**1. Introduction**

The plasminogen activation system is assembled by a cascade of proteases and their inhibitors that catalyze the conversion of the zymogen plasminogen into the two-chain protease plasmin (Figure 1). Plasminogen is a 90 kDa single-chain multidomain glycoprotein produced mainly in the liver [1] and assembled by 791 amino acids distributed in seven different structural domains: an N-terminal pre-activation peptide, 5 kringle domains and a C-terminal trypsin-like serine protease domain that harbors the catalytic triad His603, Asp646 and Ser741 [2]. Plasminogen binds to a plethora of highly heterogenous receptors on the cell surface, and this interaction not only triggers the generation of plasmin, but also activates cell signaling pathways that orchestrate a wide variety of functions including wound healing, angiogenesis, cell migration and inflammation [3]. Cleavage of plasminogen at the Arg561–Val562 bond by one of the two main plasminogen activators [tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA)] generates a two-chain plasmin molecule assembled by an N-terminal heavy-chain and a disulfidelinked C-terminal light chain containing the proteolytically active site. Importantly, the conversion of plasminogen into plasmin is enhanced when plasminogen is bound to fibrin or to the cell surface [4]. The generation of plasmin is tightly controlled at different steps of the plasminogen activating system. Accordingly, while plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) antagonize the proteolytic effect of tPA and uPA [5], <sup>α</sup>2-antiplasmin inhibits plasmin. In the intravascular space, plasmin acts not only as an effector of the fibrinolytic system by degrading fibrin, but also as an

**Citation:** Yepes, M.; Woo, Y.; Martin-Jimenez, C. Plasminogen Activators in Neurovascular and Neurodegenerative Disorders. *Int. J. Mol. Sci.* **2021**, *22*, 4380. https:// doi.org/10.3390/ijms22094380

Academic Editor: Hau C. Kwaan

Received: 18 March 2021 Accepted: 20 April 2021 Published: 22 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

<sup>1</sup> Department of Neurology, Emory University, Atlanta, GA 30329, USA

<sup>2</sup>Neuropharmacology and Neurological Diseases Section, Yerkes National Primate Research Center, Atlanta, GA 30329, USA; yena.woo@emory.edu (Y.W.);

immune regulator [6]. Likewise, on the cell surface plasmin triggers the degradation of multiple components of the extracellular matrix (ECM) and basement membrane, including collagen, vitronectin, laminin, fibronectin and proteoglycans.

**Figure 1.** The plasminogen activating system. tPA: tissue-type plasminogen activator. uPA: urokinasetype plasminogen activator. AP: antiplasmin. PAI-1: plasminogen activator inhibitor-1. FDP: fibrin degradation products.

#### **2. The Neurovascular Unit**

The concept of the neurovascular unit (NVU) describes a dynamic interaction in the central nervous system between endothelial cells ensheathed by a basement membrane, and surrounding pericytes, astrocytes, microglia and neurons (Figure 2). The nature of the interplay between these cellular and non-cellular components has led to the proposal that the NVU is a single functioning unit responsible for the maintenance of cerebral hemostasis [7].

#### **3. Plasminogen Activators in the Neurovascular Unit under Physiological Conditions**

#### *3.1. Tissue-Type Plasminogen Activator*

Tissue-type plasminogen activator (tPA) is a 70-kDa serine proteinase secreted as a single-chain form (that upon its cleavage by plasmin at Arg275-Ile276 generates an active two-chain form held together by disulfide bonds). The tPA molecule is assembled by four domains: an amino-terminal region (fibronectin-like or finger domain), an EGF-like domain, two kringles and one serine protease region that harbors the active site residues His322, Asp371 and Ser478 [8]. In the neurovascular unit (NVU), tPA is found in endothelial cells, perivascular astrocytes, microglia, pericytes and neurons [9,10].

#### *3.2. Tissue-Type Plasminogen Activator in the Neurovascular Unit*

#### 3.2.1. Cerebral Endothelial Cells

In endothelial cells tPA is stored in Weibel–Palade bodies, the specialized endothelial storage granules for von Willebrand factor [11]. The expression of tPA is increased at the transcriptional level in endothelial cells by a variety of stimuli, including vascular endothelial growth factor (VEGF), fluid shear stress, thrombin and histamine [12–14]. In turn, its release from a preformed pool is triggered by physical activity, β-adrenergic drugs, cholinergic agents, disseminated intravascular coagulation and hypoxia [15,16]. Studies with a primary monoclonal antibody that detected free and PAI-1-complexed human tPA revealed that in the non-human primate brain, tPA is found in a reduced number of endothelial cells of the microvasculature, most of them pre-capillary arterioles and postcapillary veins [17]. However, despite the relevance of these data, it is important to consider that since this report was published almost 3 decades ago no further effort has been made to characterize the expression of tPA with newer antibodies in endothelial cells of the brain. Finally, although tPA has been detected in in vitro lines of human microvascular endothelial cells [18], no in vivo studies have been published describing the expression of tPA in blood vessels of the human brain.
