RESUMEN
BACKGROUND: Historically, von Willebrand factor (VWF) activity assays utilized ristocetin despite limitations including poor limits of detection and high imprecision. Newer VWF activity assays such as the INNOVANCE® VWF Ac assay, however, do not rely on ristocetin to measure platelet-dependent VWF function. The purpose of this study was to evaluate the analytical and clinical performance of the Siemens Healthineers INNOVANCE VWF Ac Assay on the Siemens BCS® XP and the Sysmex® CS-2500 systems in a large reference laboratory setting. METHODS: Performance indicators for the INNOVANCE VWF Ac assay were the limit of quantitation (LoQ), precision, and method comparison. Method comparison studies were performed using remnant plasma patient samples from routine coagulation tests and analyzed using both the INNOVANCE VWF Ac assay and the Siemens Healthineers ristocetin-dependent BC von Willebrand Reagent. RESULTS: Evaluation of the INNOVANCE VWF Ac assay on the BCS® XP and CS-2500 systems demonstrated good precision and a lower LoQ compared to the BC von Willebrand Reagent. Method comparisons support the use of the INNOVANCE VWF Ac assay on the BCS® XP and CS-2500 systems to measure platelet-dependent VWF function. The INNOVANCE VWF Ac assay was able to further assist in von Willebrand disease classification in 6/7 (86%) samples when the result was below the LoQ for the BC von Willebrand Reagent (ristocetin cofactor activity). CONCLUSIONS: These data are consistent with the 2021 American Society of Hematology/International Society on Thrombosis and Haemostasis/National Hemophilia Foundation/World Federation of Hemophilia von Willebrand disease guidelines that suggest using newer assays such as the INNOVANCE VWF Ac assay in place of ristocetin cofactor activity assays.
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Ristocetina , Factor de von Willebrand , Humanos , Factor de von Willebrand/análisis , Pruebas de Coagulación Sanguínea/instrumentación , Pruebas de Coagulación Sanguínea/métodos , Pruebas de Coagulación Sanguínea/normas , Ristocetina/farmacología , Reproducibilidad de los Resultados , Enfermedades de von Willebrand/sangre , Enfermedades de von Willebrand/diagnóstico , Automatización de Laboratorios/instrumentación , Límite de DetecciónRESUMEN
BACKGROUND: One of the most complex risk factors for the laboratory assessment of thrombophilia is Protein S (PS). The testing algorithm for PS employs the plasma-based assays of free PS antigen, total PS antigen, and PS activity creating a complex diagnostic scheme that can lead to misdiagnosis if incorrectly used, and a potential waste of resources and money. CONTENT: This paper compares the recently published evidence-based algorithm from the International Society for Hemostasis and Thrombosis (ISTH) with several commonly performed nonevidence-based testing schemes, to demonstrate the efficiency of the evidence-based algorithm for diagnostic efficiency with improved patient care and increased cost savings for the laboratory. SUMMARY: Significant savings (31%-60%) can be realized when the evidence-based algorithm is used in place of other testing modalities of initial PS activity testing (31%) or testing with all 3 assays simultaneously (60%). This study utilizing the PS testing evidence-based algorithm as part of a thrombophilia evaluation demonstrates that the appropriate testing methods can be used to limit wasteful practices while achieving the maximum level of information in this time of limited resources and need for increase monetary savings.
