The Plasminogen Activator (PA) system in the pathogenesis of ischemic stroke

Abstract

Ischemic stroke remains a leading cause of morbidity and mortality worldwide, driven by complex pathophysiological mechanisms that make finding effective treatments challenging. Plasminogen activators (PAs) play a critical role in fibrinolysis and vascular homeostasis, and as such are important factors affecting stroke outcome. This review examines the complex relationships between ischemic stroke and PAs, highlighting their physiological, pathological, and therapeutic effects on ischemic stroke. We focus on recombinant tissue-type plasminogen activator (rtPA) as the only Food and Drug Administration (FDA) -approved thrombolytic agent, describing its clinical impact and associated obstacles impacting its wide-scale use, such as blood-brain barrier (BBB) disruption and inflammation. Furthermore, emerging PA-based therapies and combination strategies are explored to address the limitations of rtPA. By integrating mechanistic information with clinical developments, this review aims to provide insights for the advancement of PA-centered approaches to improve the safety and efficacy of stroke treatments.

Graphical Abstract

Introduction

Globally, stroke ranks as the second most common cause of mortality, and the third leading contributor to disability.¹ According to the American Heart Association, approximately 87% of strokes are ischemic, resulting from disrupted cerebral blood flow, typically caused by thrombosis or embolism.² At present, the only pharmacological approach for thrombus resolution and blood flow restoration is thrombolysis.³

Endogenous fibrinolysis is normally initiated by two PAs: tPA and urokinase type plasminogen activator (uPA).^4,5 Belonging to the serine protease family, these enzymes proteolytically activate the zymogen plasminogen to plasmin, the primary fibrinolytic enzyme.

The advent of recombinant DNA technology has enabled the development of rtPA, offering a targeted fibrinolytic solution for treating acute ischemic stroke.⁶ In 1996, the U.S. FDA authorized intravenous rtPA (Alteplase) as the first, and currently the only thrombolytic drug approved for managing acute ischemic stroke (AIS).^7,8 More recently, a variant of tPA, Tenecteplase (TNK), approved for the treatment of acute myocardial infarction, has begun to be used off label for the treatment of AIS.^9,10 Although rtPA has proven efficacy, its effectiveness, defined as survival without significant disability, is reported to be approximately 35%.¹¹ This is due in part to a significant number of patients failing to achieve successful recanalization with thrombolysis.¹² The mechanisms behind rtPA resistance are not fully understood. Thrombus size, location, and collateral circulation likely affect thrombolytic efficacy. Thrombus composition is also thought to be an important factor impacting the efficacy of rtPA, with clots having high fibrin and RBC content being more sensitive to thrombolysis than platelet-rich clots or clots with Neutrophil extracellular traps (NETs). ^13–16 Consequently, thrombolytic strategies addressing non-fibrin components such as vWF and DNA are being investigated.^17–19 rtPA also has a relatively short therapeutic window and guidelines recommend that treatment should be initiated within 4.5 hours of stroke onset.¹⁰ Thrombolysis is also associated with an increased risk of intracranial hemorrhage (ICH) that may rise with delayed treatment time beyond 4.5 hours of stroke onset.^10,20–22 This is in contrast to the only other available reperfusion therapy for ischemic stroke, endovascular thrombectomy, which also has an increased risk of ICH, but a recommended therapeutic window of 24 hours.^23,24 However, thrombectomy is only suitable for large and medium vessel occlusions and typically only available at large tertiary care medical centers. Thus, thrombolysis with rtPA remains the primary standard of care for ischemic stroke. Unfortunately, the short treatment window, and its elevated risk of bleeding complications along with the high cost, limit its availability to less than 2% of ischemic stroke patients globally²⁵ and to approximately 10% of patients in the US.²⁶

This review examines the roles of PAs in the acute and recovery phase of ischemic stroke as well as the clinical progress of PA-based therapies. We address the roles PAs play in thrombolysis, BBB disruption, ICH, neuroinflammation, and recovery angiogenesis/neurogenesis with a specific focus on the role of tPA. A significant question is why is thrombolysis limited to 4.5 hours after stroke onset when thrombectomy is efficacious up to 24 hours? Lastly, combination strategies are discussed using mechanistic insights as a framework for developing safer and more effective therapies.

