A potent antiplatelet peptide, triflavin, from Trimeresurus flavoviridis snake venom

The interaction of fibrinogen with its receptors on platelet surfaces leads to platelet aggregation. A snake-venom peptide, trigramin, has previously been demonstrated to inhibit platelet aggregation by acting as a fibrinogen-receptor antagonist. By means of gel filtration, ionic-exchange chromatography and reverse-phase h.p.l.c., a potent platelet-aggregation inhibitor, triflavin, has now been purified from the venom of Trimeresurus flavoviridis. The purified triflavin is a single-chain polypeptide, consisting of about 71 amino acid residues with a molecular mass of 7600 Da, and its N-terminal sequence is Gly-Glu-Glu-Cys-Asp. Triflavin dose-dependently inhibited human platelet aggregation stimulated by ADP, adrenaline, collagen, thrombin or prostaglandin endoperoxide analogue U46619 in preparations of platelet-rich plasma, platelet suspension and whole blood. Its IC50 ranged from 38 to 84 nM, depending on the aggregation inducer used and the platelet preparation. However, triflavin apparently did not affect the platelet shape change and ATP-release reactions caused by these agonists. Triflavin inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner, indicating that it directly interferes with the binding of fibrinogen to its receptors on platelet membranes exposed by elastase treatment. Additionally, triflavin dose-dependently blocked 125I-labelled fibrinogen binding to ADP-activated platelets. In conclusion, triflavin inhibits platelet aggregation through the blockade of fibrinogen binding to fibrinogen receptors on platelet membranes.

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Mechanism of Action of a Potent Antiplatelet Peptide, Triflavin from Trimeresurus flavoviridis Snake Venom

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646444 ◽

1991 ◽

Vol 66 (04) ◽

pp. 489-493 ◽

Cited By ~ 34

Author(s):

Tur-Fu Huang ◽

Joen-Rong Sheu ◽

Che-Ming Teng

Keyword(s):

Snake Venom ◽

Shape Change ◽

Membrane Surface ◽

Inhibitory Effect ◽

Human Platelets ◽

Free Calcium ◽

Activation Process ◽

Trimeresurus Flavoviridis ◽

Fibrinogen Binding ◽

Induced Aggregation

SummaryTriflavin, an antiplatelet peptide from Trimeresurus flavoviridis snake venom, inhibits aggregation of human platelets stimulated by a variety of agonists. However, triflavin does not affect the shape change and release reaction of platelets stimulated by thrombin and collagen. In this paper, we further investigate its effect on the intracellular events occurring after the activation of platelets. Triflavin does not inhibit the intracellular free calcium rise of Quin 2-AM loaded platelets stimulated by thrombin and it also has no significant effect on thromboxane B2 formation of platelets stimulated by thrombin. Triflavin does not affect the 3(H)-inositol monophosphate formation of the 3(H)-myoinositol loaded platelets. However, triflavin dose-dependently inhibits fibrinogen-induced aggregation and 125I-fibrinogen binding of ADP-stimulated platelets. In addition, triflavin dose-dependently blocks fibrinogen-induced aggregation of elastase-treated platelets. It is concluded that triflavin specifically inhibits fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex on platelet membrane surface without any inhibitory effect on the platelet-activation process.

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Factor Xa Like Enzyme in Snake “Habu” Venom

10.1055/s-0039-1680591 ◽

1977 ◽

Author(s):

I. Maruyama ◽

T. Kinoshita ◽

M. Kazama ◽

A. Igata ◽

T. Abe

Keyword(s):

