The A2 protein alters and is incorporated into the fibrin clot structure. (A) Representative of 3-dimensional confocal microscopy images of fibrin clots formed in whole blood from a healthy human donor mixed with either vehicle control or the A2 protein (0.5 μM) conjugated to Alexa Fluor 488 (purple). Fibrin was visualized by supplementing whole blood with 1% human fibrinogen conjugated to Alexa Fluor 647 (green). In comparison with the treatment of vehicle control, the A2 protein clearly modified the resultant fibrin structure. In addition, the A2 protein did not interact with the blood cells present in the mixture as compared with vehicle control containing dye only. Colocalization of fibrin and the A2 protein is viewed as a lighter green to white color in the right panel (red arrows). Scale bars, 10 μM. (B) A higher (300×) magnification demonstrates the incorporation of the A2 protein (top middle, purple) into the fibrin structure (top left, green). The white arrowheads point to the A2 protein bound to fibrin branching. In contrast, the A2 mutant did not increment the size of pores (bottom left, green) or formed clusters in fibrin branching (bottom middle, purple). Colocalization of fibrin and the A2 protein is viewed at 300× magnification (top right) as lighter magenta overlapping the fibrin fibers (as lighter green). In contrast, there is less colocalization or overlapping of the A2 mutant with the fibrin fibers (bottom right). Scale bars, 10 μM.