Abstract
Without conjunctive administration of an anticoagulant, endothelial injury-induced
thrombosis is resistant to thrombolysis and prone to re-thrombosis. We hypothesized
that co-delivery of recombinant tissue plasminogen activator (rtPA) with annexin V–containing
anticoagulants that specifically target the injured endothelium may passivate the
thrombogenic elements of the vascular injury site and enhance rtPA-induced thrombolysis.
In this study, the effects of conjunctive administration of Kinexins (Kunitz inhibitor–annexin
V fusion proteins) with rtPA on thrombolysis were determined in vitro and in vivo. Thromboelastometry showed that both TAP-A (tick anticoagulant peptide–annexin V
fusion protein; an inhibitor of factor Xa [FXa] and prothrombinase) and A-6L15 (annexin
V-6L15 fusion protein; an inhibitor of tissue factor/FVIIa) exerted concentration-dependent
(10–100 nM) effects on clot formation, with TAP-A being several folds more potent
than A-6L15 in whole blood. Combination of TAP-A or A-6L15 with rtPA (1 μg/mL) led
to decrease in lysis index, suggesting conjunctive enhancement of thrombolysis by
combined use of rtPA with TAP-A or A-6L15. In a rat cremaster muscle preparation subjected
to photochemical injury, conjunctive administration of rtPA and TAP-A significantly
restored tissue perfusion to 56%, which is approximately two fold of that by rtPA
or TAP-A alone. Near-infrared fluorescence images demonstrated local retention of
a fluorescent A-6L15-S288 at the injury site, suggesting a targeting effect of the
fusion protein. Pharmacokinetic analysis showed that 123I-labelled TAP-A and A-6L15 had initial distribution half-lives (T1/2α) of approximately 6 minutes and elimination half-lives (T1/2β) of approximately 2.3 hours. In conclusion, Kinexins were potentially useful adjunctive
agents with rtPA thrombolytic therapy especially for thrombosis induced by endothelial
injury.
Keywords
plasminogen activators - thrombolysis - annexin V - kunitz protease inhibitor - anticoagulants
