Morphological changes of (and microparticle release and fibrin formation by) Ca²⁺-stimulated platelets are shown in Fig. 1. When washed platelets were treated with 0.1% CaCl₂ in sample tubes and plated in culture dishes for the last 5 min of treatment (control, 2 min), platelets’ ability to adhere to the dish bottom surface increased with the duration of Ca²⁺ treatment (Fig. 1a). On the other hand, when washed platelets were treated with 0.1% CaCl₂ in sample tubes, immediately transferred onto percolated filters, and incubated for up to 15 min, the platelets were found to release microparticles and fibrinogen, which was converted to fibrin even in the absence of plasma components (Fig. 1b).

Changes in attachment area, optical thickness on average, and surface roughness of Ca²⁺-simulated platelets are shown in Fig. 2. In the control, i.e., resting platelets, the average thickness of almost all platelets was within 2 μm. Among the platelets stimulated by 0.1% Ca²⁺ for 15 min, approximately 50% of the cells increased their apparent thickness beyond 2 μm. Attachment area was also significantly enlarged by added Ca²⁺, but roughness was not changed. In this case, enlarged platelets in terms of both thickness and area definitely represent aggregated platelets, but, probably, thicker platelets also represent aggregated platelets.

Immunofluorescent (IF) evaluation of changes in surface marker expression in Ca²⁺-stimulated platelets is shown in Fig. 3. When washed platelets were treated with 0.1% CaCl₂ in sample tubes, immediately placed in culture dishes, and incubated for 15 min, CD62P and CD63, but not CD41, were substantially upregulated.

Flow cytometric analysis of changes in surface marker expression in Ca²⁺-stimulated platelets is presented in Fig. 4. When washed platelets were treated with 0.1% CaCl₂ in sample tubes for up to 30 min, CD62P⁺ platelet counts in CD41⁺ platelet populations increased time-dependently. Similarly, platelet volume, as assessed by impedance, increased with the duration of Ca²⁺ treatment.

Because these findings taken together implied that exogenously added Ca²⁺ beyond physiological in vivo levels (approximately 9.0 vs. 2.5 mM) directly activates platelets in the absence of plasma components, platelets’ direct involvement in coagulation was then examined using platelet-rich and platelet-poor plasma. Effects of CaCl₂ on fibrin clot formation on watch glasses and in polystyrene culture dishes are shown in Fig. 5. On watch glasses (panel a, control), addition of 0.1% CaCl₂ most rapidly formed loop-like substances (in a dashed-line circle) and subsequently fibrin clots in PRP at 8 min of treatment (Fig. 5a). Polystyrene culture dishes, whose surface is optimized for adherent cells, significantly delayed Ca²⁺-induced clot formation in PRP from 8 to 19 min (Fig. 5b). When the treatment was carried out in PPP, clot formation was also significantly delayed (Fig. 5c). Nonetheless, in the presence of the collagen sponge, Ca²⁺ addition caused formation of a fibrin clot in PRP in culture dishes as rapidly as in the control (Fig. 5d vs. a).