactosidase/sangue , beta-N-Acetil-Hexosaminidases/sangueAssuntos
Nucleotídeos de Adenina/sangue , Plaquetas/metabolismo , Nucleotídeos de Adenina/metabolismo , Plaquetas/enzimologia , Coleta de Amostras Sanguíneas , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Luciferina de Vaga-Lumes , Glicosídeo Hidrolases/sangue , Glicosídeo Hidrolases/metabolismo , Humanos , Indicadores e Reagentes , Luciferases , Medições Luminescentes , Valores de Referência , Serotonina/sangue , Serotonina/metabolismoRESUMO
We observed that more total inositol trisphosphate (InsP3) was formed when human platelets were stimulated with agonists (15-hydroxy-9,11-azo-prosta-5,13-dienoic acid or thrombin) in the presence of extracellular Ca2+ than in its absence. Analysis of the InsP3 by h.p.l.c. indicated that the increased InsP3 formed in the presence of extracellular Ca2+ was primarily the 1,3,4-trisphosphate [Ins(1,3,4)P3]. In addition, more inositol 1,3,4,5-tetrakisphosphate (InsP4) was formed in the presence of extracellular Ca2+. Experiments conducted with electrically permeabilized platelets demonstrated that conversion of [3H]Ins(1,4,5)P3 to [3H]InsP4 in platelets was Ca2+-dependent, with half-maximal conversion observed at approx. 2.5 microM-Ca2+. By contrast, dephosphorylation of [3H]InsP4 to [3H]Ins(1,3,4)P3 was not activated by Ca2+. A partially purified preparation of Ins(1,4,5)P3 3-kinase from human platelets was found to be insensitive to Ca2+, but addition of calmodulin restored Ca2+-sensitivity to the kinase, increasing its activity about 5-fold. These results show that in human platelets the metabolism of Ins(1,4,5)P3 is regulated by Ca2+-calmodulin, and suggest that the metabolites of Ins(1,4,5)P3 may also have important second-messenger functions in platelets, and are consistent with the hypothesis that the activation of phospholipase C is not dependent on extracellular Ca2+.
Assuntos
Plaquetas/metabolismo , Cálcio/farmacologia , Fosfatos de Inositol/sangue , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/sangue , Fosfatos Açúcares/sangue , Plaquetas/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato , Prostaglandinas H/farmacologia , Trombina/farmacologiaRESUMO
Storage pool-deficient (SPD) platelets, which have decreased amounts of dense-granule and/or alpha-granule constituents, contain normal amounts of lysosomal acid hydrolases, but in some cases exhibit impaired secretion of these enzymes. We examined this impaired secretion response in SPD patients with varying extents of granule deficiencies, and determined the effects of added dense-granule constituents. Acid hydrolase secretion was impaired in patients with severe dense-granule deficiencies, but not in patients with lesser dense-granule deficiencies, including those with alpha-granule deficiencies as well. When dense-granule constituents (ADP, ATP, serotonin, Ca+2, pyrophosphate) were added to gel-filtered platelets, ADP, but none of the other constituents, completely corrected the impairment of thrombin and A23187-induced secretion in SPD platelets. The concentration of ADP required to normalize thrombin-induced secretion varied markedly, from 0.01 to 10 microM, among the individual patients. Fixation of platelets with formaldehyde before centrifugation did not prevent the enhancement of secretion by ADP. Excess ATP, which acts as a specific antagonist of ADP-mediated responses, completely blocked this enhancement of secretion in SPD platelets by ADP, and partially inhibited acid hydrolase secretion induced by low, but not high, concentrations of thrombin in normal platelets as well. Treatment of normal platelets with acetylsalicylic acid in vivo, but not in vitro, produced an impairment of acid hydrolase secretion similar in extent to that in SPD platelets, but which could not be completely corrected by added ADP. One possible explanation of these results is that the impairment of acid hydrolase secretion may be secondary to the dense-granule deficiency in SPD platelets, and that secreted ADP may potentiate the lysosomal secretion response in normal platelets as well.