Molecular Weight ◽

Platelet Aggregation ◽

Deae Cellulose ◽

Factor Xa ◽

Coagulation Factor ◽

Suppressive Effect ◽

Induce Platelet Aggregation ◽

Platelet Suspension ◽

Trimeresurus Flavoviridis ◽

Induced Aggregation

The snake “HABU” venom was divided into subfractions and their influence on both platelet and coagulation were studied. Four fractions were obtained by gel exclusion chromatography on Sephadex G75 of which molecular weight ranged from 100,000 to 5,000. The fraction of the largest molecular weight was further divided into four subfractions; I–1, I–2, I–3 and I–4 by DEAE cellulose chromatography. The subfraction I–1 showed no activity to induce platelet aggregation but suppressed ADP, collagen and ristocetin-induced aggregation. The subfraction I–2 and I–3 equally induced the aggregation of plasma platelet and gel filtrated platelet suspension. The subfraction I–4 had the same effect on the platelet aggregation except for the specific suppressive effect on the restocetin aggregation. The subfraction I–3 was found to have Xa like activity which was revealed using chromogenic substance (S–2222). This activity was not inhibited by purified human antithrombin III, α2-macroglobulin or α1-antitrypsin. The relationship between activites of these fractions on platelet and coagulation suggested that they are different from any known coagulation factor.HABU--- Trimeresurus flavoviridis Hallowell

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Aggretin, a novel platelet-aggregation inducer from snake (Calloselasma rhodostoma) venom, activates phospholipase C by acting as a glycoprotein Ia/IIa agonist

Biochemical Journal ◽

10.1042/bj3091021 ◽

1995 ◽

Vol 309 (3) ◽

pp. 1021-1027 ◽

Cited By ~ 66

Author(s):

T F Huang ◽

C Z Liu ◽

S H Yang

Keyword(s):

Platelet Aggregation ◽

Phospholipase C ◽

Platelet Rich Plasma ◽

Shape Change ◽

Exchange Chromatography ◽

Ionic Exchange ◽

Platelet Suspension ◽

Calloselasma Rhodostoma ◽

Induced Aggregation ◽

Platelet Aggregation Inducer

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.

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Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651597 ◽

1993 ◽

Vol 69 (03) ◽

pp. 286-292 ◽

Cited By ~ 13

Author(s):

Che-Ming Teng ◽

Feng-Nien Ko ◽

Inn-Ho Tsai ◽

Man-Ling Hung ◽

Tur-Fu Huang

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Inositol Phosphate ◽

Shape Change ◽

Platelet Activating Factor ◽

Atp Release ◽

Platelet Membrane ◽

Thromboxane B2 ◽

Dependent Manner ◽

Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

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Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus binds to platelets and inhibits platelet aggregation

Thrombosis and Haemostasis ◽

10.1160/th03-05-0287 ◽

2004 ◽

Vol 91 (04) ◽

pp. 779-789 ◽

Cited By ~ 24

Author(s):

Oonagh Shannon ◽

Jan-Ingmar Flock

Keyword(s):

Platelet Aggregation ◽

Potent Inhibitor ◽

Specific Binding ◽

Binding Protein ◽

Dependent Manner ◽

Platelet Surface ◽

Putative Receptor ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Dose Dependent

Summary S. aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have shown here that Efb is a potent inhibitor of platelet aggregation. Efb can bind specifically to platelets by two mechanisms; 1) to fibrinogen naturally bound to the surface of activated platelets and 2) also directly to a surface localized component on the platelets. This latter binding of Efb is independent of fibrinogen. The specific binding of Efb to the putative receptor on the platelet surface results in a stimulated, non-functional binding of fibrinogen in a dose dependent manner, distinct from natural binding of fibrinogen to platelets. The natural binding of fibrinogen to GPIIb/IIIa on activated platelets could be blocked by a monoclonal antibody against this integrin, whereas the Efb-mediated fibrinogen binding could not be blocked. The enhanced Efb-dependent fibrinogen binding to platelets is of a nature that does not promote aggregation of the platelets; instead it inhibits aggregation. The anti-thrombotic action of Efb may explain the effect of Efb on wound healing, which is delayed in the presence of Efb.

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A New Platelet-Aggregation-Inhibiting Factor Isolated fromBothrops moojeniSnake Venom

BioMed Research International ◽

10.1155/2017/4315832 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Bruna Barbosa de Sousa ◽

Carla Cristine Neves Mamede ◽

Mariana Santos Matias ◽

Déborah Fernanda da Cunha Pereira ◽

Mayara Ribeiro de Queiroz ◽

...

