Calcium Mobilization in Freeze-Dried Human Platelets

In previous studies, we reported on a freeze-drying protocol for human platelets, using trehalose as the main lyophilization protectant. Using this protocol, we investigated calcium mobilization in rehydrated platelets upon stimulation with thrombin and the ability of rehydrated platelets to bind fibrinogen. Calcium mobilization was measured with a calcium dye, Indo-1, and the binding of fibrinogen was measured using fibrinogen conjugated with Oregon Green. Rehydrated platelets showed elevated levels of intra-cellular calcium, but that level was still below the threshold required for activation. In addition, the calcium mobilization upon stimulation with thrombin was reduced compared to that of fresh control platelets. Both populations of platelets demonstrated a dosedependent saturation of calcium mobilization when stimulated with thrombin. The diminished calcium mobilization upon stimulation with thrombin did not affect the ability of rehydrated platelets to bind fibrinogen, which suggests that freeze-dried platelets bind fibrinogen in a clinically relevant manner. Despite somewhat reduced intra- cellular calcium, freeze-dried rehydrated platelets have adequate calcium to upregulate their integrins and bind fibrinogen. Flow cytometry demonstrated that rehydrated platelets contained virtually the identical number of copies of the major platelet integrin αIIbβ3a as fresh platelets. We have demonstrated that freeze-dried rehydrated platelets maintain intra-cellular calcium levels required for long-term storage in the dry state and subsequent platelet transfusion and activation in vivo.