Assuntos
Difosfato de Adenosina/farmacologia , Transtornos Plaquetários/enzimologia , Plaquetas/efeitos dos fármacos , Hidrolases/metabolismo , Deficiência do Pool Plaquetário/enzimologia , Difosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/farmacologia , Aspirina/farmacologia , Plaquetas/enzimologia , Calcimicina/farmacologia , Cálcio/farmacologia , Relação Dose-Resposta a Droga , Epinefrina/farmacologia , Formaldeído/farmacologia , Humanos , Hidrolases/sangue , Trombina/farmacologia , beta-N-Acetil-Hexosaminidases/sangue , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Aggregation responses to low concentrations of ADP, epinephrine, collagen and cationophore A23187 in platelets from two family members with marked bleeding tendencies were virtually absent, whereas shape change with ADP was normal. The contribution to factor X activation by collagen-treated platelets was markedly decreased. Glycoproteins IIb and III were also significantly reduced. The patients' platelets had normal stores of secretable constituents, but secretion of adenine nucleotides and acid hydrolases in response to low concentrations of thrombin and A23187 was drastically reduced compared to normal platelets; secretion of platelet factor 4 was normal. Agonist concentrations that normally produce maximal responses induced only partial aggregation and secretion in the patients' platelets. After prelabelling with [3H]arachidonate thrombin caused less changes in the [3H]phosphatidylinositol, [3H]phosphatidylcholine and free [3H]arachidonate in platelets from the patients than in platelets from normals. We conclude that the patients' platelets have an impairment in part of the signal processing mechanism that is common for all agonists and responses. This platelet abnormality, which has a superficial resemblance to thrombasthenia, represents a hitherto undescribed qualitative platelet disorder.
Assuntos
Transtornos Plaquetários/genética , Glicoproteínas da Membrana de Plaquetas/deficiência , Difosfato de Adenosina/sangue , Trifosfato de Adenosina/sangue , Testes de Coagulação Sanguínea , Transtornos Plaquetários/sangue , Plaquetas/metabolismo , Feminino , Humanos , Masculino , Linhagem , Fosfolipídeos/sangue , Agregação PlaquetáriaRESUMO
1. myo-[3H]Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], when added to lysed platelets, was rapidly converted into [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], which was in turn converted into [3H]inositol 1,3,4-trisphosphate [Ins(1,3,4)P3]. This result demonstrates that platelets have the same metabolic pathways for interconversion of inositol polyphosphates that are found in other cells. 2. Labelling of platelets with [32P]Pi, followed by h.p.l.c., was used to measure thrombin-induced changes in the three inositol polyphosphates. Interfering compounds were removed by a combination of enzymic and non-enzymic techniques. 3. Ins(1,4,5)P3 was formed rapidly, and reached a maximum at about 4 s. It was also rapidly degraded, and was no longer detectable after 30-60 s. 4. Formation of Ins(1,3,4,5)P4 was almost as rapid as that of Ins(1,4,5)P3, and it remained detectable for a longer time. 5. Ins(1,3,4)P3 was formed after an initial lag, and this isomer reached its maximum, which was 10-fold higher than that of Ins(1,4,5)P3, at 30 s. 6. Comparison of the intracellular Ca2+ concentration as measured with fura-2 indicates that agents other than Ins(1,4,5)P3 are responsible for the sustained maintenance of a high concentration of intracellular Ca2+. It is proposed that either Ins(1,3,4)P3 or Ins(1,3,4,5)P4 may also be Ca2+-mobilizing agents.
Assuntos
Plaquetas/metabolismo , Fosfatos de Inositol/sangue , Fosfatos Açúcares/sangue , Plaquetas/efeitos dos fármacos , Cálcio/sangue , Cromatografia Líquida de Alta Pressão , Citosol/metabolismo , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/biossíntese , Radioisótopos de Fósforo , Trombina/farmacologia , TrítioRESUMO
Aspirinated human platelets labeled with 32PO4 showed a 1.7-fold increase in [32P]IP3 when stimulated with ADP. ADP-stimulated mobilization of internal Ca2+ and phosphorylation of myosin were enhanced in the presence of extracellular Ca2+ but the increase in IP3 was not significantly affected by external Ca2+. The Ca2+ ionophore, ionomycin, elevated internal Ca2+ and induced myosin phosphorylation without a detectable change in IP3. These results indicate that the mechanism of ADP stimulation of human platelets is similar to that of other platelet agonists and supports the theory that IP3 functions to liberate internal Ca2+.
