An important risk factor for thrombosis is the polymorphism R506Q in factor V that causes resistance of factor Va to proteolytic inactivation by activated protein C (APC). To study the potential influence of the carbohydrate moieties of factor Va on its inactivation by APC, factor V was subjected to mild deglycosylation (neuraminidase plus N-glycanase) under nondenaturing conditions. The APC resistance ratio values (ratio of activated partial thromboplastin time [APTT] clotting times with and without APC) of the treated factor V were increased (2.4 to 3.4) as measured in APTT assays. O-glycanase treatment of factor V did not change the APC resistance ratio. The procoagulant activity of factor V as well as its activation by thrombin was not affected by mild deglycosylation. Treatment of factor V with neuraminidase and N-glycanase mainly altered the electrophoretic mobility of the factor Va heavy chain, whereas treatment with O-glycanase changed the mobility of the connecting region. This suggests that the removal of the N-linked carbohydrates from the heavy chain of factor Va, which is the substrate for APC, is responsible for the increase in susceptibility to inactivation by APC. Thus, variability in carbohydrate could account for some of the known variability in APC resistance ratios, including the presence of borderline or low APC resistance ratios among patients who lack the R506Q mutation.