Reversing the deleterious effects of α2-antiplasmin on tissue plasminogen activator therapy improves outcomes in experimental ischemic stroke

Abstract

High blood levels of α2-antiplasmin have been associated with failed tissue plasminogen activator (TPA) therapy for ischemic stroke. Yet, other data suggests that α2-antiplasmin may be protective in stroke, because it defends against bleeding and excitotoxicity. To address this paradox, we examined the effects of high α2-antiplasmin levels and α2-antiplasmin inactivation in mice treated with TPA 0.5-2.5 hour after middle cerebral artery (MCA) thromboembolism. Brain infarction, swelling, hemorrhage, blood brain barrier breakdown and neuronal apoptosis were measured by a blinded observer. Thrombus dissolution was determined by gamma counting. During TPA treatment, high α2-antiplasmin blood levels increased brain infarction (2.2-fold) and swelling (3.7-fold), but decreased MCA thrombus dissolution. Conversely, α2-antiplasmin inactivation during TPA treatment reduced brain infarction, hemorrhage and swelling, but increased MCA thrombus dissolution. Inactivation of α2-antiplasmin during TPA treatment reduced neuronal apoptosis and blood brain barrier breakdown. Inactivation of α2-antiplasmin also reduced short-term mortality. Taken together these data show that α2-antiplasmin opposes the effects of TPA therapy and contributes to enhanced brain injury after experimental thromboembolic stroke. Conversely, α2-antiplasmin inactivation during TPA treatment improves thrombus dissolution and reduces brain infarction, swelling and hemorrhage. Consistent with clinical observations, these data suggest that α2-antiplasmin exerts deleterious effects that reduce the efficacy and safety of TPA therapy for ischemic stroke.

Microvascular Thrombosis, Fibrinolysis, Ischemic Injury, and Death After Cerebral Thromboembolism Are Affected by Levels of Circulating α2-Antiplasmin

“…Three different experimental groups were examined: mice with increased levels of a2AP (achieved by intravenous supplementation), normal physiologic a2AP levels (controls) or no circulating a2AP (a2AP −/− mice). 14 Intravenous supplementation increased blood a2AP levels by a median of 87.1 ug/ml (mean 79.3 ± 14.3 ug/ml) in mice measured at the end of the experiment which approximately doubled the a2AP levels found in in normal mice. 21 Laser Doppler monitoring showed that MCA thromboembolism was associated with a marked drop in hemispheric blood flow to 20% or less of the initial baseline in all three groups as expected (Fig.…”

“…15, 16, 30 The present findings are also consistent with recent reports that increased levels of a2AP may contribute to the failure of TPA treatment in humans and in mice. 13, 14 …”

“…10,11 However, recent studies indicate that high levels of circulating a2AP contribute to the failure of TPA therapy to dissolve thrombi and restore blood flow during ischemic stroke. 12-14 Moreover, genetic deletion of a2AP protects against ischemic brain injury induced by non-thrombotic permanent surgical ligation of the middle cerebral artery. 15, 16 Yet, within the neuronal and vascular compartments, a2AP and serpins that block TPA-initiated proteolytic pathways, such as the activation of matrix metalloproteinase-9 (MMP-9), may protect the brain by reducing cell death or neurotoxicity and may prevent bleeding complications.…”

Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli

“…Still, even meticulous animal models may not fully simulate the complexity of human illnesses. While these studies in pulmonary embolism, and others in ischemic stroke, show that α2-antiplasmin-I is not associated with significant bleeding risk, the safety of this approach for humans will need to be confirmed 15, 29 . Finally, these studies examined the effects of α2-antiplasmin inactivation on acute PE; its effect on subacute or chronic PE remains unknown.…”

“…After washing in PBS, sections were blocked with 10% normal donkey serum for 1 h and incubated with primary antibody in 2% normal donkey serum for 90 min at room temperature. After PBS washings, sections were probed with fluorophore-conjugated donkey secondary antibodies as we have described previously 15 . The primary antibodies include rabbit anti-mouse against tPA (ASMTPA-GF; Molecular Innovations), uPA (urokinase plasminogen activator) (ASMUPA-GF-HT; Molecular Innovations) and PAI-1 (IASMPAI-GF; Innovative Research), rabbit anti-mouse plasminogen (Abcam), goat anti-mouse α2-antiplasmin (AF1239; R&D Systems), and rat anti-mouse Ly6G (clone1A8, #127602; Biolegend).…”