Fibrin scaffold structure and platelet fibrin/fibrinogen binding in CLSM studies.

CLSM images were taken 15 minutes after stimulation of diluted PRP with thrombin (1 U/ml) in order to analyze fibrin network formation in the presence of platelets. Fluorescence images of R-6G (red), which was loaded to platelets, are shown in the upper panel. Fluorescence images of fibrinogen labeled with Alexa Fluor 647 (white) are shown in the middle panel as either fibrin network or fibrin/fibrinogen binding to platelets surface. The merged figures are shown at the bottom panel. In the control essentially all the platelets were bound to fibrin network (white) and those surfaces were coated with either fibrin or fibrinogen (white), though some of them already released rhodamine-6G as a result of substantial activation. In the presence of FK633 (30 µM), though fibrin network formation was observed, platelets were neither bound to fibrin network nor coated with fibrin/fibrinogen. In contrary, in the presence of GPRP (3 mM), fibrin network was not formed but platelets were coated with fibrin/fibrinogen. In the presence of both FK633 and GPRP, neither fibrin network formation nor the binding of fibrin/fibrinogen to platelets were observed. Ultimately, in the presence of Cyt-B (100 µg/ml) fibrin mesh formation was not disturbed, however platelets were coated with fibrin/fibrinogen in a restricted manner only. Arrows indicate localizations of platelets within the fibrin mesh. Scale bar shows 10 µm.