Biomedical subjects

T F Huang

Publications and source records attributed to T F Huang.

At least 19 recordsLinked to original sources

PubMed2001

Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells.

Apoptosis, a programmed, physiological mode of cell death, is important in tissue homoeostasis. Here we report that a new metalloproteinase, graminelysin I, purified from Trimeresurus gramineus venom, induced apoptosis of human endothelial cells as examined by electrophoresis and flow cytometry. Graminelysin I contains only a metalloproteinase domain. It is a single-chain proteinase with a molecular mass of 27020 Da. cDNA sequence analysis revealed that the disintegrin-like and cysteine-rich domains of the putative precursor protein of graminelysin I are likely to be processed post-translationally, producing the proteinase domain (graminelysin I). Graminelysin I cleaved the alpha chain of fibrinogen preferentially and cleaved the beta chain either on longer incubation or at higher concentration. Graminelysin I inhibited the adhesion of human umbilical-vein endothelial cells (HUVECs) to immobilized fibrinogen and induced HUVECs detachment in a dose-dependent manner. These effects on HUVECs were abolished when graminelysin I was pretreated with EDTA. However, graminelysin I did not inhibit the adhesion of HUVECs to immobilized collagen. HUVECs were susceptible to death after treatment with graminelysin I when they were cultured on immobilized fibrinogen. In contrast, HUVECs were rather resistant to treatment with graminelysin I if they were cultured on immobilized collagen. Furthermore, graminelysin I induced apoptosis of HUVECs in a dose-dependent manner. Similarly, its apoptosis-inducing activity was blocked if it was treated with EDTA. These results suggest that the catalytic activity of graminelysin I on matrix proteins contributes to its apoptosis-inducing activity.

Amino Acid Sequence↗

PubMed2001

Aggretin, a C-type lectin protein, induces platelet aggregation via integrin alpha(2)beta(1) and GPIb in a phosphatidylinositol 3-kinase independent pathway.

Aggretin purified from Calloselasma rhodostoma venom was previously identified as alpha(2)beta(1) agonist in triggering platelet aggregation, and exists as a heterodimer sharing a great homologous sequence to GPIb binding proteins. We show here that binding to GPIb is also required in aggregation-inducing activity of aggretin. A2-IIE10, an anti-integrin alpha(2) monoclonal antibody, delayed platelet aggregation while agkistin, a GPIb antagonist, only slightly inhibited platelet aggregation caused by aggretin. However, the aggretin-induced platelet aggregation was completely abolished by a combination of A2-IIE10 and agkistin. Either A2-IIE10 or agkistin significantly inhibited the binding of FITC-aggretin toward fixed platelets. Aggretin and collagen induced a similar signal transduction in platelets involving a time-dependent tyrosine phosphorylation of p125(FAK) and PLCgamma2, but aggretin caused a much-delayed tyrosine-phosphorylation of PI 3-kinase compared with collagen. LY294002, a PI 3-kinase inhibitor, showed a significant inhibitory effect on collagen, but not aggretin-stimulated platelet aggregation. These findings indicate aggretin induces platelet aggregation via binding of alpha(2)beta(1) and GPIb, causing phosphorylation of p125(FAK) and PLCgamma2 leading to platelet activation without the involvement of PI 3-kinase activation.

Antibodies, Monoclonal↗

PubMed2001

The role of high-resolution ultrasonography in management of calcific tendonitis of the rotator cuff.

