Effect of a peptide antagonist of the F11 Receptor on platelet adhesion to the vascular walls
We examined various parameters of platelet adhesion to the vascular walls in mice in both in the arterioles and venules in the presence of a peptide antagonist of the F11 Receptor (peptide 4D), utilizing the intravital microscopy system. To induce an inflammatory phenotype of the vascular endothelium, mice were injected intraperitoneally with both murine recombinant TNF-alpha and murine recombinant IFN-gamma prior to the initiation of the measurement of platelet adhesion. The F11R peptide 4D was injected to the peptide-treated group of animals in 3 consecutive applications. The control, vehicle- treated animal group received injections of equal volumes of saline at each time point. At the time of measurements conducted to assess platelet adhesion to the vasculature, mice were anesthetized and injected with the platelet-specific fluorescent anti-GPIbβ antibody. Video imaging was carried out at the magnification of 200x for 40 s with an exposure time of 200 ms in at least three venules and at least three arterioles in each mouse. The analysis of platelets tethering to the endothelium cells in the recorded movies was performed with the use of TrackMate plugin that is implemented in FIJI software. Three parameters, each characterizing platelet interactions with the vascular wall, were calculated: 1. the time of interaction (referred to as the ‘adhesion time’), 2. the reciprocal distance (i.e. the distance in a given view field that was overcome by a rolling platelet), which may be regarded as a measure of an overall platelet ‘immobilization’ on endothelium integrated through time, and 3. the integrated measure of these two parameters, the so called reciprocal ‘velocity’ of a platelet sliding on the vessel wall (referred to as ‘adhesion time*distance-1’). These results demonstrate that the platelets were sliding with a higher velocity in the arterioles of the peptide-treated group and suggest that peptide 4D significantly inhibits platelets’ ability to interact with endothelial layer of arterioles (Representative video is shown in Film 1, 2 and 3). In contrast to the statistically-significant differences in platelet-arteriole wall interactions observed between the peptide 4D treated group and the nontreated group, we observed that peptide 4D treatment did not have an effect on the interaction times of platelets within venules (Representative video is shown in Film 4, 5 and 6). Data from intravital microscopy demonstrate that peptide 4D blocks the adhesion of platelets to inflamed arterioles suggesting a critical role of F11R in the adhesion of platelets to cytokine-inflamed endothelium.