The intriguing role of platelets as custodians of brain-derived neurotrophic factor.

A State of the Art lecture titled "Platelets and neurotrophins" was presented at the International Society on Thrombosis and Haemostasis Congress in 2023. Neurotrophins, a family of neuronal growth factors known to support cognitive function, are increasingly recognized as important players in vascular health. Indeed, along with their canonical receptors, neurotrophins are expressed in peripheral tissues, particularly in the vasculature. The better-characterized neurotrophin in vascular biology is the brain-derived neurotrophic factor (BDNF). Its largest extracerebral pool resides within platelets, partly inherited from megakaryocytes and also likely internalized from circulation. Activation of platelets releases vast amounts of BDNF into their milieu and interestingly leads to platelet aggregation through binding of its receptor, the tropomyosin-related kinase B, on the platelet surface. As BDNF is readily available in plasma, a mechanism to preclude excessive platelet activation and aggregation appears critical. As such, binding of BDNF to α2-macroglobulin hinders its ability to bind its receptor and limits its platelet-activating effects to the site of vascular injury. Altogether, addition of BDNF to a forming clot facilitates not only paracrine platelet activation but also binding to fibrinogen, rendering the resulting clot more porous and plasma-permeable. Importantly, release of BDNF into circulation also appears to be protective against adverse cardiovascular and cerebrovascular outcomes, which has been reported in both animal models and epidemiologic studies. This opens an avenue for platelet-based strategies to deliver BDNF to vascular lesions and facilitate wound healing through its regenerative properties. Finally, we summarize relevant new data on this topic presented during the 2023 International Society on Thrombosis and Haemostasis Congress.

Alpha-2-macroglobulin prevents platelet aggregation induced by brain-derived neurotrophic factor.

The brain-derived neurotrophic factor (BDNF) has been recently shown to have activating effects in isolated platelets. However, BDNF circulates in plasma and a mechanism to preclude constant activation of platelets appears necessary. Hence, we investigated the mechanism regulating BDNF bioavailability in blood. Protein-protein interactions were predicted by molecular docking and validated through immunoprecipitation. Platelet aggregation was assessed using light transmission aggregometry with washed platelets in response to classical agonists or BDNF, in the absence or presence of alpha-2-macroglobulin (α2M), and in platelet-rich plasma. BDNF signaling was assessed with phospho-blots. As little as 25% autologous plasma was sufficient to completely abolish platelet aggregation in response to BDNF. Docking predicted two forms of BDNF binding to native or activated α2M, in parallel and perpendicular arrangements, and the model suggested that the BDNF-α2M complex cannot bind to the high-affinity BDNF receptor, tropomyosin receptor kinase B (TrkB). Experimentally, native and activated α2M formed stable complexes with BDNF preventing BDNF-induced TrkB activation and signal transduction. Both native and activated α2M inhibited BDNF induced-platelet aggregation in a concentration-dependent manner with comparable half-maximal inhibitory concentrations (IC50≈ 125-150 nM). Our study implicates α2M as a physiological regulator of BDNF bioavailability, and as an inhibitor of BDNF-induced platelet activation in blood.

Clinical Correlates Identify ProBDNF and Thrombo-Inflammatory Markers as Key Predictors of Circulating p75NTR Extracellular Domain Levels in Older Adults.

The p75NTR receptor binds all neurotrophins and is mostly known for its role in neuronal survival and apoptosis. Recently, the extracellular domain (ECD) of p75NTR has been reported in plasma, its levels being dysregulated in numerous neurological diseases. However, the factors associated with p75NTR ECD levels remain unknown. We investigated clinical correlates of plasma p75NTR ECD levels in older adults without clinically manifested neurological disorders. Circulating p75NTR levels were measured by enzyme-linked immunosorbent assay in plasma obtained from participants in the BEL-AGE cohort (n = 1,280). Determinants of plasma p75NTR ECD levels were explored using linear and non-linear statistical models. Plasma p75NTR ECD levels were higher in male participants; were positively correlated with circulating concentrations of pro-brain-derived neurotrophic factor, and inflammatory markers interleukin-6 and CD40 Ligand; and were negatively correlated with the platelet activation marker P-selectin. While most individuals had p75NTR levels ranging from 43 to 358 pg/ml, high p75NTR levels reaching up to 9,000 pg/ml were detectable in a subgroup representing 15% of the individuals studied. In this cohort of older adults without clinically manifested neurological disorders, there was no association between plasma p75NTR ECD levels and cognitive performance, as assessed by the Montreal Cognitive Assessment score. The physiological relevance of high p75NTR ECD levels in plasma warrants further investigation. Further research assessing the source of circulating p75NTR is needed for a deeper understanding of the direction of effect, and to investigate whether high p75NTR ECD levels are predictive biomarkers or consequences of neuropathology.