This article predicts the possibility of resorption of the calcific plaques in the shoulder using high-resolution ultrasonography (HRUS) and color Doppler ultrasound (CDUS), and evaluates the therapeutic effect of US-guided fine-needle multiple punctures of the calcific plaque. A total of 100 patients with calcific tendenosis were divided into 3 groups: In group 1, patients having chronic shoulder pain received conservative treatment; in group 2, patients having acute exacerbation of shoulder pain also received conservative treatment; and group 3 patients received US-guided fine-needle multiple punctures or aspiration. In CDUS, all images were classified as grade 0 (no color flow signals), grade 1 (weak spotty color flow signal), grade 2 (few rod-like color flow signals), grade 3 (many rod-like or linear color flow signals). In the follow-up study, marked improvement of patients' clinical condition with more than 50% size reduction of calcific plaque was defined as an effective treatment. There was no significant difference between group 1 and group 3 (p = 0.558) in CDUS, but there was a significant difference between group 1 and group 2 (p = 0.000), and group 2 and group 3 (p = 0.000) on the basis of classification of grade < 1 and grade > or = 1. There was also significant difference in the follow-up result of effective management between group 1 and group 3 (p = 0.000), and group 1 and group 2 (p = 0.000). In conclusion, HRUS with CDUS proved to be a good modality in evaluating the possibility of resorption of shoulder calcification and, if CDUS > or = grade 1 in calcific tendonitis, we highly recommend conservative treatment with regular follow-up. On the other hand, if CDUS < grade 1, fine-needle repeated puncture could be considered as an effective alternative treatment.

Adult↗

PubMed2001

Pharmacological characterization and antithrombotic effect of agkistin, a platelet glycoprotein Ib antagonist.

1. Agkistin, purified from the snake venom of Formosan Agkistrodon acutus, belongs to the family of C-type lectin GPIb binding proteins. It is a heterodimeric molecule, consisting of alpha- (16.5 kDa) and beta- (15.5 kDa) subunits with a molecular mass of 32,512 Daltons examined by SDS - PAGE and mass spectrometry. 2. In vitro, agkistin concentration-dependently inhibited ristocetin-induced human platelet agglutination and aggregation in the presence of vWF. It also inhibited TXA2 formation and prolonged the latent period in triggering aggregation by a low concentration of thrombin (0.03 u x ml(-1)). 3. 125I-agkistin specifically bound to unactivated human platelets in a saturable manner with a KD value of 223+/-10.6 nM. This binding reaction was rapid and reversible. Monoclonal antibodies, AP1 and 6D1 raised against platelet GPIb, almost completely blocked 125I-agkistin binding to platelets. However, monoclonal antibody 7E3 raised against GPIIb/IIIa complex, trigramin, a GPIIb/IIIa antagonist, ADP and EDTA did not affect 125I-agkistin binding reaction. 4. Agkistin (250 microg x kg(-1)) significantly prolonged the bleeding time and induced transient thrombocytopenia of mice when given intravenously. Furthermore, it markedly inhibited platelet plug formation in irradiated mesenteric venules of fluorescein-treated mice in vivo. 5. In conclusion, agkistin inhibits ristocetin induced platelet aggregation mainly through its specific binding to platelet GPIb, thereby blocking the interaction between GPIb and vWF. In addition, agkistin exhibits antithrombotic activity in vivo.

Amino Acid Sequence↗

PubMed2001

Rhodostomin, a snake venom disintegrin, inhibits angiogenesis elicited by basic fibroblast growth factor and suppresses tumor growth by a selective alpha(v)beta(3) blockade of endothelial cells.

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, although angiogenic factor and integrin-extracellular matrix interaction modulate this process. We report here that a snake venom-derived disintegrin, rhodostomin, inhibited distinct steps in angiogenesis elicited by basic fibroblast growth factor (bFGF), and also suppressed in vivo murine melanoma tumor growth. Rhodostomin dose-dependently inhibited bFGF-induced human umbilical vein endothelial cell (HUVEC) proliferation as examined by cell number count, metabolic activity, and BrdU incorporation assays with submicromolar IC(50) values. However, it apparently did not affect the viability of murine B16F10 melanoma cells, even up to 50 microM. Rhodostomin also inhibited HUVEC migration and invasion evoked by bFGF, and tube formation of bFGF-treated HUVECs in Matrigel. Moreover, rhodostomin selectively inhibited bFGF-, but not vascular endothelial growth factor-associated angiogenesis in the chick chorioallantoic membrane model. Furthermore, rhodostomin blocked both bFGF- and B16F10-induced neovascularization in murine Matrigel plug model and suppressed the growth of subcutaneously inoculated B16F10 solid tumor, leading to a prolonged survival of the rhodostomin-treated C57BL/6 mice. The antiangiogenic effect of rhodostomin on bFGF-treated HUVECs is related to the integrin alpha(v)beta(3) blockade, as evidenced by its selective inhibition on the binding of 7E3, a monoclonal antibody (mAb) raised against alpha(v)beta(3,) but not that of P1F6, an alpha(v)beta(5) mAb toward both naive and bFGF-primed HUVECs. Moreover, 7E3 specifically blocked fluorescein isothiocyanate-conjugated rhodostomin binding to HUVEC, whereas P1F6 and anti-integrin alpha(2), alpha(3), alpha(4), or alpha(5) mAbs did not.

