Shear Stress Promotes Platelet Deglycosylation Via Upregulation of Sialidase NEU1 on Platelets and Platelet-Derived Microparticles: One More Thing

Purpose of Study. Mechanical circulatory support (MCS) is a mainstay therapy for advanced heart failure. Long-term MCS is associated with persistent thrombocytopenia and severe bleeding complications, often requiring multiple transfusions of blood products. 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-apoptotic phenotype, impaired platelet aggregation, and microvesiculation - all contributors to bleeding. Recently, it has been recognized that glycosylation of platelet surface receptors plays a major role in the regulation of platelet function and lifespan. Here, we tested the hypothesis thatshear stress promotes platelet surface deglycosylation via upregulation of platelet neuraminidase activity, thus facilitating pro-apoptosis, platelet count drop, and increased microvesiculation.

Methods. Human platelets were obtained from ACD-anticoagulated blood ofhealthy volunteers via gel filtration on Sepharose 2B. Platelets were pretreated with neuraminidase inhibitors (1 mM oseltamivir acid or 1 mM DANA) and subjected to shear stress (30 & 70 dyne/cm²,10 min) in a hemodynamic shearing device. Sheared platelets were then stained with fluorophore-conjugated SNA I and RCA I lectins binding 2,6-linked sialic acids and galactose, respectively, annexin V, anti-NEU1 and anti-CD41 antibodies. Multi-colored flow cytometry was used to quantify platelet surface glycosylation, NEU1 surface expression, annexin V binding as an apoptotic marker, CD41+ platelets and microparticles; fluorescent nanobeads SPHERO^TM were used as a particle size standard. Lectin binding and NEU1 density were calculated as median fluorescence intensity normalized to forward scatter indicating particle size. Platelet neuraminidase activity was tested using fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid at pH 7.4.

Results. Exposure to shear stress induced platelet surface deglycosylation as indicated by an incremental decrease of SNA I and RCA I lectin binding on platelets with the increase of shear magnitude. Shear-mediated deglycosylation was associated with increased annexin V binding on platelets, platelet count decline, and increased generation of AnV+/CD41+ microparticles. Deciphering operative mechanisms of shear-mediated desialylation, we showed that shear stress promoted a 2-fold increase of NEU1 surface expression on platelets and an associated rise in platelet neuraminidase activity: 0.047±0.00 nM/min vs 0.387± 0.070 nM/min in resting and sheared platelet fractions, respectively. Notably, a substantial population of CD41+ microparticles generated by shear stress carried an increased density of NEU1 on their surface (0.651±0.010 AU vs 0.141±0.006 AU on sheared platelets), potentially contributing to platelet-derived neuraminidase activity. Testing the effect of neuraminidase inhibition, we found that oseltamivir carboxylate and DANA partially preserved platelet surface glycosylation compromised by shear stress, as indicated by an increase of SNA I and RCA I lectin binding on sheared platelets. Furthermore, neuraminidase inhibition resulted in platelet count recovery (23% increase vs. shear) and reduced microparticle generation (28% decrease vs. shear), specifically targeting the AnV+/CD41+ microparticle population (38% decrease vs. shear). Rather modest potency of neuraminidase inhibitors observed in our study could be explained by their limited efficacy on mammalian neuraminidases as compared to viral ones.

Conclusions. Shear stress promotes platelet surface deglycosylation, associated with pro-apoptosis, platelet count decline, and intense microvesiculation. Neuraminidase inhibition partially restores platelet count and decreases microparticle generation caused by hypershear. Shear-mediated upregulation of platelet NEU1 expression, generation of NEU1+ microparticles, and increased platelet sialidase activity is a plausible operative mechanism for shear-mediated platelet deglycosylation. Preservation of platelet sialylation via neuraminidase inhibition offers significant translational potential for pharmacologic management of MCS-related thrombocytopenia and bleeding coagulopathy.

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