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.2020 Jul 23;5(14):e139437.
doi: 10.1172/jci.insight.139437.

Intravascular hemolysis triggers ADP-mediated generation of platelet-rich thrombi in precapillary pulmonary arterioles

Affiliations

Intravascular hemolysis triggers ADP-mediated generation of platelet-rich thrombi in precapillary pulmonary arterioles

Tomasz Brzoska et al. JCI Insight..

Abstract

Patients with hereditary or acquired hemolytic anemias have a high risk of developing in situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice to assess the pathogenesis of pulmonary thrombosis following deionized water-induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbβ3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravascular ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet P2Y12 purinergic receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to our knowledge to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation, leading to thrombosis in the precapillary pulmonary arterioles, and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis-triggered pulmonary thrombosis.

Keywords: Mouse models; Platelets; Pulmonology; Thrombosis; Vascular Biology.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Intravascular hemolysis triggers acute pulmonary thrombosis in mice.
WT mice were intravascularly (IV) administered with 150 μL dH2O (n = 7 mice) to induce acute hemolysis and pulmonary circulation was imaged using quantitative fluorescence intravital lung microscopy (qFILM). (A) qFILM images of the same field of view (FOV) at 7 different time points are shown. t = 0 seconds (s) corresponds to time point before IV dH2O administration and other displayed time points are relative to IV dH2O. Pulmonary thrombosis was absent at t = 0 s. Following 150 μL dH2O, platelet-rich thrombi (white arrowheads) sequestered in the pulmonary arteriole (t = 10 s). By t = 15 s, the thrombi were trapped in the arteriolar bottlenecks causing local impairments in blood flow. Pulmonary thrombosis started to resolve by t = 23 s and completely resolved by t = 2 minutes. Data are representative of 7 independent experiments. Platelets are shown in green and pulmonary microcirculation in purple. Asterisks denote alveoli. Dotted ellipses denote arteriolar bottlenecks. White arrow mark the direction of blood flow within the feeding arteriole. The diameter of the shown arteriole is 29 μm. Scale bar: 50 μm. Complete qFILM time series corresponding toA is shown in Supplemental Video 2. (B) Pulmonary thrombi area plotted as a function of time showing changes in the total area of platelet-rich thrombi following 150 μL IV dH2O. Red arrow indicates pulmonary thrombi max area.
Figure 2
Figure 2. Acute hemolysis–induced pulmonary thrombosis is αIIbβ3-dependent.
(A) WT mice were intravascularly (IV) administrated with 150 μL dH2O to trigger acute hemolysis with or without IV administration of 10 mg/kg αIIbβ3 inhibitor (eptifibatide) 15 minutes before IV dH2O. Pulmonary circulation was imaged using quantitative fluorescence intravital lung microscopy (qFILM). qFILM images of the same field of view (FOV) at 6 different time points are shown to assess the effect of 10 mg/kg IV eptifibatide on the development of 150 μL IV dH2O–dependent pulmonary thrombosis. t = 0 seconds (s) corresponds to time point before and t > 0 s correspond to time points immediately following IV dH2O administration. Pulmonary thrombosis was absent at t = 0 s. dH2O failed to evoke pulmonary thrombosis in mouse pretreated with eptifibatide. Platelets (green) and pulmonary microcirculation (purple). Asterisks denote alveoli. White arrow marks the direction of blood flow within the feeding arteriole. The diameter of the shown arteriole is 30 μm. Scale bar: 50 μm. Also refer to Supplemental Video 3. (B) Pulmonary thrombi area plotted as a function of time for the FOV shown inA. Red arrow indicates pulmonary thrombi maximum area. (C andD) Pulmonary thrombi max area and AUC in mice with (n = 3 mice) or without (n = 7 mice) pretreatment with 10 mg/kg IV eptifibatide before 150 μL IV dH2O. Pulmonary thrombi max area and AUC were estimated as described in Methods. Pulmonary thrombi max area and AUC were compared using Wilcoxon-Mann-Whitney test. Data are shown as mean ± SEM. *P < 0.05 when comparing with and without 10 mg/kg IV eptifibatide pretreatment.
Figure 3
Figure 3. Thrombin triggers protracted and lethal pulmonary thrombosis in mice.
