Biology subjects

Slepian, M. J.

Publications and source records attributed to Slepian, M. J..

3 recordsLinked to original sources

bioRxiv2024

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

ObjectiveMechanical 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 stress in 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/583630v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@84eda2org.highwire.dtl.DTLVardef@10d3f90org.highwire.dtl.DTLVardef@c83bcborg.highwire.dtl.DTLVardef@1cd981c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Shear-Mediated Platelet Microparticles Demonstrate Phenotypic Heterogeneity as to Morphology, Receptor Distribution, and Hemostatic Function

ObjectiveImplantable cardiovascular therapeutic devices (CTD) including stents, percutaneous heart valves and ventricular assist devices, while lifesaving, impart supraphysiologic shear stress to platelets resulting in thrombotic and bleeding device-related coagulopathy. We previously demonstrated that shear-mediated platelet dysfunction is associated with downregulation of platelet GPIb-IX-V and IIb{beta}3 receptors via generation of platelet-derived microparticles (PDMPs). Here, we test the hypothesis that shear-generated PDMPs manifest phenotypical heterogeneity of their morphology and surface expression of platelet receptors, and modulate platelet hemostatic function. Approach and ResultsHuman gel-filtered platelets were exposed to continuous shear stress and sonication. Alterations of platelet morphology were visualized using transmission electron microscopy. Surface expression of platelet receptors and PDMP generation were quantified by flow cytometry. Thrombin generation was quantified spectrophotometrically, and platelet aggregation in plasma was measured by optical aggregometry. We demonstrate that platelet exposure to shear stress promotes notable alterations in platelet morphology and ejection of several distinctive types of PDMPs. Shear-mediated microvesiculation is associated with the differential remodeling of platelet receptors with PDMPs expressing significantly higher levels of both adhesion (IIb{beta}3, GPIX, PECAM-1, P-selectin, and PSGL-1) and agonist-evoked receptors (P2Y12 & PAR1). Shear-mediated PDMPs have a bidirectional effect on platelet hemostatic function, promoting thrombin generation and inhibiting platelet aggregation induced by collagen and ADP. ConclusionsShear-generated PDMPs demonstrate phenotypic heterogeneity as to morphologic features and defined patterns of surface receptor alteration, and impose a bidirectional effect on platelet hemostatic function. PDMP heterogeneity suggests that a range of mechanisms are operative in the microvesiculation process, contributing to CTD coagulopathy and posing opportunities for therapeutic manipulation.

biochemistry↗

bioRxiv2021

Shear-Mediated Platelet Activation is Accompanied by Unique Alterations of Platelet Lipid Profile

Platelet activation by mechanical means such as shear stress, is a vital driver of thrombotic risk in implantable blood-contacting devices used in treatment of heart failure. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical - shear mediated platelet activation. Here, we determined if shear-activation of platelets induced lipidome changes that differ from those associated with biochemically-mediated platelet activation. We performed high-resolution lipidomic analysis on purified platelets from four healthy human donors. For each donor, we compared the lipidome of platelets that were non-activated or activated by shear, ADP, or thrombin treatment. We found that shear activation altered cell-associated lipids and led to the release of lipids into the extracellular environment. Shear-activated platelets released 21 phospholipids and sphingomyelins at levels statistically higher than platelets activated by biochemical stimulation. Many of the released phospholipids contained an arachidonic acid tail or were phosphatidylserine lipids, which have procoagulant properties. We conclude that shear-mediated activation of platelets alters the basal platelet lipidome. Further, these alterations differ and are unique in comparison to the lipidome of biochemically activated platelets. Our findings suggest that lipids released by shear-activated platelets may contribute to altered thrombosis in patients with implanted cardiovascular therapeutic devices.

cell biology↗