Search

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.

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., (Copyright 2001 Academic Press.)

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.

Angiogenesis is a complex process consisting of the proliferation, migration and differentiation of endothelial cells, and it is essential for the progression of malignant solid tumors. In this report, we examine the effects of disintegrins (e.g. rhodostomin and accutin) and glycoprotein-lb-binding proteins (e.g. agkistin) on each step in angiogenesis using in vitro and in vivo models. Rhodostomin (but not agkistin) inhibited the viability of human umbilical vein endothelial cells (HUVECs) and capillary tube formation of HUVECs. Rhodostomin also inhibited HUVEC migration and invasion evoked by basic fibroblast growth factor (bFGF). In in vivo studies, rhodostomin inhibited bFGF-, but not vascular-endothelial-growth-factor (VEGF)- associated angiogenesis in chick chorioallantoic membrane model, blocked both bFGF and B16F10 melanoma cell-induced neovascularization, and suppressed the growth of subcutaneously inoculated B16F10 solid tumor, leading to a prolonged survival of the C57BL/6 mice treated with rhodostomin. The antiangiogenic effects of rhodostomin on bFGF-treated HUVECs may be mainly related to the blockade of the interaction of endothelial alpha(v)beta(3) and extracellular matrix., (Copyright 2002 S. Karger AG, Basel)

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.

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.

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.

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.

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.

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., (Copyright 1999 Academic Press.)

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., (Copyright 1999 Academic Press.)

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.

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.

To clarify the roles of alpha-thrombin and prostaglandin E2 (PGE2) in the healing and inflammatory processes of dental pulp, their effects on the DNA synthesis of human pulp cells were investigated by measurement of [3H]thymidine incorporation. At a concentration range of 1 to 25 units/ml, alpha-thrombin stimulated DNA synthesis of the pulp cells by 1.5 to 2.6-fold. On the contrary, PGE2 (> 0.05 microgram/ml) suppressed DNA synthesis by 24 to 39%. Using reverse transcriptase-polymerase chain reaction, thrombin receptor mRNA expression was identified in the pulp cells. Furthermore, alpha-thrombin-induced DNA synthesis could be inhibited by antithrombin III (2 units/ml) with heparin (2 units/ml) or D-Phe-Pro-ArgCH2Cl (50 micrograms/ml). PGE2 (0.1 to 0.5 microgram/ml) also inhibited the thrombin-induced DNA synthesis by 39 to 64%. These results imply that pulp cells express the thrombin receptor that is activated by the serine protease activity of thrombin. Interactions of thrombin and PGE2 are important in modulating the inflammatory and healing processes of the pulp.

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., (Copyright 1998 by The American Society of Hematology)

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.

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.

Triflavin, a 7.5-kD cysteine-rich polypeptide purified from Trimeresurus favoviridis snake venom, belongs to a family of Arg-Gly-Asp-(RGD)-containing peptides, termed disintegrins. In this study, aggregating human platelets dose-dependently induced vasoconstriction in de-endothelialized isolated rat thoracic aortas. At 5x10(7) cells per milliliter, platelets induced a peak tension averaging 65 +/- 7.2% of the tension induced by phenylephrine (10 mumol/L). The relative effectiveness of RGD-containing peptides (including venom peptides triflavin and trigramin, small RGD synthetic peptides Gly-Arg-Gly-Asp-Ser [GRGDS], Gly-Arg-Gly-Asp-Phe [GRGDF], and Gly-Arg-Gly-Asp-Ser-Pro-Lys [GRGDSPK]) was examined by testing the inhibitory effect on aggregating platelet-induced vasoconstriction in de-endothelialized aorta. Triflavin (1 mumol/L) significantly inhibited the platelet-induced vasoconstriction, whereas neither trigramin (10 mumol/L) nor small RGD peptides (2 mmol/L) (i.e., GRGDS, GRGDF, and GRGDSPK) showed any significant effect. The release of serotonin and the formation of thromboxane A2 from aggregating platelets were both significantly inhibited by triflavin (2 mumol/L), whereas trigramin and small RGD-containing peptides showed no significant effect. On scanning electron micrographs of de-endothelialized aorta, aggregating platelets adhered to the subendothelium, with loss of their discoid shape, to form irregular spheres with pseudopod extensions. Triflavin (2 mumol/L) markedly reduced the adhesion of platelets to the subendothelium in the same aorta. Furthermore, RGD-containing peptides (including triflavin, trigramin, and small RGD-containing peptides) inhibited the adhesion of 10 micrograms/mL collagen-activated platelets to extracellular matrices (i.e., fibronectin, vitronectin, and von Willebrand factor). It is concluded that the marked ability of triflavin to inhibit aggregating platelet-induced vasoconstriction in de-endothelialized aorta compared with other RGD-containing peptides (including trigramin), may be due at least partly to triflavin's efficiently preventing the activation of platelets subsequent to inhibition of serotonin release and thromboxane A2 formation. However, the different abilities of triflavin compared with other RGD-containing peptides was not related to the ability to inhibit adhesion of platelets to extracellular matrices. Therefore, from the results of this study, it appears that triflavin may be a useful therapeutic agent for the treatment of thromboembolism and its associated angiospasm.

Purpose: Cell-mediated collagen gel contraction plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). Anti-adhesion therapy has been suggested as a promising strategy in the treatment of PVR. Crovidisin, a snake venom protein isolated from Crotalus viridis, has been shown to bind selectively to collagen and to inhibit collagen-induced platelet aggregation. In the present study, the effectiveness of crovidisin in inhibiting the attachment of retinal pigment epithelial (RPE) cells to collagen, and RPE cell-mediated collagen gel contraction, was evaluated., Methods: Fluorescein isothiocyanate (FITC)-conjugated crovidisin was prepared and used to evaluate its binding affinity for collagen type I, fibronectin, vitronectin, and laminin. The inhibitory effect of crovidisin on RPE cell-mediated extracellular matrix attachment and collagen gel contraction was evaluated by cell adhesion and type I collagen gel contraction assays. The cytotoxic effect of crovidisin was examined with a cell proliferation assay, using the Alamar blue method. Flavoridin, an Arg-Gly-Asp-containing peptide from viper venom, was used for comparison., Results: FITC-conjugated crovidisin bound selectively to collagen type I with high affinity. It did not bind to other matrix proteins, including fibronectin, vitronectin and laminin, nor to RPE cells. Crovidisin inhibited RPE cell attachment to type I collagen in a dose-dependent manner. This inhibitory effect was enhanced by the presence of flavoridin. Crovidisin also dose-dependently inhibited RPE cell-mediated type I collagen gel contraction. Crovidisin was non-toxic to RPE cells., Conclusions: Crovidisin, a snake venom-derived collagen-binding protein, possessing an inhibitory activity on RPE cell-collagen interaction and RPE cell-mediated collagen gel contraction, may be a useful tool for studying cell-collagen interaction, and a potential anti-adhesion therapeutic agent for ocular disorders in which cell-collagen interaction in involved, such as PVR.

