Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Tissue-Type Plasminogen Activator

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101941


 PLAT;  tPA;  T-PA

Historical Background

Tissue-type plasminogen activator (tPA) is a serine proteinase that induces clot lysis by catalyzing the conversion of clot-bound plasminogen to plasmin. The history of tPA began with the identification in 1947 and successful extraction in 1952 of a plasminogen activator from animal tissues that was named fibrinokinase (Astrup and Stage 1952). In 1957 this activator was found in the brain, lung, heart, uterus, muscles, prostate, adrenal gland, testis, and ovaries, and in 1964 it was already known as tissue activator of plasminogen. The first purified form of human tPA was obtained from uterine tissue in 1979, and soon thereafter it was found to be identical to vascular plasminogen activator and blood plasminogen activator (Rijken et al. 1980; Pennica et al. 1983). These findings led to establish that the plasminogen activator found in blood corresponds to the vascular tPA released into the circulation by endothelial cells. Together, these data became the foundation for the first proposed model of physiological fibrinolysis, in which tPA induces fibrin-dependent conversion of plasminogen into plasmin. These discoveries rapidly steered clinicians and scientists to design pilot studies to successfully test the potential therapeutic effect of thrombolysis with recombinant tPA (rtPA) not only in animal models of pulmonary embolism and coronary artery disease but also in patients with renal vein thrombosis (Matsuo et al. 1981; Weimar et al. 1981). Since then recombinant tPA has been used to treat patients with different forms of vascular thrombosis including pulmonary embolism, deep venous thrombosis, myocardial infarction, and acute ischemic stroke. Intriguingly, in parallel with these discoveries, it was also evident that the vascular endothelium is not the only source of tPA and that the release of tPA by other cell types, particularly in the central nervous system, has pleotropic effects that do not always require the conversion of plasminogen into plasmin.


The tPA gene is located on chromosome 8 (p12-p11), contains 14 exons, and has 32.7 kb. The mRNA is 2.6 kb in length and the cDNA is 1689 bp long. The precursor and mature forms of tPA are composed of 562 and 527 amino acids, respectively, and the mature protein has a molecular mass of 68 kDa. The molecule has 17 disulfide bonds, and hydrolysis of the Arg275-Ile276 peptide bond by plasmin converts tPA into a two-chain molecule that is held together by disulfide bonds. In normal plasma, the concentration of tPA antigen is approximately 70 pM (0.005 μg/ml), and most of it is in complex with its inhibitor, plasminogen activator inhibitor-1 (PAI-1), which to function requires the sequence Lys296-His-Arg-Arg299 in tPA (Lijnen and Collen 1991). The tPA molecule contains four domains. The first (F-domain) is an N-terminal region, from residues 4 to 50, that mediates tPA binding to fibrin and its clearance from the circulation. The second (E-domain) contains residues 50 to 87, is homologous with epidermal growth factor, and also mediates tPA’s clearance. The third domain is formed by two kringles (K1 and K2) that contain residues 87 to 176 and 176 to 256, respectively. The fourth (P-domain) is the serine protease region, contains residues 276 to 527, and harbors the active site residues His322, Asp371, and Ser478.


The vascular endothelium is thought to be the main source of tPA in the intravascular space. TPA synthesis is upregulated at the transcriptional level in response to histamine, thrombin, fluid sheer stress, and incubation with vascular endothelial growth factor (VEGF), among several other stimuli. However, tPA is also released from preformed stores in a regulated manner in response to diverse factors including β-adrenergic agents, exercise, desmopressin, disseminated intravascular coagulation, bradykinin, and cholinergic agents. These observations led to identify the Weibel-Palade bodies as the storage site and main source for tPA in ECs.