Assuntos
Difosfato de Adenosina/farmacologia , Plaquetas/metabolismo , Fosfatos de Inositol/sangue , Fosfatos Açúcares/sangue , Plaquetas/efeitos dos fármacos , Cálcio/sangue , Éteres/farmacologia , Humanos , Inositol 1,4,5-Trifosfato , Ionomicina , Miosinas/sangue , Fosfatidilinositóis/sangue , Fosforilação , Trombina/farmacologiaRESUMO
Previous studies indicated that thrombin-stimulation of platelets prelabeled with [3H]inositol or [32P]orthophosphate results in an increase of radioactive inositol triphosphate, a substance thought to modulate the levels of free intracellular calcium. In the present study, we improved the method of resolution of inositol triphosphate from other compounds that are also labeled with [32P]orthophosphate using a combination of enzyme treatment and electrophoresis. We have further demonstrated that the specific activities of metabolic ATP and phosphatidylinositol diphosphate (the precursor of inositol triphosphate) are identical in [32P]orthophosphate-labeled platelets. It follows that the amount of inositol triphosphate is proportional to its radioactivity in the metabolic compartment of the cells. Using this protocol, the concentration of inositol triphosphate in resting and thrombin-stimulated platelets were determined to be 1-4 and 10-30 pmol/10(8) cells, respectively.
Assuntos
Plaquetas/metabolismo , Fosfatos de Inositol/sangue , Fosfatos Açúcares/sangue , Trifosfato de Adenosina/sangue , Plaquetas/efeitos dos fármacos , Eletroforese em Papel , Humanos , Inositol 1,4,5-Trifosfato , Lipídeos/sangue , Fosfatos/sangue , Radioisótopos de Fósforo , Trombina/farmacologiaRESUMO
Gel-filtered human platelets were stimulated with 5i.u. of thrombin/ml for times up to 1 min. The fatty acid composition of inositol-containing phospholipids, phosphatidic acid and diacylglycerol was determined by g.l.c. in control and thrombin-stimulated platelet suspensions. Inositol phospholipids were found to have similar proportions of stearic and arachidonic acids, the sum of these representing 86.6% of the total fatty acids in phosphatidylinositol (PtdIns), 76.9% in phosphatidylinositol 4-phosphate (PtdIns4P) and 85.4% in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. However, arachidonic and stearic acids were less abundant in phosphatidic acid (PtdA) and diacylglycerols in non-stimulated platelets. A transient decrease in the mass of PtdIns(4,5)P2 was observed after 5-10s of thrombin stimulation, followed by an increase after 30s. The amounts of PtdIns4P and PtdIns decreased throughout the experiment. A transient accumulation of stearoylarachidonoylglycerol was observed at 5s, whereas stearoylarachidonoylglycerol 3-phosphate (PtdA) was produced in increasing amounts throughout the experiment. The decrease in inositol-containing phospholipids was not fully compensated for by the production of diacylglycerol or PtdA [or PtdIns(4,5)P2] at 1 min. All the changes in inositol phospholipids, as well as those observed in diacylglycerols and PtdA, were due to a parallel reduction or increase in the contents of stearic and arachidonic acids, with a stoichiometry equal to 1. Taken together, this suggests an interconversion of all these lipids with the utilization of a common backbone, stearoylarachidonoylglycerol. The deacylation of this diacylglycerol could account for up to 4-5nmol of arachidonate/10(9) platelets after 1 min stimulation by thrombin.
