REVIEW ARTICLE Small-molecule modulators of zymogen activation in the fibrinolytic and coagulation systems Keiji Hasumi, Shingo Yamamichi and Tomotaka Harada Department of Applied Biological Science, Tokyo Noko University, Japan Keywords coagulation; conformation; fibrinolysis; plasma hyaluronan-binding protein; plasminogen; plasminogen activator; protease; prothrombin; urokinase; zymogen activation Correspondence K Hasumi, Department of Applied Biological Science, Tokyo Noko University, 3-5-8 Saiwaicho, Fuchu-shi, Tokyo 183-8509, Japan Fax: +81 42 367 5708 Tel: +81 42 367 5710 E-mail: hasumi@cc.tuat.ac.jp The coagulation and fibrinolytic systems are central to the hemostatic mechanism, which works promptly on vascular injury and tissue damage The rapid response is generated by specific molecular interactions between components in these systems Thus, the regulation mechanism of the systems is programmed in each component, as exemplified by the elegant processes in zymogen activation This review describes recently identified small molecules that modulate the activation of zymogens in the fibrinolytic and coagulation systems (Received 29 April 2010, revised July 2010, accepted 19 July 2010) doi:10.1111/j.1742-4658.2010.07783.x Introduction The coagulation and fibrinolytic systems are central to the hemostatic mechanism This mechanism is primarily responsible for preventing blood leakage and secondarily for tissue repair and wound healing Interactions between a variety of enzymes and nonenzyme components account for the finely regulated processes in the coagulation and fibrinolytic systems [1,2] Most of the enzymes in these systems are serine proteases circulating as either inactive proenzyme or proenzyme forms with very low activity Thus, their activation is a prerequisite for their function, whereas their inhibition or inactivation is also important in the regulation and termination of the reaction Activation of the zymogens is a consequence of a conformational change triggered by specific proteolytic cleavage(s) of the zymogen [3,4] The activated enzyme catalyzes the subsequent step or upstream reaction(s) in the cascade to amplify and ⁄ or regulate the systems The reactions in both systems operate instantly after exposure to pathophysiological stimuli Therefore, the mechanism of their regulation is programmed in the structure of each component of the systems, and modulation of the programmed function of the component can provide a novel means of pharmacological intervention in diseases associated with the coagulation and fibrinolytic systems Most extensively studied examples of zymogen modulation are nonproteolytic conformational activations of plasminogen by streptokinase, and prothrombin by staphylocoagulase (and von Willebrand factor-binding protein) These are pathogen-derived nonenzyme proteins that exploit the ‘molecular sexuality’ in the activation mechanism of plasminogen and Abbreviations AH site, aminohexyl site; EGF, epidermal growth factor; Lp(a), lipoprotein(a); PA, plasminogen activator; PAN domain, plasminogen ⁄ apple ⁄ nematode domain; PHBP, plasma hyaluronan-binding protein; scu-PA, single-chain urokinase-type plasminogen activator; SMTP, Stachybotrys microspora triprenyl phenol; tcu-PA, two-chain urokinase-type plasminogen activator; t-PA, tissue-type plasminogen activator; u-PA, urokinase-type plasminogen activator FEBS Journal 277 (2010) 3675–3687 ª 2010 The Authors Journal compilation ª 2010 FEBS 3675 Zymogen activation modulators K Hasumi et al yses of the action of such modulators have expanded the concept of zymogen modulation, leading to the identification of additional modulators that affect the coagulation system The active compounds and their targets are summarized in Table and Fig Here, we review the identification and functional characterization of such small-molecule modulators of zymogen activation in the fibrinolytic and coagulation systems Table Small-molecule zymogen modulators identified in this laboratory PHBP, plasma hyaluronan-binding protein; scu-PA, singlechain urokinase-type plasminogen activator; SMTP, Stachybotrys microspora triprenyl phenol Target Compound Reference Plasminogen Complestatin and chloropeptin I Staplabin and SMTPs Thioplabins Stachybotrydial Surfactins and iturins Glucosyldiacylglycerol Plactins Polyamines and carminic acid [43,44,52] [45–53] [52,54] [55] [68,69] [70] [84–86] [104] scu-PA ⁄ plasminogen reciprocal activation Prothrombin Pro-PHBP Plasminogen modulator The plasminogen ⁄ plasmin system The plasminogen ⁄ plasmin system plays a central role in blood clot lysis [2,9] This system is also important in other pathophysiological events in which localized proteolysis is involved [10–12] The