WT mice were administered IV with 250 U/kg (n = 5 mice) or 500 U/kg (n = 4 mice) thrombin, and pulmonary circulation was imaged using qFILM. (A andB) qFILM images of the same FOV at different time points are shown. t = 0 seconds (s) corresponds to time point before and t > 0 s correspond to time points following IV thrombin administration. Pulmonary thrombosis was absent at t = 0 s. Platelets are shown in green and pulmonary microcirculation in purple. (A) Following 250 U/kg IV thrombin, small (<500 μm2) and medium (500–1000 μm2) platelet-rich thrombi (white arrowheads) sequestered in the pulmonary arteriole (t = 10–12 s) and obstructed blood flow (t = 20 s). (B) Following 500 U/kg IV thrombin, small (<500 μm2) and medium (500–1000 μm2) platelet-rich thrombi (white arrowheads) sequestered in the pulmonary arteriole to occlude the arteriolar bottlenecks. The mouse died by t = 3 minutes, leading to arrest of pulmonary blood flow, which was evident by the reduced intensity of vascular dye (purple fluorescence) and stationary erythrocytes (Supplemental Video 5). Asterisks denote alveoli. White arrow mark the direction of blood flow. The diameters of the arterioles shown inA andB are 39 μm and 44 μm, respectively. Complete qFILM time series corresponding toA andB are shown in Supplemental Videos 4 and 5, respectively. (C andD) Pulmonary thrombi area plotted as a function of time following 250 U/kg (C) and 500 U/kg (D) IV thrombin within FOVs shown inA andB, respectively. Red and black arrows indicate pulmonary thrombi maximum area values and the time of mouse death following 500 U/kg IV thrombin, respectively. (E) Survival rate during qFILM experiments in WT mice IV administered with either 250 U/kg (n = 5 mice) or 500 U/kg (n = 4) thrombin (P = 0.046, log-rank test). (F) Pulmonary thrombi max areas in mice following 250 U/kg (n = 5 mice) and 500 U/kg (n = 4 mice) IV thrombin. Pulmonary thrombi max areas were compared using Wilcoxon-Mann-Whitney test. Data are shown as mean ± SEM. (G) Three-dimensional qFILM image of a lethal pulmonary thrombosis developed within a large pulmonary arteriole (57 μm) of a mouse administered with 500 U/kg IV thrombin. Platelets (green) and pulmonary microcirculation (purple). Refer to Supplemental Video 6. Scale bar: 50 μm.
Figure 4
Figure 4. Thrombin-triggered lethal pulmonary thrombosis is platelet-αIIbβ3 independent.
(A) WT mice were intravascularly (IV) administered 500 U/kg thrombin with or without IV administration of 10 mg/kg αIIbβ3 inhibitor (eptifibatide) 15 minutes before IV thrombin. Pulmonary circulation was imaged using quantitative fluorescence intravital lung microscopy (qFILM). qFILM images of the same field of view (FOV) at 6 different time points are shown to assess the effect of 10 mg/kg IV eptifibatide on the development of 500 U/kg IV thrombin-dependent pulmonary thrombosis. t = 0 seconds (s) corresponds to time point before and t > 0 s correspond to time points immediately following IV thrombin administration. Pulmonary thrombosis was absent at t = 0 s. Following 500 U/kg IV thrombin, medium (500–1000 μm2) and large (>1000 μm2) platelet-rich thrombi (white arrowheads) sequestered in the pulmonary arteriole and traveled down the pulmonary arterioles to occlude the arteriolar bottlenecks (t = 90 s). The mouse died at t = 3.15 minutes, leading to arrest of pulmonary blood flow, which was evident by the presence of stationary erythrocytes (Supplemental Video 7) and decrease in vascular dye (purple fluorescence) in the FOV. Platelets (green) and pulmonary microcirculation (purple). Asterisks denote the alveoli. White arrow mark the direction of blood flow. The diameter of the arteriole is 33 μm. Scale bar: 50 μm. See also Supplemental Video 7 for the complete qFILM time series. (B) Pulmonary thrombi area plotted as a function of time for the FOV shown inA. Red and black arrows indicate pulmonary thrombi maximum area and the time of mouse death, respectively. (C) Survival rate during qFILM experiments in WT mice pretreated (n = 4 mice) or untreated (n = 4 mice) with 10 mg/kg IV eptifibatide before 500 U/kg IV thrombin (P = 0.46, log-rank test).
Figure 5
Figure 5. ADP triggers reversible acute pulmonary thrombosis in mice.
