Soy Isoflavones Inhibit Both GPIb-IX Signaling and αIIbβ3 Outside-In Signaling via 14-3-3ζ in Platelet

Soy diet is thought to help prevent cardiovascular diseases in humans. Isoflavone, which is abundant in soybean and other legumes, has been reported to possess antiplatelet activity and potential antithrombotic effect. Our study aims to elucidate the potential target of soy isoflavone in platelet. The anti-thrombosis formation effect of genistein and daidzein was evaluated in ex vivo perfusion chamber model under low (300 s−1) and high (1800 s−1) shear forces. The effect of genistein and daidzein on platelet aggregation and spreading was evaluated with platelets from both wildtype and GPIbα deficient mice. The interaction of these soy isoflavone with 14-3-3ζ was detected by surface plasmon resonance (SPR) and co-immunoprecipitation, and the effect of αIIbβ3-mediated outside-in signaling transduction was evaluated by western blot. We found both genistein and daidzein showed inhibitory effect on thrombosis formation in perfusion chamber, especially under high shear force (1800 s−1). These soy isoflavone interact with 14-3-3ζ and inhibited both GPIb-IX and αIIbβ3-mediated platelet aggregation, integrin-mediated platelet spreading and outside-in signaling transduction. Our findings indicate that 14-3-3ζ is a novel target of genistein and daidzein. 14-3-3ζ, an adaptor protein that regulates both GPIb-IX and αIIbβ3-mediated platelet activation is involved in soy isoflavone mediated platelet inhibition.

Download Full-text

Phospholipase C from Clostridium Perfringens Induces Human Platelet Aggregation in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642761 ◽

1988 ◽

Vol 59 (02) ◽

pp. 236-239 ◽

Cited By ~ 2

Author(s):

Giovanna Barzaghi ◽

Chiara Cerletti ◽

Giovanni de Gaetano

Keyword(s):

Platelet Aggregation ◽

Receptor Antagonist ◽

Phospholipase C ◽

Clostridium Perfringens ◽

Human Platelet ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Platelet Activating Factor ◽

Inhibitory Effect ◽

Thromboxane Receptor Antagonist

SummaryWe studied the aggregating effect of different concentrations of phospholipase C (PLC) (extracted from Clostridium perfringens) on human platelet-rich plasma (PRP). PRP was preincubated with PLC for 3 min at 37° C and the platelet aggregation was followed for 10 min. The threshold aggregating concentration (TAG) of PLC was 3-4 U/ml.We also studied the potentiation of PLC with other stimuli on platelet aggregation. Potentiating stimuli, such as arachidonic acid (AA), ADP. Platelet Activating Factor (PAF) and U-46619 (a stable analogue of cyclic endoperoxides) were all used at subthreshold concentrations. We also studied the possible inhibitory effect of aspirin, apyrase, TMQ, a prostaglandin endoper- oxide/thromboxane receptor antagonist and BN-52021, a PAF receptor antagonist. Only aspirin and apyrase were able to reduce aggregation induced by PLC alone and PLC + AA and PLC + ADP respectively. TMQ and BN-52021 were inactive. In ex vivo experiments oral aspirin (500 mg) partially inhibited platelet aggregation induced by PLC alone, PLC + AA and PLC + ADP 2 and 24 h after administration. Aspirin 20 mg for 7 days also reduced aggregation induced by PLC + AA.

Download Full-text

Anti-CD43 Inhibits Monocyte-Endothelial Adhesion in Inflammation and Atherogenesis

Blood ◽

10.1182/blood.v90.9.3587 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3587-3594 ◽

Cited By ~ 37

Author(s):

Leslie M. McEvoy ◽

Mark A. Jutila ◽

Philip S. Tsao ◽

John P. Cooke ◽

Eugene C. Butcher

Keyword(s):