Assuntos
Plaquetas/metabolismo , Diglicerídeos/sangue , Glicerídeos/sangue , Ácidos Fosfatídicos/sangue , Fosfatidilinositóis/sangue , Ácidos Graxos/análise , Humanos , Técnicas In Vitro , Modelos Químicos , Trombina/farmacologiaRESUMO
Gel-filtered platelets prelabeled with [3H]-arachidonate and [14C]-adenine or [32P]-orthophosphate were stimulated with thrombin in the presence of various concentrations of trifluoperazine (TFP). Based on the presence of [14C]- or [32P]-labeled extracellular adenine nucleotides, TFP, above 50 microM, caused platelet lysis which reached 30-40% at 100 microM. In the non-lytic range (0-50 microM) TFP caused marked inhibition of [3H]-arachidonic acid liberation and [3H]-phosphatidylcholine breakdown which was complete at 25 microM. Breakdown of [3H]-phosphatidylinositol was partially (about 50%) inhibited at 25 microM TFP and little further inhibition occurred above this concentration. These results show that thrombin-induced liberation of [3H]-arachidonic acid occurs entirely by a TFP-sensitive mechanism, and suggest that the major portion of the arachidonate is liberated from phosphatidylcholine with a possible contribution from phosphatidylinositol. Dense granule secretion and acid hydrolase secretion were progressively inhibited by TFP, while the thiazine had only a small effect on phosphorylation of myosin. These results indicate that the inhibition of the secretory processes by TFP is not caused by action of TFP on myosin light chain kinase. It is suggested that the profound effect of TFP on arachidonic acid liberation but not myosin phosphorylation is due to different subcellular localization of these calmodulin-requiring enzymes: phospholipase A2 and myosin light chain kinase. The lipophilic TFP dissolves preferentially in the membranes where it has access to phospholipase A2 but not to myosin light chain kinase.
Assuntos
Ácidos Araquidônicos/metabolismo , Plaquetas/efeitos dos fármacos , Miosinas/metabolismo , Trombina/farmacologia , Trifluoperazina/farmacologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Humanos , Técnicas In Vitro , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/metabolismo , FosforilaçãoRESUMO
Human platelets are rich in beta-hexosaminidase and other acid hydrolases contained in organelles (lysosomes) distinct from alpha-granules and dense granules. Incubation of platelets with bovine or human thrombin (100 U/ml for 5 min at 37 degrees C) induces the secretion of 100% of the contents of alpha- and dense granules, but only 40-60% of total beta-hexosaminidase from lysosomes. Both isozymes Hex A and Hex B are secreted in the same proportion as found intracellularly. There is no selective recapture or plasma membrane binding by platelets of secreted beta-hexosaminidase. The secreted enzyme is of the low-uptake type, i.e., it is poorly recognized by the phosphomannosyl receptor-mediated uptake mechanism of fibroblasts, while the retained enzyme is a 3-fold higher uptake form. Preincubation of platelets with NH4Cl (10 mM, 2 h), followed by thrombin stimulation, results in secretion of all beta-hexosaminidase as a low-uptake form. The data support the hypothesis that there are secretory and nonsecretory forms of lysosomes. The secretory lysosomes would contain low-uptake forms of hydrolases in addition to acid phosphatase, while the nonsecretory lysosomes would contain high-uptake hydrolases and be acid phosphatase-deficient. Conditions where the contents of both lysosomal populations were released together, i.e., amine treatment followed by thrombin induction, or extraction of unstimulated cells, would result in the exposure of high-uptake phosphomannosylated hydrolases released from one population of lysosomes to acid phosphatase released from the second population of lysosomes with their subsequent conversion to low-uptake forms.
Assuntos
Plaquetas/enzimologia , Hexosaminidases/sangue , Isoenzimas/sangue , Trombina/farmacologia , Plaquetas/efeitos dos fármacos , Cromatografia DEAE-Celulose , Fibroblastos/metabolismo , Hexosaminidase A , Hexosaminidase B , Humanos , Doença de Sandhoff/metabolismo , beta-N-Acetil-HexosaminidasesRESUMO
Using a newly developed isotopic tracer technique for the measurement of 32P-labelled intermediates in glycolysis and nucleotide metabolism in platelets, we studied the variations in 32P-labelled intermediates during activation of the glycolytic flux by cyanide and platelet-activating agents. The major variations occurred in [32P]Fru-1,6-P2, dihydroxy acetone phosphate, ATP and Pi. There was a quantitative covariance between the increase in lactate production and the rise in [32P]Fru-1,6-P2 induced by different platelet-activating agents. In contrast, cyanide induced weaker activation of the flux and greater accumulation of [32P]Fru-1,6-P2. Variations in 32P-labelled intermediates were apparent 5 s after flux activation, but the major changes in [32P]Fru-1,6-P2 occurred much later and fell in periods in which a constant lactate formation was maintained. The cyanide-induced changes in 32P-labelled intermediates depended on the extracellular level of glucose, showing a predominant ATP----Pi conversion in glucose-depleted medium that shifted to an ATP----Fru-1,6-P2 conversion at excess glucose. At about 50 microM glucose, flux activation occurred without major changes in [32P]Fru-1,6-P2, dihydroxy acetone phosphate and Pi, with only a small fall in [32P]ATP. The data provide evidence for a role of the aldolase reaction in flux control and demonstrate rapid changes in Fru-1,6-P2 and ATP during flux activation with an additional role for Fru-1,6-P2 as an energy buffer during post-activation periods.