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Proteína S , Trombofilia , Algoritmos , Análisis Costo-Beneficio , Humanos , Proteína S/metabolismo , Factores de Riesgo , Trombofilia/diagnóstico , Trombofilia/etiología , Trombofilia/metabolismoRESUMEN
BACKGROUND: Indoleamine-2,3-dioxygenase (IDO) catalyzes the first step of tryptophan (Trp) catabolism, yielding kynurenine (Kyn) metabolites. The kynurenine-to-tryptophan (K/T) ratio is used as a surrogate for biological IDO enzyme activity. IDO expression is increased during Escherichia coli urinary tract infection (UTI). Thus, our objective was to develop a method for measurement of Kyn/Trp ratio in human blood and urine and evaluate its use as a biomarker of UTI. METHODS: A mass spectrometric method was developed to measure Trp and Kyn in serum and urine specimens. The method was applied to clinical urine specimens from symptomatic pediatric patients with laboratory-confirmed UTI or other acute conditions and from healthy controls. RESULTS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was linear to 500⯵mol/L for both Trp and Kyn. Imprecision ranged from 5 to 15% for Trp and 6-20% for Kyn. Analytical recoveries of Trp and Kyn ranged from 96 to 119% in serum and 90-97% in urine. No correlation was found between the K/T ratio and circulating IDO mass (râ¯=â¯0.110) in serum. Urinary Kyn and Trp in the pediatric test cohort demonstrated elevations in the K/T ratio in symptomatic patients with UTI (median 13.08) and without UTI (median 14.38) compared to healthy controls (median 4.93; pâ¯<â¯0.001 for both comparisons). No significant difference in K/T ratio was noted between symptomatic patients with and without UTI (pâ¯=â¯0.84). CONCLUSIONS: Measurement of Trp and Kyn by LC-MS/MS is accurate and precise in serum and urine specimens. While urinary K/T ratio is not a specific biomarker for UTI, it may represent a general indicator of a systemic inflammatory process.
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Quinurenina/orina , Triptófano/orina , Infecciones Urinarias/orina , Algoritmos , Biomarcadores/sangre , Biomarcadores/orina , Cromatografía Líquida de Alta Presión , Estudios de Cohortes , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/sangre , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Lactante , Quinurenina/sangre , Quinurenina/química , Quinurenina/metabolismo , Masculino , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Triptófano/sangre , Triptófano/química , Triptófano/metabolismo , Infecciones Urinarias/diagnóstico , Infecciones Urinarias/inmunología , Infecciones Urinarias/metabolismoRESUMEN
BACKGROUND: The ability to perform quantitative hCG testing in whole blood at the point-of-care is desirable. The purpose of this study was to perform an analytical validation of the Abbott i-STAT Total ß-hCG test. METHODS: Whole blood, plasma, and serum samples were prepared by the addition of hCG and were used to evaluate precision, linearity, analytical sensitivity, accuracy, the high-dose hook effect, and dilution recovery. RESULTS: Imprecision was highest with whole blood (CV = 16.0% and 6.7% at 10 and 1184 IU/l, respectively) and lowest in serum (CV = 8.1% and 4.3% at 11 and 1305 IU/l, respectively). The limits-of-quantitation were 8 and <5 IU/l for whole blood and both plasma and serum, respectively. The assay was linear between 5 and 2000 IU/l in all sample types (R(2) ≥ 0.998). i-STAT results agreed most closely with the Architect Total ß-hCG assay and with greater differences observed with Beckman DxI Total ßhCG and Roche Cobas e601 hCG+ß assays (mean differences across all sample types were 9.3% and 12.3%, respectively). A high-dose hook effect was observed at concentrations > 400,000 IU/l. Accuracy was achieved in samples diluted with serum but not saline. CONCLUSIONS: The i-STAT Total ß-hCG test demonstrates acceptable performance for quantifying hCG in whole blood, plasma and serum.
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Gonadotropina Coriónica/sangre , Inmunoensayo/normas , Pruebas de Embarazo/normas , Adulto , Calibración , Femenino , Humanos , Límite de Detección , Sistemas de Atención de Punto , Valores de Referencia , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: Measurement of human chorionic gonadotropin (hCG) and α-fetoprotein (AFP) in cerebrospinal fluid (CSF) can aid in the diagnosis of germ cell tumors (GCTs). Matrix effects can influence test results when alternative sample types are used, therefore, alternative sample types should always be validated before clinical use. Here we have validated the Advia® Centaur total hCG and AFP methods for use with CSF. We also performed a retrospective review of 5years of CSF hCG and AFP measurements sent out from our institution. DESIGN AND METHODS: Both hCG and AFP concentrations were measured using the ADVIA Centaur® total hCG or AFP assay. RESULTS: The Centaur hCG and AFP assays, performed on CSF, had intra- and inter-assay imprecisions <10.2% CV. The assays were linear over a dynamic range of 10-1000IU/L for hCG and 10-1000µg/L for AFP. Retrospective chart review confirmed that GCTs have a male predominance and are diagnosed most frequently in the second decade of life. The data also illustrate the importance of measuring both serum and CSF concentrations as CSF can be elevated in the absence of serum elevations. CONCLUSIONS: The Centaur total hCG and AFP methods accurately quantify hCG and AFP in CSF.
