: In type 2 diabetes mellitus (T2DM), increased α2-antiplasmin incorporation in fibrin and impaired fibrinolysis have been reported. Acetylsalicylic acid (ASA), used in cardiovascular prevention, modulates fibrinolysis and exerts weaker therapeutic effect in this disease. We investigated how glycation and acetylation of α2-antiplasmin affects its interaction with fibrin. Using surface plasmon resonance, we analyzed fibrin binding by α2-antiplasmin incubated with no β-D-glucose or ASA (control); incubated with β-D-glucose (5, 10, 50 mmol/l); (3) incubated with 1.6 mmol/l acetylsalicylic acid (ASA) and (4) incubated with 1.6 mmol/l ASA and 50 mmol/l β-D-glucose. Incubation with glucose decreased affinity of α2-antiplasmin for fibrin compared with control α2-antiplasmin in a glucose concentration-depending manner. α2-Antiplasmin incubation with ASA did not affect its affinity to fibrin. α2-Antiplasmin incubation with ASA and glucose resulted in 4.2-fold increased affinity to fibrin compared with α2-antiplasmin incubated with 50 mmol/l glucose (P < 0.001). In conclusion, α2-antiplasmin incubation with glucose at concentrations encountered in T2DM is associated with decreased binding affinity of α2-antiplasmin to fibrin. ASA alone does not affect the binding affinity of α2-antiplasmin to fibrin, but partly reverses the effect introduced by the incubation with 50 mmol/l glucose. This study suggests new mechanisms involved in regulating fibrinolysis efficiency in hyperglycemia.