Shear Stress Promotes Remodeling of Platelet Glycosylation via Upregulation of Platelet Glycosidase Activity
Description
This dataset contains data related to the manuscript titled "Shear Stress Promotes Remodeling of Platelet Glycosylation via Upregulation of Platelet Glycosidase Activity: One More Thing" published in the Thrombosis and Haemostasis Journal (Thromb Haemost 2025; 125(04): 317-336, DOI: 10.1055/a-2398-9532). It contains flow cytometry data (FCS-files) indicating platelet surface glycosylation, expression of glycoprotein receptors on platelets and platelet-derived microparticles, as well as glycosidase activity data (MS Excel files) in human platelets, blood plasma, and serum. We tested the hypothesis that shear stress induces remodeling of platelet surface glycosylation via upregulation of glycosidase activity, thus facilitating platelet count decline and intense microvesiculation. Human gel-filtered platelets were exposed to continuous shear stress of increasing magnitude in vitro with or without neuraminidase. Platelets and platelet-derived microparticles (PDMPs) were quantified via flow cytometry using size standard fluorescent nanobeads. Platelet surface glycosylation and NEU1 expression were evaluated using lectin- or immunostaining and multicolor flow cytometry. Platelet neuraminidase, galactosidase, hexosaminidase, and mannosidase activities were quantified using 4-methylumbelliferone-based fluorogenic substrates. We demonstrated that shear stress promotes selective remodeling of platelet glycosylation via downregulation of 2,6-sialylation, terminal galactose, and mannose, while 2,3-sialylation remained largely unchanged. Shear-mediated deglycosylation is partially attenuated by neuraminidase inhibitors, strongly suggesting the involvement of platelet neuraminidase in observed phenomena. Shear stress increased platelet NEU1 surface expression and potentiated generation of numerous NEU1+ PDMPs. Platelets exhibited high basal hexosaminidase and mannosidase activities; basal activities of platelet neuraminidase and galactosidase were rather low and were significantly upregulated by shear stress. Shear stress of increased magnitude and duration promoted an incremental decline of platelet count and immense microvesiculation, both being further exacerbated by neuraminidase and partially attenuated by neuraminidase inhibition. In summary, our data indicate that shear stress accumulation, consistent with supraphysiologic conditions of device-supported circulation, promotes remodeling of platelet glycosylation via selective upregulation of platelet glycosidase activity. Shear-mediated platelet deglycosylation is associated with platelet count drop and increased microvesiculation, thus offering a direct link between deglycosylation and thrombocytopenia observed in device-supported patients. Based on our findings, we propose a panel of molecular markers to be used for the reliable detection of shear-mediated platelet deglycosylation in MCS.
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Steps to reproduce
Blood Collection: Blood was collected from healthy volunteers via venipuncture, anticoagulated with acid citrate dextrose solution, and processed to obtain serum, platelet-rich plasma (PRP), and platelet-poor plasma (PPP). Gel-filtered platelets (GFPs) were isolated from PRP via gel-chromatography on Sepharose-2B, resuspended in modified Tyrode's buffer and platelet count was measured on Z1 Particle Counter. Platelet Exposure to Shear Stress: Recalcified GFPs (20,000 or 100,000/µL, 2.5mM CaCl2) were subjected to uniform continuous shear stress in a computer-controlled modified cone-plate-Couette viscometer. Prior to shear exposure, platelets were treated with neuraminidase inhibitors (1mM oseltamivir carboxylate, 1mM 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), and 1mM zanamivir) for 30 min. Flow Cytometry of Platelet Surface Glycosylation, Neuraminidase, and Glycoprotein Expression:To detect terminal sugars on the platelet surface, GFP (20,000 plt/μL, 2.5 mM CaCl2) were stained with fluorescein-conjugated lectins: Cy5-Sambucus Nigra agglutinin I (SNA) for α-2,6-linked sialic acids (Sias), biotinylated Maackia Amurensis lectin II (MAL) and PECy5-streptavidin (0.1 ug/ml) for α-2,3-linked Sias, FITC-Ricinus Communis agglutinin I (RCA) and FITC-Erythrina Cristagalli agglutinin (ECA) for galactose, FITC-soybean agglutinin (SBA) for N-acetylgalactosamine (GalNAc), FITC-Griffonia Simplicifolia lectin II (GSL) for N-acetylglucosamine (GlcNAc), and FITC-Lens Culinaris agglutinin (LCA) for mannose. Alternatively, recalcified GFPs were double-stained with anti-NEU1-PE and anti-CD41-APC or annexin V-FITC and anti-CD41-APC, to track platelet neuraminidase expression and phosphatidilserine exposure, respectively. Flow cytometry was conducted on a FACSCantoII cytometer. Ten thousand events were captured within the gate “Platelets + Microparticles.” Glycosidase Activity in Blood Fractions: Glycosidase activity was measured in PRP, PPP, serum, and recalcified GFPs (100,000/μL, 2.5mM CaCl2) before and after shear exposure. For intraplatelet glycosidase activity, aliquots of PRP, nonsheared GFPs, and sheared GFPs were lysed with RIPA buffer containing proteinase and phosphatase inhibitors at 1:1 ratio. Enzymatic activities of neuraminidase, galactosidase, hexosaminidase, and mannosidase were evaluated in acidic and neutral pH using corresponding 4-methylumbelliferone-based fluorogenic substrates. Kinetics of 4-methylumbelliferone release from fluorogenic substrates were recorded as an increase in fluorescence for 2 h at 37C using FilterMax F5 microplate reader (Ex:360 nm, Em:430 nm). The resulting concentration of 4-methylumbelliferone was determined using a calibration curve. Glycosidase activities were calculated as the initial velocity of 4-methylumbelliferone accumulation (nM/min).
Institutions
- University of ArizonaArizona, Tucson