title: GPVI modulation during platelet activation and storage: its expression levels and ectodomain shedding compared to markers of platelet storage lesion.
abstract: Platelet storage is associated with deleterious changes leading to the loss of platelet reactivity and response. During storage, platelets experience increased expression and shedding of P-selectin and CD40L as specific markers of platelet activation, whereas GPIbα decreases due to ectodomain shedding. As an important adhesive receptor, GPVI contributes significantly to thrombus formation while its expression and shedding levels during storage of platelet products have not been well characterized yet. This study investigated the modulation of GPVI during platelet storage. For this study, samples obtained from 10 PRP-platelet concentrates (PCs) were subjected to flow-cytometry analysis to examine the expression of platelet activation markers and GPVI on days 1, 3, and 5 post-storage. To examine the levels of etcodomain shedding of these molecules, microparticle (MP)-free supernatants were also analyzed by either ELISA or Western blot methods. According to results, the expression levels of P-selectin and CD40L as well as the amounts of their soluble forms significantly increased during storage. The expression of GPIbα and GPVI decreased whereas their shedding significantly increased post-storage. The expression and shedding levels of these two receptors were significantly correlated. Negative correlations between the expressions of GPIbα or GPVI and P-selectin have been observed whereas their shedding levels were significantly relevant together. In a control study, the use of biotinylated platelet resuspended in Tyrode's buffer in the presence of ionophore with/without EDTA, confirmed the role of calcium in receptors shedding. In citrated PRP-PCs, recalcification of platelets also enhanced shedding levels of both GPIbα and GPVI. Intriguingly, the shedding levels of GPVI in stored PRP-PCs were much higher than those of ionophore-treated controls obtained from washed platelets. The ratios of sGPVI in stored platelet to ionophore-treated controls were also at least six times higher than those of GPIbα during storage. In conclusion, here we showed significant decreases of GPVI expression associated with its increasing levels of shedding during storage, suggesting GPVI as a valid marker of platelet storage lesion. Importantly, we found higher levels of GPVI shedding in stored platelets than those of ionophore-treated non-stored control samples. This suggests whereas platelet receptor shedding is mainly modulated by calcium-dependent signals, either platelet-surface interactions with the container walls during storage or induced shear stress under long-term agitation, might be also involved in the excessive shedding of GPVI during the storage of PCs.
authors: a Blood Transfusion Research Center , High Institute for Research and Education in Transfusion Medicine , Tehran , Iran.
authors: Ehteramolsadat
authors: E
authors: Hosseini
authors: a Blood Transfusion Research Center , High Institute for Research and Education in Transfusion Medicine , Tehran , Iran.|b Australian Centre for Blood Diseases , Monash University , Melbourne , Australia.
authors: Mehran
authors: M
authors: Ghasemzadeh
authors: a Blood Transfusion Research Center , High Institute for Research and Education in Transfusion Medicine , Tehran , Iran.
authors: Fatemeh
authors: F
authors: Nassaji
authors: a Blood Transfusion Research Center , High Institute for Research and Education in Transfusion Medicine , Tehran , Iran.
authors: Zeynab Pirmohammad
authors: ZP
authors: Jamaat
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism*; D001793:Blood Preservation; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis*; D000072417:Protein Domains
chemical_list: D015415:Biomarkers; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI
keywords: CD40 ligand; P-selectin; platelet activation; platelet glycoprotein GPIb-IX Complex; platelet membrane glycoprotein VI; shedding