The brain-derived neurotrophic factor prompts platelet aggregation and secretion.

Brain-derived neurotrophic factor (BDNF) has both autocrine and paracrine roles in neurons, and its release and signaling mechanisms have been extensively studied in the central nervous system. Large quantities of BDNF have been reported in circulation, essentially stored in platelets with concentrations reaching 100- to 1000-fold those of neurons. Despite this abundance, the function of BDNF in platelet biology has not been explored. At low concentrations, BDNF primed platelets, acting synergistically with classical agonists. At high concentrations, BDNF induced complete biphasic platelet aggregation that in part relied on amplification from secondary mediators. Neurotrophin-4, but not nerve growth factor, and an activating antibody against the canonical BDNF receptor tropomyosin-related kinase B (TrkB) induced similar platelet responses to BDNF, suggesting TrkB could be the mediator. Platelets expressed, both at their surface and in their intracellular compartment, a truncated form of TrkB lacking its tyrosine kinase domain. BDNF-induced platelet aggregation was prevented by inhibitors of Ras-related C3 botulinum toxin substrate 1 (Rac1), protein kinase C, and phosphoinositide 3-kinase. BDNF-stimulated platelets secreted a panel of angiogenic and inflammatory cytokines, which may play a role in maintaining vascular homeostasis. Two families with autism spectrum disorder were found to carry rare missense variants in the BDNF gene. Platelet studies revealed defects in platelet aggregation to low concentrations of collagen, as well as reduced adenosine triphosphate secretion in response to adenosine diphosphate. In summary, circulating BDNF levels appear to regulate platelet activation, aggregation, and secretion through activation of a truncated TrkB receptor and downstream kinase-dependent signaling.

Tissue-Specificity of Antibodies Raised Against TrkB and p75NTR Receptors; Implications for Platelets as Models of Neurodegenerative Diseases.

Platelets and neurons share many similarities including comparable secretory granule types with homologous calcium-dependent secretory mechanisms as well as internalization, sequestration and secretion of many neurotransmitters. Thus, platelets present a high potential to be used as peripheral biomarkers to reflect neuronal pathologies. The brain-derived neurotrophic factor (BDNF) acts as a neuronal growth factor involved in learning and memory through the binding of two receptors, the tropomyosin receptor kinase B (TrkB) and the 75 kDa pan-neurotrophic receptor (p75NTR). In addition to its expression in the central nervous system, BDNF is found in much greater quantities in blood circulation, where it is largely stored within platelets. Levels 100- to 1,000-fold those of neurons make platelets the most important peripheral reservoir of BDNF. This led us to hypothesize that platelets would express canonical BDNF receptors, i.e., TrkB and p75NTR, and that the receptors on platelets would bear significant resemblance to the ones found in the brain. However, herein we report discrepancies regarding detection of these receptors using antibody-based assays, with antibodies displaying important tissue-specificity. The currently available antibodies raised against TrkB and p75NTR should therefore be used with caution to study platelets as models for neurological disorders. Rigorous characterization of antibodies and bioassays appears critical to understand the interplay between platelet and neuronal biology of BDNF.

Platelets Selectively Regulate the Release of BDNF, But Not That of Its Precursor Protein, proBDNF.