Assuntos
Plaquetas/metabolismo , Glicólise , Fosfatos/sangue , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Calcimicina/farmacologia , Colágeno/farmacologia , Cianetos/farmacologia , Epinefrina/farmacologia , Frutosedifosfatos/sangue , Humanos , Lactatos , Ácido Láctico , Trombina/farmacologiaRESUMO
Human platelets incubated with [32P]Pi and [3H]arachidonate were transferred to a Pi-free Tyrode's solution by gel filtration. The labile phosphoryl groups of ATP and ADP as well as Pi in the metabolic pool of these platelets had equal specific radioactivity which was identical to that of[32P]phosphatidate formed during treatment of the cells with thrombin for 5 min. Therefore, the 32P radioactivity of phosphatidate was a true, relative measure for its mass. The thrombin-induced formation of[32P]-phosphatidate had the same time course and dose-response relationships as the concurrent secretion of acid hydrolases. 125I-alpha-Thrombin bound maximally to the platelets within 13s and was rapidly dissociated from the cells by hirudin; readdition of excess 125I-alpha-thrombin caused rapid rebinding of radioligand. This binding-dissociation-rebinding sequence was paralleled by a concerted start-stop-restart of phosphatidate formation and acid hydrolase secretion. [3H]Phosphatidylinositol disappearance was initiated upon binding but little affected by thrombin dissociation and rebinding. ATP deprivation caused similar changes in the time courses for [32P]-phosphatidate formation and acid hydrolase secretion which were different from those of [3H]phosphatidylinositol disappearance. The metabolic stress did not alter the magnitude (15%) of the initial decrease in phosphatidylinositol-4,5-bis[32P]phosphate, but did abolish the subsequent increase of phosphatidylinositol-4,5-bis[32P]-phosphate in the thrombin-treated platelets. It is concluded that in thrombin-treated platelets (1) phosphatidate synthesis, but not phosphatidylinositol disappearance, is tightly coupled to receptor occupancy and acid hydrolase secretion in platelets, (2) successive phosphorylations to phosphatidylinositol-4,5-bisphosphate is unlikely to be the main mechanism for phosphatidylinositol disappearance, and (3) only a small fraction (15%) of phosphatidylinositol-4,5-bisphosphate is susceptible to hydrolysis.
Assuntos
Plaquetas/metabolismo , Hidrolases/sangue , Ácidos Fosfatídicos/biossíntese , Receptores de Superfície Celular/metabolismo , Trombina/farmacologia , Difosfato de Adenosina/sangue , Trifosfato de Adenosina/sangue , Antimetabólitos/farmacologia , Plaquetas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hirudinas/farmacologia , Humanos , Fosfatos/sangue , Ácidos Fosfatídicos/sangue , Fosfatidilinositóis/sangue , Receptores de Trombina , Trombina/metabolismoRESUMO
A method is presented for the separation and quantification of 32P-labeled carbohydrates and nucleotides in blood platelets which have been pulse-labeled with [32P]orthophosphate. The procedure is based on two-dimensional paper chromatography, identification of the spots by radioautography and enzymatic methods, and quantitation of 32P radioactivity by liquid scintillation counting. The data show that 32P is homogeneously distributed among the compounds studied so that the total radioactivity is proportional to the levels of these compounds in the metabolic compartment of the cells. Thus, this method provides a sensitive and accurate means to evaluate phosphorylated intermediates in glycolysis and nucleotide metabolism and to assess the transfer of energy-rich phosphate groups between these pathways in particular.
