Alterations in biochemical and molecular pathways in patients suffering from neurodegenerative diseases may not only emerge in the brain but also could affect blood cells and vessels. In this instance, metabolic and vascular disorders related to aging can be considered risk factors for neurodegeneration [28]. Platelets are the blood cells that not only have important roles in the regulation of hemostasis and thrombosis but are also responsible for vascular constriction, bleeding, clotting, and repair mechanisms. [5]. Besides their hemostatic function, platelets also play an important role in pathophysiological conditions such as neurodegenerative and neurological diseases, including AD [29]. There are studies showing that platelet functions change in AD, however, the results are contradictory and the molecular mechanisms underlying this change have not been fully elucidated yet [22].
In this study, we showed that ADP-induced platelet functions were decreased in Alzheimer's patients (Table 2). Similarly, Wiest et al. compared the platelet function in response to ADP in healthy controls and AD cases and found a higher platelet function in controls compared to AD [30]. Contrary to our results, Jaime Ramos-Cejudo and et.al. depicted that individuals with increased platelet response show a higher risk of dementia in their old age supporting the role of platelet function in AD development [31]. However, authors recruited individuals free of antiplatelet therapy such as acetylsalicylic acid (ASA), which is known to inhibit platelet function and this is the main cause of this contradictory result [31]. We also evaluated our results by considering ASA status in controls and AD patients and we also detected AD patients free of ASA have a significantly higher platelet function compared to controls (Table 4). In agreement with our findings, suggesting that future studies related to platelet function should be conceptualized considering the inhibitory effect of antiplatelet therapies.
Platelet adhesion and activation (platelet function) to a blood vessel wall is the initial step of vascular endothelial dysfunction or damage [5]. During vascular damage or endothelial dysfunction, von Willebrand Factor (vVF) is primarily released from endothelial cells; therefore, an increase in vWF levels is recognized as a marker of endothelial dysfunction [8]. In response to an injury to the blood vessels or to endothelial dysfunction, platelets are activated rapidly [9]. Activated platelets firmly recognize and adhere to vWF in damaged endothelium via glycoprotein 1b (GP1b) on their surface receptors [6]. GP1b is a major platelet membrane adhesion receptor that initiates platelet adhesion to VWF on endothelial surfaces and damaged subendothelial matrix and transmits signals to induce platelet activation [32]. In this study, we also investigated GP1b serum levels in AD patients as an indicator of platelet activation and discovered a significant increase in AD patients compared to controls confirming our results in ADP-induced platelet functions. Haitao et al. carried out a proteomic analysis in the platelet-rich plasma of AD patients and detected a significant association between GP1b protein and platelet activation, showing that GP1b protein levels were reduced in AD patients [33]. Also, Stellos et al. detected an increase in the levels of other biomarker proteins of platelet activation (P-selectin and glycoprotein IIb-IIIa complex) in AD with cognitive decline indicating a significant relation [34]. An increase in platelet activation and GP1b may be due to endothelial damage, therefore we also analyzed the levels of vWF which is a biomarker of endothelial dysfunction to evaluate whether the increase is associated to endothelial dysfunction or AD pathogenesis. We detected a nonsignificant difference in the vWF levels in the serum of our AD patients and control samples, supporting the notion that elevated level of platelet function is not due to endothelial dysfunction but rather may be related to AD pathogenesis. Also Yavuz B.B et al. compared the vWF antigen levels in healthy subjects and AD and they did not find significant differences in the vWF antigen levels in controls compared to AD [8]. Since vWF is a biomarker of vascular damage, the role of vWF is the focus of research studies in the pathogenesis of AD [35]. However, results of vWF levels in AD patients have been conflicting. Wei Qin et al. showed that lower MMSE scores were associated with higher levels of VWF in AD compared to controls [36]. The results of a CSF proteomics study that aimed to distinguish non-AD from AD patients have shown inconsistent data in CSF vWF levels between three independent cohorts [37]. Other studies reported cortical vWF associated with microvessel density correlates with VEGF but is normal in AD [38]. In population study shows that higher levels of vWF in blood plasma samples were detected in AD patients compared to controls [39]. Overall, the levels of proteins related to platelet function and on their receptors are differentially expressed in disease conditions [10]. The levels of vWF and GP1b in blood plasma/serum and platelet function are regulated by also