RESUMO
In patients with lupus anticoagulants (LA), acquired resistance to activated protein C (APC) is difficult to demonstrate with clot-based assays due to the presence of the anticoagulant. Via the conversion of a fluorogenic substrate (thrombinography), we monitored the complete process of thrombin formation and decay and its delimitation by the protein C system in eight consecutive LA-patients without anticoagulant therapy and non-carriers of the V Leiden polymorphism. Thrombin generation was triggered in platelet-poor and platelet-rich plasma by recalcification in the presence of a low concentration of tissue factor. In 7 out of 8 patients we observed a long lag-time before the thrombin burst (LA effect) together with a marked inability of APC to diminish the thrombin activity. The lag-phase was however prolonged to some degree by APC. The effects were more outspoken in the presence of phospholipids from patients' platelets than with added phospholipids. Thrombinography thus demonstrates APC resistance in LA-patients despite the occurrence of long lag-times (clotting times). The amount of thrombin activity generated in the presence of APC could be a better indicator of the thrombotic risk than the moment at which the thrombin burst starts.
Assuntos
Resistência à Proteína C Ativada/diagnóstico , Cumarínicos/análise , Corantes Fluorescentes/análise , Inibidor de Coagulação do Lúpus/análise , Oligopeptídeos/análise , Trombina/biossíntese , Coagulação Sanguínea/fisiologia , Plaquetas/química , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Transtornos Linfoproliferativos/sangue , Transtornos Linfoproliferativos/imunologia , Lipídeos de Membrana/sangue , Lipídeos de Membrana/imunologia , Lipídeos de Membrana/isolamento & purificação , Lipídeos de Membrana/farmacologia , Tempo de Tromboplastina Parcial , Fosfolipídeos/sangue , Fosfolipídeos/imunologia , Fosfolipídeos/isolamento & purificação , Fosfolipídeos/farmacologia , Contagem de Plaquetas , Trombina/análise , Fatores de TempoRESUMO
Activated platelets are implicated in the development of premature arterial vascular diseases, in particular ischemic stroke. Since elevated cytosolic [Ca(2+)](i) is an integrative marker of platelet activation, we determined the generation of Ca(2+) signal in stimulated platelets from 26 young patients recuperating from stroke, 20 patients with symptomatic peripheral arterial disease, and 56 healthy volunteers. Even in the presence of aspirin, the platelets from various individuals showed highly different thrombin-induced Ca(2+) responses. On average, the thrombin-induced Ca(2+) response was increased for platelets from either patient group in comparison to the controls (P <0.04). Relatively more stroke patients had high-responsive platelets (27%, 7/26) than patients with peripheral arterial disease (10%, 2/20) or healthy subjects (4%, 2/56). The average prothrombinase activities of platelets from patients and controls were similar, but 3 out of 6 patients with increased thrombin-induced Ca(2+) responses also exhibited high prothrombinase activity. In a follow-up study, the subject-dependent thrombin-induced Ca(2+) response was found to correlate strongly with the platelet response to protease-activated receptor 1 (PAR1) agonist (r = 0.91), but was not linked to the Pl(A1/2) polymorphism. It is concluded that a significant part of young patients with stroke have platelets with hyperactivity toward thrombin, which is not normalised by aspirin treatment. Furthermore, the subject-dependent variation in thrombin-induced signalling is likely to involve PAR1-mediated platelet activation.
