Utility of Platelet Endothelial Cell Adhesion Molecule 1 in the Platelet Activity Assessment in Mouse and Human Blood

Abstract

In our previous study, we introduced the platelet endothelial cell adhesion molecule 1 (PECAM-1)/thrombus ratio, which is a parameter indicating the proportion of PECAM-1 in laser-induced thrombi in mice. Because PECAM-1 is an antithrombotic molecule, the higher the PECAM-1/thrombus ratio, the less activated the platelets. In this study, we used an extracorporeal model of thrombosis (flow chamber model) to verify its usefulness in the assessment of the PECAM-1/thrombus ratio in animal and human studies. Using the lipopolysaccharide (LPS)-induced inflammation model, we also evaluated whether the PECAM-1/thrombus ratio determined in the flow chamber (without endothelium) differed from that calculated in laser-induced thrombosis (with endothelium). We observed that acetylsalicylic acid (ASA) decreased the area of the thrombus while increasing the PECAM-1/thrombus ratio in healthy mice and humans in a dose-dependent manner. In LPS-treated mice, the PECAM-1/thrombus ratio decreased as the dose of ASA increased in both thrombosis models, but the direction of change in the thrombus area was inconsistent. Our study demonstrates that the PECAM-1/thrombus ratio can more accurately describe the platelet activation status than commonly used parameters such as the thrombus area, and, hence, it can be used in both human and animal studies.

Keywords: PECAM-1; inflammation; platelet; thrombosis.

Figure 1

Effect of ASA (intravital) on (a) thrombus area and (b) PECAM-1/thrombus ratio in mouse mesentery vein. (c) Kinetics of thrombus formation at the site of laser injury. (d) Original, representative confocal microscopy images of merged channels (green, thrombus; red, PECAM-1) and thrombus (green) 10 s after thrombosis induction (bar = 10 µm; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. VEH; ^## p < 0.01, ^### p < 0.001 vs. LPS; ^^ p < 0.01, ^^^ p < 0.001 vs. LPS + ASA 3 mg/kg; n = 7−8). Data are shown as mean ± SEM.

Figure 2

Effect of ASA (ex vivo) on (a) thrombus area and (b) PECAM-1/thrombus ratio in mice without inflammation (control mice) and on (c) thrombus area and (d) PECAM-1/thrombus ratio in mice with inflammation (LPS-treated mice). (e) Top panel, control mice; bottom panel, LPS-treated mice; original, representative confocal microscopy images of merged channels (green, thrombus; red, PECAM-1, top row in each panel) and thrombus (green, bottom row in each panel) (bar = 10 µm; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. VEH; ^# p < 0.05, ^### p < 0.001 vs. LPS; ^^ p < 0.01, ^^^ p < 0.001 vs. LPS + ASA 3 mg/kg; ^$$$ p < 0.001 vs. LPS + ASA 10 mg/kg; n = 9−13). Data are shown as mean ± SEM.

Figure 3

Effect of ASA on BT in LPS-treated mice (* p < 0.05, *** p < 0.001 vs. VEH; ^### p < 0.001 vs. LPS; n = 6). Data are shown as median (interquartile range).

Figure 4

Effect of ASA (in vitro) on (a) thrombus area and (b) PECAM-1/thrombus ratio in the experiment with human blood. (c) Original, representative confocal microscopy images of merged channels (green, thrombus; red, PECAM-1) and thrombus (green) (bar = 10 µm; * p < 0.05, *** p < 0.001 vs. VEH; ^ p < 0.05, ^^^ p < 0.001 vs. ASA 0.18 µg/mL; ^$$$ p < 0.001 vs. ASA 1.8 µg/mL; n = 7). Data are shown as mean ± SEM and as median (interquartile range).