1- Serviço de Fisiologia e Bioquímica do Instituto Superior de Ciências da Saúde do Norte (ISCSN)

Most circulating "normal" platelets in a human organism, without cardiovascular lesions, are in a "resting" functional state. An heterogeneous set of stimuli may induce a transition from this "resting" to an "activated" state. Complex molecular changes are involved in this mechanism, at the platelet surface and at its interior, that determine known platelet answers: shape change, adhesion, aggregation, release reaction, procoagulant activity. During the secretion process [release reaction] intrinsic proteins of membranes, either of þ-granules (CD 62, 140 kD) or of lisossomes (CD 63, 53 kD) are translocated to the platelet surface. Therefore this two proteins may be used as markers of platelet activation. Monoclonal antibodies [Moab 2.15 (CD 62) and Moab 2.28 (CD 63), kindly supplied by HK Nieuwenhuis], recognising specifically both antigens, enabled us to detect and quantify the "in vivo" platelet activation and the "ex vivo" platelet reactivity by FCM. A secondary antibody conjugated with FITC was used and the analysis done on an EPICS C flow cytometer. We studied the "in vivo" activation and the "ex vivo" reactivity of platelets, in 10 normal persons, with no ingestion of any drugs, in the previous 15 days. Measurements of CD 62 and CD 63 expression, were obtained in the following three types of samples: 1) in platelets obtained from whole blood collected directly to anticoagulant solution (CPD) containing 1% paraformaldehyde (PFA) ("in vivo" activation). Mean frequency of platelets CD 62(+)=16.5% ñ 4.0% and CD 63(+)=17.7% ñ 7.8%. 2) in platelets obtained from whole blood anticoagulated with citrate (135 mM), washed (PBS with EDTA, 5 mM, pH=7.4) and then fixed with 1% PFA ("in vitro" activation induced by manipulation); in this group of measurements the mean frequency of platelets CD 62 (+) was 37.6% ± 15.4% and of platelets CD 63 (+) was 14.8% ± 7.8% 3) in washed platelets activated with thrombin (5 U/ml, 2 min., with stirring (1000 RPM), at 370 C) and then fixed with 1% PFA ("ex vivo" reactivity). The mean frequency of platelets CD 62 (+) was 82.4% ± 5.4% and of CD 62 (+) was 63.2% ± 15.4%. Presently, the main goal of our work is to obtain reference values for basal "in vivo" platelet activation and for "ex vivo" platelet reactivity. We plan to continue this clinical flow cytometric analysis hoping that it will provide a means to make timely therapeutic decisions based on measurements of platelet function.