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Gonadotropina Coriónica/líquido cefalorraquídeo , alfa-Fetoproteínas/líquido cefalorraquídeo , Adolescente , Adulto , Femenino , Humanos , Masculino , Neoplasias de Células Germinales y Embrionarias/líquido cefalorraquídeo , Neoplasias de Células Germinales y Embrionarias/diagnóstico , Estudios Retrospectivos , Adulto JovenRESUMEN
BACKGROUND: Maintaining appropriate glycemic control in critically ill patients reduces morbidity and mortality. The use of point-of-care (POC) glucose devices is necessary to obtain rapid results at the patient's bedside. However, the devices should be thoroughly tested in the intended population before implementation. The use of POC glucose meters in critically ill patients has been questioned both in the literature and by regulatory agencies. The aim of this study was to determine if the ACCU-CHEK® Inform II system (Roche Diagnostics) POC glucose meter demonstrated the desired accuracy and precision, as defined by Clinical and Laboratory Standards Institute guideline POCT12-A3, in a large number of critically ill patients from multiple intensive care settings at two academic medical centers. METHODS: A total of 1200 whole blood meter results from 600 patients were compared with central laboratory plasma values. Whole blood aliquots from venous samples were used to obtain duplicate meter results with the remaining sample being processed to obtain plasma for central laboratory testing within 5 min of meter testing. RESULTS: A total of 1185 (98.8%) of the new meter's glucose values were within ± 12.5% (± 12 mg/dl for values ≥ 100 mg/dl) of the comparative laboratory glucose values, and 1198 (99.8%) were within ± 20% (± 20 mg/dl for values <100 mg/dl). CONCLUSIONS: Considering the large number of patients from numerous critical care units examined, the new glucose meter system appears to have sufficient analytic accuracy for use in critically ill patients.
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Glucemia/análisis , Hiperglucemia/diagnóstico , Sistemas de Atención de Punto , Cuidados Críticos/métodos , Humanos , Unidades de Cuidados Intensivos , Sistemas de Atención de Punto/normasRESUMEN
After neoplastic cells leave the primary tumor and circulate, they may extravasate from the vasculature and colonize tissues to form metastases. ß1 integrins play diverse roles in tumorigenesis and tumor progression, including extravasation. In blood cells, activation of ß1 integrins can be regulated by "inside-out" signals leading to extravasation from the circulation into tissues. However, a role for inside-out ß1 activation in tumor cell metastasis is uncertain. Here we show that ß1 integrin activation promotes tumor metastasis and that activated ß1 integrin may serve as a biomarker of metastatic human melanoma. To determine whether ß1 integrin activation can influence tumor cell metastasis, the ß1 integrin subunit in melanoma and breast cancer cell lines was stably knocked down with shRNA and replaced with wild-type or constitutively-active ß1. When tumor cells expressing constitutively-active ß1 integrins were injected intravenously into chick embryos or mice, they demonstrated increased colonization of the liver when compared to cells expressing wild-type ß1 integrins. Rescue expression with mutant ß1 integrins revealed that tumor cell extravasation and hepatic colonization required extracellular ligand binding to ß1 as well as ß1 interaction with talin, an intracellular mediator of integrin activation by the Rap1 GTPase. Furthermore, shRNA-mediated knock down of talin reduced hepatic colonization by tumor cells expressing wild-type ß1, but not constitutively-active ß1. Overexpression in tumor cells of the tumor suppressor, Rap1GAP, inhibited Rap1 and ß1 integrin activation as well as hepatic colonization. Using an antibody that detects activated ß1 integrin, we found higher levels of activated ß1 integrins in human metastatic melanomas compared to primary melanomas, suggesting that activated ß1 integrin may serve as a biomarker of invasive tumor cells. Altogether, these studies establish that inside-out activation of ß1 integrins promotes tumor cell extravasation and colonization, suggesting diagnostic and therapeutic approaches for targeting of ß1 integrin signaling in neoplasia.
