Figure 3.
PF4-Actn1−/− mice exhibit reduced primary hemostasis and thrombosis. (A) The tail tips of the mice were amputated, and the mice were then immersed in saline. The tail transection bleeding time and bleeding volume were monitored in Actn1f/f and PF4-Actn1−/− mice (n = 20 mice per group). (B) Tail bleeding time (via the filter paper method) in Actn1f/f and PF4-Actn1−/− mice and the results of the statistical analysis are shown (n = 20 mice per group). (C) Bleeding volumes after a calibrated injury to the liver in Actn1f/f and PF4-Actn1−/− mice (n = 10 mice per group). (D) Relative quantification of the areas of bleeding in the brains of Actn1f/f and PF4-Actn1−/− mice (n = 9 mice per group). (E) Representative thromboelastography (TEG) tracings of whole blood from Actn1f/f and PF4-Actn1−/− mice. Analysis of the maximal amplitude (MA), reaction time, kinetics time, and α-angle in Actn1f/f and PF4-Actn1−/− blood via TEG (n = 6 mice per group). (F) Citrate-anticoagulated blood samples were obtained from Actn1f/f and PF4-Actn1−/− mice and subsequently transferred to collagen/ADP cartridges. The in vitro closure time (CT) was measured with a PFA-200 (n = 12 mice per group). (G) Thrombus formation of Actn1f/f and PF4-Actn1−/− platelets at shear rates of 500 or 1500 s-1. Citrate anticoagulant and recalcified whole blood were perfused at 500 or 1500 s-1 for 5 minutes. An Alexa Fluor 488–conjugated anti-CD41 antibody was used to label the platelets. Thrombus formation was observed and imaged under an inverted fluorescence microscope. The left panel shows representative images from 3 independent experiments of platelet thrombi. Arrows indicate the direction of blood flow. The scale bar is 100 μm. The right panel shows the quantitative data reflecting the percentage of surface coverage (n = 10 fields per group). The data are presented as the mean ± standard deviation (SD) from 10 randomly selected visual fields of at least 3 independent experiments. (H) Representative images of carotid artery blood flow in FeCl3-treated Actn1f/f and PF4-Actn1−/− mice obtained via laser speckle perfusion imaging (n = 12 mice per group). Blood flow was monitored for 20 minutes. (I) Representative traces of blood flow in mice with FeCl3-induced occlusive carotid artery thrombosis. (J) Quantitative analysis of the duration of complete vessel occlusion (n = 12 mice per group). The data are presented as the means ± SDs. (K) Laser injury–induced thrombus formation in the cremasteric arterioles of Actn1f/f and PF4-Actn1−/− mice (n = 5 mice per group). Platelet accumulation was visualized via intravital microscopy after laser injury using a DyLight 649–conjugated anti-GPIbβ (CD42c) antibody derivative. Representative images depicting platelet counts at the indicated time points after injury in Actn1f/f and PF4-Actn1−/− mice. The medium fluorescence intensities of the platelets over time were analyzed for all the images from the Actn1f/f and PF4-Actn1−/− mice. The area under the curve (AUC) for the platelets from each capture was plotted for the Actn1f/f and PF4-Actn1−/− mice (n = 30 captures per group). (L) Survival of Actn1f/f and PF4-Actn1−/− mice after induction of pulmonary thromboembolism via the injection of a collagen/epinephrine mixture through the tail vein (n = 14 mice per group). (M) Actn1f/f and PF4-Actn1−/− mice were intraperitoneally injected with carrageenan solution (1%, 110 μL per mouse). On days 2 and 3, thrombus length was measured in the carrageenan-induced thrombosis mice. The thrombosis rate (the ratio of tail length with thrombus to whole tail length) was calculated for the tails of carrageenan-induced thrombosis mice (n = 10 mice per group). (N) Venous thrombus formation in the deep venous thrombosis model. Thrombus formation in the inferior vena cava (IVC) was induced by partial vein ligation. Twenty-four hours after ligation of the IVC, thrombosis samples were collected to measure the weight and calculate the incidence of thrombus formation in Actn1f/f and PF4-Actn1−/− mice (n = 10 mice per group). ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. ns, not significant.

