Synergistic suppression of a disintegrin acurhagin-C in combination with AZD4547 and reparixin on terminating development for human osteosarcoma MG-63 cell.

Current therapies available for the treatment of human osteosarcoma, an aggressive bone tumor, are insufficient. To examine an alternative approach of integrin-based anti-osteosacoma strategy, acurhagin-C, a Glu-Cys-Asp (ECD)-disintegrin, was isolated and evaluated for its application in combination with two potent inhibitors of basic fibroblast growth factor (bFGF) and interleukin-8 (IL-8). The investigation of human osteosarcoma MG-63 cells pre-incubated with a FGF receptor-1 (FGFR-1) blocker AZD4547, a CXC-chemokine receptor-1/-2 (CXCR1/2) antagonist reparixin, and acurhagin-C via two given modes of separation and combination was executed. Detected by flow cytometry, integrins-α2/-α5/-αv/-ß1, FGFR-1, CXCR1 and CXCR2 constitutively express on the resting membrane. However, bFGF/IL-8-activated MG-63 cells only statistically enhanced the surface exposure of integrins-α5/-ß1, FGFR-1 and CXCR2. In activated MG-63 cells, acurhagin-C targeting integrin-α5 not only might potentiate the inhibitory effect of AZD4547 and reparixin on the surface expression of integrin-α5, FGFR-1 and CXCR2, but also acurhagin-C used alone remained effectively to diminish the surface exposure of those targeted receptors. Hence, a complicated crosstalk mechanism should be involved in the membrane interactions. Furthermore, co-administration of acurhagin-C with AZD4547 and reparixin also showed to have the synergistic suppression toward cell proliferation and the gene expression of matrix metalloproteinase-2. Also, the administration of three-in-one mode could nearly abrogate the cellular attachment onto collagen-IV- and fibronectin-coated wells, as well as penetration into Matrigel-barrier. These data supported an ECD-disintegrin acurhagin-C targeting integrin-α5 upon combined used with AZD4547 and reparixin may become a promising therapeutic approach for attenuating osteosarcoma development.

Convulxin, a C-type lectin-like protein, inhibits HCASMCs functions via WAD-motif/integrin-αv interaction and NF-κB-independent gene suppression of GRO and IL-8.

Convulxin (CVX), a C-type lectin-like protein (CLPs), is a potent platelet aggregation inducer. To evaluate its potential applications in angiogenic diseases, the multimeric CVX were further explored on its mode of actions toward human coronary artery smooth muscle cells (HCASMCs). The N-terminus of ß-chain of CVX (CVX-ß) contains a putative disintegrin-like domain with a conserved motif upon the sequence comparison with other CLPs. Importantly, native CVX had no cytotoxic activity as examined by electrophoretic pattern. A Trp-Ala-Asp (WAD)-containing octapeptide, MTWADAEK, was thereafter synthesized and analyzed in functional assays. In the case of specific integrin antagonists as positive controls, the anti-angiogenic effects of CVX on HCASMCs were investigated by series of functional analyses. CVX showed to exhibit multiple inhibitory activities toward HCASMCs proliferation, adhesion and invasion with a dose- and integrin αvß3-dependent fashion. However, the WAD-octapeptide exerting a minor potency could also work as an active peptidomimetic. In addition, flow cytometric analysis demonstrated both the intact CVX and synthetic peptide can specifically interact with integrin-αv on HCASMCs and CVX was shown to have a down-regulatory effect on the gene expression of CXC-chemokines, such as growth-related oncogene and interleukin-8. According to nuclear factor-κB (NF-κB) p65 translocation assay and Western blotting analysis, the NF-κB activation was not involved in the signaling events of CVX-induced gene expression. In conclusion, CVX may act as a disintegrin-like protein via the interactions of WAD-motif in CVX-ß with integrin-αv on HCASMCs and it also is a gene suppressor with the ability to diminish the expression of two CXC-chemokines in a NF-κB-independent manner. Indeed, more extensive investigations are needed and might create a new avenue for the development of a novel angiostatic agent.

A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbß3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbß3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbß3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to integrin αIIbß3-dependent aggregation in human platelets.

Atopic diathesis in patients with Kawasaki disease.