Thrombolytic Effect of tPA

In the first stages of thrombolysis, tPA’s F-domain and plasminogen’s aminohexyl site interact with fibrin, in areas of a fibrin protofibril where two D-domains are aligned end to end, which is an ideal site for plasmin to cleave any nearby fibrin molecules. In the presence of tPA, plasminogen is converted to plasmin, and this plasmin cleaves fibrin, exposing new lysine residues that enable their binding to more tPA and to plasminogen’s kringles 1 to 4. This is a positive feedback mechanism that ensures that plasmin is generated initially at a slow pace and throughout the fully assembled fibrin clot. This process is regulated at multiple levels, including the binding of PAI-1 to tPA and the cleavage of lysine residues exposed on fibrin by the thrombin-activatable fibrinolysis inhibitor (TAFI).

Clinical Uses of tPA as a Thrombolytic

Earlier studies with dogs and baboons demonstrated that treatment with tPA induces coronary recanalization in two different experimental models of coronary artery thrombosis. This led to a first pilot clinical study with 15 patients with acute myocardial infarction (AMI) in which 75% of those treated with recombinant tPA (rtPA) experienced successful coronary artery recanalization (Collen et al. 1984). These observations were subsequently confirmed by several larger multicentric studies that showed that treatment with rtPA induces coronary artery recanalization and protects the myocardium in patients with AMI. Since then rtPA has been used not only for the treatment of AMI but also for other thrombotic conditions such as pulmonary embolism, deep venous thrombosis, and peripheral artery occlusion. Remarkably, one of the most dramatic therapeutic effects of treatment with rtPA was observed in acute ischemic stroke (AIS) patients. Indeed, in a study performed by the National Institute of Neurological Disorders and Stroke (NINDS), treatment with rtPA within 3 hours of onset of symptoms of AIS resulted in an 11–13% absolute increase in the number of patients with complete or nearly complete neurological recovery (The National Institute of Neurological D and Stroke rt PASSG 1995). Based on these data, intravenous administration of rtPA is currently the standard of care for AIS patients presenting within 3–4.5 h of onset of symptoms.

tPA in the Central Nervous System

For a long time, it was believed that the vascular endothelium was the only source of tPA in the CNS and that, accordingly, its unique role was to catalyze the conversion of plasminogen into plasmin in the intravascular space. However, a growing body of recent experimental evidence indicates that in the CNS tPA is also expressed in neurons, astrocytes, and glia and that in these cells it has pleotropic effects, many of them mediated by its interaction with different receptors including the low-density lipoprotein receptor-related protein-1 (LRP1), annexin II, and mannose-6-phosphate and independent of tPA’s ability to catalyze the conversion of plasminogen into plasmin.

To facilitate the understanding of tPA’s role in the CNS, it is convenient to discuss it within the conceptual frame of the neurovascular unit (NVU), a highly dynamic structure assembled by endothelial cells (ECs) surrounded by a basement membrane (BM) and encased by perivascular astrocytes (PVA) (Fig. 1). With that in mind, it was soon clear that the ECs were not the main source of tPA in the CNS. Indeed, early studies with nonhuman primates detected tPA antigen in only a small fraction of ECs of the microvasculature, 90% of them precapillary arterioles and postcapillary veins (Levin and del Zoppo 1994). In contrast, tPA is abundantly expressed in perivascular astrocytes, where its release plays a central role regulating the permeability of the blood-brain barrier (BBB) and the development of cerebral edema during pathological conditions (Yepes et al. 2003; Polavarapu et al. 2007).
Tissue-Type Plasminogen Activator, Fig. 1

The neurovascular unit. The neurovascular unit (NVU) is a highly dynamic structure assembled by endothelial cells (ECs) surrounded by a perivascular basement membrane (BM), which is encased by perivascular astrocytes (PVA) in close proximity with neurons (N)

Alongside these studies, a substantial body of experimental evidence indicated that neurons constitute a large reservoir for tPA in the CNS. Accordingly, early studies using in situ zymography assays (Sappino et al. 1993) detected tPA-catalyzed proteolysis in well-defined areas of the adult brain, namely, hippocampus, hypothalamus, thalamus, amygdala, and cerebellum, and in vivo and in vitro studies indicated that the release of neuronal tPA at the growth cone mediates neuronal migration and neurite outgrowth and remodeling during development and in the ischemic brain (Shen et al. 2011; Liu et al. 2012).