By means of liquid chromatography consisting of gel filtration, anionic exchange, and C8 reverse-phase HPLC, a selective inhibitor of collagen-induced platelet aggregation, named crovidisin, has been purified to homogeneity from the venom of Crotalus viridis snake. Crovidisin is a single-chain, 53-kDa protein with a selective inhibitory activity on collagen-induced aggregation of human washed platelets without affecting those elicited by thrombin, sodium arachidonate, and ADP. Partial sequencing of tryptic digests of crovidisin reveals that partial sequence of crovidisin appears to be identical to that of catrocollastatin, a collagen antagonist occurring in the venom of Crotalus atrox snake. Crovidisin dose-dependently blocked aggregation of human washed platelets triggered by 5 and 10 microg/ml of collagen with IC50 of 0.17 and 0.47 microM, respectively. Not only platelet aggregation but also release reaction, thromboxane formation, and increase of intracellular Ca2+ level of platelets in response to collagen were all completely abolished by crovidisin. In the presence of crovidisin, the Mg2+-dependent adhesion of platelets to collagen was diminished in a dose-dependent manner, while the glycoprotein IIb/IIIa-mediated platelet-fibrinogen interaction was unaffected. When collagen was pretreated with crovidisin and followed by three washes with phosphate-buffered saline, the antiadhesion activity of crovidisin was unaffected. In addition, collagen fibers emitted fluorescence after incubation with fluorescein isothiocyanate-conjugated crovidisin, indicating that crovidisin binds directly to collagen fibers. In conclusion, crovidisin blocks the interaction between platelets and collagen fibers through its binding to collagen fibers, resulting in the blockade of collagen-mediated platelet functions such as adhesion, release reaction, thromboxane formation, and aggregation.

Platelets adhere to fibronectin and vitronectin substrates following activation with physiological concentrations of thrombin. Adhesion of activated-platelets to either substrate is dependent upon the amount of fibronectin and vitronectin, and the duration of the adhesion assay. In this study, we showed that the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, triflavin, trigramin and rhodostomin, synthetic peptides GRGDS, GRGDSPK, GRGDF, and GRGD and monoclonal antibodies, 7E3, 10E5 and AP2, raised against glycoprotein IIb/IIIa complex, inhibited the adhesion of activated-platelets to fibronectin and vitronectin-coated plates in a dose-dependent manner. In fibronectin-coated plates, GRGDF was shown to be much more efficient than GRGDS, GRGDSPK and GRGD at inhibiting the adhesion of activated-platelets to immobilized fibronectin. On the other hand, there were no marked differences in the abilities of these three peptides (GRGDF, GRGDS and GRGDSPK) to inhibit platelet adhesion to immobilized vitronectin. Furthermore, the RGD-containing venom peptide, triflavin was more effective than rhodostomin and trigramin at inhibiting the adhesion of activated-platelets to either substrates. The monoclonal antibodies raised against glycoprotein IIb/IIIa complex (i.e., 7E3, 10E5 and AP2) inhibited platelet adhesion to fibronectin and vitronectin in a similar dose-dependent manner. Interestingly, we found that 7E3 was more efficient than 10E5 and AP2 in this reaction. These studies suggest that the glycoprotein IIb/IIIa complex, present on activated-platelets, may interact with fibronectin and vitronectin substrates through the Arg-Gly-Asp-dependent mechanism. Since fibronectin and vitronectin are present in the subendothelial matrix, they may be involved in platelet-vessel wall interaction. The Arg-Gly-Asp containing peptide, especially triflavin, is an ideal therapeutic agent for inhibiting thrombus formation by interrupting platelet-platelet and platelet-subendothelium interactions.

The blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab')2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin's binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein IIb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein IIb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein IIb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin's binding at the saturation dose reflects the extent of glycoprotein IIb/IIIa blockade by 7E3. At the saturation binding concentration (5 micrograms/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein IIb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein IIb/IIIa of platelets in the 7E3 or 7E3 F(ab')2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 micrograms/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein IIb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

Integrins are a superfamily of cell surface glycoproteins that promote cellular adhesion. The interaction of integrins with extracellular matrices such as fibronectin and vitronectin has been shown to be mediated through an arginine-glycine-aspartic acid (RGD) sequence within adhesive proteins. Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins. Disintegrins have been isolated from the venom of various vipers and have been shown to be potent inhibitors of platelet aggregation. In this study, we found that human hepatoma cell adhesion to immobilized matrix proteins (i.e. fibronectin, collagen, laminin, and vitronectin) was differentially affected by various anti-integrin monoclonal antibodies (mAbs) (i.e., alpha3beta1, alpha5beta1, alpha6beta1, and alpha v beta3) as well as by the peptide GRGDS. Indirect flow cytometric analysis of hepatoma cells with anti-integrin mAbs demonstrated that alpha6beta1 was uniformly expressed at a high density, while alpha3beta1, and alpha5beta1 were moderately expressed and alpha v beta3 was expressed in small amounts on hepatoma cells, consistent with the results obtained from immunofluorescence microscopic analysis. When immobilized on plastic wells, triflavin promoted hepatoma cell attachment; this cell attachment was inhibited by either GRGDS or mAbs against integrins alpha3beta1, alpha5beta1 and alpha v beta3). In addition, the binding of FITC-conjugated triflavin to hepatoma cells was inhibited by GRGDS, anti-alpha3beta1, antialpha5beta1, and anti-alpha v beta3 mAbs. Among these mAbs, anti-alpha5beta1 exerted the most pronounced inhibitory effect (>70%) on the binding of triflavin to hepatoma cells. Taken together, these results suggest that triflavin binds via its RGD sequence to multiple integrin receptors (i.e., alpha5beta1, alpha3beta1, and alpha v beta3) expressed on the surface of hepatoma cells, resulting in inhibition of hepatoma cell adhesion to extracellular matrices (i.e., fibronectin and vitronectin).

