The effects of nitric oxide (NO) on platelet membrane receptor expression during activation with human alpha-thrombin

Nitric oxide (NO) is known as a regulator of platelet function by its anti-adhesive, anti-aggregating, and disaggregating properties. We investigated the modulating effects of the NO-releasing compound SIN-1 (3-morpholino-sydnonimine) on platelet surface glycoprotein (GP) expression during stimulation with human alpha-thrombin. Analysis was performed with two-color flow cytometry using fluoresceine-isothiocyanate (FITC) and phycoerythrin-(PE)-conjugated monoclonal antibodies (MoAbs) directed against GPIb CD42b), GP IIb-IIIa (CD41), P-selectin (CD62P), and MoAb PAC-1 directed against activated GP IIb-IIIa. Preincubation of platelets with SIN-1 (IC50: 1 microM) significantly decreased expression of both total and activated GP IIb-IIIa, and P-selectin in platelets stimulated with thrombin (ED50: 0.05 U/ml), whereas thrombin-induced downregulation of GP Ib was not attenuated. P-selectin expression increased in thrombin-stimulated platelets over time; in contrast, activated GP-IIb-IIIa decreased after an initial peak, indicating that thrombin-induced GP IIb-IIIa activation is spontaneously reversible. SIN-1 reduced P-selectin expression only when added before or at the same time as thrombin, whereas conformationally changed GP-IIb-IIIa was significantly reversed at up to 60 minutes after stimulation by SIN-1. In conclusion, NO attenuates activation marker expression in a dose and time dependent manner. GP-IIb-IIIa is highly sensitive to NO which not only prevents receptor activation but also promotes reversal of activated GP IIb-IIIa complex.