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Integrina beta1/metabolismo , Metástasis de la Neoplasia , Neoplasias/patología , Línea Celular Tumoral , Humanos , Neoplasias/metabolismoRESUMEN
Several studies over the last years have demonstrated that statins exhibit actions beyond that of lipid-lowering (pleiotropic effects) ranging from improving endothelial function, modulating the inflammatory response, maintaining plaque stability and preventing thrombus formation. Since the interplay among platelets, cells and other components of atherosclerotic lesions as well as the coagulation system play an important role in the progression of atherosclerosis and in the development of acute coronary syndromes, these non-lipid properties of statins may help to explain the early and significant reduction of cardiovascular events reported in several clinical trials of statin therapy. This review focuses on the experimental and clinical results regarding the antiplatelet/antithrombotic effects of statins.
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Antitrombinas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Humanos , Hipercolesterolemia/fisiopatologíaRESUMEN
Activation of the platelet integrin-receptor alpha(IIb)beta(3) is the final pathway of platelet aggregation, regardless of the initiating stimulus. Many studies suggest that there are several cytoplasmic proteins such as talin and beta(3)-endonexin that bind to N(744)PLY(747) and N(756)ITY(759) motif of the beta(3) cytoplasmic tail and play the major role in the receptor activation. In this study, we investigated the role of the membrane distal region of human beta(3) cytoplasmic tail and specifically the N(743)NPLYKEA(750) and T(755)NITYRGT(762) sequence that contains an NXXY motif, in platelet aggregation, secretion, alpha(IIb)beta(3) activation (PAC-1 binding) and fibrinogen binding. We synthesized two peptides corresponding to the above sequences as well as their conjugates with the Tat(48-60) cell-penetrating peptide. The capability of conjugates to penetrate the platelet membrane was investigated with confocal laser scanning microscopy using carboxyfluorescein (CF)-labeled peptides. Our results showed that the conjugated with the Tat(48-60) sequence peptides penetrate the platelet membrane and inhibit platelet aggregation in both PRP and washed platelets in a dose-dependent manner. The Tat-beta(3)743-750 conjugate exhibited similar inhibitory activity in PRP and in washed platelets whereas the Tat-beta(3)755-762 conjugate was more potent inhibitor of aggregation in washed platelets than in PRP. Both conjugated peptides were also able to inhibit P-selectin membrane expression as well as PAC-1 and fibrinogen binding to the platelets, the Tat-beta(3)755-762 conjugate being more potent than Tat-beta(3)743-750. The Tat(48-60) peptide and the peptides beta(3)743-750 and beta(3)755-762, which were not conjugated to the Tat(48-60) sequence, did not exhibit any inhibitory effect on the above parameters. In conclusion, the present study shows for the first time that the peptide analogs of the intracellular domain of the beta(3) subunit beta(3)743-750 and beta(3)755-762 conjugated to the cell-penetrating peptide Tat(48-60) are capable of penetrating the platelet membrane and expressing biological activity by inhibiting the activation of alpha(IIb)beta(3), the fibrinogen binding to the activated receptor as well as platelet aggregation. Further studies are necessary to support whether such conjugated peptides may be useful tools for the development of potent antiplatelet agents acting intracellularly through the platelet integrin alpha(IIb)beta(3).
Asunto(s)
Plaquetas , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Secuencia de Aminoácidos , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Fibrinógeno/metabolismo , Humanos , Datos de Secuencia Molecular , Selectina-P/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Péptidos/genética , Péptidos/metabolismo , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Transducción de Señal/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) catalyzes release of arachidonic acid from glycerophospholipids, leading to thromboxane A(2) (TxA(2)) production. Some platelet agonists stimulate cPLA(2)alpha, but others require fibrinogen binding to alphaIIbbeta3 to elicit TxA(2). Therefore, relationships between cPLA(2)alpha and alphaIIbbeta3 were examined. cPLA(2)alpha and a cPLA(2)alpha binding partner, vimentin, coimmunoprecipitated with alphaIIbbeta3 from platelets, independent of fibrinogen binding. Studies with purified proteins and with recombinant proteins expressed in CHO cells determined that the interaction between cPLA(2)alpha and alphaIIbbeta3 was indirect and was dependent on the alphaIIb and beta3 cytoplasmic tails. Fibrinogen binding to alphaIIbbeta3 caused an increase in integrin-associated cPLA(2)alpha activity in normal platelets, but not in cPLA(2)alpha-deficient mouse platelets or in human platelets treated with pyrrophenone, a cPLA(2)alpha inhibitor. cPLA(2)alpha activation downstream of alphaIIbbeta3 had functional consequences for platelets in that it was required for fibrinogen-dependent recruitment of activated protein kinase Cbeta to the alphaIIbbeta3 complex and for platelet spreading. Thus, cPLA(2)alpha and alphaIIbbeta3 interact to reinforce each other's functions during alphaIIbbeta3 signaling. This provides a plausible explanation for the role of alphaIIbbeta3 in TxA(2) formation and in the defective hemostatic function of mouse or human platelets deficient in cPLA(2)alpha.