OBJECTIVE: To determine the association between Kawasaki disease (KD) and atopic diathesis (atopic dermatitis [AD], allergic rhinitis, and asthma) in children younger than 5 years of age. STUDY DESIGN: In this nationwide study, we aimed to analyze the association and temporal relationship between KD and atopic diathesis. Data were obtained from the National Health Insurance Research Database of Taiwan from 1997 to 2010. In total, 200 patients with KD younger than 5 years of age and 800 age- and sex-matched control subjects were enrolled. RESULTS: In the whole study population, an increased risk of any concomitant atopic diseases was observed in patients with KD (OR 1.61, 95% CI 1.15-2.26). The risk of AD was increased in male patients between 1 and 5 years of age (OR 3.02, 95% CI 1.22-7.50). More than 60% of the patients developed atopic diseases after the diagnosis of KD. CONCLUSION: There appears to be an association between KD and risk of AD. Most of the atopic diseases occurred after the episode of KD.

Inhibition of integrins αv/α5-dependent functions in melanoma cells by an ECD-disintegrin acurhagin-C.

Acurhagin-C, a Glu-Cys-Asp (ECD)-disintegrin from Agkistrodon acutus venom, has been reported as an endothelial apoptosis inducer, previously. Here we further evaluate its potential applications in cancer therapy. In vitro assays indicated that acurhagin-C not only may influence the cell viability at higher concentration, but also can potently and dose-dependently decrease cell proliferation in murine B16-F10 melanoma. Otherwise, it also had a dose-dependent inhibition on B16-F10 cell adhesion to extracellular matrices, collagen VI, gelatin B and fibronectin, as well as disturbed transendothelial migration of B16-F10 cell. Morphological study found that acurhagin-C dramatically affected B16-F10 cell adhesion to immobilized fibronectin, leading to the formation of multicellular aggregates with rounded shape. Detected by flow cytometry, acurhagin-C was able to induce B16-F10 cell apoptosis and alter cell cycle distribution through its interactions with integrins αv/α5, and thereafter initiation the apoptotic pathways of caspase-8/-9. Furthermore, acurhagin-C could synergistically enhance the anti-proliferative activity of methotrexate in B16-F10 cells and human melanoma SK-MEL-1 cells, without diminishing the growth of human epidermal melanocytes. Taken together, acurhagin-C proved to be a potent inhibitor of integrin-based functions in melanoma cells by activating the complex apoptotic pathways.

Acurhagin-C, an ECD disintegrin, inhibits integrin alphavbeta3-mediated human endothelial cell functions by inducing apoptosis via caspase-3 activation.

BACKGROUND AND PURPOSE: Acurhagin, a member of versatile metalloproteinase disintegrins from Agkistrodon acutus venom, has been identified as a platelet aggregation inhibitor, previously. Here, acurhagin-C, the C-terminal Glu-Cys-Asp (ECD)-containing fragment of acurhagin, was evaluated for its biological activities and potential applications in anti-angiogenic therapy. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were treated with acurhagin-C to assay effects on viability, apoptosis, adhesion, migration, invasion, proliferation and angiogenesis. The recognition site and signalling involved for the interactions of acurhagin-C with HUVEC were determined using flow cytometric, electrophoresis and immunoblotting analyses. KEY RESULTS: Acurhagin-C decreased viability and induced apoptosis in HUVEC. It also dose-dependently inhibited HUVEC adhesion to immobilized extracellular matrices fibronectin, collagen I and vitronectin with respective IC(50) values of approximately 0.6, 0.3 and 0.1 microM. Acurhagin-C prevented migration and invasion of HUVEC through vitronectin- and Matrigel-coated barriers respectively. Furthermore, acurhagin-C attenuated fibroblast growth factor-2-primed angiogenesis both in vitro and in vivo, and specifically blocked the binding of anti-alphavbeta3 monoclonal antibody 23C6 to HUVEC in an ECD-dependent manner. However, purified alphavbeta3 also dose-dependently bound to immobilized acurhagin and acurhagin-C with a saturable pattern. Interference with integrin alphavbeta3-mediated functions and promotion of caspase-3 activation by acurhagin-C affected morphology of HUVEC and induced apoptosis. CONCLUSIONS AND IMPLICATIONS: Acurhagin-C elicited endothelial anoikis via disruption of alphavbeta3/focal adhesion kinase/phosphatidylinositol 3-kinase/Akt survival cascade and subsequent initiation of the procaspase-3 apoptotic signalling pathway.

