ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 6, pp. 590–605. © Pleiades Publishing, Ltd., 2014. Original Russian Text © R.B. Aisina, L.I. Mukhametova, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 6, pp. 642–657.
REVIEW ARTICLE
Structure and Function of Plasminogen/Plasmin System R. B. Aisina1 and L. I. Mukhametova Department of Chemistry, Moscow State University, Moscow, 119992 Russia Received April 24, 2014; in final form, April 30, 2014
Abstract—The main physiological function of plasmin is blood clot fibrinolysis and restoration of normal blood flow. To date, however, it became apparent that in addition to thrombolysis, the plasminogen/plasmin system plays an important physiological and pathological role in a number of other essential processes: deg radation of the extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angio genesis, inflammation, and tumor cell migration. This review focuses on structural features of plasminogen, regulation of its activation by physiological plasminogen activators, inhibitors of plasmin, and plasminogen activators, and the role of plasminogen binding to fibrin, cellular receptors, and extracellular ligands in vari ous functions performed by plasmin thus formed. Keywords: plasminogen, plasmin, plasminogen activators, inhibitors, angiostatins, fibrinolysis, inflammation, angiogenesis, oncogenesis DOI: 10.1134/S1068162014060028 1
A zymogen of plasmin (EC 3.4.21.7), plasmino gen, circulates in blood. Plasminogen is synthesized in liver and many organs and tissues and considerable amounts of plasminogen are found in extravascular fluids [1, 2]. Activation of plasminogen into plasmin is regulated by tissuetype plasminogen activators (tPA, EC 3.4.21.68), urokinasetype plasminogen activators (uPA, urokinase, EC 3.4.21.31), plasminogen activator inhibitors types 1 and 2 (PAI1 and PAI2), and inhib itors of plasmin (α2antiplasmin (α2AP) and α2 macroglobulin (α2MG)). Plasminogen activators (tPA and uPA) specifically cleave the single activation bond Arg561–Val562 in a singlechain molecule of plasminogen, which results in formation of a two chain enzyme plasmin. Recently, data on the ability of certain proteases, such as kallikrein and factors XIa and XIIa, to activate plasminogen have appeared [3, 4]. To understand the mechanisms of plasmin involve ment in various physiological and pathological pro Abbreviations: PA, plasminogen activators; 6AHA, 6amino hexanoic acid; ACE, angiotensinconverting enzyme; α2AP, α2antiplasmin; ECM, extracellular endothelium matrix; uPA, urokinasetype plasminogen activator; PAI1 and PAI2, plas minogen activator inhibitors types 1 and 2; K, kringle domain; LBS, lysinebinding site; α2MG, α2macroglobulin; MMPs, matrix metalloproteinases; scuPA, singlechain urokinasetype plasminogen activator; F, fingerlike domain; Pm, plasmin; Pg, plasminogen; GluPg and LysPg, Glu and Lysforms of plas minogen; ProMMPs, prometalloproteinases; RAS, renin– angiotensin system; uPAR, urokinase and prourokinase recep tor; VEGF, vascular endothelium growth factor; NTP, Ntermi nal peptide; tPA, tissue plasminogen activator; tAMCHA, trans (4aminomethyl)cyclohexane carboxylic acid (or tranexamic acid); PDGF, plateletderived growth factor; FGF, fibroblast growth factor; EGF, epidermal growth factorlike domain. 1 Corresponding author: phone: (495)9395417; email:
[email protected].
