Mechanism of Pulmonary Thrombosis in Hemolytic Disorders
Authors: Brzoska Tomasz , Bennewitz Margaret F. , Tutuncuoglu Egemen , Ragni Margaret V. , Neal Matthew D , Gladwin Mark T , Sundd Prithu 
Published: February 2020

Hemolysis is a hallmark of various inherited or acquired blood disorders including sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome. Epidemiological evidence suggests that in situ pulmonary thrombosis is a major pathological event contributing to cardiopulmonary morbidities affecting patients with hemolytic disorders. Intravascular hemolysis promotes the release of erythrocyte-derived damage associated molecular patterns (eDAMPs) molecules such as cell free hemoglobin (Hb), heme and adenosine diphosphate (ADP). Although these eDAMPS may activate platelets, promote endothelial dysfunction and drive sterile thrombo-inflammation, the pathogenesis of pulmonary thrombosis in hemolytic disorders and the role of platelets in this process remains unclear.
Intravascular hemolysis in C57BL/6 (WT) mice was induced by intravenous (IV) administration of deionized water (H2O). Alternatively, eDAMPs or tissue derived DAMPs relevant to hemolysis or endothelial injury were administered IV to WT mice. The pulmonary microcirculation was visualized using quantitative intravital fluorescence lung microscopy (qFILM). Fluorochrome-conjugated anti-mouse CD49b Ab and dextran were administered IV for in vivo staining of circulating platelets and visualization of blood vessels, respectively. Time series of qFILM images were collected at baseline and post IV administration of agonists to assess pulmonary thrombosis.
IV administration of H2O led to arrival of platelet-rich thrombi in the pulmonary arterioles that occluded the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries leading to their transient obstruction. DAMPs such as ADP, collagen, thrombin and tissue factor also triggered dose-dependent PT in mice. Identical to IV H2O, DAMPs triggered pulmonary thrombosis also involved entrapment of platelet-rich thrombi in the arteriolar bottle-necks, which led to ischemia in the pulmonary arteriole and the down-stream capillaries. Interestingly, pretreatment with IV heparin (300 U/kg) prevented TF-induced but not ADP-triggered pulmonary thrombosis in mice. In contrast, IV administration of αIIbβ3 receptor inhibitor eptifibatide (10 mg/kg) completely abrogated TF-, collagen and ADP-dependent PT.
Our current findings provide the first real-time in vivo visual evidence establishing that intravascular hemolysis directly induces pulmonary thrombosis, which involves entrapment of platelet-rich thrombi in the pre-capillary pulmonary arterioles. Our results are the first to identify that platelet activation and procoagulant activity contribute to pathogenesis of hemolysis induced pulmonary thrombosis.

Disclosures
Ragni: OPKO: Research Funding; Bioverativ/Sanofi: Other: Advisory Board, Research Funding; Shire/Takeda: Other: Advisory Board, Research Funding; Sangamo: Research Funding; Spark Therapeutics: Other: Advisory Board, Research Funding; Alnylam/Sanofi: Other: Advisory Board, Research Funding; BioMarin: Other: Advisory Board, Research Funding. Neal:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Gladwin:Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.