Angiogenesis Inhibitors↗

PubMed2001

Modulation of protein kinase A activation by fibronectin matrix proteins at developing neuromuscular synapses in Xenopus laevis cell cultures.

Extracellular matrix proteins, such as fibronectin, laminin, and collagen, have been implicated in a wide variety of cellular properties, which include cell adhesion, migration, differentiation, and proliferation. In this study, we investigated the modulation of protein kinase A (PKA) activity by matrix proteins at developing motoneurons. The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus laevis embryos. Spontaneous synaptic currents (SSC) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (V(h) = -60 to approximately -65 mV). Bath application of agents, which directly or indirectly activate PKA, such as forskolin (20 microM), dibutyryl cAMP (DBcAMP) (1 mM), isoproterenol (10 microM), or albuterol (10 microM), significantly increased SSC frequency in cultures grown on fibronectin (FN)-coated substratum, but not on laminin- or collagen-coated glasses. The evoked synaptic currents increased in response to forskolin in neurons grown on FN substratum. Triflavin, an Arg-Gly-Asp-dependent disintegrin, inhibited potentiating action of isoproterenol in neurons grown on FN substratum, suggesting that integrin is involved in the potentiation of the PKA pathway in the regulation of acetylcholine (ACh) release. There is collaboration of neurotrophic factors and the FN matrix in regulating synaptic transmission in response to DBcAMP. Chronic treatment with neurotrophic factors, such as ciliary neurotrophic factor (150 ng/ml), glial cell line-derived neurotrophic factor (30 ng/ml), or neurotrophin-3 (50 ng/ml), enhanced the SSC-increasing action of DBcAMP in neurons grown on FN-coated glasses. These results suggest that the FN matrix potentiates synaptic transmission in response to PKA activation. Neurotrophic factors may collaborate with FN to regulate spontaneous ACh secretion at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular synapses.

Acetylcholine↗

PubMed2001

A novel tetrameric venom protein, agglucetin from Agkistrodon acutus, acts as a glycoprotein Ib agonist.

A novel platelet agglutination inducer, agglucetin, was purified from the Formosan Agkistrodon acutus snake venom. It migrated as a single band with an apparent molecular mass of 58.8 kDa and two distinct bands of 16.2/14.5 kDa under non-reducing and reducing conditions by SDS-PAGE, respectively. Further confirmed by FPLC, electrospray ionization mass spectrometry and 2D-PAGE, native agglucetin exists as a tetramer composed of disulfide-linked alpha1, alpha2, beta1 and beta2 subunits. Partial N-terminal sequence of agglucetin subunit showed a high degree of homology to those of C-type lectin-like glycoprotein (GP) Ib binding proteins. Functional studies showed that agglucetin. in the absence of von Willebrand factor (vWF), dose-dependently induced platelet agglutination and caused a negligible elevation of intracellular Ca+2 mobilization and thromboxane B, formation in human platelet suspensions. Anti-GP Ib monoclonal antibodies (mAbs), AP1 or LJ-Ib1, specifically inhibited agglucetin-induced platelet agglutination in a dose-dependent manner. However, EDTA, arietin (a long chain RGD-containing disintegrin), 7E3 (an anti-GP IIb/IIIa mAb), heparin, hirudin, PGE1, or indomethacin exhibited no inhibitory effect on agglucetin-induced platelet agglutination. Furthermore, flow cytometric analysis revealed that FITC-agglucetin dose-dependently bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by anti-GP Ib mAb. It is concluded that agglucetin, acts specifically on an epitope of platelet membrane GP Ib overlapping with that of API, causing platelet agglutination in a Ca+2- and GP IIb/IIIa-independent manner.

