Neurotoxicity of Tissue Plasminogen Activator (tPA)
At a glance
Introduction of Neurotoxicity of tPA
Tissue plasminogen activator (tPA) is a fibrin-specific activator that converts plasminogen to plasmin, which initiates thrombolysis and rescues the ischemic brain by returning blood flow. tPA is now a clinically proven thrombolytic agent in ischemic stroke, activating apoptosis under hypoxic and ischemic conditions. But rtPA also causes neuronal damage and it is thought that interaction between rtPA and TLPs is responsible. tPA neurotoxicity can even compound ischemic damage. Therefore, we must define and learn about the pleiotropic function of tPA in the brain in detail so that combination treatments can be created to make thrombolysis safer and more effective.
Fig.1. Pleiotropic effects of tPA. (Doeppner, et al., 2011)
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tPA Neurotoxicity in Experimental Models of Ischemic Stroke
Whether tPA exacerbates brain injury has been the subject of many studies. Although the overall data are not definitive, tPA neurotoxicity has been demonstrated in cell culture experiments as well as in animal models of focal cerebral ischemia.
Neurotoxicity in In-Vitro Models
In some cases, both tPA and plasmin, which are broad-spectrum protease enzymes, are potentially neurotoxic if they reach the extracellular space. tPA knockout (KO) mice cultured with primary cortical neurons are resistant to oxygen-glucose deprivation. Exogenously added tPA effectively induces apoptosis in cultured neurons and enhances neurotoxicity after hemoglobin exposure.
Neurotoxicity in Animal Models
The neurotoxicity of tPA has been investigated using tPA knockout (tPA -/-) mice, revealing a complex role in brain injury. These mice are resistant to excitotoxic neuronal death, indicating that tPA mediates damage through plasmin proteolysis. Additionally, tPA -/- mice show reduced microglial activation after kainic acid injections, suggesting that tPA influences microglial responses via mechanisms beyond plasminogen activation.
tPA deficiency offers protection against traumatic brain injury (TBI) and leads to reduced infarction in patients with MCA occlusion, showing that tPA may accelerate stroke damage. When administered heparin in experiments, tPA-/- mice were not as hemorrhagic as wild-type mice, likely because tPA did not affect Matrix Metalloproteinase-9 (MMP-9) activity, the mediator of vascular damage and hemorrhage.
Contrasting effects of tPA are observed depending on the duration of ischemia; after mild damage, tPA KO mice show more extensive injury, while after severe damage, they fare better than WT mice. This suggests that tPA may provide protective benefits through thrombolysis in cases of minor damage, but its neurotoxicity can dominate in larger injuries.
Fig.2. tPA knockout mice have reduced infarct volumes. (Doeppner, et al., 2011)
Mechanisms of tPA Neurotoxicity
tPA Neurotoxicity Via NMDA Receptors
Multiple intracellular mechanisms have been proposed to explain neuronal damage during ischemia, such as excitotoxicity, free radical damage, and apoptosis. During ischemia, elevated extracellular glutamate levels activate receptors that lead to increased calcium inward flow and subsequent cell death. Thus, when considering tPA as a potential threat, any interaction between NMDA receptors and the exogenous serine protease tPA is associated with neurotoxicity, especially in the presence of compromised blood-brain barrier integrity, when tPA, which normally acts in an intravascular manner, will enter the extravascular nerve parenchyma.
tPA enhances neuronal cell death mediated by glutamatergic receptors by cleaving a fragment from the NR1 subunit of the NMDA receptor, which increases intracellular calcium levels and potentially leads to cell death. Additionally, tPA may promote excitotoxic cell death through receptor-mediated apoptotic pathways.
Fig.3. Deleterious effects of tPA. (Doeppner, et al., 2011)
tPA Neurotoxicity and Neurovascular Matrix
Moreover, damage to the neuron can occur as a result of perturbations in the ECM. MMPs already abundantly present in the ECM – are being used more and more in animal models of stroke. One of many MMPs, the gelatinase MMP-9, has been implicated specifically in targeting key parts of the brain's perivascular basement membrane, and activation when the brain is ischemia results in microvascular damage, BBB destruction, edema, and hemorrhagic switchover. So the increased vulnerability for hemorrhagic transformation in stroke patients on tPA might reflect tPA-induced MMP-9 upregulation.
Plasmin, a protease-activated by tPA, further complicates matters by degrading the ECM and activating MMPs, thereby increasing the risk of hemorrhage. High levels of plasmin and TLPs can trigger intracranial hemorrhage and affect cell viability. Interestingly, while plasminogen-deficient mice are resistant to excitotoxicity, studies show that the plasmin/plasminogen system can have dual effects, sometimes exacerbating injury in the central nervous system and vasculature following ischemic events. This complexity underscores the need for careful consideration of the role of tPA role in stroke treatment and its potential neurotoxicity.
Fig.4. MMP-9 may underlie tPA-induced disruption of BBB. (Doeppner, et al., 2011)
Approaches to Reduce tPA Neurotoxicity
Combination with Neuroprotection
Combine with tPA for acute ischemic stroke to maximize its efficacy and minimize side effects. The arguments for such therapies are tPA neurotoxicity reduction, hemorrhage reduction, reperfusion injury reduction, neuroprotective enhancement, and longer course of therapy.
Development of New Thrombolytic Agents
Exploring new thrombolytic therapies and techniques would also increase the effectiveness and safety of interventions for acute ischemic stroke due to the problems that are often found with traditional tPA treatment. Early trials of other thrombolytics (such as streptokinase) failed early due to mortality, but researchers are still looking at others.
Research has shown that staphylokinase (rSak) is more fibrin-specific than tPA and effectively lyses arterial emboli in animal models. Additionally, plasmin, which can dissolve thrombi without relying on plasminogen or its activators, has emerged as a potential alternative. Microplasmin, a truncated form of plasmin, has demonstrated improved outcomes and reduced hemorrhage incidence in preclinical studies.
The development of third-generation thrombolytics, such as TNK-tPA (tenecteplase), which has a longer half-life and improved fibrin specificity, shows promise. TNK-tPA has been effective in reducing neurological deficits and lesion volumes without increasing hemorrhagic transformation, even when administered beyond the standard treatment window. However, some studies have not found significant differences between TNK-tPA and traditional tPA regarding efficacy or hemorrhage rates.
Innovative approaches are being explored, including mechanical methods for thrombus removal and the use of continuous transcranial Doppler (TCD) to enhance the effectiveness of tPA by increasing clot surface area exposure, temperature, and vasodilation.
- Doeppner, T. R., et al. (2011). Acute hepatocyte growth factor treatment induces long-term neuroprotection and stroke recovery via mechanisms involving neural precursor cell proliferation and differentiation. Journal of Cerebral Blood Flow & Metabolism, 31(5), 1251-1262.
