Characterization of the binding of a glycosylated serine protease from Euphorbia cf. lactea latex to human fibrinogen.

ABSTRACT

In this study, the binding of a glycosylated serine protease (EuP-82) with human fibrinogen was investigated by isothermal titration calorimetry (ITC). ITC analysis indicated that the binding of EuP-82 to fibrinogen in the conditions with or without the activator (Ca2+ ) was an exothermic reaction (dominant negative enthalpy), which tended to be driven by hydrogen bonding and van der Waals interactions. In contrast, the binding of fibrinogen-EuP-82 in the condition with the inhibitor (Zn2+ ) was an unfavorable endothermic reaction. EuP-82 could not inhibit the platelet activity in citrated whole blood via the ADP-receptor pathways (mainly, P2Y1 and P2Y12), but it could enhance the platelet aggregation. The ITC together with whole blood platelet aggregation suggested that EuP-82 provided multiple fibrinogen-binding sites that were not related to the arginine-glycine-aspartate (RGD) and the dodecapeptide sequences of fibrinogen. In addition, EuP-82 had neither thrombin-like activity nor anticoagulant activity. The SR-FTIR spectra revealed that EuP-82 was a glycoprotein. Deglycosylation of EuP-82 did not affect its proteolytic activity. Moreover, EuP-82 did not exhibit any toxicity to the living cells (NIH-3T3). This study supports that EuP-82 may be useful for wound-healing material through stabilizing the clot via the platelet induction for the first process.