CD39 as a Master Regulator of Pulmonary Thrombosis in Sickle Cell Disease

Acute systemic painful vaso-occlusive crisis (VOC) often serves as an antecedent to acute chest syndrome (ACS), which is a type of acute lung injury and the leading cause of mortality among sickle cell disease (SCD) patients. Thrombocytopenia secondary to pulmonary thrombosis is major risk factor for ACS, however, only 20% of ACS patients are diagnosed with pulmonary thrombosis as an underlying cause of ACS. Although clinical evidence supports the presence of prothrombotic state in subset of SCD patients, the molecular, cellular and genetic mechanisms that selectively render subset of SCD patients at either higher or lower risk of developing pulmonary thrombosis remains elusive. Adenosine diphosphate (ADP) released from lysed erythrocytes can activate platelets by stimulating their purinergic P2Y1 and P2Y12 receptors, however, P2Y12 receptor antagonists have not shown any benefit in clinical trials, justifying the need for better understanding of purinergic signaling in SCD. Here, we use intravital lung microscopy in transgenic humanized SCD mice to show that intravenous administration of ADP triggered pulmonary thrombosis in control mice but failed to trigger pulmonary thrombosis in SCD mice. In contrast, collagen evoked pulmonary thrombosis identically in both control and SCD mice. Identical to intravital findings, IV ADP administration also evoked transient thrombocytopenia in control but not SCD mice, while, IV collagen led to comparable drop in platelet count in both SCD and control mice. ADP is metabolized by the ecto-nucleoside-tri-phosphate-diphosphohydrolase-1 (E-NTPDase1) CD39. IV administration of fluorescent analogue of ADP, N⁶- ethenoadenosine- 5'- O- diphosphate (ε-ADP) followed by invivo microdialysis and HPLC analysis revealed impaired ε-ADP degradation in SCD mice, suggestive of decreased CD39 activity. Our current findings suggest that loss of CD39 activity in SCD possibly prevents ADP-mediated pulmonary thrombosis. Currently, experiments are underway to identify pathways contributing to loss of CD39 activity in SCD, how that affects purinergic signaling and whether selective activation vs deactivation of this pathway is responsible for risk of pulmonary thrombosis in only 20% of ACS patients. Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Bayer Pharmaceuticals: Other: Co-investigator.