Assuntos
Carboidratos/isolamento & purificação , Nucleotídeos/isolamento & purificação , Autorradiografia , Plaquetas , Cromatografia , Glicólise , Humanos , Nucleotídeos/metabolismo , Fosfatos/isolamento & purificação , Radioisótopos de FósforoRESUMO
Previous studies have indicated different energy requirements for some platelet responses; these differences could, however, be due to inadequate methodology and differences in platelet preparation. The present study describes the effect of decreasing ATP availability on seven platelet responses measured in gel-filtered human platelets. The cells, prelabelled with 5-hydroxy[(3)H]tryptamine, [(3)H]- or [(14)C]adenine, [(32)P]P(i) or [(3)H]arachidonate, were incubated with antimycin A and 2-deoxy-d-glucose. Platelet responses induced by thrombin and collagen (secretion only), level of metabolic ATP and the adenylate energy charge (AEC) were determined at various times during incubation. Platelet aggregation was rapidly inhibited after a lag of 5-15 min and with 50% inhibition at AEC = 0.55-0.60. Secretion of 5-hydroxy[(14)C]tryptamine and ATP + ADP from dense granules and of fibrinogen and beta-thromboglobin from alpha-granules were inhibited in parallel, without a lag and with 50% inhibition at AEC = 0.65-0.70. The inhibition of secretion of platelet factor 4 from the alpha-granules followed another pattern with 50% inhibition at AEC = 0.70-0.80. Breakdown of [(3)H]-phosphatidylinositol, formation of [(3)H]- and [(32)P]-phosphatidate, liberation of [(3)H]arachidonate and secretion of acid hydrolases were inhibited in parallel and inhibition was present at the start of incubation with 50% inhibition at AEC = 0.80-0.87. These results suggest that the responses have different energy requirements, increasing in the order: aggregation < dense granule and alpha-granule secretion < acid hydrolase secretion, phosphatidylinositol breakdown, phosphatidate formation and arachidonate liberation. The powerful inhibition of phosphatidylinositol breakdown by metabolic inhibitors suggests that energy-requiring steps are involved in the activation of phospholipase C.
Assuntos
Ácidos Araquidônicos/sangue , Plaquetas/metabolismo , Ácidos Fosfatídicos/biossíntese , Fosfatidilinositóis/sangue , Agregação Plaquetária , Antimicina A/farmacologia , Ácido Araquidônico , Plaquetas/efeitos dos fármacos , Cromatografia em Gel , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Desoxiglucose/farmacologia , Metabolismo Energético , Glucuronidase/sangue , Hexosaminidases/sangue , Humanos , Agregação Plaquetária/efeitos dos fármacos , Trombina/farmacologia , beta-N-Acetil-HexosaminidasesRESUMO
A23187-treated platelets secrete dense granule constituents during centrifugation, an artifact that is presented by prior formalin fixation (Holmsen, H., and Setkowsky-Dangelmaier, C. A. (1977) Biochim. Biophys. Acta 497, 46-61). With this improved assay, A23187 induced no secretion in nonaggregating platelets and maximal secretion in aggregating platelets within 3 min. Further incubation gave a slow, submaximal secretion in nonaggregating cells. Acetylsalicylate abolished secretion in both systems. EDTA, but not ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, strongly enhanced secretion in nonaggregating platelets suspended in Mg2+-containing, Ca2+-free Tyrode's solution. Without added Mg2+, A23187 gave maximal secretion in nonaggregating platelets which was abolished by added Mg2+. Preincubation of A23187 and MgCl2 gave inhibition patterns which clearly suggested that formation of Mg.A23187 species was the cause of inhibition. Ca2+, but not Sr2+, inhibited A23187-induced secretion in the same manner as Mg2+. These findings suggest that the platelet plasma membrane has no or very little permeability for Ca2+ . and Mg2+ . A23187 species. In the physiological suspending medium, Ca2+-free Tyrode's solution, A23187-induced platelet secretion is markedly enhanced by close cell contact (aggregation) and has an absolute requirement for production of prostaglandins and/or thromboxanes. Therefore, the widely held view that secretion is directly triggered by A23187-induced increase in the cytoplasmic Ca2+ concentration is not applicable to platelets.