Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse

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Abstract

We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. We observed platelet deposition, tissue factor accumulation and fibrin generation after laser-induced endothelial injury in a single developing thrombus. The initiation of blood coagulation in vivo entailed the initial accumulation of tissue factor on the upstream and thrombus–vessel wall interface of the developing thrombus. Subsequently tissue factor was associated with the interior of the thrombus. Tissue factor was biologically active, and was associated with fibrin generation within the thrombus.

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Figure 1: Imaging of platelets, fibrin and tissue factor in arterial thrombi using fluorescent antibodies.
Figure 2: Time course and localization of platelets and fibrin during thrombus formation.
Figure 3: Confocal fluorescence imaging of the distribution of tissue factor, platelets and fibrin in a thrombus.
Figure 4: Structural organization during thrombus development.
Figure 5: Schematic diagram of the confocal–widefield high-speed microscope used for real-time intravital microscopy of the microcirculation of a living mouse.

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Acknowledgements

We thank S. Inoué for introducing us to the Yokogawa CSU-10 high-speed confocal microscope, Eric Furie for digital image capture advice, and F. Castellino and E. Rosen for demonstrating the laser injury model. This work was supported by grants from the US National Institutes of Health (HL51926 and HL69435). The confocal microscope was obtained with partial support from the National Institutes of Health (S10RR15680).

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Correspondence to Bruce Furie.

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Falati, S., Gross, P., Merrill-Skoloff, G. et al. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse. Nat Med 8, 1175–1180 (2002) doi:10.1038/nm782

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