RESUMEN
Apoptosis is a common mechanism of programmed cell death in virtually all nucleated cells. In spite of the fact that platelets and erythrocytes are the only enucleated cells in mammals they contain most of the apoptosis machinery of other cells and undergo similar apoptotic processes as nucleated cells except those connected with nuclear and chromatin transformation. Here we compare the mechanisms of platelet and erythrocytes apoptosis induced by different stimuli namely, stimulation ofthrombin and collagen receptor (T/C), inhibitor of BclX family proteins (ABT-373) for platelets, tert-butylhydroperoxide (tBH) and calcium ionophore (A-23187) for erythrocytes. Induction of platelet apoptosis by both methods (T/C and ABT-373) lead to strong phosphoetydilserine (PS) externalization, loss of the mitochondrial membrane potential (DeltaPsim), proteolytic cleavage of some cytoskeletal and regulatory proteins, and microparticle (MP) formation. However, there are clear differences between mechanisms of platelet apoptosis induced by TC and ABT-373. T/C induced apoptotic reaction is very fast (reach the maximum at 5 min), whereas ABT-373 induced reaction is more prolonged (first apoptotic evidence appears only after 30 min and reach the maximum after 3 hours). MP formation is much more pronounced in T/C than in ABT-373 stimulated platelets, whereas caspase 3 activation is much more stronger in ABT-373 than in T/C stimulated platelets. The main differences between these two apoptotic pathways are connected with aIIbp3 integrin, which activation appears only after T/C stimulation. For tBH experiments on erythrocytes we established optimal conditions (0.25x1012 cells/L, and strong, 1500 RPM stirring) for elucidation of apoptotic processes and found two independent ways of erythrocytes apoptotic processes; calcium independent, connected with met hemoglobin (metHb) formation (tBH stimulation), and calcium dependent pathway (A-23187 stimulation). Erythrocytes apoptosis induced by tBH is characterized by formation ofmetHb, cell shrinkage, fast (95 % during 3 hours) PS externalization, yield of hemoglobin, probably by vesicle (MP) formation. These cells are transformed to stomatocytes, become highly rigid, and could not be lysed even in pure water. All these reactions are calcium independent. Whereas increase of intracellular calcium concentration by A-23187 connected with formation of exinocytes, less pronounced (17 % during 3 h, 35 % during 15 h) PS externalization and rigidity (lysed in 50 mOsm buffer).
Asunto(s)
Apoptosis/genética , Plaquetas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Eritrocitos/metabolismo , Apoptosis/efectos de los fármacos , Plaquetas/citología , Plaquetas/efectos de los fármacos , Calcimicina/farmacología , Calcio/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Proteínas del Citoesqueleto/genética , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Hemoglobina A/metabolismo , Humanos , Cinética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Metahemoglobina/metabolismo , Especificidad de Órganos , Fosfatidilserinas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de Colágeno/agonistas , Receptores de Colágeno/genética , Receptores de Colágeno/metabolismo , Receptores de Trombina/agonistas , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , terc-Butilhidroperóxido/farmacologíaRESUMEN
BACKGROUND: Brazilin, isolated from the heartwood of Caesalpinia sappan L., has been shown to possess multiple pharmacological properties. METHODS: In this study, platelet aggregation, flow cytometry, immunoblotting analysis, and electron spin resonance (ESR) spectrometry were used to investigate the effects of brazilin on platelet activation ex vivo. Moreover, fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used in in vivo study. RESULTS: We demonstrated that relatively low concentrations of brazilin (1 to 10 µM) potentiated platelet aggregation induced by collagen (0.1 µg/ml) in washed human platelets. Higher concentrations of brazilin (20 to 50 µM) directly triggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an antagonist of ADP). It was not inhibited by yohimbine (an antagonist of epinephrine), by SCH79797 (an antagonist of thrombin protease-activated receptor [PAR] 1), or by tcY-NH2 (an antagonist of PAR 4). Brazilin did not significantly affect FITC-triflavin binding to the integrin αIIbß(3) in platelet suspensions. Pretreatment of the platelets with caffeic acid phenethyl ester (an antagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies raised against collagen receptor glycoprotein VI and integrin α2ß(1), respectively, abolished platelet aggregation stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the phosphorylation of phospholipase C (PLC)γ2 and Lyn, which were significantly attenuated in the presence of JAQ1 and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. An in vivo mouse study showed that brazilin treatment (2 and 4 mg/kg) significantly shortened the occlusion time for platelet plug formation in mesenteric venules. CONCLUSION: To the best of our knowledge, this study provides the first evidence that brazilin acts a novel collagen receptor agonist. Brazilin is a plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.
Asunto(s)
Benzopiranos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/tratamiento farmacológico , Animales , Benzopiranos/química , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Caesalpinia/química , Colágeno/metabolismo , Citometría de Flujo , Humanos , Ratones , Fosforilación , Agregación Plaquetaria/efectos de los fármacos , Receptores de Colágeno/agonistas , Receptores de Colágeno/metabolismoRESUMEN
Platelets have important roles in atherosclerosis and thrombosis and their inhibition reduces the risk of these disorders. There is still a need for platelet inhibitors affecting pathways that reduce thrombosis and atherosclerosis while leaving normal hemostasis relatively unaffected, thus reducing possible bleeding complications. Although combinations show progress in achieving these goals none of the present inhibitors completely fulfill these requirements. Collagen receptors offer attractive possibilities as alternative targets at early stages in platelet activation. Three major collagen receptors are assessed in this review; the alpha2beta1 integrin, responsible primarily for platelet adhesion to collagen; GPVI, the major signaling receptor for collagen; and GPIb-V-IX, which is indirectly a collagen receptor via von Willebrand factor. Several thrombosis models and experimental approaches suggest that all three are interesting targets and merit further investigation.