Dysfunction of the PI3 kinase/Rap1/integrin αIIbβ3 pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia

Blood ◽  
2013 ◽  
Vol 121 (7) ◽  
pp. 1209-1219 ◽  
Author(s):  
Samantha F. Moore ◽  
Roger W. Hunter ◽  
Matthew T. Harper ◽  
Joshua S. Savage ◽  
Samreen Siddiq ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Signaling Pathway ◽  
Essential Thrombocythemia ◽  
Clinical Observation ◽  
Ex Vivo ◽  
Pi3 Kinase ◽  
Integrin Αiibβ3 ◽  
Key Points

Key Points Platelets from essential thrombocythemia patients have an intrinsic impairment in the P13kinase/Rap1/integrin αIIbβ3 signaling pathway. This explains the clinical observation that in vitro platelet aggregation is impaired in patients with essential thrombocythemia.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

The d-Enantiomer Form of Indobufen Totally Accounts for the Anti-Cyclooxygenase and Antiplatelet Activity Ex Vivo and for the Increase in Bleeding Time by Indobufen in Man

1992 ◽  
Vol 67 (02) ◽  
pp. 258-263 ◽  
Author(s):  
Raffaele De Caterina ◽  
Rosa Sicari ◽  
An Yan ◽  
Walter Bernini ◽  
Daniela Giannessi ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Plasma Levels ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Random Sequence ◽  
Antiplatelet Agent ◽  
Antiplatelet Drug ◽  
Double Blind

SummaryIndobufen is an antiplatelet drug able to inhibit thromboxane production and cyclooxygenase-dependent platelet aggregation by a reversible inhibition of cyclooxygenase. Indobufen exists in two enantiomeric forms, of which only d-indobufen is active in vitro in inhibiting cyclooxygenase. In order to verify that also inhibition of platelet function is totally accounted for by d-indobufen, ten patients with proven coronary artery disease (8 male, 2 female, age, mean ± S.D., 58.7 ± 7.5 years) were given, in random sequence, both 100 mg d-indobufen and 200 mg dl-indobufen as single administrations in a double-blind crossover design study with a washout period between treatments of 72 h. In all patients thromboxane (TX) B2 generation after spontaneous clotting (at 0, 1, 2, 4, 6, 8, 12, 24 h), drug plasma levels (at the same times), platelet aggregation in response to ADP, adrenaline, arachidonic acid, collagen, PAF, and bleeding time (at 0, 2, 12 h) were evaluated after each treatment. Both treatments determined peak inhibition of TXB2 production at 2 h from administration, with no statistical difference between the two treatments (97 ±3% for both treatments). At 12 h inhibition was 87 ± 6% for d-indobufen and 88 ± 6% for dl-indobufen (p = NS). Inhibition of TXB2 production correlated significantly with plasma levels of the drugs. Maximum inhibitory effect on aggregation was seen in response to collagen 1.5 pg/ml (63 ± 44% for d-indobufen and 81 ± 22% for dl-indobufen) and arachidonic acid 0.5-2 mM (78 ± 34% for d-indobufen and 88 ± 24% for dl-indobufen) at 2 h after each administration. An effect of both treatments on platelet aggregation after 12 h was present only for adrenaline 2 μM (55 ± 41% for d-indobufen and 37 ± 54% for dl-indobufen), collagen 1.5 pg/ml (69 ± 30% for d-indobufen and 51 ± 61% for dl-indobufen), arachidonic acid 0.5-2 mM (56 ± 48% for d-indobufen and 35 ± 49% for dl-indobufen). The extent of inhibition of TX production and the extent of residual platelet aggregation were never significantly different between treatments. Bleeding time prolongation was similar in the two treatment groups without showing a pronounced and long lasting effect (from 7.0 ± 2.0 min to 10.0 ± 3.0 min at 2 h and 8.0 ± 2.0 min at 12 h for d-indobufen; from 6.0 ±1.0 min to 8.5 ± 2.0 min at 2 h and 8.0 ± 1.0 min at 12 h for dl-indobufen). These results demonstrate that the biological activity of dl-indobufen as an antiplatelet agent in vivo is totally accounted for by d-indobufen.

Dipyridamole Inhibits Platelet Aggregation in Whole Blood

1983 ◽  
Vol 50 (04) ◽  
pp. 852-856 ◽  
Author(s):  
P Gresele ◽  
C Zoja ◽  
H Deckmyn ◽  
J Arnout ◽  
J Vermylen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Significant Negative Correlation ◽  
Significant Inhibition ◽  
Oral Drug ◽  
Human Blood Samples ◽  
Induced Aggregation

SummaryDipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine- 5’-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p <0.001). A statistically significant inhibition of both collagen (p <0.0025) and ADP-induced (p <0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37° C with dipyridamole (3.9 μM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood.Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.

scholarly journals ROLE OF NITRIC OXIDE (NO) IN CAPSAICIN MEDIATED ANTI-PLATELET ACTIVITY IN IN VITRO, IN VIVO, EX-VIVO MODEL OF PLATELET AGGREGATION ASSAY AND ARTERIAL THROMBOSIS IN RAT: POTENTIAL THERAPEUTIC TARGET?

