Antiplatelet and Antithrombotic Activity of SL65.0472, a Mixed 5-HT1B/5-HT2A Receptor Antagonist

2001 ◽  
Vol 85 (03) ◽  
pp. 521-528 ◽  
Author(s):  
Janine Lorrain ◽  
Sylvette Lochot ◽  
Monique Delahaye ◽  
Alain Lalé ◽  
Pierre Savi ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Platelet Activation ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Shape Change ◽  
Antithrombotic Activity ◽  
Thrombosis Model ◽  
Artery Thrombosis ◽  
Ic50 Values

SummaryThe antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-dihydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 μM 5-HT + threshold concentrations of ADP (0.5-1 M) or collagen (0.3 g/ml) with mean IC50 values of 49 ± 13 and 48 ± 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p. o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p. o. SL65.0472 (30 μg/kg i. v.) virtually abolished coronary cyclic flow variations (7.2 ± 1.0/h to 0.6 ± 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 ± 0.8 ml/ min to 31.8 ± 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p. o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Abstract Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4222-4231 ◽  
Author(s):  
Anna Shcherbina ◽  
Eileen Remold-O’Donnell
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Human Platelet ◽  
Thrombus Formation ◽  
Shape Change ◽  
Vascular Wall ◽  
Human Platelets ◽  
Granule Secretion ◽  
Platelets Function

Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

Phospholipase C from Clostridium Perfringens Induces Human Platelet Aggregation in Plasma

1988 ◽  
Vol 59 (02) ◽  
pp. 236-239 ◽  
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.

Inhibition of ADP-Induced Responses in Human Platelets by Agents Elevating the Cyclic AMP Level: Comparison of Aggregation and Shape Change

1984 ◽  
Vol 52 (03) ◽  
pp. 333-335 ◽  
Author(s):  
Vider M Steen ◽  
Holm Holmsen
Keyword(s):  
Inhibitory Action ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Early Step ◽  
Stimulus Response ◽  
Sequential Relationship

SummaryThe inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses.Human platelet-rich plasma was preincubated for 2 min at 37° C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors.These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.

Comparative Studies of an Orally-active Factor Xa Inhibitor, YM-60828, with other Antithrombotic Agents in a Rat Model of Arterial Thrombosis

1998 ◽  
Vol 79 (02) ◽  
pp. 410-416 ◽  
Author(s):  
Kazuo Sato ◽  
Yumiko Sakai ◽  
Fukushi Hirayama ◽  
Hiroyuki Koshio ◽  
Yuta Taniuchi ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Arterial Thrombosis ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Test Drug ◽  
Antithrombotic Agent ◽  
Antithrombotic Activity ◽  
Thrombosis Model ◽  
Treatment And Prevention ◽  
Orally Active

SummaryWe examined the antithrombotic activity of a novel synthetic inhibitor of factor Xa, YM-60828, in an electrically-induced carotid artery thrombosis model in rats. In the first experiment, the antithrombotic activity of YM-60828 after i.v. infusion was compared with those of heparin, darteparin and argatroban. Test drug was administered by i.v. infusion from 30 min before electrical stimulation to the end of the experiment. YM-60828 at 1 mg/kg/h significantly improved patency status, prolonged the time to occlusive thrombus formation and duration of patency. Heparin at 300 U/kg/h also improved these parameters, but were accompanied by a marked increase in systemic coagulation time. In the second experiment, the antithrombotic activity of YM-60828 after oral administration was compared with those of ticlopidine, cilostazol, aspirin, beraprost, ethyl icosapentate and warfarin. Test drug was orally administered to fasted rats 60 min before electrical stimulation. YM-60828 at 30 mg/kg p.o., but not ticlopidine, cilostazol, aspirin, beraprost, ethyl icosapentate or warfarin, significantly reduced the incidence of occlusion and improved carotid arterial patency. These results suggest that YM-60828 may be a promising antithrombotic agent for the treatment and prevention of arterial thrombosis which can be given by oral as well as intravenous administration.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

scholarly journals Ultrastructural Studies of Platelet Aggregates From Human Subjects Receiving Clopidogrel and From a Patient With an Inherited Defect of an ADP-Dependent Pathway of Platelet Activation

1996 ◽  
Vol 16 (12) ◽  
pp. 1532-1543 ◽  
Author(s):  
Michel Humbert ◽  
Paquita Nurden ◽  
Claude Bihour ◽  
Jean-Max Pasquet ◽  
Joëlle Winckler ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
High Dose ◽  
Platelet Response ◽  
Platelet Surface ◽  
Ultrastructural Studies ◽  
Contact Points ◽  
Platelet Aggregates

Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti–ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca 2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.

scholarly journals Kinetics of ADP-induced human platelet shape change: apparent positive cooperativity

1980 ◽  
Vol 58 (1) ◽  
pp. 45-52 ◽  
Author(s):  
John G. Milton ◽  
W. Yung ◽  
C. Glushak ◽  
M. M. Frojmovic
Keyword(s):  
Light Transmission ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Hill Coefficient ◽  
A Value ◽  
The Hill ◽  
Kinetics Of ◽  
Type Response ◽  
Platelet Shape