PF4-Actn1−/− mice exhibit reduced primary hemostasis and thrombosis. (A) The tail tips of the mice were amputated, and the mice were then immersed in saline. The tail transection bleeding time and bleeding volume were monitored in Actn1f/f and PF4-Actn1−/− mice (n = 20 mice per group). (B) Tail bleeding time (via the filter paper method) in Actn1f/f and PF4-Actn1−/− mice and the results of the statistical analysis are shown (n = 20 mice per group). (C) Bleeding volumes after a calibrated injury to the liver in Actn1f/f and PF4-Actn1−/− mice (n = 10 mice per group). (D) Relative quantification of the areas of bleeding in the brains of Actn1f/f and PF4-Actn1−/− mice (n = 9 mice per group). (E) Representative thromboelastography (TEG) tracings of whole blood from Actn1f/f and PF4-Actn1−/− mice. Analysis of the maximal amplitude (MA), reaction time, kinetics time, and α-angle in Actn1f/f and PF4-Actn1−/− blood via TEG (n = 6 mice per group). (F) Citrate-anticoagulated blood samples were obtained from Actn1f/f and PF4-Actn1−/− mice and subsequently transferred to collagen/ADP cartridges. The in vitro closure time (CT) was measured with a PFA-200 (n = 12 mice per group). (G) Thrombus formation of Actn1f/f and PF4-Actn1−/− platelets at shear rates of 500 or 1500 s-1. Citrate anticoagulant and recalcified whole blood were perfused at 500 or 1500 s-1 for 5 minutes. An Alexa Fluor 488–conjugated anti-CD41 antibody was used to label the platelets. Thrombus formation was observed and imaged under an inverted fluorescence microscope. The left panel shows representative images from 3 independent experiments of platelet thrombi. Arrows indicate the direction of blood flow. The scale bar is 100 μm. The right panel shows the quantitative data reflecting the percentage of surface coverage (n = 10 fields per group). The data are presented as the mean ± standard deviation (SD) from 10 randomly selected visual fields of at least 3 independent experiments. (H) Representative images of carotid artery blood flow in FeCl3-treated Actn1f/f and PF4-Actn1−/− mice obtained via laser speckle perfusion imaging (n = 12 mice per group). Blood flow was monitored for 20 minutes. (I) Representative traces of blood flow in mice with FeCl3-induced occlusive carotid artery thrombosis. (J) Quantitative analysis of the duration of complete vessel occlusion (n = 12 mice per group). The data are presented as the means ± SDs. (K) Laser injury–induced thrombus formation in the cremasteric arterioles of Actn1f/f and PF4-Actn1−/− mice (n = 5 mice per group). Platelet accumulation was visualized via intravital microscopy after laser injury using a DyLight 649–conjugated anti-GPIbβ (CD42c) antibody derivative. Representative images depicting platelet counts at the indicated time points after injury in Actn1f/f and PF4-Actn1−/− mice. The medium fluorescence intensities of the platelets over time were analyzed for all the images from the Actn1f/f and PF4-Actn1−/− mice. The area under the curve (AUC) for the platelets from each capture was plotted for the Actn1f/f and PF4-Actn1−/− mice (n = 30 captures per group). (L) Survival of Actn1f/f and PF4-Actn1−/− mice after induction of pulmonary thromboembolism via the injection of a collagen/epinephrine mixture through the tail vein (n = 14 mice per group). (M) Actn1f/f and PF4-Actn1−/− mice were intraperitoneally injected with carrageenan solution (1%, 110 μL per mouse). On days 2 and 3, thrombus length was measured in the carrageenan-induced thrombosis mice. The thrombosis rate (the ratio of tail length with thrombus to whole tail length) was calculated for the tails of carrageenan-induced thrombosis mice (n = 10 mice per group). (N) Venous thrombus formation in the deep venous thrombosis model. Thrombus formation in the inferior vena cava (IVC) was induced by partial vein ligation. Twenty-four hours after ligation of the IVC, thrombosis samples were collected to measure the weight and calculate the incidence of thrombus formation in Actn1f/f and PF4-Actn1−/− mice (n = 10 mice per group). ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. ns, not significant.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal