Novel mouse hemostasis model for real-time determination of bleeding time and hemostatic plug composition

Abstract

Introduction: Hemostasis is a rapid response by the body to stop bleeding at sites of vessel injury. Both platelets and fibrin are important for the formation of a hemostatic plug. Mice have been used to uncover the molecular mechanisms that regulate the activation of platelets and coagulation under physiologic conditions. However, measurements of hemostasis in mice are quite variable, and current methods do not quantify platelet adhesion or fibrin formation at the site of injury.

Methods: We describe a novel hemostasis model that uses intravital fluorescence microscopy to quantify platelet adhesion, fibrin formation and time to hemostatic plug formation in real time. Repeated vessel injuries of ~ 50-100 μm in diameter were induced with laser ablation technology in the saphenous vein of mice.

Results: Hemostasis in this model was strongly impaired in mice deficient in glycoprotein Ibα or talin-1, which are important regulators of platelet adhesiveness. In contrast, the time to hemostatic plug formation was only minimally affected in mice deficient in the extrinsic tissue factor (TF(low)) or the intrinsic factor IX coagulation pathways, even though platelet adhesion was significantly reduced. A partial reduction in platelet adhesiveness obtained with clopidogrel led to instability within the hemostatic plug, especially when combined with impaired coagulation in TF(low) mice.

Conclusions: In summary, we present a novel, highly sensitive method to quantify hemostatic plug formation in mice. On the basis of its sensitivity to platelet adhesion defects and its real-time imaging capability, we propose this model as an ideal tool with which to study the efficacy and safety of antiplatelet agents.

Keywords: blood coagulation; blood platelets; fluorescence imaging; hemostasis; mice.

Fig. 1

Quantitative analysis of platelet accumulation, fibrin generation and bleeding time after laser injury to the saphenous vein of wild-type mice. Mice were injected with Alexa647-labeled antibodies against fibrin and Alexa488-labeled antibodies against glycoprotein (GP)IX to monitor fibrin generation and platelet accumulation, respectively. Repeated vascular injury was induced by laser ablation. (A) Representative images at the indicated times after the start of the recording. (B, C) Accumulation of platelets (B) and fibrin (C). The sum fluorescence intensity (SFI) ± standard error of the mean is shown. Total number of injuries at distinct locations: n = 15 (obtained in four experimental mice). (D) Bleeding time. *P < 0.05.

Fig. 2

Contribution of platelets to hemostasis after laser injury. Vascular lesions were induced in wild-type (WT) (black line/symbols), talin1^f/fPF4-Cre (red) and IL4R/GPIb-tg (blue) mice. (A) Representative images. (B, C) Accumulation of platelets (B) and fibrin (C). Sum fluorescence intensity (SFI) ± standard error of the mean is shown. Talin1^f/fPF4-Cre: n = 9 (four mice). IL4R/GPIb-tg: n = 10 (four mice). (D) Bleeding time. **P < 0.001, ***P < 0.0001.

Fig. 3

Contribution of the extrinsic and intrinsic coagulation pathways to hemostasis after laser injury. Vascular lesions were induced in wild-type (WT) (black line/symbols), TF^low (red) and FIX^−/− (blue) mice. (A) Representative images. (B, C) Accumulation of platelets (B) and fibrin (C). Sum fluorescence intensity (SFI) ± standard error of the mean is shown. TF^low: n = 9 (three mice). FIX^−/−: n = 13 (three mice). (D) Bleeding time. *P < 0.05, **P < 0.001, ***P < 0.0001. GP, glycoprotein; TF, tissue factor.

Fig. 4

Effect of clopidogrel on hemostasis in wild-type (WT) and TF^low mice. WT and TF^low mice were treated with the P2Y12 inhibitor clopidogrel bisulfate prior to laser injury. (A) Representative images. (B–D) Platelet accumulation (B), fibrin accumulation (C) and bleeding time (D) in WT (black line/bars) and WT/clopidogrel (green) mice. (E–G) Platelet accumulation (E), fibrin accumulation (F) and bleeding time (G) in TF^low (red line/bars) or TF^low/clopidogrel (green) mice. Sum fluorescence intensity (SFI) ± standard error of the mean is shown. *P < 0.05, **P < 0.001, ***P < 0.0001. WT/clopidogrel: n = 10 (three mice). TF^low/clopidogrel: n = 10 (three mice). Numbers in bars indicate the incidence of repeated bleeding during the 5-min observation period following laser injury. The bleeding time was recorded when occlusion of the lesion lasted for > 15 s. GP, glycoprotein; TF, tissue factor.