These findings were followed by the remarkable observation that the release of tPA from neurons has a modulatory effect on synaptic function. Accordingly, initial studies showed that the secretion of neuronal tPA mediates the development of synaptic plasticity in in vitro and in vivo models of long-term potentiation (Qian et al. 1993), learning, stress-induced anxiety (Pawlak et al. 2003), and visual cortex plasticity (Muller and Griesinger 1998). This work paved the way for the discovery that neurons release tPA at extrasynaptic sites in response to membrane depolarization and that this tPA induces the anatomical and biochemical changes in presynaptic and postsynaptic terminals required for successful synaptic transmission. More specifically, experimental work with synaptic extracts, animals genetically modified to overexpress tPA in neurons, and phosphoproteomics has shown that the release of neuronal tPA induces the recruitment of the cytoskeletal protein βII-spectrin to the synaptic release site and the phosphorylation of synapsin I at serine 9 in the presynaptic terminal. This leads to the translocation of glutamate-containing synaptic vesicles to the synaptic release site where they release their content of excitatory neurotransmitters (Wu et al. 2015). Remarkably, tPA also induces calcineurin-mediated dynamin I dephosphorylation, which is an essential step for the endocytic recovery of these synaptic vesicles from the presynaptic membrane (Yepes et al. 2016). Together, these data indicate that tPA activates the synaptic vesicle cycle in glutamatergic neurons (Yepes 2016) (Fig. 2).
Tissue-Type Plasminogen Activator, Fig. 2

Tissue-type plasminogen activator activates the synaptic vesicle cycle. (a) Glutamate (pink circles)-containing synaptic vesicles are distributed in three groups known as readily releasable (light yellow ovals), recycling (not depicted in the figure), and reserve (dark yellow ovals) pools. The synaptic vesicles of the readily releasable pool are docked to an electron-dense thickening of the presynaptic membrane, known as the active zone (orange triangles), where exocytosis of glutamate takes place. In contrast, synaptic vesicles from the reserve pool are clustered away from the active zone by homodimers of synapsin I (pink lines). (b) The release of tPA (red circles) following membrane depolarization leads to recruitment of the cytoskeletal protein βII-spectrin to the active zone, enlarging its size and population of presynaptic calcium channels (green triangles). (c) The resultant influx of calcium into the presynaptic terminal leads to calcium-mediated phosphorylation of synapsin I at serine 9 releasing it from its binding to the surface of synaptic vesicles of the reserve pool, which then are free to translocate to the active zone to release their load of glutamate (Taken with permission from Yepes (2016), with permission of Neural Regeneration Research)

A further advance in the understanding of the role of tPA in the synapse was possible, thanks to recent observations indicating that the presynaptic release of tPA has a bidirectional effect on the postsynaptic terminal of glutamatergic synapses that depends on the baseline levels of neuronal activity and the extracellular concentrations of Ca2+. Accordingly, in previously inactive neurons, tPA induces phosphorylation and synaptic recruitment of Ca2+/calmodulin-dependent protein kinase IIα at Thr286 (pCaMKIIα), followed by pCaMKIIα-mediated phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in neurons with high baseline levels of pCaMKIIα and pGluR1-containing AMPARs in the PSD, tPA induces their dephosphorylation and subsequent removal to extrasynaptic sites. Remarkably, this synaptic homeostatic effect of tPA is mediated by its ability to modulate cyclin-dependent kinase 5 (Cdk5)-dependent phosphorylation of protein phosphatase-1 (PP1) at T320 (Fig. 3). Together, these data indicate that the release of neuronal tPA is a homeostatic mechanism that modulates the response of the postsynaptic terminal to the presynaptic release of glutamate.
Tissue-Type Plasminogen Activator, Fig. 3

