RESUMO
Bacterial contamination of platelet concentrates represents the greatest post-transfusion infectious risk. Biofilm formation in this environment resulting from platelet-bacteria interactions can lead to non-uniform contaminant distribution and thus missed detection. As formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, we examined whether shielding these events would result in reduced biofilm formation by contaminant bacteria. We introduced methoxypoly(ethylene glycol) to covalently modify the platelet surface using a process termed 'PEGylation'. In the first study of its kind, we demonstrate that PEGylated platelet concentrates inoculated with Staphylococcus epidermidis display a significant reduction in bacterial binding and biofilm formation.
Assuntos
Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Plaquetas/química , Preservação de Sangue/métodos , Ativação Plaquetária/efeitos dos fármacos , Polietilenoglicóis/química , Bactérias/patogenicidade , Aderência Bacteriana/efeitos dos fármacos , Plaquetas/microbiologia , Humanos , Transfusão de Plaquetas/normas , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
Familial bleeding problems are frequently difficult to diagnose because currently used clinical tests cannot identify intracellular molecular defects of platelets. Using platelet proteomics, a comprehensive analytical tool, we diagnosed a family with severe bleeding problems of unknown origin with Quebec Platelet Disorder. Prior to proteomic analysis, we determined platelet counts, presence of glycoprotein (GP) Ib and GPIIb/IIIa, platelet aggregation, dense granule content and release, plasma levels of fibrinogen, Factor XIII and fibrin degradation products in four family members. Abnormalities were detected in platelet aggregation studies, which revealed variably reduced responses to ADP, collagen and epinephrine with concomitantly decreased ATP/serotonin secretion. In addition, D-dimer levels were significantly elevated 72 hours after in vitro thrombin stimulation of platelet-rich plasma. Together with the autosomal dominant inheritance and the delayed onset of bleeding in two of the four patients these results did not support any known platelet disorder. Therefore, the proteome of platelet lysates separated by one-dimensional SDS-PAGE was analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Platelet proteomics showed reduced amounts of alpha-granule proteins multimerin, fibrinogen and thrombospondin-1 in patient compared to control samples suggestive of Quebec Platelet Disorder. The diagnosis of Quebec Platelet Disorder was confirmed by urokinase-specific Western blots. Urokinase causes the degradation of alpha-granule proteins in this disorder. Diagnosis of rare bleeding disorders has important implications for prophylactic and acute treatment of bleeding patients. This is the first report using proteomics to identify a familial platelet defect.
Assuntos
Plaquetas/química , Proteínas Sanguíneas/análise , Grânulos Citoplasmáticos/química , Transtornos Hemorrágicos/diagnóstico , Proteômica , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Difosfato de Adenosina/farmacologia , Tempo de Sangramento , Plaquetas/enzimologia , Plaquetas/ultraestrutura , Colágeno/farmacologia , Grânulos Citoplasmáticos/enzimologia , Indução Enzimática , Epinefrina/farmacologia , Feminino , Genes Dominantes , Transtornos Hemorrágicos/epidemiologia , Transtornos Hemorrágicos/genética , Humanos , Masculino , Linhagem , Agregação Plaquetária/efeitos dos fármacos , Proteômica/métodos , Quebeque , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/sangue , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
The level of uptake and retention of amino-containing drugs in large unilamellar vesicles (LUVs) following uptake in response to a transmembrane pH gradient (DeltapH) can vary dramatically depending on the drug. For example, the anticancer drugs doxorubicin and epirubicin can be readily retained, whereas the anticancer drug vincristine and the antibiotic ciprofloxacin tend to leak out rapidly. In this investigation, we examine the influence of the hydrophobicity of the entrapped amines (that induce the DeltapH) and the anionic lipid content of the LUV on drug retention. It is shown that entrapment of increasingly hydrophobic monoamines (methylamine to amylamine) all lead to an induced DeltapH of 3 units and essentially complete drug uptake under the conditions employed, but do not lead to improved retention of vincristine and ciprofloxacin. However, significantly improved retention could be achieved by substitution of the anionic lipid distearoylphosphatidylglycerol (DSPG) for distearoylphosphatidylcholine (DSPC) in the LUV bilayer. Further, it is shown that if the induced DeltapH is reduced to 1.4 units (driven by entrapped diamine) nearly 100% accumulation of doxorubicin and epirubicin could be achieved, whereas only 25% loading for vincristine and ciprofloxacin was observed. Taken together these results provide methodology for improving (weak base) drug retention in liposomes and indicate that drugs that can partition into the lipid bilayer exhibit improved uptake and retention characteristics.