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Plaquetas/enzimología , Fosfolipasas A2 Grupo IV/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal/fisiología , Tromboxano A2/biosíntesis , Animales , Plaquetas/citología , Células CHO , Cricetinae , Cricetulus , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fibrinógeno/genética , Fibrinógeno/metabolismo , Glicerofosfolípidos/genética , Glicerofosfolípidos/metabolismo , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Fosfolipasas A2 Grupo IV/genética , Humanos , Ratones , Ratones Noqueados , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Pirrolidinas/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Tromboxano A2/genética , Vimentina/genética , Vimentina/metabolismoRESUMEN
The platelet integrin receptor alphaIIbbeta3 plays a critical role in thrombosis and haemostasis by mediating interactions between platelets and several ligands, primarily fibrinogen. We have previously shown that the synthetic peptide YMESRADRKLAEVGRVYLFL corresponding to residues 313-332 of alphaIIb, is a potent inhibitor of platelet aggregation and fibrinogen binding to alphaIIbbeta3, interacting with fibrinogen rather than the receptor. Furthermore, we have demonstrated that the biological activities of the above peptide are due to the sequence YMESRADR, which corresponds to residues 313-320. By using new synthetic peptide analogues we investigated the structural characteristics responsible for the biological activity of YMESRADR as well the possible influence of the adjacent amino acids on the peptide's biological potency. According to our results, the synthetic octapeptide YMESRADR, is a potent inhibitor of platelet aggregation and P-selectin expression. Furthermore, YMESRADR inhibits fibrinogen binding but it does not significantly influence the binding of PAC-1 to ADP-activated platelets. The inhibitory potency of YMESRADR was gradually diminished by deleting the YMES sequence from the amino terminus and prolonging the carboxyl terminus of this peptide with the KLAE sequence. Extension of YMESRADR towards the amino terminus with the GAPL sequence (GAPLYMESRADR) does not modify the biological activity of YMESRADR. Furthermore, extension of GAPLYMESRADR at its carboxy terminus with the KLAE sequence (GAPLYMESRADRKLAE) significantly diminished its biological potency. Substitution of E315 with D significantly enhances antiaggregatory potency and completely abolishes the inhibitory effect on P-selectin expression. Importantly, the D315-containing peptides inhibit to a similar extent both fibrinogen and PAC-1 binding to activated alphaIIbbeta3 in contrast to the E315-containing peptide which only inhibits fibrinogen binding. In conclusion, the present study suggests that the YMESRADR sequence 313-320 of alphaIIb, is an important functional region of the insert connecting the beta2 and beta3 antiparallel beta-strands of the W5 blade of the alphaIIb subunit. Structural changes significantly modify the biological properties of this region.
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Fibrinógeno/metabolismo , Oligopéptidos/metabolismo , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Citometría de Flujo , Humanos , Oligopéptidos/síntesis química , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Unión ProteicaRESUMEN
OBJECTIVE: Platelet-activating factor acetylhydrolase (PAF-AH) expresses a Ca2+-independent phospholipase A2 activity and hydrolyzes platelet-activating factor as well as oxidized phospholipids. Two major types of PAF-AH have been described: the plasma type, which is associated with lipoproteins, and the intracellular type II PAF-AH. METHODS AND RESULTS: We investigated the type(s) of PAF-AH expressed in human platelets as well as the mechanism and the enzyme type secreted from platelets during activation. The majority of the enzyme activity (75.1+/-14.3% of total) is found in the cytosol, whereas 24.9+/-7.3% is associated with the membranes. Immunofluorescence microscopy studies and Western blotting analysis showed that platelets contain the plasma type as well as the intracellular type II PAF-AH. Furthermore, platelets contain high levels of the mRNA of plasma PAF-AH, whereas only a small quantity of the type II PAF-AH mRNA was detected. On activation, platelets secrete the plasma type of PAF-AH mainly associated with platelet-derived microparticles (PMPs). The enzyme activity was also detected on circulating PMPs in plasma from normolipidemic healthy subjects. CONCLUSIONS: This is the first indication that in addition to lipoproteins, PAF-AH in human plasma is carried by PMPs, suggesting that the PMP-associated PAF-AH may play a role in the dissemination of biological activities mediated by these particles.
