The Molecular Mechanism Of Platelet Membrane A? Metabolic Modifications In The Formation Of Cerebral Vascular Amyloidosis

Objective:Cerebral amyloid angiopathy?CAA?is characterized by amyloid deposition in cerebral vessel walls.As the major component of these amyloid deposition,amyloid-??A??peptide is produced from a larger transmembrane protein known as the amyloid precursor protein?APP?via sequential proteolytic cleavage by?-and?-secretases,generating A?isoforms that vary in length?39⁴3?.As the more prevalent isoform found in vivo,A?_1-40 serves as a major component of CAA;In contrast,while A?_1-42makes up only 10%of the total A?,it is the dominant isoform in neuronal plaques.Evidence suggests that platelet activation can mediate the onset and development of CAA.Platelet activation and adhesion to a damaged vascular wall is the first step of vascular injury.Activated platelets contribute to more than 90%of the circulating forms of APP,which may be the major source of A?detected in whole blood.It has been demonstrated that genetic variants and/or metabolic modifications can accelerate the formation of intermediate-aggregation-state components.Meanwhile,Metabolic modifications of A?can occur in the brain and cerebral vessel walls of patients with Alzheimer's disease?AD?.As the major source of A?detected in the peripheral circulation,however,very few studies have focused on the metabolic modifications of A?-related amyloid in platelet membranes from patients with CAA.In the present study,we analyzed the metabolic modifications of A?peptide in platelet membranes from patients with CAA and healthy volunteers,and utilized the findings to characterize the underlying mechanism involved in the progression of CAA.Methods:1.According to the classic and modified Boston criteria,65 cases of patients with CAA were recruited from the neurology department of the Tianjin Medical University General Hospital as the experimental group?CAA group?;66 cases of healthy volunteers were recruited as the control group?HC?,who had no subjective or objective cognitive complaints and were physically and mentally healthy.Subjects taking any psychoactive medication with a possible impact on cognition?including chronic alcohol or drug abuse?were excluded.Whole venous blood?3 mL?for platelet analysis was collected in EDTA-2Na tubes.2.Platelets were isolated by differential centrifugation of 4 mL fresh peripheral venous blood samples drawn from study subjects,and then resuspended in Phosphate buffer saline?PBS?buffer.After washing,platelets were stained using Thioflavin-T?ThT?to confirm whether there were amyloid components in platelets from patients with CAA and HC.3.After prepared from platelets,membrane fractions were then analyzed by Western blot analysis for the identification of A?aggregates in platelet membranes from patients with CAA and HC.4.Metabolic modifications of A?peptide in platelet membranes were analyzed using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry?UPLC/ESI-Q-TOF/MS?.Results:1.The platelet count and those platelet indices were compared between patients with CAA and HC.The results showed that the platelet count and mean platelet volume were not significantly different between patients with CAA and HC?P>0.05?;while the platelet indices including platelet distribution width and platelet larger cell ratio were significantly higher?P<0.001?in patients with CAA as compared to HC.2.ThT assay results indicated that there were amyloid components in platelets from both patients with CAA and HC.3.Western blot analysis showed that different molecular-weight?MW?A?aggregates were found in platelet membranes.Proteins in platelet membrane fractions from patients with CAA were positively immunoreactive to the anti-A?antibody,with protein bands located at positions of 15 kDa,20 kDa,43kDa,55 kDa and 72 kDa;while protein bands from HC were located at positions of 15kDa,20 kDa and 43 kDa.4.LC-MS analysis were summarized as follows:1)the 15kDa protein band from patients with CAA comprised A?_1-40,while the 15 kDa protein band from HC and the 72 kDa protein band from patients with CAA comprised A?_1-40and A?_1-42;2)Metabolic modifications including methylation,phosphopantetheine,phosphorylation,Asn²77 deamidation and acetylation were identified in platelet membranes from both patients with CAA and HC;3)Met³⁵ oxidation(Met^OX)and Gln¹⁵ deamidation were identified only in platelet membranes from patients with CAA.Conclusions:1.There are different MW A?aggregates in platelet membranes between patients with CAA and HC.2.To our knowledge,this is the first study that reports UPLC/ESI-Q-TOF/MS results for the identification of metabolic modifications of A?in platelet membranes from patients with CAA.The result shows that different metabolic modifications are identified in platelet membranes from both patients with CAA and HC;Met^OX and Gln¹⁵ deamidation are identified only in platelet membranes from patients with CAA.This study considers that Met^OX might contribute to the A?aggregation in disease pathogenesis of CAA.3.Different MW A?aggregates,Met^OXX and Gln¹⁵ deamidation could serve as ancillary diagnostic bio-markers of CAA.