Assuntos
Aminas/química , Portadores de Fármacos , Lipossomos/química , Preparações Farmacêuticas/química , Sulfato de Amônio/química , Ciprofloxacina/química , Doxorrubicina/química , Epirubicina/química , Concentração de Íons de Hidrogênio , Fosfatidilcolinas , Fosfatidilgliceróis , Solubilidade , Vincristina/químicaRESUMO
Thrombocytopenic patients with acute leukemia may show high post-transfusion count increments that significantly exceed the number of transfused platelets. This study demonstrates that the automated hematology analyzer Sysmex XE-2100 reports erroneously high optical platelet counts when the blood sample contains particles in the size range of platelets or smaller. Thrombocytopenic or low-normal whole blood samples were spiked with 1 mum latex beads (n = 14) to mimic contaminants under controlled conditions. Optical and impedance measurements of spiked and control samples with the Sysmex XE-2100 were compared with the Advia 120 and the manual counts. The added beads unexpectedly increased the automated optical platelet counts in the Sysmex XE-2100 and, to a lesser extent, the Advia 120 (Wilcoxon signed ranks test, P < 0.05), while the beads were not included in the impedance or the manual microscopic platelet counts. Differential interference contrast microscopy was used to investigate samples from platelet concentrates for transfusion. Platelet concentrates (32/128) were identified as possible sources for particles that were microscopically distinct from platelets but would be included in the automated optical count. Transfusion of platelet concentrates containing contaminating particles can lead to unexpectedly high post-transfusion platelet counts and misdiagnosis of thrombocytopenic patients.
Assuntos
Automação Laboratorial/normas , Erros de Diagnóstico , Contagem de Plaquetas , Transfusão de Plaquetas , Técnicas de Laboratório Clínico/normas , Citometria de Fluxo , Humanos , Microesferas , Trombocitopenia/sangue , Trombocitopenia/diagnósticoRESUMO
Selective serotonin reuptake inhibitors (SSRIs) are a heterogeneous group of new antidepressants that cause a well documented acquired but reversible serotonin deficiency in blood platelets. Platelets are small, anucleate cells and are the only blood cells specialized in storing peripheral serotonin. Platelets are also an integral part of the hemostatic process that is initiated during pathologic thrombus formation in cardiovascular diseases. Serotonin release from platelets is important for functional hemostasis as indicated by congenital diseases with serotonin-deficient platelets that can lead to life-threatening bleeding problems. The postulate that SSRIs should have an impact on cardiovascular diseases is therefore well founded. Cardiovascular effects of SSRIs have indeed been shown in a number of studies investigating the effect of SSRIs in patients with psychosomatic comorbidity. SSRIs reduce the incidence of recurrent myocardial infarction (MI) in patients suffering from post-MI depression. In addition, SSRIs inhibit tight clot formation of platelets in vitro, which points to a direct anti-thrombotic or pro-fibrinolytic effect of SSRIs.
Assuntos
Plaquetas/efeitos dos fármacos , Doenças Cardiovasculares/etiologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Barreira Hematoencefálica , Doenças Cardiovasculares/induzido quimicamente , Proteínas de Transporte/fisiologia , Humanos , Proteínas da Membrana Plasmática de Transporte de Serotonina , Inibidores Seletivos de Recaptação de Serotonina/efeitos adversos , Inibidores Seletivos de Recaptação de Serotonina/químicaRESUMO
The role of circulating serotonin is unclear and whether or not serotonin is present in the blood of non-mammalian species is not known. This study provides the first evidence for the presence of serotonin in thrombocytes of birds and three reptilian species, the endothermic leatherback sea turtle, the green sea turtle and the partially endothermic American alligator. Thrombocytes from a fresh water turtle, American bullfrog, Yellowfin tuna, and Chinook salmon did not contain serotonin. Serotonin is a vasoactive substance that regulates skin blood flow, a major mechanism for endothermic body temperature regulation, which could explain why circulating serotonin is present in warm-blooded species. The temperature sensitivity of human blood platelets with concomitant changes in serotonin content further supports a link between circulating serotonin and thermoregulation. Phylogenetic comparison of the presence of circulating serotonin indicated an evolutionary divergence within reptilian species that might coincide with the emergence of endothermy.