Keyword(s):

Platelet Aggregation ◽

Snake Venom ◽

Platelet Rich Plasma ◽

Functional Characterization ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Single Chain ◽

Inhibiting Factor ◽

Reducing Conditions ◽

Bothrops Moojeni

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor fromBothrops moojenisnake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs fromB. moojenisnake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders.

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Both Complement- and Fibrinogen-Dependent Mechanisms Contribute to Platelet Aggregation Mediated by Staphylococcus aureus Clumping Factor B

Infection and Immunity ◽

10.1128/iai.01993-06 ◽

2007 ◽

Vol 75 (7) ◽

pp. 3335-3343 ◽

Cited By ~ 50

Author(s):

Helen Miajlovic ◽

Anthony Loughman ◽

Marian Brennan ◽

Dermot Cox ◽

Timothy J. Foster

Keyword(s):

Staphylococcus Aureus ◽

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Lag Time ◽

Dependent Manner ◽

Wild Type ◽

Factor B ◽

Fibrinogen Binding ◽

Aggregation Of Platelets ◽

Dependent Mechanism

ABSTRACT Staphylococcus aureus can stimulate activation and aggregation of platelets, which are thought to be factors in the development of infective endocarditis. Previous studies have identified clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as potent platelet aggregators. These proteins are able to stimulate rapid platelet aggregation by either a fibrinogen- or a fibronectin-dependent process which also requires antibodies specific to each protein. Slower aggregation has been seen in other systems where specific fibrinogen binding ligands are absent and platelet aggregation is mediated by complement and specific antibodies. Bacteria expressing ClfB aggregate platelets with a longer lag time than ClfA or FnBPA and FnBPB. In order to investigate whether ClfB causes platelet aggregation in a complement- or fibrinogen-dependent manner, a non-fibrinogen-binding mutant of ClfB (ClfB Q235A) was constructed. Lactococcus lactis expressing ClfB Q235A was able to stimulate platelet aggregation in platelet-rich plasma without a significant increase in lag time. The requirements for platelet aggregation were investigated using gel-filtered platelets. Fibrinogen and specific anti-ClfB antibodies were found to be sufficient to allow platelet aggregation mediated by the wild-type ClfB protein. It seems that ClfB causes platelet aggregation by a fibrinogen-dependent mechanism. The non-fibrinogen-binding ClfB mutant was unable to stimulate platelet aggregation under these conditions. However, bacteria expressing ClfB Q235A caused platelet aggregation in a complement-dependent manner which required specific anti-ClfB antibodies.

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Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation

Blood ◽

10.1182/blood.v79.8.1995.1995 ◽

1992 ◽

Vol 79 (8) ◽

pp. 1995-2003 ◽

Cited By ~ 39

Author(s):

C Legrand ◽

V Thibert ◽

V Dubernard ◽

B Begault ◽

J Lawler

Keyword(s):

Platelet Aggregation ◽

Binding Domain ◽

Platelet Glycoprotein ◽

Dependent Manner ◽

Heparin Binding ◽

Platelet Surface ◽

Amino Terminal ◽

Activated Platelets ◽

Heparin Binding Domain ◽

Induced Aggregation

Abstract We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.

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Requirement Of Fibrinogen And Calcium For Inter-Platelet Bridges In Platelet Aggregation: A Hypothesis

10.1055/s-0038-1652903 ◽

1981 ◽

Author(s):

Elizabeth Kornecki ◽

Stefan Niewiarowski

Keyword(s):

