An alternative possibility may be related to the status of platelets stored in a low-Ca²⁺ environment: platelets constantly increase thromboxane A₂ (TXA₂) production and tend to easily aggregate under the influence of the TXA₂ autocrine loop [19]. Therefore, it is thought that platelets prepared from citrated whole-blood samples continuously repeat Ca²⁺ discharge from intracellular Ca²⁺ stores and Ca²⁺ efflux across the plasma membrane [20] in response to endogenously produced TXA₂. In addition, even though the autocrine loop of TXA₂ does not function actively, intracellular free Ca²⁺ levels of platelets (~ 100 nM at actual resting levels) are maintained also by a balance between the “passive” leak of Ca²⁺ into platelets and the concurrent efflux of Ca²⁺ across the plasma membrane and accumulation in intracellular stores [21]. Therefore, in a citrated medium, it is plausible that intracellular free Ca²⁺ in platelets is depleted gradually by repeated Ca²⁺ discharge from intracellular stores and passive and active Ca²⁺ flux across the plasma membrane. We can speculate that Ca²⁺ addition may promptly enable Ca²⁺ entry via the Ca²⁺ leak pathway and subsequently activate platelets by the autocrine pathway of TXA₂ or activators stored in α-granules, such as ADP and thrombin. In support of this possibility, Aoki et al. demonstrated that added Ca²⁺ increases thrombin production by washed platelets [22]. Further investigation is needed to clarify the mechanism of Ca²⁺-induced platelet activation.

The mechanism of Ca²⁺-induced clot formation is worthier of discussion. In the human body, two types of thrombosis are known: white and red thrombi [23]. According to this definition, a fibrin mesh is deposited on platelet aggregates in a white thrombus, whereas platelets (and red blood cells) are trapped and aggregated by the fibrin mesh in a red thrombus. In this study, we found that platelet aggregates function like nuclei of clot formation. Therefore, platelets are located mainly near the center or in a deep region of a clot, and this clot may be classified essentially as a “white thrombus.” In this case, growth factors stored in platelets can be assumed to be retained for a relatively long time. It is possible that this type of clot functions as a long-lasting carrier with a better regenerative potential.