International Journal of Molecular Sciences Review Assessing Plasmin Generation in Health and Disease Adam Miszta 1,* , Dana Huskens 1, Demy Donkervoort 1, Molly J. M. Roberts 1, Alisa S. Wolberg 2 and Bas de Laat 1 1 Synapse Research Institute, 6217 KD Maastricht, The Netherlands; [email protected] (D.H.); [email protected] (D.D.); [email protected] (M.J.M.R.); [email protected] (B.d.L.) 2 Department of Pathology and Laboratory Medicine and UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; [email protected] * Correspondence: [email protected]; Tel.: +31-(0)-433030693 Abstract: Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The ki- netics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID- 19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired Citation: Miszta, A.; Huskens, D.; fibrinolytic disorders. Donkervoort, D.; Roberts, M.J.M.; Wolberg, A.S.; de Laat, B. Assessing Keywords: plasmin; plasmin generation; fibrinolysis Plasmin Generation in Health and Disease. Int. J. Mol. Sci. 2021, 22, 2758. https://doi.org/doi:10.3390/ ijms22052758 1. Introduction Academic Editor: László Muszbek Hemostasis is a process of regulated balance between clot formation and clot lysis, aimed at preventing blood loss while maintaining vascular patency. Activation of coagula- Received: 1 February 2021 tion factors and platelets leads to thrombin generation (TG) and ultimately, clot formation. Accepted: 5 March 2021 Subsequently, the fibrinolytic system is crucial for clot remodeling and lysis. Perturbation Published: 9 March 2021 of these processes can lead to bleeding disorders or thrombotic events. Although several tools to assess fibrinolysis have been developed, few methods are available to quantify the Publisher’s Note: MDPI stays neutral amount of plasmin generated. The scope of this review is to outline methods to measure with regard to jurisdictional claims in plasmin generation (PG) and highlight applications of these assays in human samples and published maps and institutional affil- mouse models of disease. iations. 2. Plasmin and the Molecular Mechanism of Plasmin Generation Plasmin is a major fibrinolytic protease generated by converting its zymogen plas- minogen by fibrinolytic activators in the presence or absence of fibrin. Plasminogen is Copyright: © 2021 by the authors. synthesized primarily in the liver and circulates in plasma at a concentration of approx- Licensee MDPI, Basel, Switzerland. imately 1.5 µmol/L [1]. Glu-plasminogen is a single chain glycoprotein with glutamic This article is an open access article acid (Glu) as the NH2 residue, consisting of an N-terminal activation peptide (NTP), five distributed under the terms and kringle domains (K1–K5), and a serine protease domain containing the catalytic triad conditions of the Creative Commons (Figure1)[1,2]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 2758. https://doi.org/10.3390/ijms22052758 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 17 Int. J. Mol. Sci. 2021, 22, 2758 2 of 17 Figure 1. Schematic representation of the primary structure of human glutamic (Glu)-plasminogen. The catalytic triad (His603, Asp646, and Ser741) within the protease domain, the activation site (Arg561–Val562), and the 24 disulfide bridges Figure 1. Schematicas well as therepresentation signal peptide areof the indicated. primary NTP, structure N-terminal of peptide; human K1–K5, glutamic kringles (Glu)-plasminogen. 1–5. Previously published The [catalytic3] and triad (His603, Asp646,adapted and from Ser741) Schaller within and Gerber the protease [4]. domain, the activation site (Arg561–Val562), and the 24 disulfide bridges as well as the signal peptide are indicated. NTP, N-terminal peptide; K1–K5, kringles 1–5. Previously published [3] and adapted from Schaller and Gerber [4]. Lys-plasminogen is a truncated plasmin-derived form that lacks the NH2-terminal 1–77 peptide and is produced during fibrinolysis [5]. Whereas Glu-plasminogen assumes a closed conformation because of intramolecular links between the NH2-terminal peptide Lys-plasminogen is a truncated plasmin-derived form that lacks the NH2-terminal 1– and kringle 5 domain, Lys-plasminogen