Figure 6.
The hemostatic potential of FVIIa-induced EVs. (A) Platelet depletion upon the administration of antiplatelet antibody (αCD42b Ab). WT mice were injected with CD42b antibodies (1 mg/kg) via the tail vein. Platelet counts in the blood were measured before the administration of the antibody and 5 hours following antibody administration. (B) Infusion of FVIIa-induced EVs to platelet-depleted mice reduces blood loss following saphenous vein injury. WT mice were injected with CD42b antibodies (1 mg/kg) via the tail vein to deplete platelets. Five hours following the administration of platelet-depleting antibodies, mice were injected with equal numbers of EVs (1 × 109/mouse) generated from endothelial cells treated with a control vehicle, FVIIa (100 nM), or saline. Immediately following the administration of EVs, mice were subjected to the saphenous vein incision, and the blood coming from the injury site was collected on filter paper. Hemoglobin was extracted from the filter paper, and values were extrapolated to the blood volume using a standard curve derived with known volumes of blood. (C) FVIIa-induced EVs support the generation of thrombin at the wound site in platelet-depleted mice. WT mice were depleted of platelets, administered with control or FVIIa-generated endothelial EVs, and subjected to saphenous vein incision as described for panel B. Blood coming from the wound site was collected periodically (up to 15 minutes; if a clot was formed, it was dislodged) directly into citrate anticoagulant, and thrombin generation was measured as the amount of TAT complexes formed in the blood. (D-H) FVIIa-generated EVs support FVIIa-induced hemostasis in hemophilia mice. FVIII−/− mice were injected with saline or EVs derived from endothelial cells treated with a control vehicle or FVIIa (1.5 × 109/mouse). Immediately following EVs, mice were administered with saline or FVIIa (0.25 mg/kg), a dose well below the threshold of FVIIa needed to correct the bleeding in itself, and subjected to the saphenous vein incision. The number of hemostatic plugs formed in a 30-minute interval was recorded (D), and the average time to achieve hemostasis (E) and blood loss (F) were determined. (G) TAT levels in the blood collected at the wound site. (H) TAT levels in the blood collected via the submandibular vein. (I) Effect of endogenously generated EVs on thrombin generation at the wound site. FVIII−/− mice were treated with a control vehicle control or rFVIIa (0.25 mg/kg) to generate EVs endogenously. After 2 hours, mice were subjected to saphenous vein incision with or without giving a second dose of rFVIIa (0.25 mg/kg) immediately prior to saphenous vein incision. Blood leaking from the wound site was collected, and levels of TAT complexes were determined as described in panel G. (J) Recruitment of FVIIa-generated EVs to the wound site. Equal numbers (1.5 × 109 EVs) of PKH67-labeled (green fluorescence) EVs derived from murine b.END3 endothelial cells treated with a control vehicle or FVIIa were injected into FVIII−/− mice via the tail vein, followed by FVIIa (0.25 mg/kg) administration. Mice were subjected to saphenous vein incision. After 5 minutes, the injured saphenous vein was excised, fixed, and processed for sectioning. Sections were stained for fibrin using antibodies against murine fibrinogen (red), and nuclei were stained with DAPI (blue) and analyzed by confocal microscopy (original magnification ×63 with 2.4× zoom) . *P < .05; **P < .01; ***P < .001; ****P < .0001.

The hemostatic potential of FVIIa-induced EVs. (A) Platelet depletion upon the administration of antiplatelet antibody (αCD42b Ab). WT mice were injected with CD42b antibodies (1 mg/kg) via the tail vein. Platelet counts in the blood were measured before the administration of the antibody and 5 hours following antibody administration. (B) Infusion of FVIIa-induced EVs to platelet-depleted mice reduces blood loss following saphenous vein injury. WT mice were injected with CD42b antibodies (1 mg/kg) via the tail vein to deplete platelets. Five hours following the administration of platelet-depleting antibodies, mice were injected with equal numbers of EVs (1 × 109/mouse) generated from endothelial cells treated with a control vehicle, FVIIa (100 nM), or saline. Immediately following the administration of EVs, mice were subjected to the saphenous vein incision, and the blood coming from the injury site was collected on filter paper. Hemoglobin was extracted from the filter paper, and values were extrapolated to the blood volume using a standard curve derived with known volumes of blood. (C) FVIIa-induced EVs support the generation of thrombin at the wound site in platelet-depleted mice. WT mice were depleted of platelets, administered with control or FVIIa-generated endothelial EVs, and subjected to saphenous vein incision as described for panel B. Blood coming from the wound site was collected periodically (up to 15 minutes; if a clot was formed, it was dislodged) directly into citrate anticoagulant, and thrombin generation was measured as the amount of TAT complexes formed in the blood. (D-H) FVIIa-generated EVs support FVIIa-induced hemostasis in hemophilia mice. FVIII−/− mice were injected with saline or EVs derived from endothelial cells treated with a control vehicle or FVIIa (1.5 × 109/mouse). Immediately following EVs, mice were administered with saline or FVIIa (0.25 mg/kg), a dose well below the threshold of FVIIa needed to correct the bleeding in itself, and subjected to the saphenous vein incision. The number of hemostatic plugs formed in a 30-minute interval was recorded (D), and the average time to achieve hemostasis (E) and blood loss (F) were determined. (G) TAT levels in the blood collected at the wound site. (H) TAT levels in the blood collected via the submandibular vein. (I) Effect of endogenously generated EVs on thrombin generation at the wound site. FVIII−/− mice were treated with a control vehicle control or rFVIIa (0.25 mg/kg) to generate EVs endogenously. After 2 hours, mice were subjected to saphenous vein incision with or without giving a second dose of rFVIIa (0.25 mg/kg) immediately prior to saphenous vein incision. Blood leaking from the wound site was collected, and levels of TAT complexes were determined as described in panel G. (J) Recruitment of FVIIa-generated EVs to the wound site. Equal numbers (1.5 × 109 EVs) of PKH67-labeled (green fluorescence) EVs derived from murine b.END3 endothelial cells treated with a control vehicle or FVIIa were injected into FVIII−/− mice via the tail vein, followed by FVIIa (0.25 mg/kg) administration. Mice were subjected to saphenous vein incision. After 5 minutes, the injured saphenous vein was excised, fixed, and processed for sectioning. Sections were stained for fibrin using antibodies against murine fibrinogen (red), and nuclei were stained with DAPI (blue) and analyzed by confocal microscopy (original magnification ×63 with 2.4× zoom) . *P < .05; **P < .01; ***P < .001; ****P < .0001.

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