Triflavin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, inhibits B16-F10 mouse melanoma cell adhesion to extracellular matrices, e.g. fibronectin, vitronectin, fibrinogen, and collagen type I. In this study, GRGDS inhibits B16-F10 mouse melanoma cell adhesion to immobilized triflavin in a dose-dependent manner. In addition, flow-cytometric analysis and the fluorescence staining method in which FITC-triflavin is utilized as a binding ligand were used. GRGDS inhibits the binding of FITC-triflavin to B16-F10 cells. Additionally, the above results suggest that triflavin directly binds to its receptors expressed on B16-F10 cell surface primarily via its RGD sequence, thereby inhibiting B16-F10 cell adhesion to extracellular matrices. Copyright 1996 S. Karger AG, Basel

Cell-mediated tractional retinal detachment (TRD) is the end result of various intraocular proliferative disorders. Interactions between cells and extracellular matrix via cellular surface receptors, integrins, play an important role. Anti-adhesion therapy has been suggested as a promising way to treat the integrin-dependent pathological events. We tested three synthetic peptides, Gly-Arg-Gly-Asp-Ser (GRGDS), derived from the fibronectin receptor binding domain; Try-Ile-Gly-Ser-Arg (YIGSR), from the laminin receptor binding domain, and Ala-Asp-Gly-Glu-Ala (ADGEA), from the collagen receptor binding domain, to evaluate their inhibitory effect on cell-mediated matrix attachment and vitreous contraction in vitro, and on cell-induced TRD in rabbit eyes in vivo. Indirect immunofluorescent stain demonstrated both bovine retinal pigment epithelial (RPE) cells and rabbit dermal fibroblasts expressed the alpha 2 beta 1, alpha 5 beta 1 and alpha 6 beta 1 integrins, the collagen, fibronectin, and laminin receptors, respectively. GRGDS exhibited a broad spectrum of inhibitory activity on RPE cell attachment to extracellular matrices. YIGSR specifically inhibited RPE cell attachment to laminin, whereas ADGEA inhibited RPE cell attachment to collagen type I and IV. ADGEA inhibited RPE cell-induced vitreous contraction in a dose-dependent manner, whereas GRGDS and YIGSR had no effect. ADGEA (1250 micrograms/mL) delayed the development of TRD but did not prevent it. ADGEA was nontoxic to cells and retina, as demonstrated by cytotoxicity tests and histological examination. The synthetic peptide, ADGEA, and its analogs may be potential candidates for the treatment of cell-mediated collagenous contraction in the ocular tissues.

The interactions between tumour cells and the microvasculature, including the adhesion of tumour cells to endothelium and extracellular matrix (ECM) as well as their migratory ability, are prerequisites for metastasis to occur. In this study we showed that thrombin is capable of enhancing in vitro tumour cell metastatic potential in terms of adhesive properties and migratory response. Following exposure to subclotting concentrations of thrombin, SW-480 human colon adenocarcinoma cells exhibited increased adhesion to both the endothelium and ECM component (i.e. fibronectin). Likewise, the pretreatment of thrombin enhanced the migratory ability of SW-480 cells. The enhanced adhesion was significantly inhibited by complexing of thrombin with its inhibitor hirudin, or by serine proteinase inhibition with 3,4-DCI, but was unaffected by pretreatment of tumour cells with actinomycin D or cycloheximide. The effect of thrombin resulted in an upregulated cell-surface expression of beta 3 integrins, a group of receptors mediating interactions between tumour cells and endothelial cells, and between tumour cells and ECM. Antibodies against beta 3 integrins effectively blocked both the enhanced adhesion and migration. This thrombin-mediated up-regulation of beta 3 integrins involved the activation of protein kinase C (PKC) as thrombin-enhanced adhesion was diminished by PKC inhibition. Rhodostomin, an Arg-Gly-Asp-containing antiplatelet snake venom peptide that antagonises the binding of ECM toward beta 3 integrins on SW-480 cells, was about 600 and 500 times, more potent that RGDS in inhibiting thrombin-enhanced adhesion and migration respectively. Our data suggest that PKC inhibitors as well as rhodostomin may serve as inhibitory agents in the prevention of thrombin-enhanced metastasis.

Purpose: Integrin-mediated extracellular matrix (ECM) attachment plays an important role in vitreous contraction of retinal pigment epithelial (RPE) cells. Disintegrins, a group of Arg-Gly-Asp (RGD)-containing peptides from viper venom, are potential anti-adhesion agents that interfere with integrin-ECM binding. This study was performed to determine whether disintegrins were effective in inhibiting RPE cell-induced matrix attachment in vitro and tractional retinal detachment in a rabbit model in vivo., Methods: Two disintegrins, echistatin from viper Echis carinatus and flavoridin from Trimeresurus flavoviridis, were used. The expression of integrins on the surface of bovine and rabbit RPE cells was examined by indirect immunofluorescent stain with specific anti-integrin monoclonal antibodies. The inhibitory effect of disintegrins on RPE cell-mediated ECM attachment and vitreous contraction was evaluated with cell adhesion and vitreous contraction assays. In the in vivo model, rabbit eyes were injected intravitreously with either homologous rabbit RPE cells alone or together with disintegrins to induce tractional retinal detachment. The cytotoxic effect of disintegrins was examined with a cell proliferation assay using the alamar blue method. Retinal toxicity of disintegrins was evaluated with electroretinograms and histologic examination of the rabbit eyes., Results: Bovine and rabbit RPE cells showed the positive staining for the integrins alpha 2 beta 1 and alpha 5 beta 1 on cell surface. Disintegrins, echistatin, and flavoridin inhibited RPE cell attachment to the ECM. The potency of disintegrins was 150 to 300 times higher than that of Gly-Arg-Gly-Asp-Ser (GRGDS) peptide. The disintegrins also inhibited RPE cell-induced vitreous contraction in a dose-dependent manner, whereas the GRGDS peptide had no effect. In the in vivo experiment, echistatin (50 microgram/ml) or flavoridin (80 microgram/ml) significantly inhibited RPE cell-induced tractional retinal detachment compared with the control group at week 2 (P< 0.05) and week 4 (P< 0.01) after surgery. Disintegrins were nontoxic to RPE cells and rabbit retina as evaluated by cytotoxicity tests, electroretinograms, and histologic examinations., Conclusions: The disintegrins were effective in inhibiting RPE cell attachment to the ECM and vitreous contraction in vitro. They also were effective in suppressing RPE cell-induced tractional retinal detachment in the rabbit eyes. They were nontoxic. Disintegrins and their analogs might be potential anti-adhesion therapeutic agents in the treatment of proliferative vitreoretinopathy.

