Skip to main content
. 2023 Sep 21;7(7):102209. doi: 10.1016/j.rpth.2023.102209

Figure 5.

Figure 5

Platelets are not necessary for the activation of the coagulation cascade and the generation of fibrin in vivo. (A) Representative images of the colocalization between platelets and fibrin (left panel) and fibrin and endothelium (right panel) in real-time laser scanning intravital confocal microscopy after laser-induced injury over time in wild-type mice. Left and right panels are independent experiments. Left panel: platelets (in red) are revealed by an antibody against the glycoprotein Ib (GPIb)β protein directly coupled to DyLight 649 (0.25 μg/g of the mouse), and the fibrin (in green) is visualized by an antibody directed against the fibrin labeled with a DyLight 488 (0.25 μg/g of the mouse). Right panel: the endothelium (in blue) is detected using an antibody against CD31 coupled to an AlexaFluor 647 (0.25 μg/g of the mouse) and the fibrin (in green) is visualized by an antibody directed against the fibrin labeled with a DyLight 488 (0.25 μg/g of the mouse). Antibodies are injected intravenously before performing the laser-induced injury and the thrombi are followed every 3 minutes for a total duration of 15 minutes. The colocalization of the 2 partners (platelet/fibrin or endothelium/fibrin) is shown in white. Scale bars: 10μm. (B) Quantitative analysis of colocalizations between fibrin and platelets and fibrin and endothelium. The graph represents the mean ± SEM of the percentages of colocalized pixels under these 2 conditions (fibrin/platelets or fibrin/endothelium at the final time point 15 minutes). Four mice per condition were analyzed and the significance was determined using an unpaired two-tailed Student’s t-test. (C) Representative images of the kinetics of fibrin generation by intravital microscopy in wild-type mice in the presence of platelets (NaCl, left panel), in mice with platelets that do not express P2Y12 receptor (ADP receptor) (P2Y12 KO, middle panel), and wild-type mice depleted in platelets with the antibody anti-GP1bα (R300, right panel). Fibrin represented in green was labeled with an anti-fibrin antibody coupled to a DyLight 488 (0.25 μg/g of the mouse). Platelets were labeled with an anti-GPIbβ antibody coupled to a DyLight 649 and administered in an amount of 0.25 μg/g of the mouse. Scale bars: 10μm. (D) Statistical analysis of the areas under the curve of integrated fluorescence intensities corresponding to the platelet accumulation (top panel) and fibrin generation (bottom panel). Data are shown as medians, and statistics were performed using a one-way analysis of variance with Mann–Whitney U-test at 95% CI. (NaCl: 45 thrombi in 4 mice; P2Y12 KO: 38 thrombi in 3 mice; and R300: 25 thrombi in 3 mice).