Background: Brain-derived neurotrophic factor (BDNF) plays a role in synaptic plasticity and neuroprotection. BDNF has well-established pro-survival effects, whereas its precursor protein, proBDNF, induces apoptosis. Thus, it has been suggested that the proBDNF/BDNF ratio could be an indicator of neuronal health. Access to neurons is, understandably, limited. Because of their similarities, platelets have been put forward as a non-invasive biomarker of neuronal health; indeed, they store large quantities of BDNF and can release it into circulation upon activation, similarly to neurons. However, whether platelets also express the precursor proBDNF protein remains unknown. We therefore sought to characterize proBDNF levels in human platelets and plasma. Methods: The presence of proBDNF was assessed by immunoblotting, cell fractionation, flow cytometry, and confocal microscopy in washed platelets from 10 healthy volunteers. Platelets from 20 independent healthy volunteers were activated with several classical agonists and the release of BDNF and proBDNF into plasma was quantified by ELISA. Results: Platelets expressed detectable levels of proBDNF (21 ± 13 fmol/250 x 106 platelets). ProBDNF expression was mainly localized in the intracellular compartment. The proBDNF to BDNF molar ratio was ~1:5 in platelets and 10:1 in plasma. In stark contrast to the release of BDNF during platelet activation, intraplatelet and plasma concentrations of proBDNF remained stable following stimulation with classical platelet agonists, consistent with non-granular expression. Conclusions: Platelets express both the mature and the precursor form of BDNF. Whether the intraplatelet proBDNF to BDNF ratio could be used as a non-invasive biomarker of cognitive health warrants further investigation.

Postoperative Administration of the Acetylcholinesterase Inhibitor, Donepezil, Interferes with Bone Healing and Implant Osseointegration in a Rat Model.

Donepezil is an acetylcholinesterase inhibitor commonly used to treat mild to moderate Alzheimer's disease. Its use has been associated with increased bone mass in humans and animals. However, the effect of postoperative administration of donepezil on bone healing remains unknown. Therefore, this study aimed to assess the impact of postoperative injection of donepezil on bone healing, titanium-implant osseointegration, and soft tissue healing. Twenty-two Sprague-Dawley rats were randomly assigned to receive a daily dose of either donepezil (0.6 mg/kg) or saline as a control. In each rat, a uni-cortical defect was created in the right tibia metaphysis and a custom-made titanium implant was placed in the left tibiae. After two weeks, rats were euthanized, and their bones were analysed by Micro-CT and histology. The healing of bone defect and implant osseointegration in the rats treated with donepezil were significantly reduced compared to the saline-treated rats. Histomorphometric analysis showed lower immune cell infiltration in bone defects treated with donepezil compared to the saline-treated defects. On the other hand, the healing time of soft tissue wounds was significantly shorter in donepezil-treated rats compared to the controls. In conclusion, short-term administration of donepezil hinders bone healing whereas enhancing soft tissue healing.

Isolation of endothelial cells, pericytes and astrocytes from mouse brain.

Primary cell isolation from the central nervous system (CNS) has allowed fundamental understanding of blood-brain barrier (BBB) properties. However, poorly described isolation techniques or suboptimal cellular purity has been a weak point of some published scientific articles. Here, we describe in detail how to isolate and enrich, using a common approach, endothelial cells (ECs) from adult mouse brains, as well as pericytes (PCs) and astrocytes (ACs) from newborn mouse brains. Our approach allowed the isolation of these three brain cell types with purities of around 90%. Furthermore, using our protocols, around 3 times more PCs and 2 times more ACs could be grown in culture, as compared to previously published protocols. The cells were identified and characterized using flow cytometry and confocal microscopy. The ability of ECs to form a tight monolayer was assessed for passages 0 to 3. The expression of claudin-5, occludin, zonula occludens-1, P-glycoprotein-1 and breast cancer resistance protein by ECs, as well as the ability of the cells to respond to cytokine stimuli (TNF-α, IFN-γ) was also investigated by q-PCR. The transcellular permeability of ECs was evaluated in the presence of pericytes or astrocytes in a Transwell® model by measuring the transendothelial electrical resistance (TEER), dextran-FITC and sodium fluorescein permeability. Overall, ECs at passages 0 and 1 featured the best properties valued in a BBB model. Furthermore, pericytes did not increase tightness of EC monolayers, whereas astrocytes did regardless of their seeding location. Finally, ECs resuspended in fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) could be cryopreserved in liquid nitrogen without affecting their phenotype nor their capacity to form a tight monolayer, thus allowing these primary cells to be used for various longitudinal in vitro studies of the blood-brain barrier.