Shear Stress Promotes Platelet Surface Desialylation, Platelet Count Drop, and Microvesiculation: Do Platelets Need to be Sugar-Coated?

Introduction. Mechanical circulatory support (MCS) devices restore hemodynamics and are lifesaving for patients with advanced heart failure and, recently, for patients with severe COVID-19. Long-term MCS is associated with bleeding complications, often requiring multiple transfusions of blood products or reoperations. To date, due to undefined etiology, device-related bleeding lacks efficient therapeutic management. We have shown that hypershear stress, existing within device-supported circulation promotes platelet dysfunction associated with pro-apoptosis, downregulation of key adhesion receptors, impaired platelet aggregation, and microvesiculation - all contributors to bleeding. Recent studies have also shown that glycosylation of platelet surface receptors, i.e. the platelet "sugar coat," plays a major role in the regulation of platelet function and lifespan. Here, we tested the hypothesis that hypershear stress promotes platelet surface desialylation, thus facilitating platelet count drop and increased microvesiculation.

Methods. Human platelets were obtained from ACD-anticoagulated blood of healthy volunteers via gel filtration on Sepharose 2B. Gel-filtered platelets were exposed to neuraminidase and continuous shear stress (30 or 70 dyne/cm²,10 min) in a hemodynamic shearing device. Optionally, platelets were pretreated with oseltamivir, a common neuraminidase inhibitor. Platelets were co-stained with fluorophore-conjugated SNA lectin, binding terminal 2,6-linked sialic acid moieties, and anti-CD41 antibody. Multi-colored flow cytometry was performed to quantify platelet surface sialylation, platelets, and microparticles; fluorescent nanobeads SPHERO^TM were used as a size standard. The surface density of platelet sialylation was calculated as median fluorescence intensity normalized to forward scatter indicating platelet size. The platelet neuraminidase activity was quantified using fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (MUNANA) and was presented as µM of substrate per min.

Results. Platelet exposure to both hypershear stress and neuraminidase-1 induced platelet surface desialylation as indicated by the significant decrease of SNA lectins' binding density on platelets (Fig.1A). Platelet desialylation induced by shear stress and neuraminidase was associated with a notable decrease in platelet count and an increase in platelet-derived microparticles (Fig.1B). Interestingly, neuraminidase reinforced shear-mediated desialylation, platelet count drop, and microvesiculation. Oseltamivir slightly inhibited shear-mediated desialylation, preserved platelet count (30% increase vs. shear), and decreased microparticle generation (29% decrease vs. shear). Exposure to 70 dyne/cm² shear stress resulted in a 9.5-fold increase in platelet neuraminidase activity as compared to basal level: 0.85 ± 0.61 µM/min vs 0.05 ± 0.03 µM/min, respectively (Mean ± SD, p < 0.01, one-way ANOVA).

Conclusions. Shear stress promotes platelet surface desialylation with the associated reduction in platelet count and increased microvesiculation. Neuraminidase inhibition restores platelet count and decreases microparticle generation caused by hypershear. Shear-mediated increase of platelet neuraminidase activity is considered a plausible mechanism for shear-mediated platelet deglycosylation. Developing therapeutic strategies for preservation of platelet sialylation offers significant translational potential for pharmacologic management of MCS-related platelet dysfunction and bleeding coagulopathy.

No relevant conflicts of interest to declare.