Agkistrodon↗

PubMed2001

Crotalin, a vWF and GP Ib cleaving metalloproteinase from venom of Crotalus atrox.

Binding of von Willebrand factor (vWF) to a variety of extracellular matrix (ECM) components and to platelet glycoprotein (GP) Ib-IX-V complex is important in mediating platelet adhesion and aggregation in the early stage of hemostasis. We previously purified a potent antithrombotic protein, named crotalin, functionally acting as a GP lb antagonist (1). In this study, we further characterized crotalin as a P-I metalloproteinase with a molecular mass of 25 kDa as determined by gel filtration and two-dimensional SDS-PAGE. Crotalin is a vWF binding and cleaving metalloproteinase. In addition, crotalin cleaved platelet GP lb as judged by flow cytometry and Western blotting. The multiple effects of crotalin on vWF and platelet GP lb antagonized ristocetin-, but not collagen and thrombin-induced platelet aggregation, suggesting that its effect is specific. We also found that crotalin autoproteolytically degraded to approximately 14 and approximately 10 kDa fragments in the presence of SDS. Interestingly, both degradation fragments, intact and reduced crotalin were able to bind vWF, suggesting the binding of crotalin to vWF is conformation-independent. In conclusion, the results presented further explain the potent antithrombotic effect of crotalin in vivo. In addition, the multiple effects of crotalin may be used as a tool to determine the binding motifs that are responsible for the vWF-ECMs or vWF-GP lb interaction.

Amino Acid Sequence↗

PubMed2000

Agkistin, a snake venom-derived glycoprotein Ib antagonist, disrupts von Willebrand factor-endothelial cell interaction and inhibits angiogenesis.

Glycoprotein (GP) Ib, an adhesion receptor expressed on both platelets and endothelial cells, mediates the binding of von Willebrand factor (vWF). Platelet GPIb plays an important role in platelet adhesion and activation, whereas the interaction of vWF and endothelial GPIb is not fully understood. We report here that agkistin, a snake venom protein, selectively blocks the interaction of vWF with human endothelial GPIb and inhibits angiogenesis in vivo. Agkistin specifically blocked human umbilical vein endothelial cell (HUVEC) adhesion to immobilized vWF in a concentration-dependent manner. Fluorescein isothiocyanate (FITC)-conjugated agkistin bound to HUVECs in a saturable manner. AP1, a monoclonal antibody (mAb) raised against GPIb, specifically inhibited the binding of FITC-conjugated agkistin to HUVECs in a dose-dependent manner, but other anti-integrin mAbs raised against alpha(v)beta(3), alpha(2)beta(1), and alpha(5)beta(1) did not affect this binding reaction. However, neither agkistin (2 microgram/ml) nor AP1 (40 microgram/ml) apparently reduced HUVEC viability. Both agkistin and AP1 exhibited a profound anti-angiogenic effect in vivo when assayed by using the 10-day-old embryo chick chorioallantoic membrane model. These results suggest endothelial GPIb plays a role in spontaneous angiogenesis in vivo, and the anti-angiogenic effect of agkistin may be because of disruption of the interaction of endogenous vWF with endothelial GPIb.

Angiogenesis Inhibitors↗

PubMed2000

The effects of temperature and oxygen content on the PCDD/PCDFs formation in MSW fly ash.