Plasminogen activators and ischemic stroke

The plasminogen activation system is a highly regulated proteolytic cascade critical for thrombolysis. The cascade results in the activation of plasminogen to plasmin, which directly degrades the fibrin matrix of a blood clot.⁵ Plasminogen activation occurs through proteolytic cleavage by the plasminogen activators tPA or uPA, which in turn are regulated primarily by plasminogen activator inhibitor 1 (PAI-1).²⁷ Both tPA and uPA are present in the circulation. While endothelial cells are a significant source of tPA in blood, other sources include vessel associated sympathetic terminals and the liver.^28–30 The uPA concentration in blood is relatively low, but uPA can be expressed by many cell types and is thought to primarily regulate cell associated plasminogen activation.³¹ tPA has higher fibrin specificity than uPA, enabling direct activation of fibrin-bound plasminogen. In contrast, uPA is often found in interaction with its cell surface receptor, urokinase-type plasminogen activator receptor (uPAR), and has been associated with basement membrane degradation and extracellular matrix turnover.³²

During the acute phase after stroke, the onset of cerebral ischemia leads to differential changes in tPA and uPA expression and activities across brain regions.^33,34 In rodent middle cerebral artery occlusion (MCAO) stroke models, parenchymal tPA activity in the ischemic brain tissue rises as early as 1 hour after stroke onset, peaks at 24 hours, and returns to baseline by 72 hours post-injury. In contrast, there is a marked increase in uPA activity by 72 hours after stroke that is primarily confined to the ischemic penumbra.^35–37 These data suggest that tPA may play a prominent role in the acute phase of cerebral ischemia, while uPA, may contribute more to post-stroke recovery and repair.^36,38 To further illustrate the functional distinctions between tPA and uPA in stroke, we summarize some of their key differences in Table 1.^39–41

Table 1.

Comparison of tPA and uPA Functions in Stroke

Feature	tPA (Tissue Plasminogen Activator)	uPA (Urokinase Plasminogen Activator)
Role in Stroke	Acute thrombolysis, affects vascular permeability & inflammation	Recovery phase, promotes synaptic recovery
Fibrin Specificity	High	Low
Site of Action	Intravascular and brain parenchyma	Brain parenchyma
Inflammation & Immune Response	Pro-inflammatory, increases BBB permeability, leukocyte infiltration, microglial activation, cytokine release	Potentially pro-inflammatory
Impact on BBB	Disrupts BBB	BBB disruption unknown
Angiogenesis	Potentially pro-angiogenic, but effects are limited by BBB disruption and hemorrhagic risk	Pro-angiogenic, may promote neovascularization
Neuronal Repair	Mixed effects, enhances synaptic plasticity but also induces excitotoxicity via NMDAR	Promotes neuronal recovery, enhances synaptic repair, and dendritic spine formation

Immediately following the onset of ischemia, a series of pathophysiological events occur, including BBB disruption, neuroinflammation, and neuronal death (Figure 1). Additionally, cerebral edema and secondary ICH can occur, both of which are potentially lethal sequelae. Reparative pathways are also activated after stroke including angiogenesis and neurogenesis with the extent of recovery depending on the balance between the injury and repair processes.^19,42–44

Figure 1. Pathophysiological processes following ischemic strokes.

The impact of tPA on neurovascular inflammation, BBB disruption, and thrombolysis-associated hemorrhagic transformation remains an area of active investigation.⁴⁵ A significant secondary source of injury leading to infarct expansion is the loss of BBB integrity, which occurs in a biphasic manner, with an early transient increase in BBB permeability that appears to be largely transcellular, followed by a secondary increase in permeability that is primarily paracellular, and is associated with the loss of tight junctions.^46,47 It is thought that this secondary phase, with the loss of tight junctions, is associated with the risk of ICH following ischemic stroke.⁴⁸ Multiple mechanisms can promote BBB dysfunction including oxidative stress and neuroinflammation.^49,50 Importantly, both phases of BBB leakage can be triggered by tPA-mediated proteolytic activation of latent platelet-derived growth factor-CC (PDGF-CC) on the parenchymal side of the BBB.^51–53 This tPA-mediated effect can occur even in the absence of cerebral ischemia.^54,55 Active PDGF-CC initiates a signaling cascade through the platelet-derived growth factor receptor-α (PDGFRα) on perivascular astrocytes that potentiates vascular endothelial cell growth factor A (VEGF) signaling in endothelial cells resulting in loss of vascular barrier integrity.^51–53 These studies show that VEGF signaling in endothelial cells stimulates protein kinase Cβ (PKCβ) phosphorylation of the tight junction protein occludin resulting in ubiquitination of occludin and the subsequent loss of the tight junction complex.⁵⁶ In murine stroke models, tPA deficient mice show reduced stroke-induced BBB disruption³⁶ and occludin phosphorylation. Blocking occludin phosphorylation, either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCβ, reduces stroke-induced permeability. Thrombolytic rtPA, when administered outside the recommended therapeutic window—when the BBB is already compromised, can intensify this signaling pathway by allowing therapeutic rtPA in the blood to cross into the brain parenchyma.^54,55 This suggests that the narrow therapeutic window for tPA-mediated thrombolysis may be due in part to activation of this pathway increasing the risk of hemorrhagic transformation with time. Understanding the pathways activated by tPA that contribute to this risk could provide new therapeutic targets, potentially expanding the safe application of thrombolytic therapy to a broader patient population. Indeed, a phase 2B clinical trial has evaluated the safety of the PDGFRα antagonist imatinib as an adjuvant treatment with thrombolysis. This trial showed that imatinib is safe and may provide benefit to ischemic stroke patients,⁵⁷ and has led to a follow-up phase 3 clinical trial (NCT03639922).

