PB0769 - The role of functional ectonucleotidases in human platelet activation signaling

Background: Circulating platelets are key mediators of thrombosis and haemostasis. Platelets dense granule contain adenine nucleotides (ATP and ADP), which are released in the surrounding medium upon agonist-stimulation. Level of these nucleotides in extracellular fluid is regulated by activities of ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73) expressed on platelet surface. After release, these nucleotides bind with their specific receptors on platelet surface to further activate and recruit additional platelets at the site of vascular damage leading to formation of platelet plug or thrombus. However, very little is known about function of CD39 and CD73 in human platelets.

Aims: To investigate the consequences of functional ectonucleotidases in human platelet activation.

Methods: Platelets were isolated from fresh human blood by differential centrifugation. Analysis of CD39 and CD73 expression on human platelet surface were carried out by flow cytometry. Inorganic phosphate (Pi) released from platelets were measured from complex formation with malachite green molybdate at 620 nm by using multimodal microplate reader. Protein expression analysis was carried out by Western blotting. Free intracellular calcium was measured in platelet-rich plasma by employing Fura-2 AM dye using Hitachi fluorescence spectrophotometer.

Results: CD39 and CD73 were found to be notably expressed on surface of resting platelets. Thrombin-stimulation significantly enhanced their surface expression, indicative of thrombin-mediated externalization of these ectonucleotidases. CD39 and CD73 activities were significantly upregulated in thrombin-activated platelets as compared to control counterparts. Inhibition of CD73 activity (by APCP, a specific inhibitor of CD73) in thrombin-treated platelets led to enhanced tyrosine phosphorylation of proteins and significant rise in intracellular free calcium, thus signifying the inhibitory role of the ectonucleotidase on agonist-mediated platelet signaling.

Conclusion(s): Our study presents compelling evidence that human platelets express functional ectonucleotidases that restrict platelet activation signaling.