circulating form of plasminogen (Glu-plasminogen) is a single-chain zymogen consisting of 791 amino acids, which form the following domain structures: a plasminogen ⁄ apple ⁄ nematode (PAN) domain, five kringle domains and a serine protease domain (Fig 2) [13,14] It is proteolytically activated to plasmin by plasminogen activators (PAs) through specific cleavage at Arg561– Val562 Urokinase-type and tissue-type PAs (u-PA and t-PA, respectively) are major physiological activators prothrombin (insertion of N-terminal Ile or Val into N-terminal binding cleft of the catalytic domain, resulting in conformational activation of the substrate binding site and oxyanion hole required for proteolytic activity) [5–8] Our laboratory has been searching for small-molecule natural products that enhance the fibrinolytic system We initially aimed at identifying a candidate small molecule that could contribute to the treatment of thrombotic and embolic complications As a result, we have identified several types of compounds, including modulators of zymogen activations Detailed anal- O R N OH O O HOOC O H N N H N O N H O Cl Cl OH HN H N O O Cl Cl OH OH O Cl HOOC O Leu O H N HN O NH NH HN Leu O O N H O D-Leu Surfactin C O N H CH2 O H N N H OH Stachybotrydial NH O OH CH2 O Thioplabins HO HO O OR1 OH OR2 O S NH HN O NH HN Asp CH2 O D-Cys Phe O OH N H D-Val O Val O S N NH2 D-Arg O O O O NH D-Leu NH O HN NH O N H N O R CH2 OH CHO O N NH CH2 O Glu NH HO CH2 Staplabin/SMTPs Complestatin HO S NH OH Cl O O HO O CHO HO N S O N H NH N O N H O D-Leu Plactin D S O H N Glucosyldiacylglycerol D-Cys O NH HN HO HO O Leu Ile OH Val NH O HN O D-Leu Malformin A1 O OH O CH3 OH COOH HO OH OH O Carminic acid Fig Structures of the modulators of zymogen activation R in the thioplabin structure corresponds to –CH3, –CH2CH3 or –CH(CH3)2 for thioplabin A, B or C, respectively R in the structure of SMTPs represents one of a variety of substituents, most of which are derived from amino acids R1 and R2 in the structure of glucosyldiacylglycerol correspond to the oleoyl or palmitoyl group Malformin A1, which enhances cellular fibrinolytic activity through a mechanism distinct from direct modulation of zymogen activation, is shown to compare its structure with that of plactin D 3676 FEBS Journal 277 (2010) 3675–3687 ª 2010 The Authors Journal compilation ª 2010 FEBS K Hasumi et al GluE1 plasminogen Zymogen activation modulators PAN K1 K2 K3 K4 K5 P Lysplasminogen K78 K1 K2 K3 K4 K5 P Plasmin K78 K1 K2 K3 K4 K5 561 562 scu-PA S1 E K tcu-PA S1 E K P R V P 158 159 A1 Prothrombin Gla K1 K P I P K2 320 321 α-Thrombin T285 Pro-PHBP F1 E1 E2 E3 F1 E1 E2 E3 K P I K PHBP R P 290 291 R I P Fig Structures of key zymogens and their active forms Each molecule is shown schematically with each domain in a colored circle The disulfide bond that connects the A- and B-chains of the mature protease is shown in green Red lines in the PHBP molecule represent the N-terminal region (NTR) PAN, PAN domain; K, kringle domain; P, protease domain; Gla, c-carboxyglutamic acid domain; E, EGF domain [2] Glu-plasminogen adopts a closed conformation because of the intramolecular binding of Lys50 and ⁄ or Lys62 to the fifth kringle domain (K5) (Fig 3) [15,16] The tight conformation renders Glu-plasminogen less sensitive to activation by PAs [17,18] The Gluplasminogen binding to fibrin or cellular receptors allows relaxation of Glu-plasminogen conformation, enabling efficient activation (Fig 3) This mechanism facilitates localized activation of plasminogen and extracellular proteolysis [19,20] Growing evidence supports the idea that cellular plasminogen binding plays roles in physiological processes such as macrophage recruiting, leukocyte migration and liver regeneration [21–24] During the course of fibrinolysis, Lys-plasminogen (Fig 2), a truncated form of Glu-plasminogen, predominantly with an N-terminal Lys78, appears through autoproteolysis by the resulting plasmin [25,26] The molecule no longer has the PAN domain and therefore adopts a relaxed conformation [27] because of the lack of intermolecular binding between the PAN domain and K5 (Fig 3) Lys-plasminogen is highly sensitive to activation even in the absence of fibrin or cells PAN–K5 binding is mediated via the aminohexyl (AH) site in K5 [28,29] (Fig 3) Unlike lysine-binding sites in K1, K2 and K4 of plasminogen, the AH site can bind an internal lysine residue in addition to the C-terminal lysine [30], which is a preferred ligand for the lysine-binding site in K1, K2 and K4 Thus, aminohexyl or lysine analogs can interfere with PAN–K5 binding and relax Glu-plasminogen conformation, rendering Glu-plasminogen susceptible to activation by PAs [31,32] Some proteins, as well as cell-surface