Shear Stress Promotes Remodeling of Platelet Glycosylation via Upregulation of Platelet Glycosidase Activity: The Ulterior Cause of MCS-Related Thrombocytopenia?

Abstract

ABSTRACTObjectiveMechanical circulatory support (MCS) is a mainstay of therapy for advanced and end-stage heart failure. Accompanied by systemic anticoagulation, contemporary MCS has become less thrombogenic, with bleeding complications emerging as a major cause of readmission and 1-year mortality of device-supported patients. Shear-mediated platelet dysfunction (SMPD) and thrombocytopenia of undefined etiology are primary drivers of MCS-related bleeding. Recently, it has been demonstrated that deprivation of platelet surface glycosylation is associated with the decline of hemostatic function, microvesiculation, and premature apoptosis. 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.Approach and ResultsHuman gel-filtered platelets were exposed to continuous shear stressin vitro. Platelets and platelet-derived microparticles were quantified via flow cytometry using size standard fluorescent nanobeads. Platelet surface glycosylation was evaluated using lectin staining and multicolor flow cytometry; lectin blotting was utilized to verify glycosylation of individual glycoproteins. 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 DANA and zanamivir, strongly suggesting involvement of platelet neuraminidase in observed phenomena. 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 potentiated an incremental decline of platelet count and immense microvesiculation, both being further exacerbated by neuraminidase.ConclusionsOur 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.GRAPHICAL ABSTRACT