The in vitro haemostatic functions of fresh whole blood (FWB) are well preserved after cold storage. This study aimed to determine whether platelets derived from FWB and stored whole blood (SWB) contribute to clot formation in tissue injury after transfusion into coagulopathic rats with polytrauma/haemorrhage (T/H). The rats were resuscitated 1 h after trauma with FWB or SWB collected from green fluorescence protein (GFP) transgenic rats. After transfusion, a liver incision was made and the tissue was collected 10 min after injury to identify GFP platelets by immunohistochemistry. In comparison to FWB, platelet aggregation to adenosine diphosphate and protease-activated receptor-4 was reduced by 35% and 20%, and clotting time was shortened by 25% in SWB. After transfusion, SWB led to a significant increase in platelet activation as measured by an elevation of CD62P and phosphatidylserine expression. The platelets from SWB were in a higher activation state, and showed higher clearance rate and formation of platelet-leucocyte aggregates than those from FWB after transfusion. Platelets from both FWB and SWB were equivalently incorporated into the clot at the incisional site, as determined by co-localization of CD61 and GFP. This study suggests that SWB contributes to haemostatic function and is an effective alternative resource to treat trauma patients.