Sialic Acid Content on Platelet Surface Glycoproteins Modulates Thrombin-Induced Activation

Platelets are fundamentally important in normal hemostasis and pathological thrombosis (i.e. cardiovascular diseases, stroke, etc.). Platelets mediate the initial first-step in hemostasis through surface glycoproteins like the GPIb-IX-V complex and integrin αIIbβ3 (GPIIbIIIa). Although the functions of platelet surface glycoproteins are well known, the roles of posttranslational modifications on those surface glycoproteins are poorly understood. We have recently shown that sialic acid is a key regulator of platelet survival. As platelets circulate and age in blood, they lose sialic acid and are rapidly cleared by the hepatocytes where they stimulate liver TPO production and consequently regulate thrombopoiesis. Here, we investigated the importance of glycosylation to platelet function by measuring the impact of sialic acid content on platelet responses to thrombin activation. Freshly isolated wild-type washed platelets were treated with a2-3, -6, -8 sialidase (neuraminidase, NA) to remove sialic acid from the platelet surface glycoproteins or with a competitive NA inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) to prevent sialic acid loss by the action of sialidases. After treatment with both NA and DANA, platelets were activated with Thrombin (THR, 0.1U/mL). As controls, aliquots of freshly isolated wild-type washed platelets were left untreated (Rest), only treated with neuraminidase (NA) or activated with Thrombin (THR). First, we measured b-galactose exposure using RCA-I lectin to test the efficacy of treatments. As expected, NA treated platelets showed significantly higher RCA-I binding when compared to Rest, THR and DANA treated platelets. Noteworthy, RCA-I binding to THR activated platelets was higher than Rest or DANA + THR platelets. We next investigated the effect of NA and DANA treatments of platelet degranulation. Thrombin activated platelets showed high level of P-selectin surface exposure when compared to Rest platelets. NA treatment alone caused low P-selectin exposure (~18% positive platelets) and NA + THR treated platelets showed high levels of P-selectin similar to THR only treatment. Interestingly, DANA +THR platelets showed a lower percentage of P-selectin positive platelets when compared to THR activation only (~50% compared to ~85%). In platelets, thrombin signaling is mediated by PARs, G-protein-coupled receptors that trigger several intracellular pathways, including phosphorylation of several proteins. We next investig