PREPARATION AND CHARACTERIZATION OF A DISULFIDE-LINKED BIOCONJUGATE OF ANNEXIN-V WITH THE B-CHAIN OF UROKINASE - AN IMPROVED FIBRINOLYTIC AGENT TARGETED TO PHOSPHOLIPID-CONTAINING THROMBI

A conjugate of annexin V and the B-chain of urokinase was prepared and its fibrinolytic properties were studied. First, a mutant of annexin V was constructed with an N-terminal extension of six amino acids (Met -Ala-Cys-Asp-His-Ser) and with Cys(316) mutated to Ser; this molecule was expressed in Escherichia coli. The urokinase B-chain was prepared by limited reduction of the interchain disulfide bond between the A- a nd B-chains of urokinase. These two molecules were then connected by a disulfide bond and purified to yield a 1:1 stoichiometric conjugate. The conjugate had the same catalytic activity as urokinase against a s ynthetic substrate, Glt-Gly-Arg-MCA, and a similar plasminogen activat ing activity. The conjugate showed the same binding affinity for phosp hatidylserine-containing membranes as annexin V. The in vitro fibrinol ytic activity of the conjugates on clots prepared from platelet-rich p lasma was comparable to that of urokinase. However, the conjugate show ed 3-4-fold stronger in vivo thrombolytic activity than urokinase in a rat pulmonary embolism model, while having essentially the same plasm a clearance rate as urokinase or B-chain. These results show that anne xin V is a useful agent for targeting plasminogen activators to phosph olipid-containing thrombi.