Platelet Aggregation ◽

Binding Sites ◽

Proteolytic Enzymes ◽

Divalent Cations ◽

Platelet Surface ◽

Fibrinogen Binding ◽

Platelet Aggregates ◽

High Concentrations ◽

Induced Aggregation ◽

Aggregation Of Platelets

Fibrinogen and calcium are required for the aggregation of platelets stimulated by ADP or pre-treated with proteolytic enzymes. Specific platelet surface fibrinogen binding sites (receptors) are exposed after platelets are stimulated by ADP or pre-treated with Chymotrypsin or pronase. It has previously been shown in our laboratory that an intact, symmetrical fibrinogen molecule is essential for fibrinogen binding and fibrinogen-induced aggregation of both ADP-stimulated and proteolytically-treated platelets. Here we propose that the mechanism by which fibrinogen and calcium aggregate platelets is by forming inter-platelet bridges linking the fibrinogen receptors of adjacent platelets together. In support of this proposition are the following new lines of evidence: 1) The fibrinogen-induced aggregations of ADP-stfiliulated or proteolytically-treated platelets are inhibited by high concentrations of fibrinogen (Ki=2.6 and 8.5 × 10 5M, respectively). The fibrinogen binding sites on adjacent platelets, at these concentrations, would be saturated by fibrinogen and therefore no inter-platelet fibrinogen bridges could be formed to hold the platelets together. 2) ADP-stimulated or chymotrypsin-treated platelets aggregated by fibrinogen are deaggregated by Chymotrypsin or pronase and this deaggregation coincides with the loss of 125I-fibrinogen from the platelet surface. 3) Preincubation of platelets with EDTA results in inhibition of both platelet aggregation and 125I-fibrinogen binding. Following the aggregations of ADP-stimulated or of chymotrypsin-treated platelets by fibrinogen, the addition of EDTA to the platelet aggregates results in both their deaggregation and their loss of bound 125I-fibrinogen. Thus it appears that divalent cations, especially calcium, are essential for the formation of fibrinogen-linked platelet aggregates.

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Recognition of platelet-associated fibrinogen by polyclonal antibodies: correlation with platelet aggregation

Blood ◽

10.1182/blood.v79.8.2028.bloodjournal7982028 ◽

1992 ◽

Vol 79 (8) ◽

pp. 2028-2033

Author(s):

EI Peerschke

Keyword(s):

Platelet Aggregation ◽

Time Course ◽

Polyclonal Antibodies ◽

Surface Membrane ◽

Adenosine Diphosphate ◽

Dependent Manner ◽

Membrane Structures ◽

Platelet Surface ◽

Fibrinogen Binding ◽

Qualitative Changes

Progressive decreases in platelet-bound fibrinogen accessibility to antibody and enzymes were recently reported to occur after adenosine diphosphate (ADP)-induced fibrinogen binding. Because previous studies also indicated that platelets that are activated but not aggregated by ADP in the presence of fibrinogen lose their ability to aggregate in a time-dependent manner despite negligible changes in fibrinogen binding, the present study examined the relationship between platelet aggregation and accessibility of platelet-bound fibrinogen to specific polyclonal antibody F(ab')2 fragments over a 60-minute time course. Although 125I-fibrinogen binding remained virtually unchanged, comparison of antifibrinogen antibody F(ab')2 binding and platelet aggregation 5 minutes and 60 minutes after platelet stimulation with ADP or thrombin showed decreases in F(ab')2 binding of 62% +/- 13% and 73% +/- 7% (mean +/- SD, n = 5), respectively, and decreases of 65% +/- 16% and 60% +/- 10% in platelet aggregation. In contrast, platelets stimulated with A23187 or chymotrypsin retained 87% +/- 16% and 76% +/- 12% of their ability to aggregate over the same time course, and lost only 39% +/- 14% and 36% +/- 12% of their ability to bind antifibrinogen antibody F(ab')2 fragments, respectively. Pretreatment of ADP-stimulated platelets with chymotrypsin largely prevented the progressive loss of platelet aggregability and the accompanying decreased recognition of bound fibrinogen by antifibrinogen F(ab')2 fragments. Preincubation of platelets with cytochalasin D (30 micrograms/mL) also inhibited the decrease in platelet aggregation after exposure of ADP-treated platelets to fibrinogen over a 60-minute time course. This was accompanied by only a 25% +/- 18% decrease in antifibrinogen antibody F(ab')2 binding. Present data support the hypothesis that qualitative changes in platelet-bound fibrinogen correlate with loss of the ability of platelets to aggregate, and implicate both the platelet cytoskeleton and chymotrypsin-sensitive surface membrane structures in modulating qualitative changes in bound fibrinogen on the platelet surface.

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