Temporal changes in distribution of VWF and fibrinogen in thrombi generated under high shear rate conditions.
Whole blood without mepacrine labeling from healthy controls was perfused over a collagen surface under a high shear rate (1500 s−1). Thrombi generated at several time points (1, 3, 5, and 7 minutes of perfusion) were fixed, double-stained with Cy3-labeled anti-VWF and Cy2-labeled antifibrinogen antibody, and viewed by CLSM. (A) Merged CLSM images were obtained by the superimposition of 2 images of the identical portion and slice of thrombi; orange or green colors indicate VWF and fibrinogen, respectively, within thrombi. (B) CLSM images displayed in rows 1, 2, and 3 (left) are cross-sections at a height of 30, 15, and 0 μm from the collagen surface, respectively (right; original magnifications, × 400). Images are representative of 3 independent perfusions using blood from 3 individual donors. Images in row 4 taken at time points corresponding to those of the upper rows in a real-time observation with epifluorescence microscopy are displayed as a reference, in which platelet thrombi are visualized by mepacrine green fluorescence (original magnification, × 200). Note the gradual accumulation of fibrinogen at the inner areas of thrombi, as they grow, whereas the outer areas, including the portions adjacent to the collagen surface, are constantly occupied by VWF.