cesses, structural features of plasminogen, its activa tors, activator inhibitors, and plasmin inhibitors, all involved in regulation of the plasminogen/plasmin system activity, and their interactions with each other upon binding fibrin, cell receptors, and extracellular ligands, are to be analyzed. In this paper, we use the term “plasminogen/plas min system” to refer to the complex effect of plasmi nogen, its activators, activator inhibitors, and plasmin inhibitors (Table 1); that is, the role of plasmin in not only fibrinolysis, but also a number of other physiolog ical and pathological processes, in which fibrin either is not involved or does not dominate, is considered. The next sections of the review are dedicated to the analysis of structure of individual key components of the plasminogen/plasmin system and the effect of their binding to fibrin, cell receptors, and ligands of the extracellular endothelial matrix (ECM) on their specific interactions. PLASMINOGEN Structure of plasminogen. Native Gluplasmino gen is a singlechain glycoprotein (Glu is the Ntermi nal amino acid; 2% carbohydrates; 93 kDa), which contains the Nterminal peptide (NTP), five homolo gous kringle domains (K1–K5), and the protease domain (PD) (Fig. 1). Each kringle domain contains approximately 80 aa connected by three disulfide bonds [7, 8]. In the presence of trace amounts of plas min, NTP (residues 1–77) is cleaved and Lysplasmi nogen is formed (85 kDa, LysPg); Nterminal amino acid of plasmin is lysine or methionine. After cleavage of the Arg561–Val562 specific bond by plasminogen
590
STRUCTURE AND FUNCTION OF PLASMINOGEN/PLASMIN SYSTEM
591
Table 1. Biochemical characteristics of the major components of plasminogen/plasmin system [5, 6] Aa
Mol. weight, kDa
Plasma concentration
Plasma halflife time
Plasminogen
791
93
0.2 mg/mL
53 h (GluPg) 19 h (LysPg)
His603 Asp646 Ser741
Tissue plasminogen activator (tPA)
530
70
5–10 ng/mL
3–5 min
His322 Asp371 Ser478
Urokinase plasminogen activa tor (uPA)
411
55/33
1 ng/mL
5–10 min
His204 Asp255 Ser356
α2Antiplasmin (α2AP)
452
67
0.07 mg/mL
50 h
Arg364 Met365
4 × 160
2.5 mg/mL
–
–
Component
α2Macroglobulin (α2MG)
Active site
Plasminogen activator inhibitor type 1 (PAI1)
379
52
50 ng/mL
5–7 min
Arg346 Met347
Plasminogen activator inhibitor type 2 (PAI2)
393
46/60
107 М–1 s–1) [34]. At the second stage, Arg346–Met347 (P1 P '1 ) bond in the reactive center of PAI1 is cleaved with formation of a covalent bond between the Arg346 residue of the inhibitor and serine active sites of uPA (Ser356) or tPA (Ser478) resulting in tight covalent complexes uPA–PAI1 and tPA–PAI1 RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
595
[33]. PAI1 inactivates single and twochain tPA with equal power, but inhibits uPA much more strongly than scuPA. PAI1 is synthesized in various types of cells, including hepatocytes, platelets, endothelial cells, smoothmuscle cells, monocytes, and macrophages. It is present in plasma and ECM. A unique feature of PAI1 is the absence of disulfide bonds, therefore it circulates in plasma in three forms: active, inactive, and latent [35]. Secreted in the active form, PAI1 molecule is easily (τ1/2 = 5 min) transformed into latent inactive form due to conformational changes that result in the reactive center being shielded by a molecule loop [36]. Latent form can be reactivated under the action of negatively charged phospholipids. The third, inactive form PAI1 is formed as a result of its cleavage by proteases via the substrate pathway. In plasma, in complex with vitronectin, as well as in the matrix, PAI1 remains active much longer than in the free state [33]. Under pathological conditions, including throm bosis, inflammatory processes, etc., PAI1 expression by various cells may be stimulated by cytokines, angio tensinII, growth factor, hormones, lipids, aldosterol, interleukin1, tumor necrosis factor, etc. [35], which leads to local concentrating of the inhibitor. This is particularly important in the case of platelets, which contain large amounts of PAI1 in latent form [34]. Upon activation and aggregation of platelets, PAI1 is released; it binds to fibrin, which explains the relative resistance to lysis of plateletrich clots. Fibrin contains two types of PAI1binding sites: small number of highaffinity sites with Kd < 1 nM and high number of lowaffinity sites with Kd = 3.8 μM [37]. α2Antiplasmin. At the first stage of plasmin (Pm) reaction with α2AP, a noncovalent enzyme–inhibitor complex Pm*α2AP (ki = 2–4 × 107 М–1 s–1) is formed due to interactions between lysine residues in Ctermi nal domain of α2AP molecule with the complementary lysinebinding site in kringle 1 of plasmin molecule [13]. At the second stage, Arg364–Met365 (P1 P '1 ) bond is cleaved in the reactive center of the inhibitor with for mation of a covalent bond between Arg364 residue of the inhibitor and Ser741 residue of plasmin active site. Since Сterminal domain of the inhibitor molecule remains noncovalently bound to kringle 1 of plasmin, an extremely stable covalent complex Pm–α2AP is formed, in which plasmin has at least one open LBS [38]. α2AP is synthesized in liver and platelets [34]. It has regions of specific binding with plasmin and fibrin [38]. In contrast to PAI1, α2AP is crosslinked with fibrin and approximately 20% of the plasmin inhibitor is incorporated into a clot [34]. NTerminal domain of α2AP binds to fibrin; the middle domain, containing a loop with reactive center, interacts with plasmin active site, while Cterminal domain comprising 51 aa is unique for serpins and serves for binding to the heavy chain of Vol. 40