plasminogen receptors, interact with kringles and modulate plasminogen activation [33,34] It is postulated that the fibrinolytic process proceeds as follows [35]: at an initial phase, the K5–fibrin interaction accumulates Glu-plasminogen on fibrin Subsequently, partially degraded fibrin, which has C-terminal lysines, plays a role in fibrinolytic propagation by serving as more efficient binding sites for plasminogen The high-affinity lysine-binding sites in K1 and K4 may be involved in this propagation process Thus, plasminogen binding is essential for the fibrinolytic process The kringle ligands, however, inhibit plasminogen binding to fibrin and cellular receptors and, therefore, suppress fibrinolysis This is the basis of the antihemorrhagic action of lysine analogs such as tranexamic acid and 6-aminohexanoic acid [36] Competition in the binding between plasminogen and fibrin or cellular receptors can occur via physiologic molecules Lipoprotein(a) [Lp(a)], a risk factor for coronary heart disease [37,38], is a plasma lipoprotein related to low-density lipoprotein In the Lp(a) molecule, apolipoprotein B-100, which is a sole protein component of low-density lipoprotein, is covalently modified with apolipoprotein(a), a protein closely related to plasminogen, consisting of multiple (11–50 repeats) K4-like domains, a K5-like domain and a pseudo-protease domain [39,40] Lp(a) can compete with plasminogen for binding to fibrin or cell-surface receptors and attenuate localized plasminogen activation [41] and conversion of Glu-plasminogen to Lysplasminogen [42] These findings suggest a possible link between thrombosis and atherosclerosis Our laboratory has screened up to 10 000 microbial cultures for their ability to enhance Glu-plasminogen binding to fibrin or culture cells This attempt is based on the hypothesis that relieving the plasminogen binding competition might enhance fibrinolytic activity The active compounds identified include complestatin FEBS Journal 277 (2010) 3675–3687 ª 2010 The Authors Journal compilation ª 2010 FEBS 3677 Zymogen activation modulators K5 K50 PAN Plasmin P K3 K2 K78 E1 K1 PA K2 K3 Glu-plasminogen (Closed conformation) K1 P K4 K Hasumi et al K4 K5 Lys-plasminogen (Open conformation) PAN (Open conformation) K1 P PA K2 K3 D57 D55 E1 W62 K78 F36 I35 K2 K4 K5 Fibrin or cellular receptor Y72 W64 K1 K3 K4 Plasmin K5 L71 H33 P H31 V562 R561 K5 structure Fig A model of the conformational regulation of plasminogen activation The plasminogen and plasmin molecules are shown as in Fig The schematic conformation shown is speculative, because detailed experimental data are not yet available Key disulfide bonds are shown in green The molecular structure K5 is shown in the right-hand box The electrostatic surface image, in which areas of neutral, negativeand positive potential are depicted in white, red and blue, respectively, was constructed using the coordinate deposited in the RCSB Protein Data Bank with the code number 2KNF The labeled amino acids are those implicated in the binding of the lysine analog tranexamic acid [29] [43] and its isomer chloropeptin I [44], staplabin [45] and its congener Stachybotrys microspora triprenyl phenols (SMTPs) [46–53], thioplabins [54] and stachybotrydial [55] Complestatin and chloropeptin I Tachikawa et al [43] isolated complestatin, a chlorinecontaining peptide metabolite from Streptomyces sp., as an active principle that enhanced Glu-plasminogen binding to cultured U937 monocytoid cells Complestatin enhances the binding by several fold at 1–10 lm The enhancement is observed in either the absence or presence of Lp(a) The compound is also effective in elevating Glu-plasminogen–fibrin binding The actions of complestatin are canceled by a lysine analog Thus, the target of complestatin is Glu-plasminogen, and the agent promotes its binding to fibrin and cells via the AH site or the lysine-binding sites However, the initial characterization does not clarify the exact mechanism of the complestatin actions The current understanding is that the action of complestatin modulates Glu-plasminogen conformation [52], but verification of this has awaited the characterization of staplabin and SMTPs However, the concept that small molecule-mediated augmentation of plasminogen binding results in increased plasmin formation on cell surfaces has been confirmed by experiments using complestatin [43] Chloropeptin I, an isomer of complestatin, has a similar but slightly different effect on plasminogen binding compared with complestatin [44] Chloropeptin I is 3–10 times more active than complestatin in enhancing cellular binding of Glu-plasminogen, cell-surface plasmin formation, and whole-blood 3678 fibrinolysis assessed by