In addition to the PDGF-CC-PKCβ pathway, another important process impacted by rtPA is neutrophil recruitment and inflammation. TPA is reported to enhance neutrophil recruitment,^58,59 a process that not only increases local inflammation, but may also contribute to thrombolysis resistance.¹⁷ Administration of tPA is reported to upregulate Nod-like receptor protein 3 inflammasome expression, driving neutrophil recruitment.⁵⁸ tPA can also increase neutrophil infiltration by upregulating adhesion molecules such as ICAM and VCAM, leading to the release of myeloperoxidase and neutrophil proteases, which can cause oxidative stress and vascular damage, potentially exacerbating vascular injury and further heightening the risk of hemorrhagic transformation.⁵⁹ Additionally, tPA and TNF-α synergistically upregulate LRP-1 expression on neutrophils, establishing a feedback loop that can sustain inflammation.^41,60,61 Neutrophil NETs from infiltrated neutrophils also activate the inflammatory pathways in microglia,^62,63 and studies have shown that tPA can induce the expression of MIP-1α and adhesion molecule ICAM-1 in brain endothelial cells, further enhancing leukocyte recruitment and polarizing microglia toward a pro-inflammatory phenotype.^64,65 In addition to effects on neutrophils, recent studies have shown that tPA can also mobilize T cells, enhancing their adhesion and infiltration into the brain. ⁴¹ In contrast, other studies showed that tPA reduced lymphocyte, monocyte and dendritic cell counts, while simultaneously increasing pro-inflammatory cytokines like TNF-α, and immunosuppressive cytokines like IL-10, thus suggesting that tPA can have both pro- and anti-inflammatory activities.⁶⁶ While these findings may seem divergent, they likely reflect different facets of tPA’s immunomodulatory effects. The observed increase in inflammatory cytokines alongside immune cell depletion suggests that tPA may induce both activation and suppression in distinct immune compartments. This highlights the dynamic nature of tPA’s immune effects and underscores the potential for therapeutic strategies aimed at modulating this balance.

Additionally, tPA’s effects on the BBB have been suggested to involve increased MMP activity, potentially contributing to extracellular matrix degradation and BBB disruption.^67–70 However, whether the action of tPA on the BBB involves MMP activity or expression is controversial.^36,71 Finally, tPA can activate the complement cascade, exacerbating vascular permeability and cerebral edema.⁷² Taken together, all these studies suggest a varied and complex response to rtPA treatment after stroke well beyond its established role in fibrinolysis.

The effects of PAs during the recovery phase after stroke are not well characterized. It is thought that following ischemic stroke, angiogenesis is coupled to axonal outgrowth and neurogenesis, and that this process can promote functional recovery (reviewed in ⁷³). Consistent with this hypothesis, studies have suggested that endogenous tPA or uPA can promote recovery by enhancing angiogenesis and/or neurogenesis. For example, tPA has been shown to promote neovascularization and tissue regeneration by mobilizing bone marrow-derived proangiogenic myeloid cells via VEGF and Kit ligand signaling.⁷⁴ As noted above, tPA can also enhance VEGF signaling locally the cerebrovascular bed, and although this activity was tied increased BBB permeability ⁴⁸, it might also influence recovery angiogenesis. Studies have also suggested that uPA plays an angiogenesis-independent role in repair following ischemic injury.^38,75 Neuron-derived uPA bound to its receptor uPAR on astrocytes during post-ischemic recovery can activate the ERK1/2-STAT3 pathway to promote synaptic repair.⁷⁵ Additionally, uPA-uPAR signaling supports new dendritic spines and branches to enable synaptic repair.³⁸