PC-3 cells, a metastatic human prostate adenocarcinoma line, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma (PRP). PC-3 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) and limited by increasing concentrations of apyrase. This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2. PC-3 cell suspension caused marked, dose-dependent decreases in plasma recalcification times using normal, Factor VIII-deficient and Factor IX-deficient, but not Factor VII-deficient, human plasma. This effect was potentiated in cell lysates, but was inhibited in intact cells preincubated with sphingosine. Overall, these data suggest that PC-3 TCIPA arises from PC-3 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides which antagonise the binding of fibrinogen to platelet membrane glycoprotein IIb-IIIa, prevented PC-3 TCIPA. Similarly, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoproteins IIb-IIIa and Ib prevented PC-3 TCIPA, which was unaffected by control peptide GRGDS. On a molar basis, trigramin (IC50, 0.11 microM) and rhodostomin (IC50, 0.03 microM) were approximately 5000 and 18000 times, respectively, more potent than GRGDS (IC50, 0.56 mM).

In static condition, 6F1, an anti-glycoprotein Ia/IIa monoclonal antibody, almost completely prevented initial platelet adhesion to fibrillar collagen, but markedly lost its action with prolonged incubation. The platelet adhesion and spreading at the later stage were prevented by adding the peptide GRGDS, aspirin, and apyrase, suggesting that after initial recognition of platelet glycoprotein Ia/IIa with collagen the activation of glycoprotein IIb/IIIa and the release of thromboxane A2/ADP would promote platelet spreading, thus strengthening the stability of adhesion. Both initial platelet adhesion and platelet spreading were prevented by cytochalasin B, an inhibitor of actin polymerization. In contrast, BAPTA (an intracellular Ca2+ chelator) only inhibited platelet spreading. Inhibition of protein kinase C or protein tyrosine kinase by staurosporine or genistein, respectively, had only little effect on platelet adhesion. These data suggest that actin polymerization and intracellular Ca2+ mobilization are involved in the regulation of platelet spreading after initial platelet contact with collagen.

Ancrod-generated fibrin has been shown to stimulate prostacyclin synthesis of human umbilical vein endothelial cells (Chang et al., 1994, Biochem. Biophys. Res. Commun. 203, 1920). We further investigated its mechanism of action. The increment of 6-keto prostaglandin F1 alpha stimulated by ancrod-generated fibrin was almost completely inhibited when endothelial cells were either pretreated with 50 microM 8-(N,N'-diethylamino)octyl-3,4,5- trimethoxybenzoate (TMB-8) or preloaded with 15 microM 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). 6-Keto prostaglandin F1 alpha production during 2 and 10 h incubation period was also inhibited by 1.2 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraace tic acid (EGTA) (41 +/- 12 and 53 +/- 17% inhibition, respectively). Further, ancrod-generated fibrin caused a rapid-onset increase in [3h]inositol monophosphate (IP1) formation in endothelial cells. This increase in IP1 was significantly inhibited by 1 mM Gly-Pro-Arg-Pro, 1 mM neomycin or 100 ng/ml pertussis toxin. At the same time, neomycin and pertussis toxin also significantly inhibited 6-keto prostaglandin F1 alpha synthesis of endothelial cells stimulated by ancrod-generated fibrin. Additionally, the increment of IP1 production as well as prostacyclin production were significantly inhibited by monoclonal antibodies directed against alpha v beta 3. These results suggest that intra- and extra-cellular Ca2+ participate in prostacyclin synthesis stimulated by ancrod-generated fibrin. Ancrod-generated fibrin stimulates pertussis toxin-sensitive G-protein regulated phosphoinositide breakdown, which is responsible for prostacyclin synthesis. This augmentation in prostacyclin synthesis and phosphoinositide breakdown caused by ancrod-generated fibrin area, at least in part, mediated by fibrin binding to integrin alpha v beta 3 on endothelial cells.

Triflavin, an Arg-Gly-Asp-containing peptide from Trimeresurus flavoviridis snake venom (M(r) of 7500 Da) inhibits platelet aggregation through the blockade of fibrinogen binding to activated platelets. The present study demonstrated that the intravenous injection of triflavin (0.1 and 0.25 mg/kg) significantly prolonged the bleeding time about 1.8- to 2.4-fold as compared with control (normal saline) of severed mesenteric arteries in rats, whereas the injection of Gly-Arg-Gly-Asp-Ser (GRGDS) (2-8 mg/kg) failed to increase the bleeding time in this model. Continuous infusion of triflavin (0.08 mg/kg/min) significantly increased the bleeding time about 2.6-fold, and the bleeding time returned to normal within 20 min after the cessation of triflavin infusion. Triflavin (10-20 mg/kg) significantly prolonged the occlusion time of platelet plug formation induced by irradiation of mesenteric venules of fluorescein sodium-pretreated mice. In contrast, trigramin (10-20 mg/kg) and GRGDS (500 and 1000 mg/kg) showed no significant effect. These results suggest that triflavin has an effective antiplatelet effect in vivo and this peptide may be a useful therapeutic agent for arterial thrombosis.

