[Show abstract][Hide abstract] ABSTRACT: SMTP-7 is a small molecule that promotes the proteolytic activation of plasminogen by relaxing its conformation. SMTP-7 has excellent therapeutic activities against thrombotic stroke in several rodent models. The objective of this study was to elucidate detailed mechanism of the action of SMTP-7 in vitro. We report here that the action of SMTP-7 requires a cofactor with a long-chain alkyl or alkenyl group, and that the fifth kringle domain (kringle 5) of plasminogen is involved in the SMTP-7 action. In this study, we found that the SMTP-7 action to enhance plasminogen activation depended on the presence of a certain type of surfactant, and we screened biologically relevant molecules for their cofactor activity for the SMTP action. As a result, phospholipids, sphingolipids, and oleic acid were found to be active in assisting the SMTP-7 action. On the contrary, stearic acid and bile acids were inactive. Thus, a certain structural element, not only the surface-activating potential, is required for a compound to act as a cofactor for the SMTP-7 action. The plasminogen molecule consists of a PAN domain, five kringle domains, and a serine protease domain. The cofactor-dependent effects of SMTP-7 was observed with plasminogen species including kringle 5 such as intact plasminogen (Glu-plasminogen), des-PAN plasminogen (Lys-plasminogen), and des-[PAN - (kringles 1-4)] plasminogen (mini-plasminogen). However, SMTP-7 effect was not observed with the smallest plasminogen species des-[PAN - (kringles 1-4) and a half of kringle 5)] plasminogen (micro-plasminogen). Thus, kringle 5 is crucial for the action of SMTP-7.
Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis 06/2014; 25(4):316-321. · 1.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coccid-derived natural food colorants contain active ingredients that potentiate inhibition of tissue proteolysis mediated by activation of plasma hyaluronan-binding protein (PHBP). In the present study, we examined the effect of lac color (LC) and cochineal extract (CE), representative coccid-derived colorants containing laccaic acid and carminic acid as active ingredients, in an intracapsular invasion model of experimental thyroid cancers using rats. One week after initiation with N-bis(hydroxypropyl)nitrosamine, male F344/NSIc rats were fed a powdered diet containing 5.0% LC or 3.0% CE during promotion with 0.15% sulfadimethoxine (SDM) in the drinking water for 13 weeks. Capsular invasive carcinomas (CICs) and lung metastases were decreased by LC treatment and accompanied by transcript downregulation on angiogenesis and PHBP-related tissue proteolysis in CICs. In contrast, CE upregulated angiogenesis-related genes in CICs. PHBP was expressed in capsular macrophages and thyroid proliferative lesions with increased intensity in CICs, and LC decreased PHBP-expressing CICs. The size of CICs and their proliferation activity, however, were unchanged compared with those treated with SDM alone. Suppression of cancer by invasion by LC was more evident after an eight-week treatment, exhibiting a profound decrease in tenascin-C-positive early invasive foci and marked reductions in capsular inflammation and fibrosis. These results suggest that LC and CE exerted dissimilar effects on CIC development, the former suppressing the initial step of neoplastic cell invasion into the capsule by targeting PHBP activity of macrophages and neoplastic cells on tissue proteolysis involving inflammatory responses and angiogenesis, and the latter promoting angiogenesis of developed CICs at later stages.
Experimental Biology and Medicine 06/2012; 237(6):728-38. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plasma hyaluronan-binding protein (PHBP), an activator of factor VII and prourokinase, is a serine protease circulating as a single-chain proenzyme (pro-PHBP). Pro-PHBP converts to the active two-chain form through autoproteolysis, and effectors that modulate autoactivation can regulate PHBP-mediated processes. Here, we show that histone promotes pro-PHBP autoactivation in vivo. Histone bound to pro-PHBP and promoted intermolecular pro-PHBP binding. Histone-mediated pro-PHBP activation in plasma leads to the formations of bradykinin and PHBP-α(2)-antiplasmin complex as well as histone degradation. Pro-PHBP activation was observed in the circulation of mice after injection of histone or lipopolysaccharide, which induced septic response accompanying extracellular histone release. Our results suggest pathophysiological relevance of histone-dependent pro-PHBP activation in hyperinflammatory process.
