The Antiplatelet Effects of a New Nitroderivative of Acetylsalicylic Acid - An In Vitro Study of Inhibition on the Early Phase of Platelet Activation and on TXA2 Production

Abstract

SummaryWe studied in vitro the antiplatelet activity of a new nitroderivative chemically related to acetylsalicylic acid: 2 acetoxybenzoate 2-[l-nitroxy-methyl]-phenyl ester (NCX 4016), in order to identify any effects due to the release of nitric oxide and the blockade of cyclooxygenaseThe effects of scalar doses of NCX 4016 on the early phase of platelet activation, platelet aggregation and thromboxane A2 production were investigated. We observed inhibitory effects of NCX 4016 on platelet adhesion (IC50 = 7.3 × 10−5 M), platelet cytosolic calcium concentration, assayed by fluorescent probe Fura 2, and the expression of glycoprotein IMIIa (CD41 / αIIbβ3) (IC50 = 3.4 × 10−5 M) and P-selec-tin (CD62 / GMP-140) (IC50 = 4.9 × 10−5 M) measured by flow cytometry. NCX 4016 also prevented thrombin-induced platelet aggregation (IC50 = 3.9 × 10−5 M). None of these parameters were affected by acetylsalicylic acid. These inhibitory activities of NCX 4016 were abolished by oxyhaemoglobin and methylene blue. Intracellular cyclic GMP observed during thrombin-induced aggregation was increased by incubation with NCX 4016. These results appear to be attributable to the release of nitric oxide, which activates soluble platelet guanylyl-cyclase and promotes intracellular cyclic GMP increase. NCX 4016 almost completely inhibited platelet thromboxane A2 production and arachidonic acid-induced platelet aggregation. This also occurred in the presence of oxyhaemoglobin and methylene blue, indicating that its antiplatelet activity can be attributed not only to nitric oxide release but also to cyclo-oxygenase inhibition.

NCX4016 (NO‐Aspirin) has multiple inhibitory effects in LPS‐stimulated human monocytes

“…This is dependent on cell activation, since not observed in non-stimulated cells, and is accompanied by increased expression of COX-2 (Hempel et al, 1994). The inhibitory activity of NCX4016 on platelet COX has been shown to be irreversible since not reduced when the drug is washed away from the cell (Lechi et al, 1996). In the present study, while the eects of NCX4016 and ASA are similar when these drugs are added to adherent monocytes before stimulation and incubation with LPS lasts 6 h, the eects of 10 ± 100 mmol l 71 NCX4016 appear weaker than ASA after a more prolonged period of incubation (16 h), thus suggesting that the anti-COX activity of NCX4016 might be reversible.…”

“…Similarly to ASA, NCX4016 has been shown in vitro to prevent the release of thromboxane (TX) B 2 from activated human platelets through the irreversible inhibition of platelet COX (Lechi et al, 1996). When repeatedly administered in rat, NCX4016 reduced ex vivo platelet release of TXB 2 to an extent similar to that observed with ASA (Cuzzolin et al, 1996).…”

“…In fact, NCX4016, unlike ASA, has been shown to inhibit platelet adhesion as well as platelet aggregation induced by thrombin. These eects were entirely dependent on the release of NO, since not being achievable through the inhibition of platelet COX (Lechi et al, 1996). When tested in vivo NCX4016 proved to have anti-thrombotic activity even better than ASA, as observed in experimental animals (Momi et al, 2000;Wallace et al, 1999).…”

“…This compound belongs to a new class of non-steroidal anti-in¯ammatory agents (NSAIDs) able to release nitric oxide (NO) and to inhibit cyclo-oxygenase (COX) (del Lechi et al, 1996). Due to chemical similarity, NCX4016 and ASA may share antithrombotic properties.…”

Effects of nitric oxide-releasing aspirin versus aspirin on restenosis in hypercholesterolemic mice

“…Therefore, in this clinical setting, a more effective therapeutic approach is necessary. NCX-4016 possesses two additional actions, namely antiinflammatory and antiplatelet adhesion (20), which are well known to be beneficial in treating restenosis (1)(2)(3).…”