Annexin A2: a tissue plasminogen activator amplifier for thrombolytic stroke therapy

Abstract

Hemorrhagic transformation, incomplete reperfusion, neurotoxicity, and the short treatment time window comprise major challenges for thrombolytic therapy. Improving tissue plasminogen activator therapy has become one of the highest priorities in the stroke field. Recent efforts have been aimed at identifying new strategies that might enhance the thrombolytic efficacy of tissue plasminogen activator at the same time as reducing its associated complications related to hemorrhage and neurotoxicity. We believe that the combination of low-dose tissue plasminogen activator with recombinant annexin A2 (a tissue plasminogen activator and plasminogen coreceptor) might constitute a promising approach. Our pilot study using a focal embolic stroke model in rats supports this hypothesis.

Figure 1

Working model of fibrinolytic assembly on cell surfaces. In association with its partner protein, p11, annexin A2 is linked to the cell surface via calcium-dependent phospholipid-binding sites located within the four core domain repeats. The A2₂p11₂ heterotetramer binds tPA at the tail domain of annexin A2, while plasminogen appears to bind to residues within the fourth core domain of A2. Fibrinolytic assembly is a precisely orchestrated process that enhances the catalytic efficiency of plasmin generation.

Figure 2

Recombinant annexin A2 accelerates tPA-dependent plasminogen activation in vitro. A range of concentrations of tPA (1, 2.5, 5, 10 μg /ml) with or without the indicated concentrations of rA2 (0, 1, 2.5, 5 μg/ml) were added to wells of a 96-well plate. Plasmin activity was represented as fold plasmin activity relative to 1 μg /ml of tPA alone. The data are expressed as mean + s.e.m., n=4 per group. Reprinted with permission from J Cereb Blood Flow Metab. Copyright 2010, Nature Publishing Group.

Figure 3

tPA-rA2 combination therapy reduces hemorrhage volume in rats with ischemic stroke. Rats subjected to focal embolic stroke were treated at 4 hours with saline, standard high-dose tPA (10 mg/kg, H-tPA), or low-dose tPA (2.5 mg/kg, L-tPA) plus rA2 (5 mg/kg). The volumes of intracerebral hemorrhage ware quantified by hemoglobin assay at 24 hours after stroke. The data were expressed as mean + s.e.m., n=10, *p<0.05. Reprinted with permission from J Cereb Blood Flow Metab. Copyright 2010, Nature Publishing Group.

Figure 4

Neurologic scores and infarction volume are improved in rats receiving combination therapy. Two groups of rats were treated intravenously at 4 hours after stroke with either saline or a combination of low-dose tPA (2.5 mg/kg, L-tPA) plus rA2 (5 mg/kg). A. Three days after the onset of stroke, neurological scores of two group rats were evaluated. B. Ischemic infarction size on hematoxylin and eosin-stained sections were examined in each group, *p<0.05, n=8 for saline and n=11 for the combination. Reprinted with permission from J Cereb Blood Flow Metab. Copyright 2010, Nature Publishing Group.

Figure 5

Working models for tPA-related thrombolytic stroke therapies. A. Conventional therapy with tPA alone. When delivered into the circulation, tPA complexes immediately with its high affinity inhibitor, plasminogen activator inhibitor-1 (PAI-1). Free tPA circulates only when the inhibitory capacity of PAI-1 has been exceeded. Free tPA can then bind to fibrin, where plasminogen will already be bound. Fibrin markedly accelerates the catalytic efficiency of tPA-dependent plasminogen activation, generating plasmin, which degrades the thrombus, and generates fibrin degradation products (FDPs). B. Combination therapy with tPA and recombinant annexin A2 (rA2). We postulate that injection of tPA with rA2 leads to the formation of a circulating complex. Two scenarios seem plausible. In the first, the tPA-rA2 complex would also bind plasminogen and activate it to generate rA2-associated plasmin. This complex could then interact with fibrin in a thrombus, and initiate its degradation, forming FDPs. Alternatively, rA2-associated tPA might interact directly with fibrin within the thrombus, and the tPA might then activate fibrin-bound plasminogen, and generate plasmin, which would then dissolve the thrombus, generating FDPs. Both tPA and plasmin would be relatively protected from their circulating inhibitors, plasminogen activator inhibitor-1 (PAI-1) and alpha₂-antiplasmin (alpha₂-AP), respectively. Because, in both scenarios, there would be less circulating free tPA that that seen with conventional therapy, the potential for restoration of blood flow without hemorrhage or neurotoxicity would be much greater.