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1-Alquil-2-acetilglicerofosfocolina Esterasa/sangre , Plaquetas/enzimología , Plaquetas/ultraestructura , Membranas Intracelulares/enzimología , Membranas Intracelulares/ultraestructura , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Citosol/enzimología , Humanos , Isoenzimas/sangre , Isoenzimas/genética , Tamaño de la Partícula , Fosfolipasas A2 , Activación Plaquetaria , ARN Mensajero/sangreRESUMEN
The antiplatelet potency of clopidogrel may be attenuated by short-term co-administration of lipophilic statins metabolized through the cytochrome P-450, isoform 3A4. We investigated whether the co-administration of atorvastatin (20?mg/day) for 5 weeks, in patients with acute coronary syndromes (ACS) could affect the antiplatelet activity of clopidogrel. Fifty-one patients with the first episode of an ACS were included in the study. All patients underwent percutaneous coronary intervention (PCI) and received a loading dose of 375 mg of clopidogrel, followed by 75 mg/day for at least 3 months. Twenty-six of them presented with low density lipoprotein (LDL) cholesterol levels >100?mg/dl (2.6 mmol/l) (measured within 24 h from the onset of symptoms) and received daily 20 mg/day of atorvastatin. The ADP- or TRAP-induced platelet aggregation, as well as P-selectin and CD40L surface expression, were studied at baseline (within 30 min after admission) and 5 weeks afterwards. Atorvastatin did not influence either the clopidogrel-induced inhibition of platelet aggregation initiated by 5 or 10 microM ADP or the clopidogrel-induced reduction of the membrane expression of P-selectin and CD40L induced by ADP. In conclusion, atorvastatin, even at a dose of 20 mg/day does not affect the antiplatelet efficacy of clopidogrel when co-administered for 5 weeks in ACS patients.
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Adenosina Difosfato/antagonistas & inhibidores , Anticolesterolemiantes/farmacología , Plaquetas/efectos de los fármacos , Enfermedad Coronaria/tratamiento farmacológico , Ácidos Heptanoicos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Pirroles/farmacología , Ticlopidina/análogos & derivados , Enfermedad Aguda , Adenosina Difosfato/farmacología , Anciano , Angioplastia Coronaria con Balón , Anticolesterolemiantes/administración & dosificación , Atorvastatina , Plaquetas/metabolismo , Ligando de CD40/biosíntesis , Ligando de CD40/sangre , Colesterol/sangre , LDL-Colesterol/sangre , Clopidogrel , Enfermedad Coronaria/sangre , Enfermedad Coronaria/terapia , Esquema de Medicación , Interacciones Farmacológicas , Femenino , Citometría de Flujo , Ácidos Heptanoicos/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Selectina-P/biosíntesis , Selectina-P/sangre , Fragmentos de Péptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Pirroles/administración & dosificación , Ticlopidina/administración & dosificación , Ticlopidina/farmacología , Triglicéridos/sangreRESUMEN
Lipoprotein(a) [Lp(a)] may be an independent risk factor for coronary artery disease (CAD). Lp(a) is enriched in platelet activating factor acetylhydrolase (PAF-AH), an enzyme which hydrolyzes and inactivates platelet activating factor (PAF) and oxidized phospholipids that are implicated in atherogenesis. We determined the mass and catalytic properties of the Lp(a)-associated PAF-AH in 28 CAD patients in relation to the LDL-associated enzyme ones. Results were then compared to those of 30 control subjects and 16 unrelated patients with primary hypercholesterolemia (Type IIA dyslipidemia) before and after atorvastatin therapy. The mass, the specific activity and kinetic constants of the Lp(a)-associated PAF-AH were significantly lower in CAD patients compared to those of either controls or hypercholesterolemic patients, a phenomenon not observed for LDL-associated PAF-AH. The enzyme specific activity and kinetic constants were significantly increased after removal of apo(a) from Lp(a) by reductive cleavage, which was not found in the control population, suggesting that the apo(a) moiety of Lp(a) from CAD patients may play an important role in the observed lower catalytic efficiency of PAF-AH. The reduced PAF-AH mass and specific activity on Lp(a) is a feature characteristic of this lipoprotein in CAD patients and may lead to a diminished capability of Lp(a) to degrade proinflammatory phospholipids. The consequences of this phenomenon as regards the pathophysiological role of Lp(a) in atherosclerosis remain to be established.