Alternative and Adjunctive Therapeutic Strategies for the PA System

Current research aimed at improving thrombolytic therapy primarily focuses on optimizing lytic drugs, and/or identifying adjuvant therapies to mitigate the off-target effects of thrombolysis. Currently, Tenecteplase (TNK), Reteplase (rPA), and recombinant human prourokinase (rhPro-UK) are in active clinical trials. TNK is a variant of tPA with a longer half-life and increased fibrin specificity compared to Alteplase. It can be administered by a single bolus infusion, avoiding the need of a 60-minutes infusion required for alteplase. ⁷⁶ Reteplase, a derivative of tPA that like TNK has a longer half-life and does not require continuous infusion, but unlike TNK, Reteplase has a lower affinity for fibrin. The RAISE trial indicates better functional outcomes with Reteplase compared to alteplase, but a higher risk of bleeding complications.^77,78 Another variant of tPA, Desmoteplase (rDSPA), a recombinant plasminogen activator with high homology to tPA derived from vampire bat saliva, did not reach its primary endpoint of demonstrating clinical benefit compared to placebo when administered 3–9 hours after stroke onset, however it did show remarkable safety with no increase in symptomatic ICH or cerebral edema even with a median time to treatment of 7 h.⁷⁹ Compared to tPA, uPA has been less thoroughly investigated, primarily due to its lower specificity for fibrin.⁸⁰ The TRUST trial of urokinase in patients with mild stroke (NIHSS score ≤ 5) did not show improved outcomes.⁸¹ In another recent study of uPA in patients with a median NIHSS score of 10 similarly showed no benefit and no increased risk of symptomatic ICH; however, there was an increased risk of extracranial bleeding. ⁸² Recently, the PROST trial of recombinant human prourokinase (rhPro-UK) indicated that rhPro-UK was noninferior to alteplase with a similar risk of symptomatic ICH.^83,84 Based on meta-analyses, we have compiled Table 3 as a general reference for recent clinical trials.^85–87

Table 3.

Comparison of Key Characteristics of Different Thrombolytic Agents

Factor	Alteplase (rtPA)	Tenecteplase (TNK)	Reteplase (rPA)	Urokinase (uPA)
Fibrin specificity 104	✓✓✓	✓✓✓✓	✓	No specificity
Half-life 105	<5 min	20–24 min	13–16 min	10–20 min
Administration 87	Bolus and infusion over 90min	Single bolus	Double bolus 30min apart	Infusion
ICH risk	+	+85	+87	+87
Recanalization	+	++++ 85	NR	NR
Long-term recovery	+	+++ 85	++87	+87

NR: Not reported

Long-term recovery refers to the percentage of patients achieving a modified Rankin Scale (mRS) score of 0–1 at 90 days.

The “+” notation represents the relative effect size based on the Odds Ratio (OR):

+ (OR ≈ 1.0–1.5)

++ (OR ≈ 1.5–2.0)

+++ (OR ≈ 2.0–3.0)

++++ (OR > 3.0)

Adjunctive therapies are another approach to enhancing thrombolytic efficacy and mitigating risks. Targeting the identified mechanisms underlying PA-associated adverse effects with combination therapies holds significant potential for improving safety and therapeutic outcomes. A partial list of some of the promising approaches is presented in Table 2. Numerous clinical trials have explored adjunctive therapies for tPA, with many phase II neuroprotective drug trials and multiple phase III randomized controlled trials failing to demonstrate clear clinical efficacy.⁸⁸ For instance, the MOST trial (NCT03735979) investigated the addition of argatroban and eptifibatide to tPA but found no reduction in post-stroke disability and an association with increased mortality.⁸⁹ Among the remaining promising candidates, in addition to imatinib, discussed above, Fingolimod and Uric Acid have gained attention. Exploratory studies suggest that Fingolimod may protect the blood-brain barrier and reduce hemorrhage,⁹⁰ and the URICO-ICTUS trial demonstrated that uric acid therapy may improve functional outcomes by mitigating oxidative stress and reperfusion injury.⁹¹ Studies have also been exploring physical adjunctive therapies to enhance thrombolysis. Examples include ultrasound-assisted microbubble delivery to reduce tPA dosage,⁹² low-frequency vibration environments to augment thrombolytic efficacy,⁹³ and hypothermia therapy to reduce tPA side effects.⁹⁴

Table 2.