Lymph Node ◽

Ex Vivo ◽

Inhibitory Effect ◽

Lymphoid Tissues ◽

Monocyte Adhesion ◽

Blood Monocytes ◽

High Endothelial Venules ◽

Aortic Endothelial Cells ◽

Novel Target ◽

And Migration

Abstract Recruitment of blood monocytes into tissues is a central event in the inflammatory response and in atherogenesis. The mechanisms leading to monocyte adhesion and migration through endothelium are not completely defined. We recently reported that MAb L11, against the leukocyte sialomucin CD43, blocks T-lymphocyte binding to lymph node and Peyer's patch high endothelial venules (HEV) and inhibits T-cell extravasation from the blood into organized secondary lymphoid tissues. We have now assessed the ability of L11 to inhibit monocyte-endothelial (EC) interactions and trafficking. L11 blocks binding of WEHI78/24 cells, a murine monocytoid cell line, to inflamed lymph node HEV and inhibits recruitment of monocytes and neutrophils to thioglycollate-inflamed peritoneum. Because monocyte adhesion to the endothelium and diapedesis in lesion-prone regions of the vasculature is among the earliest events in atherogenesis, leading to formation of lipid-laden foam cells, the ability of L11 to block monocyte recognition of aortic endothelial cells was assessed in a novel ex vivo assay of monocyte binding to intact rabbit aortic endothelium. Cholesterol feeding of rabbits induces enhanced aortic adhesiveness for monocytes and WEHI78/24 monocytoid cells, and this adhesion is inhibited by L11. The inhibitory effect of L11 is additive with that of a cocktail of anti–L-selectin and anti-α4 and β2 integrin monoclonal antibodies. Thus, CD43 represents a novel target for manipulation of monocyte recruitment in inflammation and atherogenesis.

Download Full-text

Ex vivo evaluation of anti-GPVI antibody in Cynomolgus monkeys: Dissociation between anti-platelet aggregatory effect and bleeding time

Thrombosis and Haemostasis ◽

10.1160/th06-05-0266 ◽

2006 ◽

Vol 96 (08) ◽

pp. 167-175 ◽

Cited By ~ 27

Author(s):

Yutaka Matsumoto ◽

Hisao Takizawa ◽

Kazuhiro Nakama ◽

Xiaoqi Gong ◽

Yoshihisa Yamada ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Thrombus Formation ◽

Inhibitory Effect ◽

Bleeding Tendency ◽

Fab Fragment ◽

Dose Escalation Study ◽

Cynomolgus Monkeys ◽

Induced Aggregation

SummaryRecent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (>80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0. 2 mg/kg with a slight prolongation of bleeding time (1. 3 times baseline value). Furthermore, at 18. 8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1. 9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5. 0 times at 0. 35 mg/kg, the lowest effective dose on platelet aggregation. Ina pharmacodynamic study,a bolus injection of OM2 at 0. 4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exerta potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.

Download Full-text

Effect of Sulphinpyrazone (SP) Aspirin (ASA) and Dipyridamole (DP) on Plateletvessel Wall Interaction after Oral Administration to Rabbits

10.1055/s-0039-1684734 ◽

1979 ◽

Author(s):

J.A. Davies ◽

V.C. Menys

Keyword(s):

Clinical Trials ◽

Platelet Aggregation ◽

Vessel Wall ◽

Platelet Adhesion ◽

Ex Vivo ◽

Inhibitory Effect ◽

Inhibitory Effects ◽

Human Dose ◽

Wall Interaction ◽

Adhesion Of Platelets

Clinical trials of anti-platelet drugs have suggested that they may be useful in the prevention of thrombotic disease. While such drugs inhibit platelet function, those which act on cyclooxygenase also reduce PGI2 synthesis and may interfere with tne natural antithrombotic properties of the vessel wall. We studied the effects of SP, ASA and OP ex vivo on the platelet-vessel wall interaction. Rabbits were dosed by mouth with drug (at about twice the weight-adjusted human dose) or placebo for 5 days, then exsanguinated and aortas removed. Washed platelets prepared from the blood were labelled with 51Cr. and their adhesion to everted aortapr epared from treated or control rabbits was measured in a perfusion device. PGI2-like activity in aortic rings was assayed by its inhibitory effect on platelet aggregation to ADP. Adhesion of platelets to aort as from SP- treated rabbits was i ncreased (p < 0.025), PGI2 - like activity was partially inhibited, but over all adhesion of SP-treated platelets to aor tas f rom SP-treated animals reduced by 30% (p < 0.02). Adhesion to aortas of ASA- treated rabbits was sliahtly inc r ea=-.ed (p > 0 . 1) , PGI 2 - l ike act ivi ty abolished , and no overall reduc tion in platelet adhesion seen. DP had no effecton adhesion or PGI-like activity. These results support the evidence that cyclo-oxygenase inhibitors reduce the inherent resistance of the vessel wall to platelet adhesion. However with SP, inhibitory effects on platelets appear to be more important.