WT mice were IV administered either 0.5 mg/kg ADP (n = 4 mice) or 2.5 mg/kg ADP (n = 7 mice), and pulmonary circulation was imaged using qFILM. (A) qFILM images of the same field of view (FOV) at 8 different time points are shown. t = 0 seconds (s) corresponds to time point before IV ADP administration, and other displayed time points are relative to IV ADP. Pulmonary thrombosis was absent at t = 0 s. Following 2.5 mg/kg IV ADP, medium (500–1000 μm2) and large size (>1000 μm2) platelet-rich thrombi (white arrowheads) sequestered in the pulmonary arteriole (t = 36 s). The thrombi obstructed the arteriolar bottlenecks (t = 55 s), resulting in loss of the pulmonary blood flow, which was evident by the absence of the vascular dye (purple fluorescence) in the capillaries downstream of the embolized arteriole. Pulmonary thrombosis resolved and the capillary blood flow recovered (purple fluorescence was back) by t = 115 s. Platelets (green) and pulmonary microcirculation (purple). Asterisks denote alveoli. White arrows mark the direction of blood flow. The diameters of the arteriole is 41 μm. Scale bar: 50 μm. Complete qFILM time series corresponding toA is shown in Supplemental Video 9. (B) Pulmonary thrombi area plotted as a function of time to show changes in the total area of platelet-rich thrombi following 2.5 mg/kg IV ADP within FOV shown inA. Pulmonary thrombi maximum area value marked by red arrow. (C andD) Pulmonary thrombi max area and AUC were estimated to compare pulmonary thrombosis development in mice following 0.5 mg/kg IV ADP (n = 4 mice) and 2.5 mg/kg IV ADP (n = 7 mice). Pulmonary thrombi max area and AUC were compared using Wilcoxon-Mann-Whitney test. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 for 0.5 mg/kg vs. 2.5 mg/kg IV ADP.
Figure 6
Figure 6. ADP-induced pulmonary thrombosis in mice is αIIbβ3 dependent.
(A) WT mice were IV administered 2.5 mg/kg ADP with or without IV administration of 10 mg/kg αIIbβ3 inhibitor (eptifibatide) 15 minutes before IV ADP. Pulmonary circulation was imaged using quantitative fluorescence intravital lung microscopy (qFILM). qFILM images of the same field of view (FOV) at 6 different time points are shown to assess the effect of eptifibatide on the development of IV ADP-dependent pulmonary thrombosis. t = 0 seconds (s) corresponds to time point before and t > 0 s correspond to time points immediately following IV ADP administration. Pulmonary thrombosis was absent at t = 0 s. ADP failed to evoke pulmonary thrombosis in mouse pretreated with eptifibatide. Platelets (green) and pulmonary microcirculation (purple). Asterisks denote alveoli. White arrow mark the direction of blood flow within the arterioles. The diameter of the arteriole shown is 38 μm. Scale bar: 50 μm. (B) Pulmonary thrombi area plotted as a function of time for the FOV shown inA. Red arrow indicates pulmonary thrombi maximum area. (C andD) Pulmonary thrombi max area and AUC in mice with (n = 4 mice) or without (n = 7 mice) pretreatment with eptifibatide before IV ADP. Pulmonary thrombi max area and AUC were estimated as described in Methods. Pulmonary thrombi max area and AUC were compared using Wilcoxon-Mann-Whitney test. Data are shown as mean ± SEM. **P < 0.01 when comparing with and without eptifibatide pretreatment.
Figure 7
Figure 7. Inhibition of platelet P2Y12 receptor abrogates hemolysis-induced pulmonary thrombosis in mice.
(A) WT mice were administered 10 mg/kg prasugrel by oral gavage and then IV challenged with 150 μL dH2O. Pulmonary circulation was imaged using quantitative fluorescence intravital lung microscopy (qFILM). qFILM images of the same field of view (FOV) at 6 different time points are shown. t = 0 seconds (s) corresponds to time point before and t > 0 s correspond to time points immediately following IV dH2O administration. Pulmonary thrombosis was absent at t = 0 s. dH2O failed to evoke pulmonary thrombosis in mouse pretreated with prasugrel. Platelets (green) and pulmonary microcirculation (purple). Asterisks denote alveoli. White arrows mark the direction of blood flow within the feeding arterioles. The diameter of the arteriole shown is 30 μm. Scale bar: 50 μm. Also refer to Supplemental Video 11. (B) Pulmonary thrombi area plotted as a function of time for the FOV shown inA. Red arrow indicates pulmonary thrombi maximum area. (C andD) Pulmonary thrombi max area and AUC in mice with (n = 6 mice) or without (n = 7 mice) pretreatment with prasugrel before IV dH2O. Pulmonary thrombi max area and AUC were estimated as described in Methods. Pulmonary thrombi max area and AUC were compared using Wilcoxon-Mann-Whitney test. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01 when comparing with and without prasugrel pretreatment.
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