Tissue-type plasminogen activator is a homeostatic regulator of synaptic function. (a) A silent synapse with synaptic NMDA (blue triangles) but not AMPA (orange triangle) receptors and high protein phosphatase-1 (PP1) activity (purple triangle). (b) The release of tPA from the presynaptic terminal in response to membrane depolarization (1, red circles) induces Cdk5 activation (2, magenta rectangle) and Cdk5-induced PP1 inactivation by phosphorylation at T320 (3, purple triangles). This leads to phosphorylation and synaptic recruitment of CaMKIIα (4, yellow circles) and pCaMKIIα-induced phosphorylation and synaptic recruitment of GluR1-containing AMPA receptors (5, orange triangle). The final outcome is that the synapse becomes active by the synaptic recruitment of AMPA receptors

tPA as a Neuroprotectant

The observations that tPA is induced as an immediate-early gene during seizures and long-term potentiation and the identification of a functional link between tPA and N-methyl-D-aspartate receptors (NMDAR) were two of the fundamental observations for a role for tPA in the CNS. Shortly thereafter, it was found that membrane depolarization induces the rapid release of neuronal tPA by a mechanism that does not involve tPA mRNA or protein synthesis but requires the influx of Ca+2. Few years later, studies with animal models of experimental cerebral ischemia showed that there is a transient increase in tPA activity in the ischemic tissue during the early phases of the ischemic injury (Yepes et al. 2000). These reports were rapidly followed by the intriguing observation that mice genetically deficient in tPA (tPA−/−) in all the cellular components of the NVU have an ∼ 41% decrease in the volume of the ischemic lesion following the induction of experimental cerebral ischemia (Wang et al. 1998). This led to postulate a neurotoxic effect for tPA in the ischemic brain, which was in open contradiction with clinical studies indicating that treatment with rtPA leads to complete or nearly complete neurological recovery in a significant number of AIS patients (The National Institute of Neurological D and Stroke rt PASSG 1995). The translational impact of this disagreement between basic researchers and clinicians was further heightened by the observation that following its intravenous administration, rtPA crosses the BBB and permeates the ischemic tissue. These observations led to develop the concept that in contrast with the intravascular space where tPA has a beneficial thrombolytic effect, in the brain parenchyma, tPA induces cell death. Since then several research groups have attempted to develop therapeutic strategies to antagonize this purported neurotoxic effect of tPA without interfering with its beneficial thrombolytic role in the intravascular space.

The clinical importance of these basic sciences studies warranted a second analysis. And this occurred when more recent studies showed that either neuronal overexpression of tPA or treatment with rtPA protects the brain from the deleterious effects of cerebral ischemia (Echeverry et al. 2010; Wu et al. 2012), via tPA’s ability to induce 5′ AMP-activated protein kinase (AMPK) activation in the postsynaptic terminal, promoting neuroglial coupling (An et al. 2014) and enhancing the uptake of glucose by neurons in the ischemic tissue (Wu et al. 2012). Furthermore, since then, it has also been showed that tPA is an effective inductor of homeostatic plasticity and that this contributes to attenuate the harmful effect of the ischemic injury on neuronal survival. These data not only agree with the clinical observation that millions of acute ischemic stroke patients have benefited from treatment with rtPA but also suggest that the therapeutic effect of rtPA in these patients may not be limited to its thrombolytic role but also to a neuroprotective effect currently under investigation.


tPA is a serine proteinase released from endothelial cells into the intravascular space where it promotes thrombolysis via its ability to generate plasmin in fibrin-rich clots. This has led to the successful use of rtPA to treat millions of patients worldwide suffering a variety of thrombotic conditions including pulmonary embolism, deep venous thrombosis, coronary artery disease, renal artery thrombosis, and acute ischemic stroke. Alongside these important observations, a growing body of in vivo and in vitro data indicate that tPA also has important effects that do not require the generation of plasmin. This is particularly true in the CNS where recent studies indicate that tPA protects the synapse from the harmful effects of cerebral ischemia by its ability to promote the detection and adaptation to metabolic stress.


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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of NeurologyEmory UniversityAtlantaUSA
  2. 2.Division of NeurosciencesYerkes National Primate Research CenterAtlantaUSA