Measurements

Platelet count was measured in the clinical lab, while platelet aggregation testing was assessed via point of care testing in a non-clinical research lab. Platelet aggregation was assessed using the Multiplate® multiple electrode aggregometer (Verum Diagnostica GmbH; Munich, Germany) immediately after sample collection as previously described (15). Specifically, 0.3mL of whole blood was diluted in warmed normal saline containing 3mM CaCl₂ and incubated for 3 minutes at 37°C with continuous stirring in a Multiplate® test cell. Each test cell contains two sets of 3 mm silver-coated copper wires, across which electrical resistance is measured at 0.57 second intervals. Platelet activation was induced by agonists arachidonic acid (ASPI, final concentration 0.5mM; via the cyclooxygenase pathway), adenosine diphosphate (ADP, final concentration 6.5µM; via P2 receptors), collagen (COL, final concentration 3.2µg/mL; via GpIa/IIa and GpVI receptors) or thrombin receptor activating peptide-6 (TRAP, final concentration 32µM; via PAR receptors). Platelet adhesion to the electrodes was detected as increasing electrical impedance, measured by duplicate sets of sensor wires in each test cell. Agonist responses are reported as area under the aggregation curve in units (U) over a 6-minute measurement period. Reference ranges were provided by the manufacturer based on studies of healthy control subjects. To study the relationship between ARDS and platelet aggregation, we dichotomized each aggregation test at the lower limit of normal, delineating impaired platelet aggregation, based on prior studies of impedance based platelet aggregometry in the setting of trauma (15).

Plasma cotinine, a biomarker with a half-life of 16 hours that accurately quantifies cigarette smoke exposure (23), was measured on emergency department (0 hour) samples when available via liquid chromatography-tandem mass spectrometry (24, 25). The limit of quantification for plasma cotinine was 0.02 ng/ml.