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

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 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.

Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity

“…Preparation of FITC-Labeled Annexin V. Human annexin V, C316S, was prepared by a procedure similar to that for annexin V-N6 (18). Annexin V was labeled with FITC according to the manufacturer's instruction (Molecular Probes).…”

The annexins: spatial and temporal coordination of signaling events during cellular stress

“…Coupled to thrombolytic enzymes, annexin A5 has localized intravascular thrombi [210]. Technetium-labeled annexin A5 has been used as a marker for apoptotic cells in cardiovascular medicine [211], and its specific binding to phosphatidylserine has made this protein a potential candidate for predicting the effectiveness of cancer chemotherapy [212].…”

Reversible Covalent Chemistry in Drug Delivery

“…Urokinase consists of two protein chains (A and B) linked by a disulfide bond; of these two chains, chain B is responsible for the catalytic conversion of plasminogen. An annexin Vurokinase (B chain) conjugate was produced by reducing the disulfide bond between the two urokinase chains and linking the B chain to the modified annexin V protease through a disulfide bond [47].…”