Biochemical and Biophysical Research Communications 06/2011; 409(3):483-8. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plasma hyaluronan-binding protein (PHBP), a serine protease that can activate coagulation factor VII and prourokinase, circulates in a single-chain form (pro-PHBP) and autoproteolytically converts to an active two-chain form with the aid of an effector such as spermidine and heparin. It has been postulated that PHBP plays roles in regulating inflammation, vascular function, fibrosis and atherosclerosis. From the comprehensive screening of natural sources for inhibitors of spermidine-induced pro-PHBP autoactivation, we identified several compounds with a polyphenol feature. Of these inhibitors, tannic acid (IC(50)=0.020 µM), delphinidin (IC(50)=0.079 µM), hamamelitannin (IC(50)=0.19 µM), (-)-epicatechin gallate (IC(50)=0.24 µM), and 3,5-di-O-caffeoylquinic acid (IC(50)=1.0 µM) were potent and selective, and did not inhibit heparin-induced pro-PHBP autoactivation and the active form of PHBP at concentrations 100 times higher than the respective IC(50) values. From evaluation of the activities of related compounds, it has been suggested that a compound with multiple aromatic rings with plural phenolic hydroxyl substituents exhibits potent activity. The inhibitory actions of delphinidin, hamamelitannin, (-)-epicatechin gallate and 3,5-di-O-caffeoylquinic acid were attenuated by catechol, a minimum polyphenol unit. Thus, it is likely that pro-PHBP binds these potent inhibitors through its site(s) that recognize a catechol-like structure. Our results would facilitate understanding of the molecular mechanism of pro-PHBP autoactivation and rational design of a compound for suppressing unregulated pro-PHBP activation.
[Show abstract][Hide abstract] ABSTRACT: Plactin, a family of cyclopentapeptides of fungal origin, enhances fibrinolytic activity by promoting of single-chain urokinase-type plasminogen activator (scu-PA) activation on the cell surface. For this activity, factor(s) in the blood plasma is absolutely required. In the previous studies, we identified prothrombin as a plasma cofactor involved in this mechanism, while the presence of another independent cofactor was suggested. The objective of this study was to identify the second cofactor and investigate the mechanism involved. Using plactin-affinity and ion-exchange chromatographies, we purified plasma hyaluronan-binding protein (PHBP) ~4,000-fold from human plasma as an independent plactin cofactor. PHBP, at ~10nM, was effective in plactin-dependent promotion of scu-PA activation by U937 cells. PHBP is a serine protease that is produced as a single-chain proenzyme (pro-PHBP) and autoproteolytically converted to an active two-chain form. Pro-PHBP was comparable to PHBP in activity to promote plactin-dependent scu-PA activation by U937 cells. Plactin enhanced both cellular binding and autoproteolytic activation of pro-PHBP. The two activities were obtained with a plactin concentration at ~30μM, which resulted in a significant increase in intrinsic fluorescence and self association of pro-PHBP. Thus, it is suggested that such changes account for both enhanced cellular binding and autoactivation of pro-PHBP, resulting in an enhancement of scu-PA activation.
Thrombosis Research 11/2010; 126(5):406-13. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Plasma hyaluronan-binding protein (PHBP), a protease implicated in extracellular proteolysis, consists of multiple domains: an N-terminal region (NTR), three epidermal growth factor (EGF)-like domains, a kringle domain, and a protease domain. PHBP circulates as a single-chain proenzyme (pro-PHBP), which is converted to an active, two-chain form through autoproteolysis.
To understand the mechanism of autoactivation. Here, we report that polyamine induces the formation of pro-PHBP autoactivation complex, in which an intermolecular interaction between NTR and the third EGF-like domain (E3) plays a role.
Using a series of pro-PHBP mutants that partially lack functional domains, polyamine-induced pro-PHBP autoactivation was investigated in terms of enzyme activity, protein interaction, and inhibition by carminic acid, an anthraquinone compound identified in this study.
Polyamine enhanced intermolecular binding of pro-PHBP, but not of mutant pro-PHBP that partially lacked NTR (DeltaN). Carminic acid inhibited intermolecular pro-PHBP binding and specifically abolished polyamine-induced autoactivation. NTR bound to pro-PHBP and DeltaN, but its binding was minimal to a mutant that lacked E3. The NTR-DeltaN binding was inhibited by a combination of polyamine and carminic acid, but each compound alone was ineffective.
We infer from the data that (i) polyamine modulates intramolecular NTR-E3 interaction to allow intermolecular binding between NTR and E3 in another pro-PHBP molecule to form an autoactivation complex, and (ii) carminic acid inhibits polyamine-modulated intermolecular NTR-E3 binding. Polyamine concentrations are higher in cells and tissues with inflammation and malignancy. Polyamine leakage from legions through cell death or tissue injury may account for physiologically relevant pro-PHBP activation.
Journal of Thrombosis and Haemostasis 10/2009; 8(3):559-66. · 6.08 Impact Factor