thromboelastography, although the agent is less active in promoting fibrin binding of Glu-plasminogen Although the structural difference between complestatin and chloropeptin I is only at the position of the C–C bond that connects the indole ring of the tryptophan residue and the aromatic ring of the 3,4-dihydroxyphenylglycine residue, a large difference in conformation between the two compounds is evident from the nuclear Overhauser effect in NMR spectroscopy It is speculated that this difference may account for the distinct activities of the two compounds [44] Staplabin and SMTP Shinohara et al [45] discovered a novel triprenyl phenol compound, named staplabin, from a culture of the fungus Stachybotrys microspora, as an active principle that enhanced Glu-plasminogen binding to fibrin Later, several new staplabin congeners, SMTPs, were isolated by Kohyama et al [46], Hasumi et al [48,53], and Hu et al [49,51] These are named after Stachybotrys microspora triprenyl phenol The staplabin ⁄ SMTP molecule consists of a tricyclic c-lactam moiety, an isoprene side chain and an N-linked side chain (Fig 1) Most of the congeners differ in the N-linked side chain moiety, which is essential for their activity The N-linked side chain can be derived from an amine present in the culture medium, and this finding enabled selective, efficient production of SMTP congeners through an amine-feeding cultivation of S microspora [50,51,53] Staplabin enhances Glu-plasminogen binding to both fibrin and cultured U937 cells [45] These FEBS Journal 277 (2010) 3675–3687 ª 2010 The Authors Journal compilation ª 2010 FEBS K Hasumi et al K50 PAN E1 P K5 K4 P K3 K2 Modulator K1 (Conformational change) K5 K4 Glu-plasminogen K3 K50 PAN E1 K1 K2 E1 PAN PA K78 K1 P bindings are mediated by the lysine-binding site or the AH site, because a lysine analog inhibits the bindings These activities are quite similar to those of complestatin and chloropeptin I Takayasu et al [47] show that staplabin promotes PA-dependent Glu-plasminogen activation The two staplabin activities, the elevation of Glu-plasminogen-fibrin binding and the promotion of Glu-plasminogen activation, are observed at the same range of staplabin concentrations Thus, a common mechanism may govern the two effects The fact that the activation of Glu-plasminogen is a conformationally regulated process is the key to understanding this mechanism The effect of staplabin on the activation of Lys-plasminogen, which adopts an open conformation, is less prominent than its effect on Gluplasminogen Similarly, smaller effects of staplabin on Glu-plasminogen activation are observed in the presence of the lysine analog 6-aminohexanoic acid or fibrinogen fragments, both of which relax Glu-plasminogen conformation The molecular elution time of both Glu-plasminogen and Lys-plasminogen is slightly but significantly shortened in the presence of staplabin These results support the idea that the staplabin effects are related to the conformational status of plasminogen, and Takayasu et al [47] have concluded that staplabin modulates plasminogen conformation, rendering the molecule susceptible to proteolytic activation and to binding to cells and fibrin The reason why the effects of staplabin on Glu-plasminogen is larger than its effect on Lys-plasminogen can be explained by the fact that the impact of the conformational effect depends on the initial conformational status of plasminogen With respect to the conformational modulation that leads to an elevated Gluplasminogen activation, the staplabin effect is similar to that of lysine analogs, whereas the effects of each compound on plasminogen binding are quite different This implies that staplabin acts as a plasminogen modulator that works through a mechanism distinct from the lysine-binding site (or AH site) occupancy Staplabin congener SMTPs may act on plasminogen activation and binding similarly to staplabin, whereas some congeners, such as SMTP-7 and -8, are far more potent than staplabin [49] The action of staplabin ⁄ SMTP is shown schematically in Fig Pharmacological evaluations of SMTP-7 suggest that the compound is a promising candidate drug for treating thrombotic and embolic complications Detailed investigations are now under way Plasmin formation in the presence of SMTP is transient in an incubation of Glu-plasminogen with u-PA [52] A decrease in plasmin activity follows a rapid increase in plasmin formation This is because of auto- Zymogen activation modulators K2 K3 K4 K5 K1 K2 P K3 K4 Fibrin or cellular receptor K5 V562 R561 Plasmin Fig A model of the action of the plasminogen modulator staplabin ⁄ SMTP Staplabin and SMTP alter plasminogen conformation and enhance both PA-dependent plasminogen