Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and alpha(2)Macroglobulin (alpha M-2) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and alpha M-2 levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, alpha M-2 plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of alpha M-2 to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII.