Since Marx’s report [1], platelet-rich plasma (PRP) and subsequently modified PRP derivatives have been widely applied in regenerative dentistry. Unlike self-clotted platelet-rich fibrin (PRF), for better handling efficiency and minimizing the loss of growth factors to diffusion, PRP and some other derivatives in liquid form are usually clotted by addition of exogenous coagulation factors, such as thrombin and/or CaCl₂. For example, in the case of plasma-rich in growth factors (PRGF) (the most successful PRP derivative) [2], venipuncture is performed with anticoagulants, usually citrate or acid citrate dextrose (ACD), to chelate plasma Ca²⁺ [3]. Somewhat excessive amounts of Ca²⁺ are recommended for addition to citrated PRGF preparations to reconstitute plasma by recovering free Ca²⁺ levels on a watch glass at 37 °C [4, 5].

Behind this clot formation, there is the intrinsic coagulation pathway, which is activated at the level of factor XII by the glass surface and proceeds in the presence of Ca²⁺ to convert prothrombin to thrombin, subsequently fibrinogen to fibrin, and consequently facilitates fibrin polymerization and cross-linking [6]. In this process, thrombin converted from prothrombin is known to activate platelets via specific subtypes of protease-activated receptors [7, 8]. Therefore, it is likely that added CaCl₂ indirectly activates platelets through activation of a coagulation pathway in citrated PRP in glassware. The resulting fibrin fibers are thick and well cross-linked and are almost identical to those formed in a preparation of PRF [9].

In contrast to Ca²⁺, when an alternative coagulation factor, e.g., thrombin, is added to citrated PRP, the resulting fibrin fibers are thin and often fused together turning into a sheet-like matrix. Because this thrombin-induced fibrin matrix is relatively easily degradable [9], to stabilize its existence at an implantation site and to retain its growth factors longer, it is better to use Ca²⁺ as a coagulation factor and to employ glassware for activation of the intrinsic coagulation pathway. This approach is not limited to PRP and PRGF and can be extended to PRF preparation from stored whole-blood samples. Nonetheless, direct effects of exogenously added Ca²⁺ on platelets in vitro have been poorly investigated and understood.