The kinetics of ADP-induced human platelet shape change have been examined. Initial velocities of platelet shape change were estimated by two methods: (1) the slope of the initial decrease in light transmission through stirred, citrated platelet-rich plasma, and (2) direct examination of platelet morphologies by phase-contrast microscopy. In both cases, a value of the Hill coefficient, NH, significantly greater than 1 is obtained (2.0 ± 0.2 and 1.8 ± 0.2, respectively). The observed elevated value of NH is not due to a substantial fraction of the ADP being platelet bound, the presence of factors in the plasma, platelet heterogeneity, or the influence of the rate of platelet shape change reversion. Our observations suggest that ADP-induced platelet shape change may be a positively cooperative or "threshold" type response.

scholarly journals Human platelet activation in the absence of aggregation: a calcium- dependent phenomenon independent of thromboxane formation

Blood ◽  
1982 ◽  
Vol 59 (5) ◽  
pp. 1078-1085 ◽  
Author(s):  
S Levy-Toledano ◽  
J Maclouf ◽  
P Bryon ◽  
E Savariau ◽  
RM Hardisty ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Ultrastructural Change ◽  
Creatine Phosphokinase ◽  
Light Transmission ◽  
Human Platelet ◽  
Shape Change ◽  
Creatine Phosphate ◽  
Intracellular Membrane ◽  
Calcium Dependent ◽  
Intracellular Membrane Transport

Abstract In response to ionophore A 23187, thrombasthenic and EDTA-treated control platelet-rich plasmas (PRP) undergo a change in light transmission (LT) accompanied by a normal 14C-serotonin (5HT) release and thromboxane (TX) synthesis in the absence of aggregation. Ultrastructural qualitative electron microscopy revealed central apposition of organelles and loosely packed platelets in both models, while a central gel mass appeared only in thrombasthenic patients. Quantitative analysis of this ultrastructural change showed an increase in the elongation and a decrease in the circularity coefficients of thrombasthenic platelets, indicating a shape change with pseudopod formation, while EDTA-treated platelets underwent a shape change in the absence of pseudopod formation. Morphometric analysis showed that the ionophore caused extensive degranulation in both types of platelets (decrease of the granule volume), which occurred in the presence of contraction of thrombasthenic PRP (decrease of the SCS plus granule volume) but in its absence in EDTA-treated platelets. The change in LT was not inhibited by aspirin, suggesting a dissociation between release of 14C-5HT and TX formation. Moreover, it was not inhibited by creatine phosphate plus creatine phosphokinase, prostaglandin E1, or cytochalasin and/or colchicine. It was not dependent on ADP, cAMP, or the integrity of microfilaments and microtubules. However, chlorpromazine, TMB 8, and dibucaine, which interfere with intracellular membrane transport of Ca2+, inhibited this platelet activation (change in LT, 14C-5HT release and TX synthesis.

EXPERIMENTAL THROMBUS FORMATION AND HAEMOSTASIS OF DIFFERENT LOW MOLECULAR WEIGHT HEPARINS AND DOSAGES

1987 ◽  
Author(s):  
R A Zimmerman ◽  
C T Rieger ◽  
K Hübner ◽  
C W Harenber ◽  
W Kübler
Keyword(s):  
Molecular Weight ◽  
Thrombus Formation ◽  
Factor Xa ◽  
Bleeding Complications ◽  
Maximum Effect ◽  
Low Molecular Weight ◽  
Antithrombotic Effect ◽  
Low Molecular Weight Heparins ◽  
Antithrombotic Activity ◽  
Thrombosis Model

Low molecular weight heparin induces a higher anti factor Xa (a-Xa) and a lower antithrombin activity in plasma in comparison to conventional heparin. From this constellation a more pronounced antithrombotic effect and a minor incidence of bleeding Complications has been suggested.Therefore the antithrombotic activity of heparins was studied in a standardized experimental thrombosis model in rabbits. Three low molecular weight heparins with a mean molecular weight of 4.200 (heparin I),4.000 (heparin II),4.600 Dalton (heparin III) and standard heparin were tested at different dosages in 120 experiments. In the first series the dose of 60 anti Xa units (a-Xa U) given initially and 60 a-Xa U/kg/h induced a reduction of the thrombus size by 40 % (heparin I),37 % (heparin II) and 53 % (heparin III) and a prolongation of the aPTT to 45 (heparin I),66 (heparin II) and 79 sec (heparin III). The a-Xa activity was minor than 0.1 U/ml. In the second series heparins were given to aim at an a-Xa activity of 0.2-0.3 U/ml. Thereby the thrombus formation could be reduced by 84 % (heparin I), 62 % (heparin II) and 39 % (heparin III). aPTT and a-Xa activity were measured at 65.5 sec and 0.22 a-Xa U/ml (heparin I),67.3 sec and 0.3 a-Xa U/ml (heparin II) and 67.5 and 0.31 a-Xa U/ml (heparin III),respectively. In the third series the increase of the a-Xa activity to more than 0.3 U/ml showed no further reduction of the thrombus formation by heparin I, while heparins II and III already at this level reachedthe antithrombotic activity of heparin I.Our data on three different low molecular weight heparins demonstrate that already a heparin level ranging at a minimal a-Xa activity induces a clear and statistically significant antithrombotic effect. A higher heparin dosage with higher a-Xa activity increases the antithrombitic effect. At a level of 0.2-0.3 a-Xa U/ml an obvious and maximum effect could be reached, but the further elevation of the a-Xa activity produced no further antithrombotic action.

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