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Enfermedad de la Arteria Coronaria/enzimología , Lipoproteína(a)/metabolismo , Lipoproteínas LDL/metabolismo , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: Lipoprotein (a) [Lp(a)] is considered an atherogenic lipoprotein, which is also implicated in thrombosis. Lp(a) binds to platelets and modulates the effects of various platelet agonists. Platelet activating factor (PAF) is a potent platelet agonist degraded and inactivated by PAF-acetylhydrolase (PAF-AH), which in plasma is associated with lipoproteins. Lp(a) is enriched in PAF-AH, thus a functional characteristic of this lipoprotein is its capability to hydrolyze and inactivate PAF. In the present study, we investigated the effect of Lp(a) on PAF-induced platelet activation. The potential roles of the apo(a) moiety and especially of the PAF-AH content of Lp(a) on the above effect were also addressed. METHODS: Lp(a) was isolated by affinity chromatography from plasma of apparently healthy fasting donors with serum Lp(a) concentrations >/=20 mg/dl. Reduced Lp(a) [Lp(a-)] was prepared by incubation of Lp(a) with dithiothreitol (DTT), whereas inactivation of Lp(a)-associated PAF-AH was performed by incubation of Lp(a) with pefabloc [pefa-Lp(a)]. Platelet-rich plasma (PRP) or washed platelets were prepared from peripheral venous blood samples of normolipidemic, apparently healthy fasting donors with their serum Lp(a) levels lower than 0.8 mg/dl. The surface expression of the platelet integrin-receptor alpha(IIb)beta3 and the fibrinogen binding to the activated alpha(IIb)beta3 was studied by flow cytometry. RESULTS: Lp(a), at doses higher than 20 microg/ml, inhibits PAF-induced platelet activation in a dose-dependent manner. Pefa-Lp(a), lacking PAF-AH activity, exhibited a similar to Lp(a) inhibitory effect. Importantly, the Lp(a) inhibitory effect was not influenced by the apo(a) isoform size, whereas Lp(a-) was a more potent inhibitor compared to Lp(a). Similarly to PAF, Lp(a) inhibits platelet aggregation induced by ADP or Calcium ionophore A23187. Lp(a), pefa-Lp(a) or Lp(a-) effectively inhibited PAF- or ADP-induced surface expression of alphaIIbbeta3, the Lp(a-) being more potent compared to Lp(a) or to pefa-Lp(a). Finally, Lp(a) significantly inhibited fibrinogen binding to platelets activated with PAF. CONCLUSIONS: Lp(a) inhibits PAF-induced platelet activation in a non-specific manner. The Lp(a)-associated PAF-AH does not play any important role in this effect, whereas the apo(a) moiety of Lp(a) significantly reduces its inhibitory effect. The inhibition of alpha(IIb)beta3 activation and fibrinogen binding to the activated platelets may represent the major mechanism by which Lp(a) inhibits PAF-induced platelet aggregation.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Lipoproteína(a)/farmacología , Factor de Activación Plaquetaria/metabolismo , Activación Plaquetaria/efectos de los fármacos , Plaquetas/efectos de los fármacos , Depresión Química , Fibrinógeno/metabolismo , Humanos , Técnicas In VitroRESUMEN
BACKGROUND: The antiplatelet effect of clopidogrel may be attenuated by short-term coadministration of lipophilic statins. We investigated whether the coadministration of atorvastatin for 5 weeks in patients with acute coronary syndromes (ACS) could affect the antiplatelet potency of clopidogrel. METHODS AND RESULTS: Forty-five hypercholesterolemic patients with the first episode of an ACS were included in the study. Patients were randomized to receive daily either 10 mg of atorvastatin (n=21) or 40 mg of pravastatin (n=24). Thirty patients who underwent percutaneous coronary intervention (PCI) received a loading dose of 375 mg of clopidogrel, followed by 75 mg/d for at least 3 months. In the remaining 15 patients who refused to undergo PCI, clopidogrel therapy was not administered. Eight normolipidemic patients with the first episode of an ACS were also included and received only clopidogrel. The serum levels of soluble CD40L and the adenosine 5'-diphosphate- or thrombin receptor activating peptide-14-induced platelet aggregation, as well as P-selectin and CD40L surface expression, were studied at baseline (within 30 minutes after admission) and 5 weeks later. Neither atorvastatin nor pravastatin significantly influenced the clopidogrel-induced inhibition of platelet activation, nor did clopidogrel influence the therapeutic efficacy of atorvastatin. CONCLUSIONS: Atorvastatin does not affect the antiplatelet potency of clopidogrel when coadministered for 5 weeks in ACS patients.