Agglucetin, a tetrameric C-type lectin-like venom protein, regulates endothelial cell survival and promotes angiogenesis by activating integrin alphavbeta3 signaling.

Agglucetin, a platelet glycoprotein (GP)Ib binding protein from Formosan Agkistrodon acutus (A. acutus) venom, could sustain human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC adhering to immobilized agglucetin showed extensive spreading, which was strongly abrogated by integrin antagonists 7E3 and triflavin. Flow cytometric analyses confirmed the expression of GPIb complex on HUVEC is absent and fluorescein isothiocyanate (FITC)-agglucetin binds to HUVEC in a dose-dependent and saturable manner. Furthermore, native agglucetin specifically and dose-dependently inhibited the binding of FITC-23C6, an anti-alphavbeta3 monoclonal antibody (mAb), but not antibodies against alpha2 and alpha5, toward HUVEC and purified alphavbeta3 also bound to immobilized agglucetin-beta in a dose-dependent manner. Moreover, agglucetin exhibited a pro-angiogenic effect in vitro, as well as the focal adhesion kinase (FAK)-associated signaling molecules responsible for HUVEC activation were initiated by agglucetin. In conclusion, agglucetin, acting as a survival factor, promotes endothelial adhesion and angiogenesis by triggering alphavbeta3 signaling through FAK/phosphatidylinositol 3-kinase (PI3K)/Akt pathway.

Purification and functional characterization of AAV1, a novel P-III metalloproteinase, from Formosan Agkistrodon acutus venom.

AAV1, an alkaline glycoprotein (GP), was purified from Agkistrodon acutus venom by two chromatographic steps on successive DEAE-Sephadex A-50 and Superdex 75 FPLC columns. AAV1 on SDS-PAGE under non-reducing conditions migrated as a monomeric and a polymeric forms with apparent molecular mass of 57 and 180 kDa, respectively. Upon reduction, it appeared as a single broad band with a mass of 50.3 kDa corresponding to the size of a typical P-III metalloproteinase acurhagin. The N-terminal sequence of an autoproteolytical 30 kDa-fragment of AAV1 showed a high homology to that of venom proteins with Metalloproteinase, Disintegrin-like, and Cysteine-rich (MDC) domains. Although it was devoid of cleaving activity toward gelatin, fibronectin and prothrombin, AAV1 preferentially digested the Aalpha chain of fibrinogen and followed by the Bbeta chain, leading to the inhibition of fibrinogen-induced platelet aggregation in elastase-treated human platelets. However, the proteolytic activity of AAV1 was completely inactivated by the chelating agent but not serine proteinase inhibitor. Furthermore, AAV1 could concentration-dependently inhibit platelet aggregation and suppress tyrosine phosphorylation of intracellular proteins in collagen- and convulxin-stimulated platelets, respectively. The interaction of MDC domains in AAV1 molecule with platelet GPVI was responsible for the inhibitory effect of AAV1 on collagen- and convulxin-induced platelet aggregation. Taken together, these pieces of evidence suggest that AAV1 from Formosan viper venom belongs to a new member of high-molecular mass metalloproteinase family and functions as a GPVI antagonist.

Primary structure and antiplatelet mechanism of a snake venom metalloproteinase, acurhagin, from Agkistrodon acutus venom.