No. 6
2014
596
AISINA, MUKHAMETOVA
plasmin. α2AP regulates fibrinolysis via three mecha nisms: (1) neutralizes plasmin in plasma; (2) inhibits plasmin(ogen) binding to fibrin; and (3) increases the stability of clot to lysis being incorporated in the fibrin network with the aid of factor XIIIa (a transglutami nase), catalyzing the formation of peptide bonds between Gln14 residue of α2AP Nterminal domain and Сterminal region in fibrin αchain [39]. BIOLOGICAL FUNCTIONS OF PLASMINOGEN/PLASMIN SYSTEM Plasmin specifically cleaves peptide bonds formed by carboxylic groups of Llysine and Larginine in proteins [40]. Apart from fibrin, active plasmin can destroy fibrinogen, factors V, VIII [41], and X [42] and activate some zymogens, including tPA1, scuPA [43], and matrix prometalloproteinases (proMMPs) [44, 45]. Binding of plasminogen through LBS in its kring les to fibrin and cell receptors promotes its activation into plasmin and protects the bound enzyme thus formed from inactivation with α2AP. Localization of plasminogen on surface of cells performing various biological functions leads to formation of cell surfaces with a wide range of plasmin proteolytic activity [46]. Therefore, the plasminogen/plasmin system is involved in multiple physiological and pathological processes, including thrombolysis, degradation of extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, oncogenesis, and metastasis [47, 48]. Plasminogen/plasmin system and fibrinolysis. A zymogen of plasmin, plasminogen, circulates in blood at a concentration of 2 μM. The main physiological function of plasmin is degradation of fibrin. Fibrin is not a passive substrate of plasmin. On one hand, bind ing to fibrin transforms the closed conformation of plasminogen into the open, easily activated one, and on the other, fibrin protects plasmin thus formed from inhibition by α2AP. When a blood clot (thrombus) is formed, blood cells and proteins are crosslinked with fibrin fibers. Under physiological conditions, fibrin formation initiates activation of plasminogen by its activators, which specifically interact with each other on the clot surface, generating plasmin, which in turn efficiently breaks the fibrin clot down to soluble frag ments. The effect of fibrin is manifested the most upon plasminogen activation by tPA, which, similarly to plasminogen, possesses high affinity to fibrin. Such a colocalization on the fibrin surface considerably (by a factor of 1000) increases the rate of plasmin activation by tPA [24, 49]. Besides, the plasmin generated by lys ing fibrin creates on its surface an increasing number of Сterminal lysines—new sites for plasminogen and tPA binding, which in turn leads to a considerable increase in their concentration on fibrin [50]. The main role in thrombus lysis is played by tPA [51], while uPA, possessing no affinity to fibrin, acti vates plasminogen preferably in plasma and ECM
[29]. Formation of high concentrations of plasmin in circulation under conditions of fibrinolysis inhibitors’ deficiency may lead to the degradation of fibrinogen, factors V, VIII, and X, causing severe coagulation defects and, consequently, uncontrollable bleeding. In the case of insufficient expression of plasminogen activators or high level of their inhibitors leading to plasmin deficiency, thrombus can block the vessel lumen partially or completely, which is the reason for myocardial infarction, stroke, and other thromboem bolic diseases. Relationship between plasminogen/plasmin and renin–angiotensin (RAS) systems in circulation. Angiotensin converting enzyme (ACE) is the key enzyme of RAS catalyzing the transformation of inac tive angiotensin1 (AT1) decapeptide into a powerful vasoconstrictor ATII. ACE inhibitors are known to be used as hypotensive agents in therapy of cardiovascular diseases (myocardial infarction, ischemia, and athero sclerosis). There are data on some indirect links between plasminogen/plasmin system and RAS in cir culation. Through binding to receptor type I, ATII induces vessel narrowing and PAI1 secretion by endothelial cells, thus promoting thrombosis progres sion [52]. Another main substrate of ACE is bradyki nin, which causes vessel widening and stimulates tPA secretion by endothelial cells, thus enhancing the fibrinolytic potential [53]; degradation of bradykinin under the action of ACE increases the risk of thrombo sis. ACE inhibitors decrease ATII production and prevent bradykinin degradation and platelet aggrega tion [53, 54]. We performed an in vitro investigation of cross effects of synthetic plasmin inhibitors (6AHA and tANCHA) and ACE (captopril, lisinopril, and enala pril) used in medicine as antifibrinolytic and hypoten sive agents, respectively, on the activity of the two sys tems [55]. ACE inhibitors were found to be capable of inhibition, stimulation, or producing no effect on plasminogen activation and plasmin activity. The modulatory effect depended on the type of side groups in the inhibitor molecule and specificity to fibrin of the plasminogen activator [55]. Therapeutic concentra tions of ACE inhibitors in plasma are relatively low (
