CD59 is a membrane protein inhibitor of the membrane attack complex (MAC) of complement. mCd59 knockout mice reportedly exhibit hemolytic anemia and platelet activation. This phenotype is comparable to the human hemolytic anemia known as paroxysmal nocturnal hemoglobinuria (PNH), in which platelet activation and thrombosis play a critical pathogenic role. It has long been suspected but not formally demonstrated that both complement and nitric oxide (NO) contribute to PNH thrombosis. Using mCd59a and mCd59b double knockout mice (mCd59ab−/− mice) in complement sufficient (C3+/+) and deficient (C3−/−) backgrounds, we document that mCd59ab−/− platelets are sensitive to complement-mediated activation and provide evidence for possible in vivo platelet activation in mCd59ab−/− mice. Using a combination of L-NAME (a NO-synthase inhibitor) and NOC-18 or SNAP (NO-donors), we further demonstrate that NO regulates complement-mediated activation of platelets. These results indicate that the thrombotic diathesis of PNH patients could be due to a combination of increased complement-mediated platelet activation and reduced NO-bioavailability as a consequence of hemolysis. Am. J. Hematol. 2009. © 2009 Wiley-Liss, Inc.