Previous studies have demonstrated the presence of

Previous studies have demonstrated the presence of soluble NTPDases, 5′-nucleotidase and adenosine deaminase in rat blood serum as well as in human blood (Doleski et al., 2016; Oses et al., 2004; Yegutkin, 1997; Yegutkin et al., 2007). In addition, our earlier reports have shown that these membrane-bound enzymes may mediate and modulate purinergic system in lymphocytes and platelets of sickle cell anemia patients (Castilhos et al., 2015, 2016). However, there is dearth of information on the activities and levels of the soluble NTPDases in SCA. Thus, we speculated that soluble NTPDases, 5′-nucleotidase and adenosine deaminase may also participate in the regulation of Tirapazamine nucleotides in serum of SCA patients. To accomplish this, we investigated the possible changes in adenine nucleotides and nucleoside metabolizing enzymes as well as the levels of nucleotides and nucleosides in serum of SCA patients.
Materials and methods
Results
Discussion Pathologic conditions such as inflammation and ischemia occur in sickle cell disease and are directly associated with the extracellular release of nucleotides, particularly of ATP and ADP (Jackson et al., 1996; Sun and Xia, 2013). Previous studies have shown that elevated ATP levels may occur due to nucleotide release from the red blood cells in response to low oxygen tension and hemoglobin deoxygenation (Ellsworth, 2004; Ellsworth et al., 1995). In addition, its levels and effects could be attenuated by soluble NTPDase activity. To our best knowledge, this is the first report where the relationship between adenine nucleotides hydrolyzing enzymes and its levels in serum of SCA patients was studied. Certain amounts of soluble NTPDase activity are constitutively circulated in human bloodstream (Yegutkin et al., 2007). In this study, the observed elevations in ATPase and ADPase activities in serum of SCA patients may indicate the pathophysiological conditions associated with SCA and the attempt to maintain the ATP and ADP levels at physiological concentration (Ralevic and Burnstock, 1998; Yegutkin et al., 2006). High concentrations of ATP and ADP in the extracellular medium activate the pro-inflammatory purinergic P2X7 receptors and contribute to tissue damage (Di Virgilio, 1995), platelets induction aggregation and thrombus formation, respectively (Puri, 1999). These findings corroborate with previous results obtained from our research group where increased E-NTPDase activity for ATP and ADP hydrolyses were also observed in lymphocytes (Castilhos et al., 2015) and platelets of SCA patients (Castilhos et al., 2016). Soluble NTPDase enzyme may thus be fundamentally importance as a diagnostic marker in SCA. In this study, the non-significant change in ATP and ADP levels between SCA patients and healthy individuals may suggest a compensatory mechanism initiated by high ATPase and ADPase activity in serum in order to maintain normal physiological concentrations of ATP and ADP in SCA patients. Earlier studies have revealed that physiological concentrations of extracellular nucleotides are usually in nanomolar range under normal conditions (Yegutkin et al., 2006) and the presence of high ATP concentrations are considered cytotoxic (Ralevic and Burnstock, 1998). Oses et al. (2004) reported that soluble NTPDase with 5′-nucleotidase from rat blood serum may act by lowering ATP, ADP and AMP, as well as increasing the concentration of adenosine, which is a potent vasodilator. Besides the increased ATPase and ADPase activities observed in this study, the AMPase activity was also significantly higher in SCA patients when compared to healthy subjects. This result agrees with the study of Mohamed et al. (1993), where an increase in 5′-nucleotidase activity in serum of patients with sickle cell anemia was reported. The increased AMPase activity observed in SCA patients may be a compensatory mechanism that could be directly related to the amount of AMP nucleotide produced by high ATPase and ADPase activity and typified by the extracellular normal nanomolar range of AMP measured by HPLC in this study. Cellular release of ATP/ADP may result in feed-forward inhibition of 5′-nucleotidase and delay of extracellular adenosine formation until their extracellular levels have been reduced to low micromolar levels by nucleotidases (James and Richardson, 1993). The 5′-nucleotidase catalyzes the formation of adenosine from extracellular AMP and activation of P1 adenosine receptors (Zimmermann, 1996). Adenosine is an endogenous regulatory metabolite and inhibitor of platelet aggregation (Galen et al., 1992). Adenosine released from different cells or produced through the activity of cell surface ectoenzymes, exerts its effects through the binding of four different G-protein-coupled adenosine receptors. In platelets, binding of A2 subtypes (A2A or A2B) leads to consequent elevation of intracellular cyclic adenosine monophosphate, an inhibitor of platelet activation (Johnston-Cox and Ravid, 2011).