The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did not cause morphologic alteration of HUVEC by itself at concentrations ranging from 0.01 to 10 U/ml. However, when 0.02 U/ml of ancrod was added to cultured HUVEC monolayers in the presence of citrated plasma, it caused pronounced morphologic change of HUVEC after 6-10 h incubation period. Gly-Pro-Arg-Pro (4 mg/ml), an inhibitor of fibrin polymerization, prevented the morphologic alteration, indicating that the morphologic alteration was caused by the polymerized fibrin. The morphologic change of HUVEC caused by ancrod-generated fibrin was not observed in the presence of an intracellular calcium mobilization inhibitor TMB-8 (50 microM), and the morphologic alteration was also less pronounced with BAPTA(15 microM)-loaded HUVECs and HUVECs pretreated with EGTA (1.2 mM). Ancrod (in Medium 199) itself did not stimulate phosphoinositide breakdown of HUVEC. However, when ancrod was present in plasma, it caused an increase of [3H]IP1 of HUVECs preloaded with [3H]myoinositol. This IP1 increment was inhibited by Gly-Pro-Arg-Pro. The increase of IP1 was significantly inhibited by the pretreatment of monoclonal antibodies 23C6 and 7E3 directed against alpha v beta 3 integrin. Neomycin (1 mM) and pertussis toxin (100 ng/ml), but not aspirin or mepacrine, blocked this enhanced phosphoinositide breakdown. The morphologic change was also prevented by the monoclonal antibodies, 23C6 and 7E3. These results suggest that both intra- and extra-cellular calcium participate in the event of morphologic change of HUVEC caused by ancrod-generated fibrin, and the morphologic change is mediated, at least in part, by fibrin binding to integrin alpha v beta 3 on HUVECs, causing the subsequent activation of the endogenous G-protein coupled phospholipase C.

A potent platelet aggregation inhibitor in the venom of Crotalus viridis snake was purified to homogeneity by gel filtration chromatography and reverse phase high-performance liquid chromatography. This purified principle, named crotavirin, is a single-chain polypeptide with a mol. wt of 9200 as estimated by SDS-polyacrylamide gel electrophoresis. It inhibited the aggregation of human washed platelets induced by collagen, thrombin and thomboxane analogue (U46619) with a similar IC50 (approximately 1.0 micrograms/ml, 0.11 microM). The binding of fluorescein isothiocyanate-conjugated crotavirin to platelets was abolished in the presence of divalent cation chelator, EDTA, indicating that divalent cation is essential for crotavirin's binding. A monoclonal antibody, 7E3, raised against platelet glycoprotein IIb-IIIa complex blocked the binding of fluorescein isothiocyanate-conjugated crotavirin to platelets, whereas the other monoclonal antibody against glycoprotein IIb-IIIa, 10E5, had no inhibitory effect. In addition, crotavirin inhibited in a concentration-dependent manner the binding of fluorescein isothiocyanate-conjugated rhodostomin, a member of the disintegrin family, to platelets. Its binding to platelets was blocked by disintegrins, e.g. trigramin and rhodostomin. It is concluded that crotavirin is a potent platelet aggregation inhibitor, which acts specifically on an epitope of glycoprotein IIb-IIIa, leading to the blockade of fibrinogen binding to glycoprotein IIb-IIIa and eventually the blockade of platelet aggregation.

A potent platelet aggregation inducer, aggretin, was purified from Malayan-pit-viper (Calloselasma rhodostoma) venom by ionic-exchange chromatography, gel-filtration chromatography and HPLC. It is a heterodimeric protein (29 kDa) devoid of esterase, phospholipase A and thrombin-like activity. Aggretin (> 5 nM) elicited platelet aggregation with a lag period in both human platelet-rich plasma and washed platelet suspension. EDTA (5 mM), prostaglandin E1 (1 microM) and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester ('TMB-8'; 100 microM) abolished its aggregating activity, indicating that exogenous bivalent cations and intracellular Ca2+ mobilization are essential for aggretin-induced platelet aggregation. Neomycin (4 mM) and mepacrine (50 microM) completely inhibited aggretin (33 nM)-induced aggregation; however, creatine phosphate/creatine phosphokinase (5 mM, 5 units/ml) and indomethacin (50 microM) did not significantly affect its aggregating activity. Aggretin caused a significant increase of [3H]InsP formation in [3H]Ins-loaded platelets, intracellular Ca2+ mobilization and thromboxane B2 formation. Neomycin, a phospholipase C inhibitor, completely inhibited both the increase of [3H]InsP and intracellular Ca2+ mobilization of platelets stimulated by aggretin. A monoclonal antibody (6F1) directed against glycoprotein Ia/IIa inhibited platelet shape change and aggregation induced by aggretin. 125I-aggretin bound to platelets with a high affinity (Kd = 4.0 +/- 1.1 nM), and the number of binding sites was estimated to be 2119 +/- 203 per platelet. It is concluded that aggretin may act as a glycoprotein Ia/IIa agonist to elicit platelet aggregation through the activation of endogenous phospholipase C, leading to hydrolysis of phosphoinositides and subsequent intracellular Ca2+ mobilization.

We investigated the adhesion of human umbilical vein endothelial cells (HUVECs) to fibrin(ogen) molecule of varying structure for identifying sites that mediate cell attachment. Fibrin was prepared either with ancrod which liberates only FPA from fibrinogen, or with thrombin, which liberates both FPA and FPB. Both fibrin preparations equally supported HUVEC attachment. GRGDS, RGD-containing peptides of snake venoms, and monoclonal antibodies against alpha v beta 3 (23C6 and 7E3) inhibited the attachment of HUVECs to fibrin by 65-75%. In contrast, the attachment of HUVECs to fibrinogen was less effective and was almost completely inhibited by both RGD-containing peptides and by antibodies against integrin alpha v beta 3 (85-95% inhibition). The C-terminal dodecapeptide of fibrinogen gamma chain (residues 400-411) inhibited minimally the attachment of HUVECs to fibrin. Additionally, the binding of RGD-containing snake venom peptides to HUVECs was both RGD- and divalent-cation-dependent. The IC50S for inhibition of HUVEC attachment to fibrin were 0.09 microM (rhodostomin), 1.54 microM (trigramin) and 1.64 microM (halysin). These results indicate that fibrin mediated support of cell attachment is independent of the cleavage of FPB from fibrinogen. HUVEC attachment to fibrinogen was almost completely inhibited by RGD-containing peptides and by antibodies against alpha v beta 3. In contrast, the attachment to fibrin was partially resistant to RGD-containing peptides and to the monoclonal antibodies against integrin alpha v beta 3. However, alpha v beta 3 is the major receptor mediating HUVEC attachment to fibrin.

