Peripheral Artery Disease Increases Platelet Activity and Promotes a Prothrombotic Platelet Phenotype

Background: Peripheral artery disease (PAD), characterized by lower extremity atherosclerosis, increases the risk of cardiovascular morbidity and mortality. Among their many functions, platelets modulate hemostasis, thrombosis, inflammation, and angiogenesis. Previously, we noted the potent pathogenic role of platelets in PAD. However, how vascular dysfunction impacts the platelet phenotype is not fully understood. Using a mouse model of PAD (hindlimb ischemia), we investigated the platelet phenotype and transcriptome. Methods: Eighteen C57BL/6J mice (10 weeks) underwent femoral artery ligation (HLI) or sham surgery. Platelet activity, P-selectin, JON/A expression, and platelet-leukocyte aggregation, was assessed at day 0, 3, 7, 10, 14, and 21. At day 21, we performed the FeCl3-induced thrombosis model and assessed time to vessel occlusion. To investigate platelet-relevant mechanisms, we induced HLI or sham surgery in 20 mice and collected platelets for RNA sequencing (day 12). Results: Our acute PAD model resulted in >90% drop in blood flow after the procedure with ≈50% recovery at day 21. Following the procedure, platelet activity, defined by PAR4 agonist stimulated P-selectin expression (P=0.001), αIIbβ3 activation (JON/A expression, P=0.002), platelet-neutrophil aggregation (P<0.001) and platelet monocyte aggregation (P=0.047) increased over time in the HLI vs sham group. Mice in the HLI group had faster occlusive carotid thrombosis than the sham group (P=0.026). Comparing the platelet transcriptome in the HLI vs sham group noted 895 differentially expressed transcripts (P<0.05) between groups (516 up-, 79 down-regulated). Pathway analysis between groups demonstrated enrichment of inflammatory pathways, including interleukin-1 mediated signaling (P=0.02), apoptotic process (P=0.02), noncoding RNA processing (P=0.02), and signaling receptor binding (P=0.03). Conclusion: In our murine model of acute PAD, we found increased platelet activity, a proinflammatory platelet transcriptome, and a prothrombotic state. These data highlight the feasibility of the HLI model to further investigate platelet-associated mechanisms that underlie PAD.