Abstract P1172: Shear-mediated Remodeling Of Platelet Surface Glycosylation: How Can Platelets Trim Their Sugar Coats?

Abstract

Mechanical circulatory support (MCS) is associated with bleeding complications often requiring multiple transfusions or reoperation. Hypershear stress within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, impaired aggregation, and microvesiculation - all contributors to bleeding. Recent studies showed that glycosylation of surface receptors, i.e. “ sugar coat ,” plays a major role in the regulation of platelet function and lifespan. We tested the hypothesis that shear stress promotes platelet surface deglycosylation by increasing platelet glycosidase activity. Methods: Human platelets were obtained from ACD-anticoagulated blood via gel filtration on Sepharose 2B. Platelets were exposed to neuraminidase and continuous shear stress in a hemodynamic shearing device. Platelets were stained with fluorophore-conjugated lectins binding terminal sialic acids (SNA I, MAL II) and galactose (ECA), anti-CD41 and anti-CD42a a/b. Multi-colored flow cytometry was used to quantify platelet surface glycosylation. Neuraminidase and galactosidase activities of intact and lysed platelets were measured using enzyme-specific fluorogenic substrates. Results: Exposure to shear stress and neuraminidase induced a significant decrease in platelet 2,6-sialylation and galactosylation, while 2,3-sialylation remained unaltered. Neuraminidase reinforced shear-mediated platelet desialylation, causing a prominent decrease in SNA binding even at low shear, and increased platelet galactose levels by unmasking the galactose residues capped by sialic acids. Shear stress resulted in a 9.5-fold increase in platelet neuraminidase and a 1.4-fold increase in galactosidase activities vs their basal levels. Neutral neuraminidase and galactosidase were upregulated by shear, while acidic isoforms did not increase, suggesting that other than lysosomal glycosidase pools are involved. Shear stress promotes platelet deglycosylation via upregulation of platelet neuraminidase and galactosidase activities. Developing therapeutic strategies for the preservation of platelet glycosylation offers significant translational potential for the pharmacologic management of MCS platelet dysfunction and bleeding.