The primary therapeutic goal for patients with AIS is to timely restore blood flow to the regions of brain that are ischemic but not yet infarcted. However, not all AIS patients benefit from reperfusion therapy. Despite successful recanalization of occluded large cerebral vessels, further infarct growth and neurological worsening, called ischemia/reperfusion (I/R) injury, can be observed [25][26]. The occlusion of the middle cerebral artery (MCA) and its branches account for ~ 70% of human strokes. Among several animal models of ischemic stroke, the transient MCAO model that affects the MCA territory is very close to the human situation and therefore widely used in experimental stroke research [27]. In this model, complete recanalization is achieved by the removal of the suture filament, while cerebral microvascular perfusion is incomplete, only about 50 ~ 70% restored to the baseline [27]. The rapid recanalization of the occluded MCA with incomplete reperfusion to downstream microvessels provide clinical relevance for this model to endovascular reperfusion therapy (mechanical removal of thrombi) for AIS patients [28]. In this study, the suture MCAO model was used to investigate the role of PI3Kβ in cerebral I/R injury.
It is widely accepted that activated platelets serve as critical modulators of I/R injury in various organs including brain. Experimental data (mostly using the suture MCAO model) show that circulating platelet activation and interactions with each other (homotypic aggregation) or with circulating leukocytes (heterotypic aggregation) contribute substantially to the pathophysiology of downstream microvascular thrombosis after cerebral I/R injury [29][30][31]. However, no clinical trials have yet demonstrated reliable and safe treatments with currently available antithrombotic drugs for AIS patients, mainly due to increased risk of brain hemorrhage [32]. Thus, there is an urgent need for developing new adjuvant anti-thrombotic therapies for AIS patients. The present study demonstrated the following: (1) PI3Kβ protein levels in platelets were significantly elevated at 24h after tMCAO and (2) inhibition of PI3Kβ with its selective inhibitor TGX-221 administered immediately before the recanalization of the occluded MCA significantly reduced infarct volume and improved neurological function without increased risk of bleeding at 3 days after stroke. Further, we demonstrated that PI3Kβ inhibition with TGX-221 profoundly reduced downstream microvascular thrombosis and improved microvascular reperfusion, and mechanistically, these effects appear to be mediated by inhibition of circulating platelet activation and aggregation and platelet-leukocyte (neutrophil, monocyte) aggregation by flow cytometry analysis of whole blood at 24h after stroke. These findings support an important role for PI3Kβ in mediating platelet thrombotic activity and secondary microvascular thrombosis elicited by focal cerebral I/R. While PI3Kβ is ubiquitously expressed in different types of cells, it is predominantly found in platelets. Thus, the present study focused on understanding the changes of PI3Kβ expression in platelets and the role of PI3Kβ in platelet activation and downstream microvascular thrombosis following cerebral I/R injury. However, whether and how PI3Kβ expressed by other types of cells in the brain and the periphery contribute to cerebral I/R injury warrant further investigation. Moreover, further research is also needed to investigate whether and how therapeutic blocking PI3kβ can improve long-term neurological outcomes after cerebral I/R injury.
PI3Kβ has emerged as a new target for antithrombotic therapy [12]. Among the class I PI3Ks (p110α, -β, -γ, -δ) that all are expressed in platelets, PI3Kβ is the most abundant isoform in platelets and plays a dominant role in regulating platelet adhesion and aggregation [33]. With either PI3Kβ inhibitors [34] and PI3Kβ deficient mice [35][36], previous studies have shown that PI3Kβ regulates platelet aggregation and thrombosis mainly through modulating signals downstream of the Gi-coupled P2Y12 receptor and sustained integrin αIIbβ3 activation. Integrin αIIbβ3 is a major membrane protein expressed on the surface of platelets and plays a central role in normal hemostasis and pathological thrombosis. On unactivated platelets, αIIbβ3 has little affinity for soluble fibrinogen [37]. Platelet activation leads to activation of αIIbβ3 through inside-out signaling, resulting in a significant increase in the affinity for fibrinogen [37]. On stimulated platelets, αIIbβ3 serves as a specific receptor for fibrinogen (and fibrin). Fibrinogen is converted into the insoluble protein fibrin during coagulation. Platelet aggregation and thrombus formation is determined by the integrin αIIbβ3-mediated interactions of platelets with fibrinogen (and fibrin), presumably a fibrinogen molecule acts as a bridge between αIIbβ3 molecules on different platelets [37]. PI3Kβ inhibitors undermine stable platelet aggregation in vitro and platelet thrombus formation in vivo, affording protection from thrombotic occlusion [12][36]. The present study demonstrated that PI3Kβ inhibition with TGX221 significantly reduced circulating platelet activation (determined by platelet P-selectin expression) and platelet aggregation (assessed by platelet-fibrinogen binding assay) after I/R, and ADP-induced platelet aggregation in vitro. These findings support an important role for PI3Kβ in mediating thrombotic activity of platelets, a key contributing factor to secondary microvascular thrombosis after I/R injury. As a consequence, PI3Kβ inhibition with TGX-221 profoundly reduced the deposition of platelets and fibrin in the cerebral microvessels after I/R.
Elevated levels of platelet activation and platelet-leukocyte aggregates contribute importantly to inflammatory and thrombotic events in ischemic stroke [38]. Experimental studies have shown that microvascular lumina were obstructed with platelets, leukocytes, and fibrin-rich aggregates during early I/R [39][40][41]. Thus, preventing secondary microvascular thrombosis should improve microvascular reperfusion in stroke [18]. It has been widely recognized that the formation of platelet-leukocyte aggregates is mediated via platelet P-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) on the leukocyte [42]. The present study demonstrated that inhibition of PI3Kβ with TGX-221 profoundly inhibited circulating platelet P-selectin expression and reduced circulating platelet-leukocyte (neutrophil, monocyte) aggregates elicited by tMCAO.
Thromboxane A2 (TxA2) is a member of the family of lipids known as eicosanoids. It is produced mainly by activated platelets via a cyclooxygenase (COX) enzyme in the platelet cytosol [43]. TxA2 synthesis by platelets increases in response to a variety of stimuli (e.g. thrombin, collagen, and ADP) during the times of tissue injury. The released TxA2 acts as a positive feedback mediator that stimulates platelet activation and promotes recruitment of more platelets into growing thrombi, thereby contributing to the pathophysiology of thrombus formation in ischemic stroke [43]. TxA2 is highly unstable, with a half-life of about 30 seconds, and rapidly hydrated to thromboxane B2 (TxB2), an inactive stable metabolite). PI3Kβ has been shown to mediate ADP-induced TxA2 generation by activated platelets in vitro [44]. In this study, we measured TxB2 instead of TxA2 and found that blocking of PI3Kβ with TGX221 significantly decreased plasma TxB2 levels after stroke.
In summary, our study shows that increased platelet expression of PI3Kβ is associated platelet activation and aggregation, and downstream microvascular thrombosis after ischemic stroke and blocking of pi3Kβ has potential to protect against I/R injury in acute ischemic stroke.