activation and binding to fibrin or cellular receptors Although these compounds can also modulate the functions of Lys-plasminogen, the magnitude of the effects is small, possibly because Lys-plasminogen adopts a more relaxed conformation compared with Glu-plasminogen The schematic model shows only the modulation of Glu-plasminogen function proteolytic degradations of the catalytic domain of the plasmin molecule Ohyama et al speculate that the conformational change brought about by SMTP affects susceptibility to autoproteolytic cleavage 6-Aminohexanoic acid does not lead to autoproteolysis, but enhances plasminogen activation Thus, the difference between conformational modulation by SMTP and that by the lysine analog is also evident from these results Similar promotion of plasmin autoproteolysis is observed with complestatin [52] This effect is obtained with a concentration range identical to that effective in enhancing Glu-plasminogen binding, suggesting complestatin’s action as a plasminogen modulator As expected, complestatin-mediated enhancement of Glu-plasminogen activation has been confirmed Thioplabins Ohyama et al [54] identified thioplabins (or antibiotic A10255) (Fig 1), a family of thiopeptide metabolites from Streptomyces sp., as modulators of plasminogen binding Thioplabin B enhances the binding of both Glu-plasminogen and Lys-plasminogen to fibrin It also promotes PA-dependent activation of Glu- and Lys-plasminogen Like staplabin, the effect of thioplabin B is smaller on conformationally relaxed Lys-plasminogen Thioplabin B alters patterns of proteolytic degradation of Glu- and Lys-plasminogen upon FEBS Journal 277 (2010) 3675–3687 ª 2010 The Authors Journal compilation ª 2010 FEBS 3679 Zymogen activation modulators K Hasumi et al incubation with elastase The agent also increases autoproteolytic degradation of the plasmin catalytic domain [52] These features conform to the concept of the nonlysine-analog plasminogen modulator established by staplabin ⁄ SMTP Thioplabin analogs that lack the terminal carboxyl group are inactive in plasminogen modulation [54] Stachybotrydial Stachybotrydial (Fig 1) is a tripreny phenol metabolite from Stachybotrys sp., structurally distinct from staplebin ⁄ SMTPs Sasaoka et al [55] show that stachybotrydial has a specific effect on Glu-plasminogen It enhances the activation and fibrin binding of Glu-plasminogen, but not Lys-plasminogen Unlike the modulation of plasminogen function by other small molecules described above, the action of stachybotrydial involves covalent modification of Glu-plasminogen Because covalent stachybotrydial modification is observed even with Lys-plasminogen, the selective effects on Glu-plasminogen are related to its conformational status Thus, stachbotrydial represents another class of plasminogen modulators in addition to complestatin ⁄ chloropeptin I, staplabin ⁄ SMTP and thiplabins, which reversibly modulate the function of Glu- and Lys-plasminogen Modulator of the reciprocal activation of single-chain u-PA and plasminogen Reciprocal activation of single-chain u-PA and plasminogen Of the two major physiological plasminogen activators, t-PA is postulated to play a role in fibrin dissolution in the circulation, whereas u-PA is involved in pericellular proteolysis [2] t-PA has a significant affinity for fibrin and exhibits activity more than two orders of magnitude higher in the presence of fibrin through the formation of a cyclic ternary complex with plasminogen and fibrin [56,57] u-PA, which consists of an epidermal growth factor (EGF) domain, a kringle domain (which does not contain a lysine-binding site) and a protease domain (Fig 2), has little affinity to fibrin and utilizes a distinct mechanism to regulate localized proteolysis u-PA is synthesized as a single-chain zymogen (scu-PA), and binds to the cell-surface receptor u-PAR via its EGF domain [58,59] in an autocline manner to facilitate the activation of cell-bound plasminogen for pericellular proteolysis involved in a variety of conditions including tissue remodeling, macrophage function, ovulation and tumor invasion [60,61] scu-PA is specifically cleaved at Lys158– 3680 Ile159 by plasmin, affording a two-chain derivative (Fig 2) that has a full protease activity [62] Thus, the reciprocal activation of scu-PA and plasminogen constitutes a mechanism of localized initiation and propagation of pericellular proteolysis [63–66] and fibrinolysis on platelets [67] The mechanism of the initiation of the reaction (how the two zymogens activate each other in the initial phase), however, remains to be fully elucidated A screen of natural sources, including microbial cultures and plant extracts, for a modulator of the reciprocal activation