A thrombin-like enzyme, grambin, was purified to homogeneity by gel filtration, affinity and ion-exchange chromatography from the venom of Trimeresurus gramineus. Its mol. wt was estimated to be 45,400 by SDS-PAGE under reduced conditions. The mass of neutral sugars in grambin is estimated to be 20.7% of total mass. Grambin's NH2-terminal ten amino acid residues show a high homology to other venom thrombin-like enzymes. It clots human fibrinogen with a specific activity of 220-250 NIH thrombin-equivalent units/mg protein. It preferentially releases fibrinopeptide A accompanied by a slow release of trace amounts of fibrinopeptide B as monitored by HPLC following enzyme treatment of fibrinogen. EDTA, aprotinin, hirudin and heparin did not affect the fibrinogen-clotting activity of grambin in purified human fibrinogen solution. Diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and leupeptin inhibited the clotting activity of grambin whereas iodoacetamide did not affect its activity, indicating that grambin is a serine protease rather than a cysteine protease. In addition, it caused defibrinogenation and showed a marked antiplatelet effect when administered intravenously to mice. It also significantly prolonged the time lapse of platelet-rich thrombus formation in the irradiated mesenteric venules of fluorescein sodium-treated mice. Therefore, grambin may be used as a therapeutic agent not only in treatment of venous thrombosis but also in prevention of arterial thrombosis.

Ancrod, a thrombin-like enzyme purified from the venom of Calloselasma rhodostoma, was administered to rabbits intravenously, and blood samples were obtained at 1, 3, 6, 10, and 24 hours after infusion. Ancrod caused a rapid and sustained defibrinogenation within the first 6 hours, with production of fibrinogen degradation products (FDPs) peaking at 1 hour and declining to background level at 6 hours. No significant changes in platelet count, white cell count, or hematocrit was observed. Citrated PRP prepared 1, 3, and 6 hours after ancrod infusion showed diminished aggregation, adenosine triphosphate (ATP) release, and thromboxane B2 formation on the addition of collagen. Although platelet suspension prepared from defibrinogenated platelet-rich plasma (PRP) at 3 hours showed no significant change in aggregation and ATP-releasing activity, the latent period of platelet aggregation was prolonged. When the remaining platelet-poor plasma obtained from defibrinogenated PRP at 3 hours was used to suspend the normal washed platelets prepared from PRP before ancrod infusion, the platelets showed a similar defect in aggregation and release action. Addition of fibrinogen (200 micrograms/ml to 2 mg/ml) to the above preparation partially restored aggregation but not capacity for secretion and thromboxane formation. When normal washed platelets were suspended with the defibrinogenated plasma, prepared by mixing ancrod with normal plasma in vitro and removing the formed fibrin, the platelet suspension showed impaired platelet aggregability, and the aggregability could be restored to the normal level by the addition of exogenous fibrinogen to this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

MCF-7 cells, a metastatic human breast carcinoma line, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma (PRP). MCF-7 tumor cell-induced platelet aggregation (TCIPA) was almost blocked by apyrase (0.5 U/ml) and completely inhibited by hirudin (5 U/ml). This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2. MCF-7 cell suspension caused marked, dose-dependent decrease in plasma recalcification times using normal, Factor VIII-deficient, and Factor IX-deficient human plasma. This effect was potentiated in cell lysates but was inhibited in intact cells preincubated with sphingosine. MCF-7 cell suspension did not affect the recalcification time of Factor VII-deficient plasma. Taken together, these data suggest that MCF-7 TCIPA arises from MCF-7 tissue factor activity expression. Trigramim and rhodostomin, RGD-containing snake venom peptides which antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented MCF-7 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against human tissue factor, platelet membrane glycoprotein IIb/IIIa and Ib prevented MCF-7 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50, 0.1 microM) and rhodostomin (IC50, 0.03 microM), were about 5,000 and 18,000 times, respectively, more potent than GRGDS (IC50, 0.54 mM).

Saos-2 cells, derived from a primary human osteosarcoma, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma. Saos-2 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin but unaffected by apyrase. The cell suspension shortened the plasma recalcification times of normal, factor VIII-deficient and factor IX-deficient human plasmas in a dose-dependent manner. However, the cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, a monoclonal antibody (MAb) against human tissue factor completely abolished TCIPA. Flow cytometric analysis using anti-integrin MAbs as the primary binding ligands demonstrated that the integrin receptors alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1 were present of Saos-2 cells, which might mediate tumour cell adhesion to extracellular matrix. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide which antagonises the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented Saos-2 TCIPA as well as tumour cell adhesion to vitronectin, fibronectin and collagen type I. Likewise, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) showed a similar effect. On a molar basis, rhodostomin was about 18,000 and 1000 times, respectively, more potent than GRGDS in inhibiting TCIPA and tumour cell adhesion.

Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi and named echistatin beta and gamma. These proteins were found to be about 85% similar in amino acid sequence to echistatin alpha which has been well studied. The disulphide pattern of echistatin gamma appeared to be identical with that of echistatin alpha. They all contain the adhesive recognition sequence Arg-Gly-Asp (RGD) but inhibit the aggregation of platelets from human and other mammals with different potencies. Echistatin beta and alpha are far more effective on platelets from humans and guinea pigs than those from rabbits and rats whereas echistatin gamma is less discriminating of the platelets of the species tested. This species-dependent platelet sensitivity to echistatin beta and gamma could be attributed to the variations in residues 15, 21, 22 and 27, which are close to or within the RGD loop, rather than to the C-terminal variations after residue 46. Taking advantage of the presence of methionine residues flanking both sides of the ARGDDM motif in echistatin gamma, we deleted this hexapeptide by CNBr cleavage to produce des-(23-28)-echistatin gamma. The modified protein showed c.d. and fluorescent spectra grossly similar to the intact echistatin but its antiplatelet potency decreased more than 200-fold. We thus propose that a favourable conformation of the RGD region is responsible mainly for the high-affinity binding of echistatin to the platelet glycoprotein IIb-IIIa as shown previously for the binding of medium-size disintegrin.