has a more open conformation. The conformational 77 peptidechange and of Glu-plasminogenis produced during to Lys-plasminogen fibrinolysis during[5]. Whereas fibrinolysis Glu-plasminogen not only renders Lys- assumes a closed plasminogenconformation more because readily of activated intramolecular by plasminogen links activators,between butthe also NH endows2-terminal Lys- peptide and kringleplasminogen 5 domain, with a higher Lys-plasminogen affinity for fibrin than has its intacta more precursor. open Of theconformation. five kringle The conformationaldomains in change plasminogen, of Gl kringlesu-plasminogen 1, 4, and 5 are to reported Lys-plasmino to be thegen major during mediators fibrinolysis of the not plasminogen/fibrin interaction [6,7]. only rendersTissue Lys-plasminogen plasminogen activator more (tPA) readily and urokinase-typeactivated by plasminogen plasminogen activator activators, (uPA but also endowsor Lys-plasminogen urokinase) are plasminogen with a activators. higher affinity TPA is secreted for fibrin predominantly than its intact from endothelialprecursor. Of the five kringlecells triggered domains by localin plasminogen, stimuli, including kringles thrombin 1, activity, 4, and bradykinin 5 are reported (a product to of be high the major mediatorsmolecular of the weight plasminogen/fibrin kininogen cleavage interaction by kallikrein), [6,7]. and shear stress. TPA-mediated PG is initiated when thrombin, the central enzyme in coagulation, cleaves fibrinopeptides TissueA and plasminogen B from fibrinogen activator and enables (tPA) fibrin and network urokinase-type formation (Figureplasminogen2). This processactivator (uPA or urokinase)exposes cryptic are domainsplasminogen in fibrin(ogen) activators. that allows TPA for binding is secreted of both plasminogen predominantly and a from endothelialplasminogen cells triggered activator tPA by to local fibrin stimuli, via C-Lysine including binding sites.thrombin Exposure activity, of these bradykinin sites is (a productdriven of high primarily molecular by the interaction weight betweenkininogen complementary cleavage sitesby ofkallikrein the D and), E and regions shear [1]. stress. TPA-mediatedThe catalytic PG efficiency is initiated of plasminogen when thrombin, activation the by central tPA is 3 ordersenzyme of magnitude in coagulation, greater cleaves in the presence of fibrin than it is in its absence and 2 orders of magnitude greater in the fibrinopeptidespresence of A fibrinogen. and B from uPA fibrinogen is expressed and by endothelialenables fibrin cells, network macrophages, formation and renal (Figure 2). This processepithelial exposes cells and cryptic activates domains plasminogen in independentfibrin(ogen) of fibrin,that allows when it for interacts binding with of both plasminogenthe cellular and receptor a plasminogen urokinase protease-activated activator tPA receptorto fibrin (uPAR) via [8C-Lysine,9]. uPA catalytic binding sites. Exposureefficiency of these for sites plasminogen is driven activation primarily is similar by the in interaction the presence between and absence complementary of fibrin or sites fibrinogen. of the D and E regions [1]. The catalytic efficiency of plasminogen activation by tPA is 3 orders of magnitude greater in the presence of fibrin than it is in its absence and 2 orders of magnitude greater in the presence of fibrinogen. uPA is expressed by endothelial cells, macrophages, and renal epithelial cells and activates plasminogen independent of fibrin, when it interacts with the cellular receptor urokinase protease-activated receptor (uPAR) [8,9]. uPA catalytic efficiency for plasminogen activation is similar in the presence and absence of fibrin or fibrinogen. Int. J. Mol. Sci. 2021, 22, 2758 3 of 17 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 17 FigureFigure 2. 2.A A schematic schematic representation representation of of the the fibrinolytic fibrinolytic system. system. Fibrinolysis Fibrinolysis is is initiated initiated when when the the product product of of coagulation, coagulation, thrombin,thrombin, cleaves cleaves fibrinopeptides fibrinopeptides A A and and B B from from fibrinogen, fibrinogen,