Acurhagin has been characterized as a P-III hemorrhagic metalloproteinase. We herein report the complete sequence of acurhagin by molecular cloning. Analysis of the cDNA-predicted amino acid sequence encoding acurhagin precursor revealed that this mosaic Asn-linked glycoprotein possesses a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domains (189/205/102/114 residues), with an overall 87% identity to that of jararhagin, an integrin alpha2beta1-cleaving metalloproteinase. Acurhagin has a Ser-Glu-Cys-Asp sequence in the disintegrin-like domain instead of the typical Arg-Gly-Asp motif. In contrast to inhibiting fibrinogen-integrin alphaIIbbeta3 interaction by disintegrins, acurhagin selectively showed a dose-dependent inhibition on platelet aggregation induced by collagen, and suppression on tyrosine phosphorylation of several signaling proteins in convulxin-stimulated platelets. Although the immobilized acurhagin was shown to bind platelet GPVI and collagen in a primary structure- and steric conformation-dependent manner, respectively, the mechanism of acurhagin under short incubation is mainly through its binding to GPVI and collagen, instead of binding to alpha2beta1, or cleaving platelet membrane glycoproteins. Moreover, the molecular conformation maintained by divalent cations is required for the proteolytic activity of acurhagin toward extracellular matrix fibronectin. Taken together, these results suggest that all the three domains in mature acurhagin may cooperatively contribute to its biological function.

A novel P-I class metalloproteinase with broad substrate-cleaving activity, agkislysin, from Agkistrodon acutus venom.

The venom of Viperdae is a rich source of metalloproteinases, which have potential clinical applications for lowering plasma fibrinogen or dissolving thrombi. Recently, we purified a novel proteinase from Formosan Agkistrodon acutus venom using DEAE-Sephadex A-50 and Mono-Q HR 5/5 column chromatography. The purified getatinolytic component, agkislysin, is a 22kDa-monomeric protein without Asn-linked sugar. Functional characterization showed that agkislysin possessed both fibronectin- and type IV collagen-cleaving activities. In addition, agkislysin preferentially cleaved the Aalpha chain of fibrinogen, followed by the Bbeta chain, while the gamma chain was finally affected. Furthermore, agkislysin was also capable of cleaving prothrombin into various fragments, as well as suppressing ristocetin-induced platelet aggregation by hydrolyzing von Willebrand factor. However, the proteolytic activity of agkislysin was slightly enhanced by divalent metal ions and completely inhibited by chelating agents, but not serine proteinase inhibitor. These findings suggest that agkislysin is a P-I class metalloproteinase with unique biological properties.

A tetrameric glycoprotein Ib-binding protein, agglucetin, from Formosan pit viper: structure and interaction with human platelets.

Agglucetin, a tetrameric agglutination inducer from the Formosan pit viper, has been identified as a platelet membrane glycoprotein (GP) Ib agonist and directly agglutinated fixed-platelets in the absence of von Willebrand factor (vWf). Here, we resolved the complete cDNA sequences of agglucetin subunits (alpha(1), alpha(2), beta(1) and beta(2)) by molecular cloning. Each cloned cDNA encoding the leader peptide (23 residues) and the mature subunit (131/135/123/126 residues) shares a high degree of homology to each other and the C-type lectin-like GP Ib-binding proteins (BPs). Furthermore, agglucetin specifically caused platelet agglutination and surface exposure of integrin alpha(IIb)beta(3) with a GPIb-dependent manner in washed platelets, based on the observation that the enhanced expression of functional alpha(IIb)beta(3) was suppressed by a GPIb-cleaving metalloproteinase, crotalin. Pretreating platelets with staurosporine or BAPTA-AM also completely blocked the exposure of functional alpha(IIb)beta(3), suggesting that the activation of protein kinase C and intracellular calcium mobilization are involved in the GPIb-dependent signaling. In human platelet-rich plasma (PRP), agglucetin elicited sequential biphasic responses of platelet agglutination and aggregation in a GPIb- and alpha(IIb)beta(3)-dependent manner, respectively, implying that other cofactors may amplify platelet activation to trigger aggregation.

Purification and characterization of a novel metalloproteinase, acurhagin, from Agkistrodon acutus venom.

Acurhagin, a high-molecular mass hemorrhagic metalloproteinase, was purified from the crude venom of Agkistrodon acutus using anion-exchange and hydrophobic interaction chromatography. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other high-molecular mass metalloproteinases from snake venoms. It preferentially cleaved Aalpa chain of fibrinogen, followed by Bbeta chain, while gamma chains was minimally affected. Monitored by RP-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa fragment at 37 degrees C. The N-terminal sequence of the 30 kDa fragment of acurhagin showed a high homology to those proteins consisting of disintegrin-like and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of platelet-rich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWF, respectively.