RESUMEN
We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.
Asunto(s)
Actinas/química , Dalteparina/química , Proteínas de Microfilamentos/química , Physarum/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/metabolismo , Northern Blotting , Western Blotting , Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Dalteparina/inmunología , Electroforesis en Gel de Poliacrilamida , Eliminación de Gen , Regulación de la Expresión Génica , Biblioteca de Genes , Glutatión Transferasa/metabolismo , Cinética , Proteínas de Microfilamentos/fisiología , Datos de Secuencia Molecular , Péptidos/química , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Quinasas Asociadas a Fase-S , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Treonina/química , Factores de TiempoRESUMEN
Delayed hypersensitivity to heparins and heparinoïd is a problem for prophylaxis of thrombo embolic diseases. The hirudins did not seem to have any cross-reactivity with the two others groups of anticoagulants. We present two clinical cases of delayed type reactions to heparins and heparinoïd and we reviewed the literature about adverse reactions to low molecular weight heparins and the alternative possibilities.
Asunto(s)
Hipersensibilidad a las Drogas/etiología , Heparina de Bajo-Peso-Molecular/efectos adversos , Heparina/efectos adversos , Hirudinas/análogos & derivados , Hipersensibilidad Tardía/inducido químicamente , Anciano , Anticoagulantes/efectos adversos , Anticoagulantes/inmunología , Sulfatos de Condroitina/efectos adversos , Sulfatos de Condroitina/inmunología , Reacciones Cruzadas , Dalteparina/efectos adversos , Dalteparina/inmunología , Dermatán Sulfato/efectos adversos , Dermatán Sulfato/inmunología , Combinación de Medicamentos , Enoxaparina/efectos adversos , Enoxaparina/inmunología , Femenino , Heparina/inmunología , Heparina de Bajo-Peso-Molecular/inmunología , Heparitina Sulfato/efectos adversos , Heparitina Sulfato/inmunología , Terapia con Hirudina , Humanos , Nadroparina/efectos adversos , Nadroparina/inmunología , Proteínas Recombinantes/uso terapéutico , Pruebas CutáneasRESUMEN
UNLABELLED: We investigated the efficacy, safety and relation of dose to plasma anti-Xa activity of the low molecular weight heparin (LMWH) dalteparin in prophylaxis and therapy of arterial and venous thrombosis in pediatric patients. A total of 48 children were enrolled: 10 received dalteparin for prophylaxis (group I), 8 for reocclusion prophylaxis following successful thrombolysis (group II), 5 following failed thrombolysis (group III) and 23 for primary antithrombotic therapy (group IV). Two children were treated with dalteparin for pulmonary veno-occlusive disease (PVOD) and for primary pulmonary hypertension (PPH), respectively. OUTCOME: In group I no thrombo-embolic event occurred. In group II recanalization was maintained or improved, in group III vascular occlusion persisted under dalteparin. In group IV we saw complete recanalization in 7/23 (30%), partial recanalization in 7/23 (30%) and no recanalization in 9/23 (40%) cases. The child with PVOD had recanalization proven by lung biopsy; the clinical condition of the child with PPH also improved. Minor bleeding occurred in 2/48 (4%) children. For prophylaxis 95 +/- 52 (mean and SD) anti-Xa IU/kg BW, for therapy 129 +/- 43 (mean and SD) anti-Xa IU/kg BW proved effective. For both prophylaxis and therapy the required dose per kg BW was inversely related with age (r2 = 0.64, P = 0.017; r2 = 0.13, P = 0.013). CONCLUSION: Dalteparin proved to be an effective and well tolerated drug for prophylaxis and therapy of thrombosis in pediatric patients. Dose requirement for effective treatment was higher in younger children and decreased with age.
Asunto(s)
Anticoagulantes/uso terapéutico , Dalteparina/uso terapéutico , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Adolescente , Factores de Edad , Anticoagulantes/inmunología , Niño , Preescolar , Dalteparina/inmunología , Relación Dosis-Respuesta a Droga , Factor Xa/inmunología , Femenino , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Lactante , Recién Nacido , Modelos Lineales , Masculino , Enfermedad Veno-Oclusiva Pulmonar/tratamiento farmacológico , Resultado del TratamientoRESUMEN
During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide-protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.