Intravascular hemolysis triggers ADP-mediated generation of platelet-rich thrombi in precapillary pulmonary arterioles
Tomasz Brzoska, Ravi Vats, Margaret F. Bennewitz, Egemen Tutuncuoglu, Simon C. Watkins, Margaret V. Ragni, Matthew D. Neal, Mark T. Gladwin, Prithu Sundd
Tomasz Brzoska, Ravi Vats, Margaret F. Bennewitz, Egemen Tutuncuoglu, Simon C. Watkins, Margaret V. Ragni, Matthew D. Neal, Mark T. Gladwin, Prithu Sundd
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Research Article Pulmonology Vascular biology

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

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.

Authors

Tomasz Brzoska, Ravi Vats, Margaret F. Bennewitz, Egemen Tutuncuoglu, Simon C. Watkins, Margaret V. Ragni, Matthew D. Neal, Mark T. Gladwin, Prithu Sundd

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Figure 3

Thrombin triggers protracted and lethal pulmonary thrombosis in mice.

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Thrombin triggers protracted and lethal pulmonary thrombosis in mice. WT...
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 and B) 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 in A and B are 39 μm and 44 μm, respectively. Complete qFILM time series corresponding to A and B are shown in Supplemental Videos 4 and 5, respectively. (C and D) Pulmonary thrombi area plotted as a function of time following 250 U/kg (C) and 500 U/kg (D) IV thrombin within FOVs shown in A and B, 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.