The surface microstructures of the formed fibrin clots are depicted in Fig. 6. The loop-like substances that initially formed in PRP on watch glasses were composed of abundant aggregated platelets, and relatively smaller amounts of fibrin fibers were deposited around platelet aggregates (Fig. 6a). By contrast, in the final version of fibrin clots, platelet aggregates were hardly detectable and most parts consisted of fibrin fibers (Fig. 6b). Compared with the control clots, those formed in culture dishes were enriched in platelets although either thickness or cross-link density of fibrin fibers was apparently similar to that in the control clots (Fig. 6c). Fibrin clots formed from PPP on watch glasses and from PRP in culture dishes with collagen sponges were similar to the control clots.

Finally, effects of storage time on coagulation activity and platelet functions were examined. Effects of Ca²⁺ on prothrombin time and clot formation of stored whole-blood samples are shown in Fig. 7a, b. Because we preliminarily confirmed that reconstitution of citrated blood with CaCl₂ can recover the conditions applicable to the prothrombin time assay, we evaluated prothrombin time of citrated whole-blood samples stored for up to 6 days. Prothrombin time increased with the duration of storage. Effects of Ca²⁺ on CD62P and CD63 expression in platelets isolated from citrated whole-blood samples stored for up to 6 days are presented in Fig. 7c, d. The responsiveness to Ca²⁺ in terms of both CD62P and CD63 at 15 min seemed to somewhat decrease with storage time; however, even after 6-day storage, platelets maintained their response to added Ca²⁺: upregulation of CD62P and CD63 (vs. control levels).