Atomic force microscopy of fibrin networks and plasma clots during fibrinolysis

We have used atomic force microscopy (AFM) in order to study the ultrastruc ture of fibrin fibre dissolution in real time. Thin purified fibrin gels an d plasma clots were prepared on glass surfaces and overlaid with isotonic s aline or heparinized plasma in an AFM fluid-cell. Fibrinolysis was initiate d by introducing plasmin or recombinant tissue-type plasminogen activator ( rt-PA) into the solution bathing the clots. Microscopy was performed serial ly in real time on the Nanoscope III Atomic Force Microscope operating in t he tapping or contact mode. The acquisition time for a single image was 2-8 min and the clots were imaged for up to 1 h with fields of view ranging fr om 128 x 128 mu m to 0.7 x 0.7 mu m with a resolution of 512 x 512 pixels. In the smallest fields of view fibrin fibres were seen to be composed of gl obules 40-70 nm in diameter. The diameter of composite fibrin fibres in pur ified gels depended on the concentration of NaCl in the fibrinogen solution : 250 +/- 155 nm in 150 mmol/l NaCl vs. 1.42 +/- 0.19 mu m in 50 mmol/l NaC l. Plasma clots were composed of thick fibres with interspersed thinner fib res. In clots from platelet-rich plasma both the thick and the thin fibres had significantly smaller diameters than the corresponding fibre types in c lots from platelet-depleted plasma (620 +/- 195 nm vs. 965 +/- 200 nm, and 195 +/- 30 nm vs. 260 +/- 60 nm, P < 0.001 for both comparisons). Fibrinoly sis of both thick and thin fibres proceeded predominantly by lateral sectio n of the whole fibre thickness at a given site, regardless of whether it wa s initiated by plasmin or by rt-PA. The time to complete fibre section by 2 .5 U/ml of plasmin did not differ between thin and thick fibrin fibres (7.6 +/- 3.7 min vs. 6.4 +/- 4.2 min). With a low concentration of plasmin (0.1 7 U/ml) some fibrin fibres became thinner along their entire observed lengt h before they were cleaved. The rate of fibre thinning was S-times faster i n the thicker fibres than in the thinner ones. We conclude that the 'cut-th rough' pattern is the predominant way of fibrinolysis in purified gels and in plasma clots, and that proteolysis leading towards fibre cleavage procee ds more efficiently in thick than in thin composite fibrin fibres. (C) 2000 Harcourt Publishers Ltd.