Disintegrins are a group of molecules containing Arg-Gly-Asp (RGD) sequence which can interfere with cell-matrix interaction. Rat mesangial cells are known to express an RGD-sensitive integrin receptor. We investigate the effect of a potent disintegrin, triflavin, on rat mesangial cell adhesion and proliferation. While synthetic RGD-containing peptide (GRGDS) inhibits mesangial cell adhesion to fibronectin, type I and type III collagen, triflavin has a similar but much more potent effect. Triflavin, when added to the medium, inhibits serum-stimulated rat mesangial cell proliferation in both growing and growth-arrested cells. In serum-free medium, platelet-derived growth factor promoted mesangial cell growth is also inhibited by triflavin. When coated as a substratum, both GRGDS and triflavin inhibit mesangial cell growth. The inhibition of proliferation by exogenously added triflavin and GRGDS is, at least in part, due to their antiadhesive effect. We conclude that triflavin can modulate both adhesion and proliferation of rat mesangial cells.

The antiplatelet activity of DC-015, a newly synthesized quinazoline derivative was determined in human platelet-rich plasma. From the binding studies, the Ki values of DC-015 for alpha 1-, alpha 2-adrenoceptors and 5-HT1, 5-HT2 receptors were about 0.21 (nM), 0.59 (microM), 0.16 (microM) and 0.38 (microM), respectivity. On the other hand, the Ki values of prazosin for alpha 1- and alpha 2-adrenoceptors were about 0.19 (nM) and 4.8 (microM), respectivity. Experimental results indicated that DC-015 dose-dependently inhibited noradrenaline (10 microM)-induced platelet aggregation in human platelet-rich plasma. At 20 microM, DC-015 would completely inhibit platelet aggregation induced by noradrenaline. A high concentration of prazosin (> 30 mM) caused sligh inhibition of aggregation. Furthermore, DC-015 (2 microM) significantly increased the cyclic AMP level in human platelet-rich plasma, whereas, prazosin significantly increased cyclic AMP level only at higher concentrations (100 microM). We can conclude that DC-015 inhibited noradrenaline-induced platelet aggregation mainly through binding to alpha 2-receptor on platelets, resulting in inhibiting platelet aggregation.

Integrins are a superfamily of cell surface glycoproteins that mediate cell-extracellular matrix (ECM) and cell-cell adhesion. Immunofluorescence microscopy and flow cytometric analysis using anti-integrin mAbs as the primary binding ligands demonstrated that the platelet integrin receptor alpha IIb beta 3, as well as alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1, are present on the surface of SW-480 human colon adenocarcinoma cells. Monoclonal antibodies (mAbs) against alpha IIb beta 3 and alpha 5 beta 1 inhibited unstimulated basal adhesion to fibronectin by approximately 30% and 40%, respectively. The surface immunoreactivity of tumor cells for alpha IIb beta 3 was enhanced by pretreatment (5 min) with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or a lipoxygenase metabolite of arachidonic acid, 12-hydroxyeicosatetraenoic acid (12-HETE) in a dose- and time-dependent manner. SW-480 cells possess a large intracellular pool of alpha IIb beta 3, from which the receptor complex translocates to the cell surface following pretreatment with TPA or 12(S)-HETE. This pretreatment enhances adhesion to fibronectin, which is mediated exclusively by alpha IIb beta 3 integrins. Staurosporine was found to block alpha IIb beta 3 up-regulation and enhanced-adhesion. TPA and 12(S)-HETE also facilitated the redistribution of alpha IIb beta 3 during the enhanced-spreading process. Rhodostomin, an Arg-Gly-Asp- (RGD) containing antiplatelet snake venom peptide, was about 400-times more potent than RGDS at inhibiting control, TPA- or 12(S)-HETE-enhanced adhesion of SW-480 cells to fibronectin. The binding of mAbs against alpha IIb beta 3, alpha v beta 3 and alpha 5 beta 1 was inhibited by pretreatment with rhodostomin, suggesting that rhodostomin binds via its RGD sequence to multiple integrin receptors (i.e., alpha IIb beta 3, alpha v beta 3, alpha 5 beta 1) expressed on the SW-480 cell surface, inhibiting cell adhesion to ECM.

Disintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein IIb-IIIa (GPIIb-IIIa) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-IIIa complex on platelet membrane with a very high affinity (Kd, 10(-7)-10(-8) M). In this study, we analyzed GPIIb-IIIa complex on platelet membrane by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were. The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy) and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-IIIa. ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used, whereas it had little effect on that of FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case of FITC-Rn as the probe. The platelets from three patients with Glanzmann's thrombasthenia were probed with FITC-disintegrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Triflavin, an Arg-Gly-Asp-containing snake venom peptide, inhibits platelet aggregation through the blockade of fibrinogen binding to the activated platelets. In this study, platelet thrombus formation was induced by irradiation of the mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. Electron microscopy reveals moderately damaged endothelial cells, as well as aggregates consisting almost exclusively of platelets with pseudopod formation, and degranulated appearance. Triflavin (10-20 micrograms/g) significantly prolonged the lag period of inducing platelet plug formation in mesenteric venules when it was intravenously infused. Triflavin (20 micrograms/g) prolonged the occlusion time about 2-fold (from control 112 +/- 23 to 240 +/- 47 s). Furthermore, PGE1 briefly prolonged the occlusion time about 1.5-fold (from 105 +/- 21 to 168 +/- 20 s) when it was given by continuous infusion (40 micrograms/kg/min). On the other hand, triflavin was also effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at dose of 2-4 micrograms/g. Heparin (1.5 U/g) and indomethacin (200 micrograms/g) had no significant effect in prolonging the occlusion time or in reducing ADP-induced pulmonary embolism in mice. Therefore, triflavin is an effective antithrombotic agent in preventing the thromboembolism in these two in vivo models.