PA Variants and Adjunct Therapies

Type	Agent	Target	Effect	Stage	Ref
Adjunct to rtPA therapy	Imatinib	PDGFRα	Protects the BBB	Phase IIB clinical trial complete and Phase III ongoing	57
	Fingolimod	S1PR	Protects the BBB, reduces hemorrhage	Exploratory clinical study	90
	Uric Acid	ROS, Peroxynitrite, NF-κB	Neuroprotection, reduces oxidative stress, protects BBB	Phase IIB/III clinical trial complete	91
	PKCβ Inhibitor LY-333531	Occludin	Reduces ICH	Preclinical	48
	PAI-1 Inhibitor	PAI-1	Enhances fibrinolysis	Preclinical	98
	PAItrap4	PAI-1	Improves thrombolysis	Preclinical	99
	FSAP-SPD	Cleaves fibrin directly	Thrombolysis	Preclinical	100
	SCE5-scuPA	Targets glycoprotein IIb/IIIa receptors on activated platelets	Enhances thrombolysis in platelet rich clots	Preclinical	101
Different targets	Microlyse	vWF	Enhances thrombolysis in platelet rich clots	Preclinical	18
	LT3001	Clears free radicals and improves endothelial function	Reduces edema and ICH	Preclinical	102
	AZD9684	Carboxypeptidase U	Reduces fibrin deposition	Preclinical	103

In addition, personalized therapy is emerging as a promising strategy. The development of an individualized PA-based treatment plan is critical to address the impact of gender, age, comorbidities, and imaging assessment on stroke prognosis. Comorbidities such as diabetes and metabolic syndrome significantly impact the efficacy of rtPA therapy. Patients with these conditions exhibit increased resistance to fibrinolysis and a higher risk of hemorrhagic transformation following rtPA treatment.⁹⁵ Therefore, carefully selecting treatment options based on imaging techniques such as CT to assess embolus characteristics may be more appropriate for these patients.⁹⁶ Notably, findings from the CHABLIS-T II trial suggest that if imaging confirms the presence of a favorable penumbral profile, administering Alteplase (rtPA) within a 4.5–24-hour window can still provide thrombolytic benefits without increasing the risk of hemorrhage.⁹⁷

Conclusions

PAs are central to both the pathogenesis and therapeutic management of ischemic stroke. This review emphasizes their multifaceted roles, focusing on contributions of PAs to thrombolysis, BBB integrity, inflammation, and neurovascular recovery. While rtPA remains the clinical gold standard, its limitations, including thrombolytic resistance, bleeding risks, inflammatory responses, and a restricted therapeutic window highlight the urgency of finding innovative strategies to address these shortcomings. Emerging approaches, including novel thrombolytics and multimodal therapies, hold the potential to overcome these challenges, paving the way for safer and more effective treatments. By integrating current advancements and identifying critical gaps, this review lays the groundwork for optimizing stroke management through refined PA-based interventions.

Highlights.

• Review of the roles of PAs in the acute and recovery phase of ischemic stroke.

• Focus on recombinant tissue plasminogen activator as the only FDA approved thrombolytic.

• Discuss PA-mediate blood-brain barrier (BBB) disruption and inflammation.

• Describe emerging PA-based therapies and combination strategies to improve treatment and mitigate off target activities.

Non-standard Abbreviations and Acronyms

PA

Plasminogen Activator

rtPA

Recombinant Tissue- Type Plasminogen Activator

FDA

Food and Drug Administration

BBB

Blood-Brain Barrier

AIS

Acute Ischemic Stroke

TNK

Tenecteplase

MCAO

Middle Cerebral Artery Occlusion

ICH

Intracranial Hemorrhage

PAI-1

Plasminogen Activator Inhibitor-1

uPA

Urokinase-Type Plasminogen Activator

uPAR

Urokinase-Type Plasminogen Activator Receptor

VEGF

Vascular Endothelial Growth Factor

PDGF

Platelet-Derived Growth Factor

PDGFR

Platelet-Derived Growth Factor Receptor

PKC

Protein Kinase C

NETs

Neutrophil Extracellular Traps

ICAM

Intercellular Adhesion Molecule

VCAM

Vascular Cell Adhesion Molecule

LRP-1

Low-Density Lipoprotein Receptor-Related Protein 1

MIP-1α

Macrophage Inflammatory Protein-1α

MMP

Matrix Metalloproteinase

rDSPA

Recombinant Desmoteplase

rhPro-UK

Recombinant Human Prourokinase

NIHSS

National Institutes of Health Stroke Scale

CT

Computed Tomography