Download Full-text

Inhibitory effect of a selective thromboxane A2 receptor antagonist, EP 092, on platelet aggregation in whole blood ex vivo and in vivo

British Journal of Pharmacology ◽

10.1111/j.1476-5381.1989.tb11830.x ◽

1989 ◽

Vol 96 (2) ◽

pp. 395-405 ◽

Cited By ~ 2

Author(s):

R.F.G. Booth ◽

A.C. Honey ◽

N. Lad ◽

D.P. Tuffin ◽

P.J. Wade

Keyword(s):

Platelet Aggregation ◽

Receptor Antagonist ◽

Whole Blood ◽

Ex Vivo ◽

Thromboxane A2 ◽

Inhibitory Effect ◽

Thromboxane A2 Receptor

Download Full-text

Inhibitory Effect of Piracetam on Platelet-rich Thrombus Formation in an Animal Model

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614243 ◽

1998 ◽

Vol 79 (01) ◽

pp. 222-227 ◽

Cited By ~ 8

Author(s):

F. Stockmans ◽

W. Deberdt ◽

Å. Nyström ◽

E. Nyström ◽

J. M. Stassen ◽

...

Keyword(s):

Platelet Aggregation ◽

Ex Vivo ◽

Peak Plasma ◽

Thrombus Formation ◽

Plasma Concentrations ◽

Inhibitory Effect ◽

Human Platelets ◽

Cell Deformability

SummaryIntravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 ± 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 ± 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests.In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50’s of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 ± 0.3 mM.A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced “spontaneous” platelet aggregation in human whole blood.It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.

Download Full-text

GUARANA (Paullinia cupana) INHIBITS AGGREGATION IN WHOLE BLOOD

10.1055/s-0038-1644553 ◽

1987 ◽

Author(s):

D A F Chamone ◽

M Ivany-Silva ◽

C Cassaro ◽

G Bellotti ◽

C Massumoto ◽

...

Keyword(s):

Platelet Aggregation ◽

Whole Blood ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Oral Intake ◽

Inhibitory Effect ◽

Impedance Method ◽

Paullinia Cupana ◽

Induced Aggregation

Guarana, a methylxanthine obtained from the seeds of Paullinia cupana has been largely used in the Amazon region by native indians during centuries as stimulant. We evaluated the effect of guarana on ex-vivo and in vitro platelet aggregation induced by adenosine-5-diphosphate (ADP) in human and rat whole blood with an impedance (Chrono-Log, model 500) and in their platelet rich plasma (PRP) with an optical aggregometer (Chrono-Log, model 440). Ex-vivo studies were carried out after single oral intake of guarana. Seven healthy volunteers (5 male and 2 female) aged 19-26 years who had taken no drugs for 10 days before, ingested 8gm of crude powder of guarana. Blood samples were drawn before and 1 hour after guarana intake. We observed a significative inhibition of platelet aggregation in whole blood meanwhile PRP was un changed as compared to basal values. In vitro studies were performed in whole blood and PRP from human volunteers and male Wis-tar rats. The combined effect of guarana and adenosine was also studied. A control aggregation was always run with saline. The results demonstrated an inhibition statistically significative (p < 0.001) of platelet aggregation in whole blood. Differently from whole blood the PRP with the same concentration of guarana did not result in inhibition of ADP induced aggregation when eva luated with the impedance method. The blood incubation with adenosine and guarana resulted in synergistic inhibitory effect that was much more strinking in whole blood than in PRP. Guarana fails to inhibit aggregation of rat platelets.Our results demonstrate that guarana prevents platelet aggregation in whole blood which depends on red blood cells, probably involving adenosine.