Asunto(s)
Enfermedad Coronaria/tratamiento farmacológico , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Pirroles/farmacología , Ticlopidina/análogos & derivados , Ticlopidina/farmacología , Enfermedad Aguda , Adenosina Difosfato/farmacología , Anciano , Angioplastia Coronaria con Balón , Atorvastatina , Ligando de CD40/sangre , Clopidogrel , Terapia Combinada , Enfermedad Coronaria/sangre , Enfermedad Coronaria/cirugía , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Esquema de Medicación , Interacciones Farmacológicas , Femenino , Ácidos Heptanoicos/administración & dosificación , Ácidos Heptanoicos/farmacocinética , Ácidos Heptanoicos/uso terapéutico , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Inactivación Metabólica , Masculino , Persona de Mediana Edad , Selectina-P/sangre , Fragmentos de Péptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/farmacocinética , Inhibidores de Agregación Plaquetaria/uso terapéutico , Pravastatina/administración & dosificación , Pravastatina/farmacología , Pravastatina/uso terapéutico , Pirroles/administración & dosificación , Pirroles/farmacocinética , Pirroles/uso terapéutico , Stents , Ticlopidina/administración & dosificación , Ticlopidina/antagonistas & inhibidores , Ticlopidina/farmacocinética , Ticlopidina/uso terapéutico , Resultado del TratamientoRESUMEN
The platelet integrin receptor alphaIIbbeta3 plays a critical role in thrombosis and haemostasis by mediating interactions between platelets and several ligands but primarily fibrinogen. It has been shown previously that the YMESRADR KLAEVGRVYLFL (313-332) sequence of the alphaIIb subunit plays an important role in platelet activation, fibrinogen binding and alphaIIbbeta3-mediated outside-in signalling. Furthermore, we recently showed that the 20-residue peptide (20-mer) alphaIIb 313-332, is a potent inhibitor of platelet aggregation and fibrinogen binding to alphaIIbbeta3, interacting with fibrinogen rather than the receptor. In an effort to determine the sequence and the minimum length required for the biological activity of the above 20-mer, we synthesized seven octapeptides, each overlapping by six residues, covering the entire sequence and studied their effect on platelet activation as well as fibrinogen binding to activated platelets. We show for the first time that octapeptides containing the RAD sequence are capable of inhibiting platelet aggregation and secretion as well as fibrinogen binding to the activated alphaIIbbeta3, possibly interacting with the ligand rather than the receptor. This suggests that the RAD sequence, common to all the inhibitory peptides, is critical for their biological activity. However, the presence of the YMES sequence, adjacent to RAD, significantly increases the peptide's biological potency. The development of such inhibitors derived from the 313-332 region of the alphaIIb subunit may be advantageous against the RGD-like antagonists as they could inhibit platelet activation without interacting with alphaIIbbeta3, thus failing to further induce alphaIIbbeta3-mediated outside-in signalling.
Asunto(s)
Fibrinógeno/metabolismo , Oligopéptidos/genética , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/farmacología , Adenosina Difosfato/farmacología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Colágeno/farmacología , Relación Dosis-Respuesta a Droga , Fosfatasa 2 de Especificidad Dual , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Proteína Fosfatasa 2 , Proteínas Tirosina Fosfatasas/metabolismo , Trombina/farmacologíaRESUMEN
alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.