In this study, the effects of the temperature, oxygen content in the gas stream and carbon content in ash particles on PCDD/Fs formation on the fly ash surface were investigated. The optimum temperatures for dioxin formation were found at 350 degrees C for boiler ash, 300 degrees C for cyclone ash and 250 degrees C for ESP ash, respectively. Preliminary results indicate that the optimum temperature will decrease as the particle size decreases. When the O2 concentration is varied between 0% and 100%, the optimum oxygen content for PCDD/Fs formation is found to be at 7.5% for cyclone ash, and the PCDD/PCDF ratio increases with the increase of oxygen content. Dioxin formation is observed even for the gas containing no oxygen passed through the fly ash. Hence, other reacted routes that do not need O2 for dioxin formation take place on fly ash. The carbon content in fly ash is varied between 0% and 20% in this study, and the results have indicated that the maximum dioxin formation is to be found at 5%. The precursors are not injected into the fly ash or gas stream in all formation experiments, however, dioxin is still formed in fly ash. Consequently, other chlorinated routes besides Deacon reactions may take place on the fly ash surface.

Benzofurans↗

PubMed2000

Rhodostomin inhibits the transforming growth factor-beta1-enhanced adhesion activity of ROS 17/2.8 osteosarcoma cells.

We have investigated the effect of transforming growth factor-beta1 (TGF-beta1) on the in vitro adhesion activity of the rat osteosarcoma cell lines (ROS 17/2.8) to extracellular matrix substrata, including fibronectin, type I and IV collagen, as well as laminin. The interaction of Arg-Gly-Asp (RGD) and rhodostomin, an RGD containing snake venom, with TGF-beta1 on the cell adhesion was also evaluated. The results showed that incubation with various concentration of TGF-beta1 (1-15 ng/ml) significantly increased the adhesion activity (1.4 to 2.5 folds) of ROS 17/2.8 to fibronectin and type I collagen (p<0.01), whereas the adhesion activity to laminin and type IV collagen was slightly elevated (1.1 to 1.5 folds). The peak effect of TGF-beta1 on the cell adhesion occurred after pretreatment of ROS 17/2.8 with TGF-beta1 for 6 hours. Treatment with Arg-Gly-Asp-Ser (RGDS) and rhodostomin effectively suppressed the TGF-beta1-enhanced adhesion activity to fibronectin and type I collagen. This study demonstrated that the up-regulated cell adhesion activity of ROS 17/2.8 cells by the TGF-beta1 can be inhibited by the rhodostomin.

Animals↗

PubMed1999

Molecular cloning and sequence analysis of aggretin, a collagen-like platelet aggregation inducer.

A cDNA library derived from the Malayan-pit-viper (Calloselasma rhodostoma) venom gland was constructed in the phagemid vector. Using the information of the N-terminal amino acid sequences of two subunits of aggretin, synthetic mixed-base oligonucleotides were employed as a screening probe for colony hybridization. Separate cDNA clones encoding for the alpha and beta chains of aggretin were isolated and sequenced. The results revealed that mature alpha and beta chains contain 136 and 123 amino acid residues, respectively. Aggretin subunits show high degrees of identity with respective subunits (50-60% for alpha, 49-58% for beta) of C-type lectin-like snake venoms. The identity to rattlesnake lectin is relatively lower (i.e., 39 and 30%). All cysteine residues in each chain of aggretin are well conserved and located at the positions corresponding to those of C-type lectins. Thus, three intracatenary disulfide bridges and an interchain disulfide bond between Cys83(alpha) and Cys75(beta) may be allocated. This is the first report regarding the entire sequence of venom GPIa/IIa agonist. According to the alignment of amino acid sequences, hypervariable regions among these C-type lectin-like proteins were revealed. These hypervariable regions are proposed to be the counterparts directly interacting with different receptors or different domains of a receptor on the surface of platelet.

Amino Acid Sequence↗

PubMed1999

Cytokines modulate integrin alpha(v)beta(3)-mediated human endothelial cell adhesion and calcium signaling.

