Recombinant Annexin II Modulates Impaired Fibrinolytic Activity In Vitro and in Rat Carotid Artery

Abstract

Abstract-Fibrinolytic activity has been reported to be decreased in atherosclerosis. Recently, annexin II was identified as a coreceptor on endothelial cells for plasminogen and tissue plasminogen activator. In this study, we examined whether recombinant annexin II (rAN II) protein can modulate fibrinolytic activity on vascular endothelium in vitro and in vivo. Key Words: annexin II Ⅲ rat carotid artery Ⅲ thrombus V ascular endothelial cell luminal surfaces maintain a "thromboresistant status" by dynamic balancing between the coagulation and fibrinolytic systems; however, the damage to vessel walls often seen in ischemic syndromes leads to thrombus formation. The fibrinolytic characteristics of vascular endothelium are modulated by the activity of plasminogen activators that facilitate conversion of plasminogen to active plasmin, 1 and the binding of plasminogen to endothelial cells significantly enhances the catalytic efficiency of its activation by tissue plasminogen activator (t-PA). 2,3 Annexins are a superfamily of calcium-dependent phospholipid binding proteins, 4 which have a highly conserved core domain preceded by a more variable amino terminal tail domain. 5 Recently, annexin II, a member of this superfamily, has been identified on endothelial cells and found to bind t-PA and plasminogen, as well as to enhance plasmin generation. 6,7 The expression and cell surface transport of annexin II is not fully understood; however, several important features of this molecule have been reported, including calciumdependent high-affinity binding to phospholipid. 8,9 Annexin II is now considered to be one of the key fibrinolytic modulators on the surface of endothelial cells. 7Considering the hypercoagulable state of most vascular diseases, including atherosclerosis, the possibility of modulation of fibrinolytic activity by annexin II is of great interest. Recently, annexin V, another annexin family member, was shown to be effective in reducing thrombus formation in a rat carotid artery injury model by using a mechanism that may involve inhibition of platelet activity in the injured vessel. 10 In this study, we examined the potential role of annexin II in a rat carotid artery model utilizing a recombinant annexin II (rAN II) protein. Our results indicate that intravenous administration of annexin II significantly inhibits thrombus formation in vivo without affecting systemic hemostatic parameters, and may demonstrate its potential as a novel therapeutic tool for vascular diseases.

Efficacy of recombinant annexin 2 for fibrinolytic therapy in a rat embolic stroke model: A magnetic resonance imaging study

“…Interestingly, there were no significant differences in ADC lesion volume at 180 min after stroke among groups B, C and D. These results are consistent with our previous observation that rAN II-promoted plasmin generation may have been saturated at higher concentrations (Ishii et al, 2001). …”

“…We have previously shown that annexin 2 is attached to endothelial cells in a calcium-dependent manner and fibrinolytic activity disappears in the absence of calcium (Ishii et al, 2001). rAN II restored impaired fibrinolytic activity and sustained carotid flow when rAN II was present on the endothelium.…”

“…rAN II restored impaired fibrinolytic activity and sustained carotid flow when rAN II was present on the endothelium. Deletion of the C-terminal 224 amino acid results in a lost fibrinolytic activity of annexin 2 (Ishii et al, 2001), suggesting that C-terminal 224 amino acids in annexin 2 plays important in phospholipid binding to plasminogen. Moreover, rAN II inhibits the formation of thrombus and overexpressed annexin 2 protein on intima of rat carotid artery can be detected by fluorescent immunochemistry.…”

“…The method used to prepare rAN II was previously described in details (Ishii et al, 2001). Animals were treated with various doses of rAN II or saline in a blinded manner and investigated after embolization in rAN II-treated group, rAN II was administered intravenously less than 5 min before the induction of embolic stroke.…”

“…Intravenous administration of recombinant annexin 2 (rAN II) significantly inhibits thrombus formation in vivo without affecting fibrinolytic and coagulation parameters in a rat carotid artery model (Ishii et al, 2001), suggesting rAN II could potentially be used in antithrombolytic treatment of stroke with minimal side effects.…”