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

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Abstract
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 Cys316 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- and 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 synthetic substrate, Glt-Gly-Arg-MCA, and a similar plasminogen activating activity. The conjugate showed the same binding affinity for phosphatidylserine-containing membranes as annexin V. The in vitro fibrinolytic activity of the conjugates on clots prepared from platelet-rich plasma was comparable to that of urokinase. However, the conjugate showed 3−4-fold stronger in vivo thrombolytic activity than urokinase in a rat pulmonary embolism model, while having essentially the same plasma clearance rate as urokinase or B-chain. These results show that annexin V is a useful agent for targeting plasminogen activators to phospholipid-containing thrombi.