Assuntos
Serotonina/sangue , Vertebrados/sangue , Jacarés e Crocodilos , Animais , Evolução Biológica , Aves , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Temperatura Corporal , Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Elefantes , Peixes , Humanos , Microscopia Eletrônica de Transmissão , Filogenia , Rana catesbeiana , Leões-Marinhos , Especificidade da Espécie , TartarugasRESUMO
Because the initial decrease in light transmission in platelet aggregometry is attributed to platelet shape change, it is widely held that platelet shape change is a prerequisite for platelet aggregation. We conducted this study to determine the basis of this initial optical effect in aggregometry. Platelets were activated with ADP, thrombin, or the thrombin receptor agonist peptide SFLLRN (TRAP(1-6)). In every case the initial decrease in light transmission occurred with the concomitant formation of microaggregates. This was also seen when preactivated platelets, which cannot undergo further morphological changes, were used, and when platelets were activated in the presence of shape-change inhibitors such as cytochalasin D and vincristine. Microscopy analysis of samples fixed at minimum light transmission in the aggregometer, which is generally assumed to signal shape change, always showed the presence of microaggregates. Microaggregation appeared to be distinct from full aggregation, as it was not inhibited by the addition of CD61, an antibody to the beta(3) integrin. To model these findings, fibrinogen-coated latex spheres, which cannot change shape, were aggregated with thrombin; the initial decrease in light transmission was still seen, and microaggregates formed at this time. These results indicate that platelet shape change is not a prerequisite for aggregation and that the signal widely believed to represent shape change reflects platelet microaggregation instead. We conclude that platelet aggregation occurs independently of shape change and that shape change is not necessarily followed by aggregation. These observations suggest an alternative role for platelet shape change of single platelets.
Assuntos
Plaquetas/citologia , Agregação Plaquetária/fisiologia , Difosfato de Adenosina/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Plaquetas/fisiologia , Tamanho Celular/fisiologia , Citocalasina D/farmacologia , Fibrinogênio/farmacologia , Hemostáticos/farmacologia , Humanos , Microesferas , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Temperatura , Trombina/farmacologia , Vincristina/farmacologiaRESUMO
Temperatures ranging from room temperature (20 degrees C) to 42 degrees C are generally not considered to have an activating effect on platelets. However, this assumption is not supported by clinical phenomena that result in hemostatic failure related to hypothermia. In this study, we investigated the effect of temperatures between room temperature (20 degrees C) and 42 degrees C on human blood platelets and found that room temperature causes marked activation of platelets. Major changes in platelet morphology were seen at 20 degrees C compared to resting platelets at 37 degrees C. Platelet morphology was investigated with noninvasive live cell techniques (light microscopy and dynamic and static light scattering), as well as with transmission and scanning electron microscopy. The changes in platelet morphology correlated with the expression of the activation marker, activated glycoprotein (GP) IIb-IIIa, measured by flow cytometry. Twenty-five percent to 30% of platelets expressed activated GPIIb-IIIa after exposure to 20 degrees C for 10 minutes. In the presence of serotonin re-uptake inhibitors, the serotonin content of platelets at 20 degrees C was twice that of resting platelets. In comparison, moderate heat shock conditions (42 degrees C for 10 minutes) caused no signs of platelet activation as indicated by the absence of morphological alterations, no expression of activated GPIIb-IIIa, and no changes in serotonin content. These results show that room temperature by itself significantly activates platelets and has an effect on the platelet serotonin content. This may contribute to both the functional lesion associated with 22 degrees C storage of platelets for transfusion and the in vivo hemostatic failure after hypothermia.
Assuntos
Ativação Plaquetária , Temperatura , Plaquetas/química , Plaquetas/ultraestrutura , Citometria de Fluxo , Humanos , Luz , Microscopia Eletrônica , Microscopia de Contraste de Fase , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , Espalhamento de Radiação , Serotonina/análise , Serotonina/sangueRESUMO
The binding of 125I-C1q to anionic liposomes was studied as a function of protein concentration, pH, ionic strength, and anionic lipid composition. The maximum amount of protein bound per micromole of lipid was very sensitive to electrostatic factors, increasing strongly with decreased pH and ionic strength or increased anionic lipid content. The apparent association constant was independent of these electrostatic factors, however, in marked contrast to studies on basic peptide binding to anionic lipid vesicles. Microscopic observations of large unilamellar liposomes containing fluorescently labeled C1q or phosphatidylglycerol demonstrated, under conditions causing strong electrostatic interactions, that C1q and anionic lipids colocalized into domains whose radii of curvature were higher than that of the surrounding lipid. These domains were observed to bud and pinch off into brightly fluorescent vesicles. We propose a model for all of these observations in which the line tension or edge energy at the boundary of the domain resists its increase in circumference as the domain grows by electrostatic effects on binding, eventually resulting in vesiculation. We propose that under favorable electrostatic conditions, as larger domains form the edge energy balances the increases in the electrostatic contribution to binding, resulting in a net binding energy independent of electrostatic factors.