Under normal physiologic conditions, platelets circulate in an inactivated state and require only minimal stimulation ensuring their immediate availability for hemostasis. Previous research has shown that during platelet pheresis and storage, platelets undergo early activation resulting in expression of the surface activation marker CD62, release of α granule contents, and a decrease in inducible activation. In addition to regulating vascular tone, nitric oxide (NO) groups in plasma may provide tonic inhibition to circulating platelets thereby preventing the inappropriate adhesion and aggregation seen in stored platelets. S-Nitrosothiols (RSNOs) may serve as a reservoir for nitric oxide (NO) in the normal circulation. We hypothesize that platelets are prematurely activated during the process of apheresis in part due to the lack of RSNOs in the microenvironment during collection and storage. Furthermore, RSNO metabolism by stored platelets is changed by the process of apheresis itself. To test this hypothesis, we monitored the activation status of platelets using CD62 flow cytometry and quantified intracellular NO content and uptake using the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA). Both measurements were made on freshly isolated platelets (Fl-Plts) and on apheresis platelets (A-Plts). In addition, we employed these techniques to evaluate the responsiveness of Fl-Plts and A-Plts to agonists [thrombin receptor activating peptide (TRAP), ADP, and collagen] and to NO-donating molecules after varying storage times. In preliminary studies we have found that Fl-Plts bind RSNOs and demonstrate steroselectivity in their degree of uptake. Fl-Plts incubated with L-RSNOs produce over 2-fold the fluorescence compared to Fl-Plts incubated with D-RSNOs. A-Plts lose this stereoselectivity in RSNO uptake following the collection and storage process. We have also found that A-Plts have up to a 10-fold increase in uptake of nitric oxide from RSNOs compared to Fl-Plts. This significant increase and loss of stereoselectivity in RSNO uptake in A-Plts correlates with the loss of inducible activation over storage time. These data suggest RSNO metabolism in platelets is fundamentally altered during apheresis, collection, and storage and provides a novel target with therapeutic potential for further investigation into the loss of platelet function during storage.