Angiogenesis is a complex process regulated by the interactions of endothelial cells with cytokines and the adhesive protein matrix. The cytokines basic fibroblast growth factor (bFGF) and tumor necrosis factor-alpha (TNF-alpha) are two of the modulators of angiogenesis. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor activity, in particular, alpha(v)beta(3). In this study, we examined the ability of these angiogenic factors to modulate the adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized disintegrins (i.e., rhodostomin and arietin), which are specific in antagonizing integrin alpha(v)beta(3) in cells. As these disintegrins were immobilized as substrates, they acted as agonists to induce HUVEC adhesion in a dose- and alpha(v)beta(3)-dependent manner. In addition, adhesion also triggered a sustained increase of intracellular free calcium. Furthermore, bFGF-primed HUVECs potentiated, but TNF-alpha primed cells attenuated, about 50% adhesion events and calcium signaling triggered by immobilized disintegrin compared to naive cells, respectively. The mechanisms of modulating alpha(v)beta(3)-dependent HUVEC adhesion by cytokines may be related to changes of integrin alpha(v)beta(3) conformation, as demonstrating the antagonistic effect of Mn(2+) on decreased adhesion by TNF-alpha pretreatment, and confirmed with flow cytometric analysis probed by anti-LIBS1 mAb. However, cytokine pretreatment did not alter the expression of this integrin on the cell surface, as determined by flow cytometry. Phosphoinositide-3 kinase may be one of the signaling molecules involved in the enhanced adhesion of bFGF-primed cells.

Androstadienes↗

PubMed1999

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation.

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg-Gly-Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100-1000 microM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 microM) with various concentrations of BN52021 (4-1000 microM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8+/-1.5 ng/10(6) cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.

Animals↗

PubMed1998

A new short chain RGD-containing disintegrin, accutin, inhibits the common pathway of human platelet aggregation.

A new short-chain disintegrin, accutin, was purified from the Formosan Agkistrodon acutus venom by using of gel filtration, ion exchanger and reverse phase HPLC. The homogeneous protein is a 47-residue polypeptide with a molecular mass of 5241 Da containing an Arg-Gly-Asp sequence and seven cysteinyl residues at positions highly homologous to other disintegrins. Accutin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, thrombin or the thromboxane analogue U46619 in platelet suspension with IC50 values of 66-267 nM. It was also active in inhibiting platelet aggregation of platelet-rich plasma. However, accutin apparently did not affect the shape change caused by these agonists. Accutin also inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner. Furthermore, accutin dose-dependently inhibited the binding reaction of fluorescein isothiocyanate (FITC)-conjugated arietin, a member of the disintegrin family, to human platelets. In addition, the binding of FITC-conjugated accutin to platelets was almost completely blocked by a monoclonal antibody, 7E3, raised against the platelet glycoprotein IIb/IIIa complex. On the other hand, accutin as well as other disintegrins, rhodostomin and arietin, exhibited an inhibitory effect on 7E3 binding toward platelets and endothelial cells in a dose-dependent manner. It is concluded that accutin, a new platelet aggregation inhibitor belonging to the short-chain disintegrin family, acts specifically on a binding epitope of GPIIb/IIIa overlapping with that of 7E3, leading to the blockade of fibrinogen binding to its receptor.

Amino Acid Sequence↗

PubMed1998

Accutin, a new disintegrin, inhibits angiogenesis in vitro and in vivo by acting as integrin alphavbeta3 antagonist and inducing apoptosis.

Endothelial integrins play an essential role in angiogenesis and cell survival. Accutin, a new member of disintegrin family derived from venom of Agkistrodon acutus, potently inhibited human platelet aggregation caused by various agonists (eg, thrombin, collagen, and, adenosine diphosphate [ADP]) through the blockade of fibrinogen binding to platelet glycoprotein IIb/IIIa (ie, integrin alphaIIbbeta3). In this report, we describe that accutin specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin alphavbeta3, to human umbilical vein endothelial cells (HUVECs), but not those of other anti-integrin MoAbs such as alpha2beta1, alpha3beta1, and alpha5beta1. Moreover, accutin, but not the control peptide GRGES, dose-dependently inhibited the 7E3 interaction with HUVECs. Both 7E3 and GRGDS, but not GRGES or Integrelin, significantly blocked fluorescein isothiocyanate-conjugated accutin binding to HUVEC. In functional studies, accutin exhibited inhibitory effects on HUVEC adhesion to immobilized fibrinogen, fibronectin and vitronectin, and the capillary-like tube formation on Matrigel in a dose- and RGD-dependent manner. In addition, it exhibited an effective antiangiogenic effect in vivo when assayed by using the 10-day-old embryo chick CAM model. Furthermore, it potently induced HUVEC apoptotic DNA fragmentation as examined by electrophoretic and flow cytometric assays. In conclusion, accutin inhibits angiogenesis in vivo and in vitro by blocking integrin alphavbeta3 of endothelial cells and by inducing apoptosis. The antiangiogenic activity of disintegrins might be explored as the target of developing the potential antimetastatic agents.