Ancrod, a thrombin-like enzyme, has been used as defibrinogenating agent in prevention of venous thrombosis. This study showed ancrod in citrated plasma elevated 6-keto PGF1 alpha production of human umbilical vein endothelial cells; this increment of 6-keto PGF1 alpha was completely inhibited by Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The enhanced prostacyclin production, but not basal level of prostacyclin, was inhibited by actinomycin D and cycloheximide. In washed aspirin-pretreated cells, ancrod-formed fibrin induced restoration of prostacyclin production by a cycloheximide- and actinomycin D-sensitive process. Ancrod-formed fibrin stimulated synthesis of cyclooxygenase as probed by Western blotting and this enhancement was blocked by actinomycin D and cycloheximide. In conclusion, we first report that ancrod-formed fibrin stimulates prostacyclin production of human endothelial cells and this event is dependent on de novo synthesis of cyclooxygenase.

SW-480 cells, derived from a primary human colon adenocarcinoma, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma. SW-480 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) but unaffected by apyrase (10 U/ml). This TCIPA was also unaffected by cysteine proteinase inhibition with E-64 (10 microM) but was limited by cell pretreatment with phospholipase A2. SW-480 cell suspension caused marked dose-dependent decreases in plasma recalcification times using normal, factor VIII-deficient and factor IX-deficient human plasma. This effect was potentiated with cell lysates but inhibited in intact cells pretreated with sphingosine. SW-480 cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, monoclonal antibody against human tissue factor completely abolished SW-480 TCIPA. Taken together, these data suggest that SW-480 TCIPA arises from SW-480 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides, which antagonize the binding of fibrinogen to platelet membrane glycogen IIb/IIIa, prevented SW-480 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoprotein IIb/IIIa and Ib prevent SW-480 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50 0.09 microM) and rhodostomin (IC50 0.03 microM) were about 6000 and 18,000 times, respectively, more potent than GRGDS (IC50 0.56 mM).

The antiplatelet and vasorelaxing actions of 14-acetoxycedrol, an acetyl derivative of the sesquiterpene 8,14-cedranediol isolated from Juniperus squamata Hayata, were investigated in washed rabbit platelets and rat aorta, respectively. 14-Acetoxycedrol inhibited the aggregation and ATP release of rabbit platelets induced by ADP, arachidonic acid, platelet-activating factor (PAF), collagen, and thrombin. Prolongation of the incubation time of 14-acetoxycedrol with platelets did not cause further inhibition and the aggregability of the treated platelets could be restored after washing of the platelets. It inhibited thromboxane B2 formation of washed platelets caused by arachidonic acid, collagen, and thrombin in a concentration-dependent manner. The formation of inositol phosphate caused by collagen and PAF was inhibited by 14-acetoxycedrol, while that caused by thrombin was not affected. 14-Acetoxycedrol markedly inhibited the intracellular calcium rise caused by PAF, and slightly inhibited that caused by thrombin in quin-2/AM-load platelets. In rat thoracic aortae, 14-acetoxycedrol inhibited the high K+ (60 mM) and Ca2+ (0.03-3 mM) induced cumulative contractions in a concentration-dependent manner, while it did not affect the phasic and tonic contractions elicited by norepinephrine. The tonic contractions elicited by KCl (60 mM) and Bay K 8644 were also relaxed by 14-acetoxycedrol. It is concluded that the antiplatelet effect of 14-acetoxycedrol is due to the inhibition of thromboxane formation and phosphoinositides breakdown and the vasorelaxing action of 14-acetoxycedrol is due to inhibition of Ca2+ influx through the voltage-dependent Ca2+ channel.

By means of gel filtration, ionic exchange chromatography and DEAE-column HPLC, an acidic phospholipase A2 (PLA2) was purified from beaded lizard (Heloderma horridum) venom. The purified PLA is a single-chain polypeptide, consisting of about 163 amino acid residues with a molecular mass of 19,000 Da as calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analysis. HHV-PLA showed a rather specific inhibitory effect on platelet aggregation induced by U46619 and epinephrine in human platelet-rich plasma in a dose- and time-dependent manner, whereas it had little effect on collagen- and ADP-induced aggregation. ATP-release reaction induced by various agonists were dose- and time-dependently inhibited by HHV-PLA, even though platelet aggregation was apparently not affected in human washed platelets. When HHV-PLA was chemically modified with p-bromophenacyl bromide, both of its enzymatic activity and antiplatelet activity were lost. Furthermore, exogenous lysophosphatidylcholine and HHV-PLA treated phosphatidylcholine inhibited platelet aggregation induced by U46619 in human washed platelets. In conclusion, PLA enzyme from H. horridum venom inhibits exclusively U46619- or thromboxane-induced platelet aggregation of human platelet-rich plasma probably by virtue of their PLA enzymatic activity on plasma phospholipids, converting phospholipids (e.g., phosphatidylcholine) into lysophospholipids, which in turn interfere with the coupling of TXA2 receptor and its signalling transduction system.

The interaction of tumor cells with extracellular matrix components such as laminin, fibronectin, and collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid amino acid sequence within each protein. Triflavin, a 7.5 kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of arginine-glycine-aspartic acid-containing peptides termed disintegrins that have been isolated from the venoms of various vipers and shown to be potent inhibitors of platelet aggregation. In this study, we showed that triflavin inhibited adhesion of human hepatoma J-5 cells to extracellular matrices (fibronectin, vitronectin, fibrinogen, and collagen type I) in a dose-dependent manner. On the other hand, triflavin exerted a limited inhibitory effect on cell attachment to collagen type IV and laminin (< or = 40%). Triflavin is approximately 1000 times more potent than glycine-arginine-glycine-aspartic acid-serine at inhibiting cell adhesion. When immobilized on plate, triflavin promoted J-5 cell attachment; this attachment was inhibited by glycine-arginine-glycine-aspartic acid-serine. In addition, triflavin labeled with iodine 125 binds to J-5 cells in a saturable manner and its binding was also inhibited by glycine-arginine-glycine-aspartic acid-serine. Its Kd value was estimated to be 3.9 x 10(-7) mol/L and the number of binding sites was around 60,000 per cell. Furthermore, triflavin did not affect tritiated thymidine uptake during a 3-day incubation.(ABSTRACT TRUNCATED AT 250 WORDS)