system has led to the identification of surfactins [68], iturins Cs [69] and glucosyldiacylglycerol [70] Surfactins and iturins Surfactin C (Fig 1), a cyclic heptapeptide with a fatty acid ester in the cyclic structure, is a metabolite from Bacillus sp Kikuchi and Hasumi [68] show that surfactin C modulates the reciprocal activation of Gluplasminogen and scu-PA Upon incubation with Glu-plasminogen and scu-PA, the spontaneous activation of both Glu-plasminogen and scu-PA proceeds slowly Surfactin C markedly enhances the concomitant formation of tcu-PA and plasmin The surfactin C action involves modulation of Glu-plasminogen activation through relaxing the conformation of the protein Surfactin C increases the intrinsic fluorescence of Glu-plasminogen, shortens molecular elution time in size-exclusion chromatography, and enhances fibrinbinding and activation of Glu-plasminogen catalyzed by tcu-PA or t-PA [68] Thus, surfactin C can be a plasminogen modulator, but the mechanism by which the agent promotes the initiation of the reciprocal activation is not fully understood A possible explanation would be that the modulation of Glu-plasminogen conformation allows cleavage by scu-PA, which has very low intrinsic activity toward native Glu-plasminogen (< 0.4% of tcu-PA [71]) Surfactin C, in combination with scu-PA, significantly enhances thrombolysis in a rat pulmonary embolism model [68] Surfactin C belongs to a large family of lipopeptides that includes surfactins (heptapeptides consisting of two acidic and five aliphatic amino acids), iturin As (heptapeptide consisting of six polar amino acids and a proline) and iturin Cs (heptapeptide consisting of five polar and an acidic amino acids as well as a proline) All the surfactins and iturin As tested are effective in enhancing the reciprocal activation and tcu-PA-catalyzed Glu-plasminogen activation, whereas iturin Cs, which contain no carboxyl group, are inactive [69] FEBS Journal 277 (2010) 3675–3687 ª 2010 The Authors Journal compilation ª 2010 FEBS K Hasumi et al Glucosyldiacylglycerol Wu et al [70] identified glucosyldiacylglycerol (Fig 1), a cellular constituent of the seaweed Sargassum fulvellum, as a stimulator of the reciprocal activation of scuPA and Glu-plasminogen The apparent action of glucosyldiacylglycerol is similar to that of surfactin C in that it leads to the mutual activation of scu-PA and Glu-plasminogen Unlike surfactin C, however, glucosyldiacylglycerol minimally affects Glu-plasminogen activation catalyzed by tcu-PA and t-PA Thus, the agent likely represents a different class of zymogen modulator than the plasminogen modulators Glucosyldiacylglycerol markedly enhances scu-PA-mediated Glu-plasminogen activation in the absence of the conversion of scu-PA to tcu-PA Upon incubation with glucosyldiacylglycerol, the intrinsic fluorescence of scu-PA, but not tcu-PA or Glu-plasminogen, increases significantly Thus, glucosyldiacylglycerol may act on scu-PA to draw intrinsic PA activity in the zymogen The agent enhances fibrin dissolution mediated by scu-PA and Glu-plasminogen Modulator of the activation of prothrombin Prothrombin activation The formation of thrombin from its zymogen prothrombin is the central event in the coagulation cascade In addition to the conversion of fibrinogen to fibrin, thrombin orchestrates the coagulation and fibrinolytic processes through activation of factors V, VIII, XI and XIII [1], as well as protein C [72] and thrombin-activatable fibrinolysis inhibitor [73] after binding to thrombomodulin In addition, thrombin triggers a variety of cellular responses by binding to and specifically cleaving the extracellular domain of the family of G-protein-coupled receptors, protease-activated receptors [74] Prothrombin, a 579-amino-acid glycoprotein, consists of a c-carboxyglutamic acid domain, two kringle domains and a protease domain (Fig 2) [75] Prothrombin activation is due to proteolytic cleavage at Arg320–Ile321, followed by cleavage at Arg271–Thr272 [76–78] An additional cleavage at Arg284–Thr285 by thrombin itself affords mature a-thrombin At the site of vascular injury, prothrombin is rapidly activated to thrombin by the coagulation factor Xa which is Ca2+-dependently assembled with factor Va on acidic phospholipid membranes of damaged vascular endothelium or activated platelet aggregates [76,79–82] Activation of prothrombin by the complex (prothrombinase complex) is more than 105 times faster than that by free Xa [83] Zymogen activation modulators Therefore, physiological coagulation is essentially catalyzed by the prothrombinase complex Dual modulation of prothrombin activation by plactin Inoue et al [84] discovered a family of novel cyclic pentapeptides after screening