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 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.
Research Division, Green Cross Company, Osaka, Japan.
K
K
Tanaka
K
K
Einaga
H
H
Tsuchiyama
J F
JF
Tait
K
K
Fujikawa
D000595:Amino Acid Sequence; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D013927:Thrombosis / Q000188:drug therapy* / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology*
D017304:Annexin A5; D005343:Fibrinolytic Agents; D010743:Phospholipids; D014568:Urokinase-Type Plasminogen Activator