Investigation into the effect of platelet-released molecules on the brain

The brain is a highly fragile organ kept separated in its unique environment by specialised brain barriers, mainly the aptly named blood-brain barrier (BBB). Apart from being a physical barrier, the BBB also functions as a transport barrier to strictly govern the balance of ions and molecules that traverse between the blood and the brain. In a number of neuropathological conditions, particularly in the highly prevalent and debilitating conditions of stroke and traumatic brain injury (TBI), the integrity of the BBB becomes compromised leading to the dysregulated migration of blood constituents such as platelets, into the brain. Platelets are best known for their primary role in haemostasis. However, it is also known that platelets partake in several non-haemostatic events including immune regulation, angiogenesis, wound healing as well as in the propagation and migration of cancerous cells. The association of platelets with multiple roles has been attributed to the extensive inventory of bioactive molecules that are released by platelets or exposed on the platelet surface upon platelet activation. Due to its participation in wound repair, platelets in the form of “platelet concentrates” have been exogenously applied in clinical settings to accelerate the recovery of various organs such as skin, tendon, muscle and bone. Surprisingly, despite its widespread use, there is a clear lack of mechanistic insight into precisely how platelets aid in the recovery of these organs. With regard to the role of platelets in the brain, intriguingly, published studies to date have focussed solely on the thrombotic actions of platelets. Therefore, the purpose of this study was to assess the non-thrombotic roles of platelets in the brain. This project builds upon the understanding that upon BBB breakdown, platelets again unimpeded access into the brain where they become activated and release hundreds of bioactive molecules. These bioactive platelet-released molecules (PRMs) have direct contact with injured neurons and thereby may influence brain injury. To address this aim, we show that cerebrospinal fluid from human TBI patients contain elevated levels of soluble GPVI (a platelet marker) compared to cerebrospinal fluid from non-TBI patients; with the mean levels of soluble GPVI measured at 35.42ng/mL versus 0ng/mL, respectively. Interestingly, the amount of GPVI detected within cerebrospinal fluid of TBI patients is comparable to that observed in the plasma of patients with disseminated intravascular coagulation. Next, using a mouse model of TBI, we document that platelet deposition is not confined within cerebral vasculature but that platelets also extravasate in considerable number into the surrounding brain parenchyma. The deposition of platelets within the brain after TBI coincides with BBB breakdown and plasma extravasation. Moreover, we demonstrate for the first time that platelets that deposit in the brain after TBI become activated thereby intimating the presence of bioactive PRMs with the injured brain. Having established that PRMs do indeed come in contact with injured neurons, we used primary neuronal cultures to study the effects of PRMs on injured neurons. We find that PRMs profoundly protect neurons against injuries induced by etoposide or linsidomine. Contrastingly, PRMs have no influence on the viability of neurons that are challenged with glutamate or subjected to oxygen-glucose deprivation conditions. In our neuronal culture system, both etoposide and linsidomine induce apoptosis whereas glutamate and oxygen-glucose deprivation induce necrosis. Therefore, we show that PRMs selectively protect neurons from undergoing apoptosis but have no effect on necrotic injuries. We further demonstrate that the introduction of PRMs after the induction of apoptosis dampens neuronal injury, highlighting the presence of a therapeutic window for the cytoprotective capabilities of PRMs. This remarkable neuroprotective effect of PRMs has never been previously described. Altogether, our novel finding of a selective cytoprotective role of PRMs in neurons extends current understanding of the non-thrombotic role of platelets in the brain. Due to the paucity in efficacious cytoprotective therapies for neuropathological conditions, the isolation of neuroprotective molecule(s) coupled with the identification of how PRMs protect neurons will certainly be beneficial. Studies into these areas will open novel avenues to develop therapeutic strategies that may ultimately impact on the treatment and recovery of patients that sustain brain injury. Accordingly, we further pursued two avenues of investigation: 1) to determine which molecule(s) within the plethora of bioactive PRMs are responsible for its cytoprotective actions and 2) to determine the neuronal signal transduction events that mediate the cytoprotective actions of PRMs. To address the first objective, we separated PRMs into fractions using ion-exchange chromatography and assessed the capacity of these fractions to inhibit apoptosis. We uncovered several fractions that significantly prevent apoptosis. Within these fractions, we identified 6 candidate anti-apoptotic proteins namely: haemoglobin, profilin-I, GAPDH, aldolase A, immunoglobulin and CXCL7. Importantly, these identified molecules have no known thrombotic actions. Consistent with this notion, we show that 5 out of the 7 cytoprotective fractions do not cause platelet aggregation. Thus, we have not only narrowed the identities of cytoprotective molecules within PRMs to a number of candidates, but also uncoupled the cytoprotective function of platelets from their pro-thrombotic role; thereby substantially increasing the therapeutic potential of PRMs. To address the second objective, we used a phospho-protein microarray to show that in etoposide-injured neurons, PRMs cause differential up-regulation of 25 molecules and down-regulation of 44 molecules. Based on this, we find that neuronal activation of the following kinases: p38α, JNK1, JAK1 and DNA-PK, mediates the cytoprotective actions of PRMs. Interestingly, the anti-apoptotic action of PRMs is not restricted to neurons, as we find that PRMs also protect human U937 cells (a leukaemic monocyte lymphoma cell line) from chemotherapy-induced apoptosis. The ability of PRMs and its fractions to inhibit apoptosis in U937 cells denotes that in addition to the other known contributions of platelets to tumour growth and progression, platelets also confer significant protection to cancerous cells against chemotherapy. As such, efforts to counteract the anti-apoptotic effect of PRMs represent an interesting prospect to reduce chemoresistance of tumour cells and thereby improve cancer prognosis. Taken together, our findings of an anti-apoptotic role of platelets have implications not only for brain injury where there is a clear lack of neuroprotective therapies; but also bears substantial consequences to treatment of cancer whereby chemoresistance of cancer cells remains a major challenge. Given that platelet concentrates are currently used in clinical settings to aid recovery, this highlights the clinical applicability of our findings. Altogether, we provide novel evidence supporting the applicability of platelet concentrates in any situation where platelet activation and widespread apoptosis coincide.