Abstract Body (Do not enter title and authors here): Background: Platelet activation plays a key role in peripheral artery disease (PAD), but the relevant platelet activation mechanism in chronic limb threatening ischemia remains unclear. Moreover, multi-center study of platelet activation remains difficult due to methodologic limitations. We report the feasibility of a novel method to measure platelet activation using samples sent via overnight mail. The primary objective was to report baseline markers of platelet activation from samples sent by overnight mail compared to an age-matched population recruited locally without known atherosclerosis.

Methods: Peripheral venous blood was drawn from 190 participants with severe peripheral artery disease at 43 sites in the BEST-CLI (Best Endovascular Versus Best Surgical Therapy in Patients with Chronic Limb Ischemia) Trial and shipped by Federal Express cold pack from study site locations within the continental United States to Vanderbilt University Medical Center. Fifty-six volunteers with and without diabetes were recruited locally as time and age-matched control subjects. Samples were collected using a platelet activation inhibitor. Thromboxane B2, dilysyl-malondialdehyde (MDA), p-selectin, WEDE 15, and SPAN 12 as markers of platelet activation were measured using liquid chromatography with tandem mass spectrometry and flow cytometry.

Results: Compared with control subjects, patients from the BEST-CLI trial were more likely to be male (72.6% vs 51.8%, p<0.01), have prior atherosclerotic cardiovascular disease (22.1% vs 0%, p<0.01), and be prescribed aspirin (69.4% vs 35.7%, p<0.01) or clopidogrel (20.0% vs 0%, p<0.01). Thromboxane B2 (0.5 vs 2.9 ng/mL, p<0.01) and dilysyl-MDA (4.7 vs 9.7 ng/mg, p<0.01) were significantly higher in BEST-CLI patients than control subjects while p-selectin (592.0 vs 420.5 MFI, p<0.01) was lower. Levels of thromboxane B2 (figure 1A) and dilysyl-MDA (figure 1B) varied significantly and as expected across the range of antithrombotic prescription (p<0.01).

Conclusions: Measurement of platelet activation after sample shipment on cold pack is feasible and yields reliable results that match expected clinical values.