Inhibitory effect of nicked β2-GPI on plasmin generation. (A) Plasmin generation was measured by parabolic rate assay using synthetic substrate S-2251 in the presence of tPA, Glu-plasminogen, and fibrin monomer. Nicked β2-GPI (•), intact β2-GPI (○), β2-GPI domain I-IV mutant (▪), or BSA (□) was added to the reaction in the indicated concentrations. After 12 hours of incubation, absorbance at 405 nm was measured and expressed as tPA activity (U/mL) using tPA as standard. (B) Fibrinolytic activity was measured using fibrin plate assay. Solution reaction containing tPA, Glu-plasminogen, and nicked (•) or intact β2-GPI (○) were placed onto fibrin plates. After 36 hours of incubation, the ring area of lysis was measured. Assays were performed in triplicate. Error bars indicate SDs. D indicates domain.