microbial cultures for agents that enhanced cellular fibrinolytic activity in an incubation of U937 cells with plasma The cyclopentapeptides, designated plactins, consist of an aromatic (Phe or Tyr), a basic (d-Arg) and three bulky aliphatic amino acids (d-Val, Leu and d-Leu or d-allo-Ile) (see Fig for the structure of plactin D) The structure– activity relationships of 50 synthetic plactins demonstrate that a sterically restricted arrangement of four hydrophobic amino acids and a basic amino acid is essential for their activity [85] Plactin increases U937 cell-mediated fibrin degradation, which depends on the presence of plasma The profibrinolytic action of one of the promising compounds, plactin D, has been demonstrated in animal experiments [85] The action of plactin involves an increase in cellular u-PA activity [85] In this mechanism, the presence of plasma is an absolute requirement The plasma dependency is characterized in detail by Harada et al [86], because plasminogen alone cannot substitute for plasma, and the presence of a cofactor for the plactin action is suggested On cultured U937 cells, most of the u-PA molecules exist in the zymogen form, scuPA Upon treatment with plactin in the presence of plasma, scu-PA converts to the two-chain form Thus, plactin, in combination with a plasma cofactor, aids proteolytic activation of cellular scu-PA It is interesting that malformin A1 (Fig 1), which belongs to another family of cyclopentapeptides, has plasmadependent activity to promote cellular fibrinolytic activity [87] Although the structural features of malformin partially resemble that of plactin, the mechanisms involved in their actions are quite different The malformin action does not involve the increase in cellular u-PA activity [87] Using plactin-affinity gels, Harada et al [86] identified prothrombin as a candidate for a cofactor from plasma The actions of plactin and prothrombin that lead to the activation of scu-PA are explained as follows: (a) plactin binds to prothrombin, alters its conformational status and therefore modulates prothrombin activation by factor Xa; (b) the consequence of the modulation under the conditions of the assay for cellular fibrinolytic activity is the promotion of prothrombin activation; (c) the resulting thrombin can cleave scu-PA at Arg156–Phe157 (two residues FEBS Journal 277 (2010) 3675–3687 ª 2010 The Authors Journal compilation ª 2010 FEBS 3681 Zymogen activation modulators K Hasumi et al Prothrombin Plactin Xa DPP-I-like peptidase α-Thrombin S1 E K 156 R F K I S1 E K 156 157 R I K F S1 E 156 K R 159 I P P P scu-PA (inactive) Thrombin-cleaved tcu-PA derivative (inactive) tcu-PA (active) Fig Prothrombin-mediated pathway to cellular scu-PA activation promoted by plactin The scu-PA molecule is shown schematically as in Fig The amino acids involved in proteolytic cleavages are given in white circles DPP-I, dipeptidyl peptidase I proximal to the activation cleavage site, Lys158– Ile159) to form an inactive two-chain u-PA species; and (d) the tcu-PA derivative, in turn, is processed to active tcu-PA by dipeptidyl peptidase I-like activity of U937 cells, possibly through the removal of Phe157–Lys158 from the newly formed N-terminus (Fig 5) The plactin-modulation of prothrombin activation leads to different outcomes depending on the form of the catalyst factor Xa [86] Plactin inhibits prothrombin activation by factor Xa associated with acidic phospholipid membranes, especially by the prothrombinase complex, which accounts for the physiological coagulation reaction By contrast, plactin enhances prothrombin activation by membrane-free Xa, resulting in increased formation of a-thrombin The activation of prothrombin is conformationally regulated The specificity of prothrombinase for prothrombin is mediated by exosites, which are physically separated from the catalytic site It has been postulated that substrate recognition by prothrombinase involves a twostep mechanism with an initial docking of prothrombin to the exosites, followed by a conformational change to engage the Xa catalytic site [88] The plactin-mediated dual modulation of prothrombin activation may be related to the alteration of prothrombin conformation induced by the agent Modulator of the activation of plasma hyaluronan-binding protein Plasma hyaluronan-binding protein activation Plasma hyaluronan-binding protein (PHBP; alternatively designated factor VII activating protease) is a protease that is implicated in both the coagulation and 3682 fibrinolytic systems, because the enzyme catalyzes the activation of factor VII and scu-PA [89,90] It is suggested that PHBP plays a role in the regulation of inflammation [91], vascular function [92], neointima formation [93], liver fibrosis [94,95] and atherosclerosis [92,96] PHBP exists in plasma as a single-chain zymogen form (pro-PHBP) at a concentration of  170 nm Pro-PHBP consists of 537 amino acids which form the following domains: an N-terminal region, three EGF domains, a kringle domain and a protease domain (Fig 2) [97] The pro-PHBP activation occurs via cleavage at Arg290–Ile291 No physiologically relevant protease that can activate pro-PHBP has been found Alternatively, pro-PHBP can be activated autoproteolytically Negatively charged molecules such as heparin and RNA accelerate pro-PHBP autoactivation [98– 101], possibly by serving as a scaffold for the accumulation of pro-PHBP, whereas pro-PHBP activation is observed only after hepatic injury, partial hepatectomy [102] or inflammation [103] Thus, pro-PHBP activation may be a highly regulated process and a particular mechanism should be involved in pathophysiological pro-PHBP activation Polyamines: promotion of pro-PHBP autoactivation complex formation Yamamichi et al [104] searched for inflammation-associated factors that promote pro-PHBP activation and identified polyamines as potential candidates The polyamines, for example, putrescine, spermidine and spermine, are cationic small molecules that accumulate in cells undergoing rapid growth and play a role in the regulation of proliferation, differentiation and programmed cell death [105,106] Spermidine markedly enhances intermolecular association of pro-PHBP to form the ‘autoactivation complex’ [104] The importance of the complex formation is supported by the result that a ‘pro-PHBP decoy’, with its active site Ser486 replaced by Ala (S486A), efficiently inhibits the autoactivation The experiments aided by a series of domain-deletion mutants prepared based on the S486A mutant show that: (a) the mutant lacking the third EGF domain (DE3) cannot form the autoactivation complex; (b) heparin, which binds the third EGF domain, inhibits the complex formation; (c) N-terminal region binds to the mutant lacking N-terminal region (DN) and this binding is inhibited by heparin; and (d) spermidine binds to pro-PHBP but not to the DN mutant Thus, the N-terminal region participates in the formation of the pro-PHBP autoactivation complex, and this function is regulated by spermidine (Fig 6) [104] FEBS Journal 277 (2010) 3675–3687 ª 2010 The Authors Journal compilation ª 2010 FEBS K Hasumi et al Zymogen activation modulators Polyamine E1 P E2 E3 P Pro-PHBP K P Carminic acid on the physiological supply of the enzyme that activates the zymogen E2 E1 K E3 K E3 E1 E2 PHBP Autoactivation complex Fig A model of the modulation of pro-PHBP autoactivation by polyamine and carminic acid The pro-PHBP molecule is shown as in Fig The schematic conformation shown is speculative Carminic acid: specific inhibition of polyamine-mediated pro-PHBP autoactivation On the basis of the specific effects of polyamines, Nishimura et al screened natural sources for an inhibitor of spermidine-induced pro-PHBP activation [107] and identified several small molecules including carminic acid [104], an anthraquinone derived from the cochineal insect Carminic acid inhibits spermidinepromoted pro-PHBP autoactivation selectively, and does not affect the autoactivation in the absence of spermidine or that induced by negatively charged molecules such as heparin or RNA It also has no effect on the catalytic activity of the active form of PHBP This specific effect is due to the inhibition of autoactivation complex formation (Fig 6) Carminic acid may modulate the polyamine-dependent N-terminal region function, because the agent inhibits binding between the N-terminal region and DN only in combination with spermidine [104] The features of carminic acid, as well as of polyamines, conform to the idea of the zymogen modulator Conclusions and perspectives The activation of zymogens, particularly those in the coagulation and fibrinolytic systems, are regulated by fine mechanisms programmed in their molecules The small molecules described here act by utilizing or modulating such embedded mechanisms and not affect the catalytic activity of the mature enzymes These features discriminate zymogen modulators from inhibitors or activators that simply act on mature enzymes Zymogen activation is an allosteric process, and the zymogen modulators are allosteric effectors acting during zymogen activation Selective inhibitors or antagonists ⁄ agonists have been used as a pharmacologically powerful means to treat a variety of diseases Zymogen modulators will contribute to the development of novel classes of drugs, as their actions are an ideal ondemand system, which operates where 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