Use of flow cytomery to differentiate between activation and reactivity of blood platelets - methodological report

摘要

Amongst a plethora of methods, which enable us to monitor platelet function and response to activating agents, flow cytometry is the only one which ensures the possibility to reliably monitor both platelet activation occurring in circulation under in vivo conditions, and platelet reactivity as a function of the consumption and/or exhaustion of circulating platelets. In the present study we elaborated the experimental protocol which might serve to evaluate platelet function in specific clinical settings and in model scientific approaches. The extent of platelet activation was monitored using flow cytometry either immediately after blood withdrawal or under the following experimental conditions: a) in the course of the spontaneous anticoagulant-dependent platelect activation (static model), b) following platelet stimulation with various agonists (thrombin, TRAP, ADP), and c) under the simulated flow conditions (dynamic model). We verified the usefulness of the above protocol to distinguish between platelet activation and rectivity in the group of type 2 diabetic patients. Hypothesis: In patients with diabetes, as a result of platelet hypersensitivity, the circulating blood platelets undergo more frequent episodes of the release from platelet granule contents. Such an augmented relase may lead to the accelerated platelet consumption, the formation of platelet size gradient, and it contributes to the increased platelet turnover and the reduced platelet survival in diabetic individuals. Results: We showed that the enhanced activation of blood circulating platelets (the decreased expression of GPIbα) and the enhanced platelet consumption (the reduced expression of P-selectin in platelets activated ex vivo with agonists and the increased number of platelet microparticles), due to frequent platelet relase episodes, may result in their depressed reactivity and reduced responce to activating agents. Conclusions: All the employed model systems for the monitoring of platelet activation used inthe present study, i.e. the spontaneous time-driven, the agonist-induced, as well as the agitation-induced platelet activation, in the conjunction with flow cytometry technique appeared promising and might be recommended for the routine use in the investigation of platelet responsiveness and reactivity - both the parameters being greatly informative in clinical studies.