Abciximab↗

PubMed1998

Antithrombotic effect of crotalin, a platelet membrane glycoprotein Ib antagonist from venom of Crotalus atrox.

A potent platelet glycoprotein Ib (GPIb) antagonist, crotalin, with a molecular weight of 30 kD was purified from the snake venom of Crotalus atrox. Crotalin specifically and dose dependently inhibited aggregation of human washed platelets induced by ristocetin with IC50 of 2.4 microg/mL (83 nmol/L). It was also active in inhibiting ristocetin-induced platelet aggregation of platelet-rich plasma (IC50, 6.3 microg/mL). 125I-crotalin bound to human platelets in a saturable and dose-dependent manner with a kd value of 3.2 +/- 0.1 x 10(-7) mol/L, and its binding site was estimated to be 58,632 +/- 3, 152 per platelet. Its binding was specifically inhibited by a monoclonal antibody, AP1 raised against platelet GPIb. Crotalin significantly prolonged the latent period in triggering platelet aggregation caused by low concentration of thrombin (0.03 U/mL), and inhibited thromboxane B2 formation of platelets stimulated either by ristocetin plus von Willebrand factor (vWF), or by thrombin (0.03 U/mL). When crotalin was intravenously (IV) administered to mice at 100 to 300 microg/kg, a dose-dependent prolongation on tail bleeding time was observed. The duration of crotalin in prolonging tail bleeding time lasted for 4 hours as crotalin was given at 300 microg/kg. In addition, its in vivo antithrombotic activity was evidenced by prolonging the latent period in inducing platelet-rich thrombus formation by irradiating the mesenteric venules of the fluorescein sodium-treated mice. When administered IV at 100 to 300 microg/kg, crotalin dose dependently prolonged the time lapse in inducing platelet-rich thrombus formation. In conclusion, crotalin specifically inhibited vWF-induced platelet agglutination in the presence of ristocetin because crotalin selectively bound to platelet surface receptor-glycoprotein Ib, resulting in the blockade of the interaction of vWF with platelet membrane GPIb. In addition, crotalin is a potent antithrombotic agent because it pronouncedly blocked platelet plug formation in vivo.

Animals↗

PubMed1998

What have snakes taught us about integrins?

Snake venoms contain unique components that affect cell-matrix interactions. Disintegrins represent a class of low molecular weight, Arg-Gly-Asp (RGD)-containing, cysteine-rich peptides purified from the venom of various snakes among the Viperidae and Crotalidae. They bind with various degrees of specificity to integrins alpha IIb beta 3, alpha 5 beta 1 and alpha V beta 3 expressed on cells. Snake venom metalloproteases (high molecular mass haemorrhagins) also contain disintegrin-like domains, in addition to zinc-chelating sequences. Membrane-anchored ADAMs (A Disintegrin And Metalloprotease domain), multidomain molecules consisting of metalloprotease, disintegrin-like, cysteine-rich, and epidermal growth factor domains, a transmembrane domain and a cytoplasmic tail, are a new family of proteins. In the light of the large number and wide distribution of ADAMs, they may participate in cell-cell fusion events, including sperm-egg binding and fusion, myoblast fusion and other cell-cell and cell-matrix interactions. The structure-function relationship of these molecules is discussed.

Amino Acid Sequence↗