It is well known that platelets are activated by adenosine diphosphate (ADP), thrombin, epinephrine, thromboxane A₂, collagen, and many other compounds and thus aggregate through binding of fibrinogen and glycoprotein IIb/IIIa receptors and upregulate surface antigens known as “platelet activation markers”: CD62P and CD63 [13, 14]. During activation, platelet morphology generally changes from a disc-shaped appearance (resting) to a rolling ball-shaped appearance, hemisphere-shaped appearance, and finally spreading adhesion appearance [15]. To our knowledge, however, increased extracellular free Ca²⁺ levels have not been sufficiently studied as an activator of platelets probably because free Ca²⁺ levels are maintained within some range, but not depleted, under pathological conditions of plasma and tissues.

Compared with the in vivo settings, citrated whole blood provides totally different conditions to platelets: extracellular free Ca²⁺ is chelated by citrate. The primary purpose of addition of Ca²⁺-chelating anticoagulants is to inhibit serial reactions of the coagulation pathway. Nevertheless, it is known that this chelation also suppresses various platelet functions related to aggregation [16]. Platelet activators mentioned above elevate intracellular Ca²⁺ concentrations, either by releasing Ca²⁺ from intracellular stores or by increasing Ca²⁺ influx across the plasma membrane [17]. Conversely, acute chelating of extracellular Ca²⁺ by EGTA inhibits the platelet ability to adhere to glass and to aggregate in response to ADP or other platelet activators [16].

The mechanism of Ca²⁺-induced platelet activation is discussed below. We preliminarily postulated expression of Ca²⁺-sensing receptors (CaSR) but failed to detect this type of receptor by the methods of flow cytometry and IF staining with a mouse monoclonal anti-CaSR antibody (cat. # ab19347; Abcam). No appreciable specific binding was observed in IF staining, and fewer than 5% of platelets tested positive in the flow cytometric analysis. Therefore, because this CaSR antigen is known to be expressed in the brain, kidneys, lungs, liver, heart, skeletal muscle, and placenta [18], but not in platelets, it is reasonable to rule out the participation of CaSR in Ca²⁺-induced platelet activation in our study.