Cyclophilin D Mediated Platelet Necrosis Regulates Ischemic Stroke Outcomes in Mice

Background: Anti-platelet agents are used clinically to prevent ischemic stroke but are incompletely effective. Emerging evidence highlights that platelets contribute to ischemic stroke through mechanisms and pathways that are not targeted by classic anti-platelet agents. Besides their role in thrombosis, platelets also mediate inflammation through the formation of heterotypic leukocyte aggregates. In particular, platelet-neutrophil interactions are known to promote brain injury following ischemic stroke. However, the mechanism by which platelets interact with neutrophils and promote thromboinflammation in ischemic stroke remains elusive. Recently, cyclophilin D (CypD)-mediated platelet necrosis emerged as a potential mediator of detrimental platelet-neutrophil interactions.

Aims: To investigate the role of CypD-mediated platelet necrosis in the setting of acute ischemic stroke.

Methods: Mice harboring a floxed allele of CypD were crossed to animals expressing Cre recombinase controlled by the Pf4 promoter to generate platelet-specific CypD deficient mice (KO). KO mice and littermate controls (WT)were subjected to a transient middle cerebral artery occlusion (tMCAO) model with 1h of cerebral ischemia followed by 23h of reperfusion or a permanent MCAO model with 24h of ischemia. Neurological and motor outcomes and brain infarct size were measured 24h after stroke onset. In addition, we examined both circulating and cerebral platelet-neutrophil aggregates 24h after stroke. Cerebral blood flow was monitored via blood laser doppler flowmetry. Neutrophils were depleted 24h before stroke onset using a neutrophil-depleting antibody to examine the contribution of neutrophils in ischemic stroke.

Results: Loss of CypD in platelets significantly improved neurological (p<0.001) and motor (p<0.005) functions and reduced ischemic stroke infarct volume (p<0.0001) after cerebral transient ischemia/reperfusion injury compared to WT controls. To determine whether improved stroke outcomes in KO mice was associated with improved local cerebral blood flow (CBF) during reperfusion, CBF was measured at time points during and after stroke induction. During ischemia, and immediately after reperfusion, CBF was similar in WTand KO mice. Nevertheless, 3h after stroke onset, CBF was significantly greater (48±15% vs 31±10%; p<0.05) in KO mice compared to WTmice. This difference in CBF persisted and was even more pronounced at 24h (54±12% vs 27±8%; p<0.01). To further investigate whether platelet necrosis was contributing to brain infarction during cerebral reperfusion, we employed a permanent MCAO model. With permanent occlusion, no differences were observed in infarct volume, neurological functions, or motor functions between WT or KO mice, suggesting platelet CypD specifically mediates reperfusion injury following transient cerebral ischemia.

These detrimental effects of platelet necrosis were attributable, in part, to platelet-neutrophil interactions. Twenty-four hours after stroke, significantly (p<0.01) fewer circulating platelet-neutrophil aggregates were found in KO mice. Underscoring the role of platelet necrosis in this process, we observed that 33±4% of platelets in platelet-neutrophil aggregates expressed phosphatidylserine (PS) on their surface in WTmice. In contrast, only 17.8±5.1% of platelets in platelet-neutrophil aggregates were PS-positive in KO counterparts (p<0.01). Furthermore, KO mice had less neutrophils recruited to their brain relative to WT controls, and cerebral platelet-neutrophil aggregates were virtually absent in KO mice. To determine whether the protective effect observed in KOmice was due to reduced interactions between necrotic platelets and neutrophils, we depleted circulating neutrophils 24h before induction of stroke. Depletion of neutrophils significantly (p<0.05) reduced infarct size and neurological damage following ischemic stroke in WTmice. However, neutrophil depletion conferred no additional protective effect in KOanimals.

Conclusions: These results suggest 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 following ischemic stroke.