Platelets, circulating tissue factor, and fibrin colocalize in ex vivo thrombi: real-time fluorescence images of thrombus formation and propagation under defined flow conditions

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Abstract
Although it is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of tissue factor (TF) to blood, there is still little agreement about the mechanisms of thrombus propagation and the identities of the molecular species participating in this process. In this study, we characterized the thrombotic process in real-time and under defined flow conditions to determine the relative contribution and spatial distribution of 3 components of the thrombi: circulating or blood-borne TF (cTF), fibrin, and platelets. For this purpose, we used high-sensitivity, multicolor immunofluorescence microscopy coupled with a laminar flow chamber. Freshly drawn blood, labeled with mepacrine (marker for platelets and white cells), anti-hTF1Alexa.568 (marker for tissue factor), and anti-T2G (marker for fibrin) was perfused over collagen-coated glass slides at wall shear rates of 100 and 650 s−1. A motorized filter cube selector facilitated imaging every 5 seconds at 1 of 3 different wavelengths, corresponding to optimal wavelengths for the 3 markers above. Real-time video recordings obtained during each of 10 discrete experiments show rapid deposition of platelets and fibrin onto collagen-coated glass. Overlay images of fluorescent markers corresponding to platelets, fibrin, and cTF clearly demonstrate colocalization of these 3 components in growing thrombi. These data further support our earlier observations that, in addition to TF present in the vessel wall, there is a pool of TF in circulating blood that contributes to the propagation of thrombosis at a site of vascular injury.