Abstract 176: Platelet Cyclophilin D Mediates Neutrophil Recruitment and Ischemic Stroke Outcomes in Mice

Abstract

Rationale: Besides their role in thrombosis, platelets also mediate inflammation through platelet-neutrophil aggregates (PNA). Recently, cyclophilin D (CypD)-mediated platelet necrosis emerged as a potential mediator of detrimental PNA during thrombosis. However, the role of platelet CypD in ischemic stroke has never been examined. Objective: Here, we investigate the contribution of platelet CypD following ischemic stroke. Methods: We generated mice lacking CypD specifically in platelets (KO). Both wild-type (WT) and KO mice were subjected to a 1h transient middle cerebral artery occlusion (tMCAO) stroke model. Twenty-four hours after occlusion, neurological and motor function, and stroke infarct size were determined. We also examined if the CypD pathway was altered in human platelets after ischemic stroke. Results: Loss of platelet CypD significantly improved neurological (p<0.001) and motor (p<0.005) functions compared to WT mice after tMCAO. Furthermore, platelet CypD deficient significantly reduced ischemic stroke infarct volume (39.1±15.7mm 3 vs. 78.6±27.7mm 3 , n=15; p<0.0001). Smaller infarcts in KO mice was not due to difference in blood flow during the ischemia stage. Twenty-four hours after stroke, a greater than 2-fold reduction in neutrophils was observed in the brains from KO mice (p<0.0001). In addition, we observed significantly fewer circulating and cerebral PNA (p<0.01). Depletion of neutrophils significantly (p<0.05) reduced infarct size and neurological damage following ischemic stroke in WT mice, however, no additional protective effect was observed in KO mice, suggesting necrotic PNAs are critical during ischemic stroke. RNA-sequencing on platelets isolated from ischemic stroke patients (n=8) and healthy aged, gender matched controls (n=7) revealed significant increases in several targets involved in CypD-mediated necrosis, including MCUR1, TMEM16F and calpain2 (p<0.005). Conclusion: Our results demonstrate necrotic platelets interact with neutrophils to exacerbate brain injury following ischemic stroke. As inhibiting platelet necrosis does not compromise hemostasis, targeting platelet CypD may be a potential therapeutic strategy to limit brain damage after ischemic stroke.