Suppression of plasminogen activator inhibitor 1 (PAI-1) activity levels in rats by monoclonal antibodies

Abstract

Summary Background : Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with several important thrombotic diseases. Rat models have been shown to be suitable for in vivo investigations on thrombolysis and fibrinolysis. In the present study, monoclonal antibodies (MA) against rat PAI-1 are used to reduce PAI-1 levels in endotoxin-treated rat plasma. Results : A large panel of murine monoclonal antibodies was raised against recombinant rat PAI-1. Out of 237 monoclonal antibodies, six antibodies showed a strong inhibition towards recombinant rat PAI-1 activity in vitro resulting in 70–100% inhibition of PAI-1 activity at a 16-fold molar excess of MA over PAI-1. Relative epitope analysis revealed that this panel represented at least two different epitopes. Subsequent selection of two monoclonal antibodies (MA-124K1 and MA-159M12) and evaluation of their PAI-1 neutralizing properties in vitro in rat plasma, supplemented with recombinant rat PAI-1 and in plasma from endotoxin-treated rats, revealed that MA-124K1 was capable of inhibiting native rat PAI-1 in a plasma environment. Based on these data, MA-124K1 was selected for evaluation in in vivo experiments. Rats were pre-treated with endotoxin (0.5 mg/kg, ip, 90 min prior to MA administration) to obtain high levels of native rat PAI-1 in vivo. Levels of PAI-1 antigen and activity in plasma were measured by specific ELISAs. Injection of a single dose of MA-124K1 (3 mg/kg body weight) resulted in a ∼60% inhibition of PAI-1 activity without affecting PAI-1 antigen levels. No changes were observed upon administration of MA-32K3, a control monoclonal antibody. Conclusion : MA-124K1 may serve as a useful tool in studies on the evaluation of the role of PAI-1 in cardiovascular disease using rat models.