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 5

ADP triggers reversible acute pulmonary thrombosis in mice.

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ADP triggers reversible acute pulmonary thrombosis in mice. WT mice were...
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 to A 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 in A. Pulmonary thrombi maximum area value marked by red arrow. (C and D) 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.