microRNAs (miRNAs) [11], [12]. miRNAs are recognized as a group of short non-coding RNAs that regulates gene expression negatively at the post-transcriptional level and have crucial roles in several biological pathways such as differentiation, proliferation, cell cycle, and celllular functions [13]. The differential expressions of miRNAs have been extensively studied in a variety of disorders including AD indicating the promising roles of miRNAs as therapeutic targets and biomarkers [14]. Previous studies already reported the comparison of levels of GP1b and vWF in AD [33], [34], [35], [36], [37], [38], [39], however, the relationship between platelet function of the regulatory mechanism of these two proteins in AD pathogenesis has not been elucidated yet. Therefore, in this study, we not only compared the levels of GP1b and vWF proteins in AD patients but also to clarify the molecular regulation mechanism of these two proteins, we compared the expression levels of hsa-miR-24-3p and hsa-miR-26a-5p to investigate the association with platelet function and AD. It has been suggested that hsa-miR-24-3p can regulate vWF [15] and reported AD [16, 17]. hsa-miR-26a-5p was previously reported to be differentially expressed in the blood of AD patients [40]. In an animal model, over-expression of hsa-miR-26a-5p inhibited the phosphorylation of Tau protein and the accumulation of Aβ, indicating a role of hsa-miR-26a-5p in AD pathogenesis [41]. Xie et al. also reported a significant elevation of hsa-miR-26a-5p in the occurrence and development of AD [42]. According to our results, the relative expression levels of both miRNAs mir24 and hsa-miR-26a-5p were found to be lower in AD patients compared to the control group, and we showed that the value was statistically significantly low in hsa-miR-26a-5p. İn this study, we detected the expression of hsa-miR-26a-5p was significantly down-regulated and the serum levels of GP1b was up-regulated in the blood samples of AD patients. We performed correlation analysis to examine the effect of this significant decrease in hsa-miR-26a-5p on platelet functions and the levels of GP1b and vWF we investigated. According to the correlation analysis, a positive significant correlation was found between the relative expression values of hsa-miR-26a-5p and hsa-miR24-3p in both healthy control group and AD patients. (Fig. 1A), (Fig. 2A), (Table 3). Also the result of the correlation analysis of the control group and AD patients indicated a significant positive correlation between both hsa-miR-24-3p and hsa-miR-26a-5p and platelet aggregation (Slope Ω) data. In addition, a significant positive correlation between platelet aggregation % Amplitude values and hsa-miR-26a-5p levels was detected in AD patients. These results suggest that hsa-miR-24-3p and hsa-miR-26a-5p both play a role in a molecular pathway related to the adhesion and secretion functions of platelets. Similarly, Linsey et al. demonstrated that hsa-miR-26b, another member of the same miRNA-26 family attenuates platelet adhesion and aggregation, possibly through Src and EGFR signaling in mice a model [11]. Further studies involving the targeting signaling pathways would elucidate the role of hsa-miR-26a-5p in platelet function.
Additionally, in this study, we demonstrated the inhibitory effect of ASA on the platelet function. Therefore, we evaluated the effect of ASA on the relative expression levels of hsa-miR-24-3p and hsa-miR-26a-5p. We identified reduced levels of hsa-miR-24-3p expression but not hsa-miR-26a-5p as a consequence of ASA usage, however, the reduction was nearly significant. Only one study showed the decreasing level of hsa-miR-24-3p in response to ASA and other antiplatelet therapy drugs clopidogrel, and prasugrel in diabetes mellitus (DM) patients, however, a comparison with a control group without ASA treatment was not included in the study [43]. Similarly, Peter Willeit et al. showed that few abundant platelet miRNAs, such as miR-24 levels significantly decreased with antiplatelet therapy in their study [44]. Appropriate evaluation of patients receiving antiplatelet therapy can identify low responders or patients at risk for a variety of potentially life-threatening disorders [45].
Thus, the molecular mechanism of this inhibitory effect of ASA on hsa-miR-24-3p levels should be clarified in other neurodegenerative diseases.
In the literature review, we did not find any publications including miRNAs that we studied in the molecular pathway in which the adhesion and secretion functions of platelets were investigated in AD. Our results will pave the way for further detailed studies involving the role of both that hsa-miR-24-3p and hsa-miR-26a-5p associated with the adhesion and secretion functions of platelets. The present study implicates that increased expression of serum GP1b and decreased relative expression levels of hsa-miR-26a-5p in AD. Our findings show that GP1b and hsa-miR-26a-5p have an essential role of platelet function in AD. As a conclusion, we suggest investigating the role of platelet function and two miRNAs (hsa-mir24-3p and hsa-miR-26a-5p) in the molecular mechanisms of AD, and other neurodegenerative diseases.