Arteriolar Bottle-Necks in the Lung Are the Site of Pulmonary Thrombo-Embolism

Acute Pulmonary Thrombo-Embolism (PTE) is a major vaso-occlusive pulmonary complication associated with high morbidity. Despite advances in diagnosis and treatment, the pathophysiology of PTE remains incompletely understood and anticoagulant therapy is the only available treatment. Based on computed tomography in humans and accumulation of radiolabeled platelets in mice, PTE is believed to be primarily triggered by embolic platelet aggregates arriving from the venous circulation and sequestering in the pulmonary arterioles. However, new clinical evidence suggest that in situ pulmonary arteriole micro-thrombosis may also promote PTE. Studies that can visualize PTE in real time in vivo in mice are highly warranted to identify different mechanisms contributing to PTE. Improved understanding of the biophysical, cellular and molecular mechanism would enable development of new therapies for PTE.

ADP, collagen or thrombin were administered intravascularly (IV) to wild type (WT) mice and the pulmonary microcirculation was studied using multi-photon-excitation (MPE) enabled quantitative intravital fluorescence lung microscopy (qFILM). Fluorochrome-conjugated anti-mouse CD49b Ab and dextran was administered IV for in vivo staining of circulating platelets and visualization of blood vessels. Time series of qFILM images were collected at baseline and immediately following IV administration of ADP, collagen or thrombin. Images were analyzed to identify PTE, which was defined as occlusion of blood vessels with platelet aggregates leading to loss of blood flow.

ADP (0.5-2.5 mg/kg), collagen (0.2-0.5 mg/kg) and thrombin (250-500 U/kg) triggered dose-dependent PTE in mice. Remarkably, large embolic platelet aggregates were observed to arrive in the pulmonary arterioles and occlude the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries. The occlusion of arteriolar bottle-necks led to stasis of blood flow in the arteriole and the down-stream capillaries. Surprisingly, unlike collagen or thrombin, ADP induced PTE was transient and resolved over a time period of 10 min. However, thrombin (500 U/kg) and collagen (0.5 mg/kg) triggered stable emboli within the arteriolar bottle-necks.

We introduce an intravital microscopy approach to probe agonist-dependent platelet aggregation in the lung microcirculation of live mice. We provide the first real-time visual in vivo evidence establishing that PTE indeed involves entrapment of embolic platelet aggregates in the pre-capillary pulmonary arterioles. Our model has potential application in investigating the molecular determinants of pulmonary thrombosis in diverse pulmonary disorders as well as evaluating efficacy of new antithrombotic drugs.