Download Full-text

Lu 23051, A New Potent Inhibitor Of Platelet Aggregation

10.1055/s-0038-1653265 ◽

1981 ◽

Author(s):

H D Lehmann ◽

J Gries ◽

D Lenke

Keyword(s):

Platelet Aggregation ◽

Ex Vivo ◽

Platelet Rich Plasma ◽

Coronary Vessels ◽

Inhibitory Effect ◽

Spontaneously Hypertensive ◽

Dependent Inhibition ◽

Induced Aggregation

6- [p-(2-(Chiorpropionylamino)phenyl] -4.5-dihydro-5-methyl-3(2H)-pyridazinone, LU 23051, is primarily characterized by its strong inhibition of platelet aggregation under in vitro and in vivo conditions. In vitro there is a concentration-dependent inhibition of ADP and collagen induced aggregation in platelet rich plasma of man, rat and dog. The inhibitory concentration EC 33 % is 0.0010-0.030 mg/1 (man: ADP-0.030, col 1.-0.013 mg/l) depending on species and type of aggregation. When administered orally in ex vivo experiments on rats and dogs the substance is found to have a dose-dependent antiaggregatory effect in the range from 0.1-3.16 mg/kg. The ED 33 % is 0.27-0.63 mg/kg.-In addition after oral administration the substance has a good inhibitory effect in models being based on intravascular platelet aggregation. Thus, a dose of 1 mg/kg inhibits laser-induced aggregation in mesenteric venules of rats. Mortality after i.v. injection of collagen in mice is reduced by 50 % after a dose of 0.02 mg/kg. A dose of 0.039 mg/kg prolongs the bleeding time of rats by 50 %. The aggregation-inhibiting action is of long duration (0.1 mg/kg p.o.∼24 h). The substance does not interfere with clotting.Besides its effect on platelet aggregation LU 23051 acts as vasodilatator as well. Dilatation of coronary vessels by 100 % is seen in isolated guinea-pig hearts at a concentration of 0.1 mg/l. In spontaneously hypertensive rats the substance has an anti hypertensive effect. The ED 20 % is 0.36 mg/kg p.o.The combination of antiaggregatory and vasodilatatory effects opens up interesting aspects with respect to the pharmacotherapeutic use of the new substance

Download Full-text

Abstract 10018: High on Aspirin Platelet Reactivity in Children Undergoing the Fontan Procedure

Circulation ◽

10.1161/circ.132.suppl_3.10018 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Jason T Patregnani ◽

Yaser Diab ◽

Darren Klugman ◽

Michael Spaeder ◽

Robert Freishtat ◽

...

Keyword(s):

Platelet Aggregation ◽

Patient Population ◽

Ex Vivo ◽

Platelet Reactivity ◽

Fontan Procedure ◽

Significant Proportion ◽

Platelet Inhibition ◽

Platelet Aggregation Inhibition ◽

Increased Risk ◽

Aggregation Inhibition

Introduction: Despite routine thromboprophylaxis with aspirin, thromboembolism (TE) continues to be a significant complication after the Fontan procedure. Data suggest that suboptimal ex-vivo aspirin-induced platelet inhibition-known as High on-Aspirin Platelet Reactivity (HAPR) is an independent risk factor for breakthrough TE. The occurrence of HAPR may potentially increase the risk of aspirin prophylaxis failure in children who underwent Fontan procedure. However, HAPR has not been adequately studied in this patient population. Hypothesis: There is a high incidence of HAPR in children after the Fontan procedure. Methods: We conducted a single-center, prospective, observational pilot study of children requiring Fontan procedure. Thromboelastography with platelet mapping (TEG-PM), platelet CD62P expression by flow-cytometry and urinary 11-dehydro-thromboxane B2 (11-dhTxB2) were measured at baseline prior to starting aspirin at 5 mg/kg/day and after at least 5 days of continuous therapy. HAPR was defined as less than 30% Arachidonic Acid (AA)-induced platelet aggregation inhibition as measured by TEG-PM. Results: 17 patients (11 males and 6 females, median age 3 years) were enrolled. HAPR was identified in 9/17 (53%) with 3/9 (33%) patients exhibiting no detectable AA-induced platelet aggregation inhibition. There was no significant effect of aspirin on percentage of activated platelets (i.e.: platelets expressing CD62P marker) in children with or without HAPR. In contrast to children with HAPR, aspirin therapy in children without HAPR led to significant reduction in urinary 11-dhTxB2 levels (Table 1). Conclusions: A significant proportion of children with single ventricle physiology exhibited HAPR in the immediate post-operative period after Fontan procedure. Further studies are needed to examine association of HAPR with increased risk of TE in this patient population.

Download Full-text