2018 ◽  
Vol 10 (4) ◽  
pp. 44
Author(s):  
Mihir K Patel ◽  
Kiranj K. Chaudagar ◽  
Anita A. Mehta
Keyword(s):  
Platelet Aggregation ◽  
Arterial Thrombosis ◽  
Ex Vivo ◽  
Platelet Activity ◽  
Aggregation Assay ◽  
Thrombosis Model ◽  
Platelet Aggregation Assay

Objective: Although recent advances in the treatment of congestive heart disease, mortality among patients’ remains a questionable remark. Therefore, we evaluated the role of capsaicin on in vitro and ex vivo platelet aggregation induced by Adenosine Di-Phosphate (ADP) as well as in in vivo thrombosis models and role of NO, KATP was also identified in the capsaicin-induced anti-platelet animal model as well as in vivo model of arterial thrombosis.Methods: According to body weight wistar rats were divided into five groups. Group I and Group II was treated with saline and capsaicin (3 mg/kg, i. v), while animals from Group III were treated with N(ω)-nitro-L-arginine methyl ester (L-NAME) (30 mg/kg, i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group IV animals were treated with glibenclamide (10 mg/kg,i. v) 30 min before administration of capsaicin (3 mg/kg, i. v). Group V was considered as a positive control and administered clopidogrel (30 mg/kg, p. o). Animals were subjected for in vitro, ex-vivo platelet aggregation assay. ADP (30µM) was utilized as an aggregating agent in these experiments. After these assays; animals of each group were subjected for subaqueous tail bleeding time in a rat model and FeCl3-induced arterial thrombosis model in rats.Results: In ADP-induced in vitro platelet aggregation, a significant reduction in % platelet aggregation was observed at 50µM (64.35±4.641) and 100µM (52.72±4.192) concentration of capsaicin as compared to vehicle control (85.82±3.716). Capsaicin (3 mg/kg, i. v) also showed a significant reduction (49.53±4.075) in ex-vivo ADP-induced platelet aggregation as compared to vehicle control (89.38±2.057). In FeCl3 induced arterial thrombosis model, Capsaicin (3 mg/kg, i. v) exhibited an increase in time to occlusion in this rodent model and presence of the L-NAME and glibenclamide had inhibited the activity of capsaicin.Conclusion: In our study, capsaicin (50 µM, 100µM) exhibited potent anti-platelet activity in ADP-induced platelet aggregation, similarly capsaicin exhibited significant anti-platelet action in the ex-vivo study. Moreover, the presence of L-NAME and glibenclamide inhibited the anti-thrombotic and anti-platelet action of capsaicin. Therefore, it was concluded that NO and KATP may be involved in the anti-thrombotic action of capsaicin.

Comparison of the effect of coagulation and platelet function impairments on various mouse bleeding models

2014 ◽  
Vol 112 (08) ◽  
pp. 412-418 ◽  
Author(s):  
Nima Vaezzadeh ◽  
Ran Ni ◽  
Paul Y. Kim ◽  
Jeffrey I. Weitz ◽  
Peter L. Gross
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Saphenous Vein ◽  
Ex Vivo ◽  
Arterial Injury ◽  
Inhibitory Antibody ◽  
Study Objective ◽  
Tail Tip

SummaryHaemostatic impairments are studied in vivo using one of several murine bleeding models. However it is not known whether these models are equally appropriate for assessing coagulation or platelet function defects. It was our study objective to assess the performance of arterial, venous and combined arterial and venous murine bleeding models towards impaired coagulation or platelet function. Unfractionated heparin (UFH) or αIIbβ3 inhibitory antibody (Leo.H4) were administered to mice, and their effects on bleeding in saphenous vein, artery, and tail tip transection models were quantified and correlated with their effects on plasma clotting and ADP-induced platelet aggregation, respectively. All models exhibited similar sensitivity with UFH (EC50 dose = 0.19, 0.13 and 0.07 U/g, respectively) (95% CI = 0.14 – 0.27, 0.08 – 0.20, and 0.03 – 0.16 U/g, respectively). Maximal inhibition of ex vivo plasma clotting could be achieved with UFH doses as low as 0.03 U/g. In contrast, the saphenous vein bleeding model was less sensitive to αIIbβ3 inhibition (EC50 = 6.9 µg/ml) than tail transection or saphenous artery bleeding models (EC50 = 0.12 and 0.37 µg/ml, respectively) (95% CI = 2.4 – 20, 0.05 – 0.33, and 0.06 – 2.2 µg/ml, respectively). The EC50 of Leo.H4 for ADP-induced platelet aggregation in vitro (8.0 µg/ml) was at least 20-fold higher than that of the tail and arterial, but not the venous bleeding model. In conclusion, venous, arterial and tail bleeding models are similarly affected by impaired coagulation, while platelet function defects have a greater influence in models incorporating arterial injury.

scholarly journals Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme fromLachesis muta rhombeataSnake Venom

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Frank Denis Torres-Huaco ◽  
Cláudio C. Werneck ◽  
Cristina Pontes Vicente ◽  
Talita Vassequi-Silva ◽  
Ana Cláudia Coelho Nery-Diez ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Proteinase Inhibitors ◽  
Ex Vivo ◽  
Serine Proteinase ◽  
Dependent Manner ◽  
Amidolytic Activity ◽  
Venom Component ◽  
Purification Method ◽  
Rapid Purification

We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) fromLachesis muta rhombeatavenom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts,in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation

Blood ◽  
2013 ◽  
Vol 122 (23) ◽  
pp. 3825-3831 ◽  
Author(s):  
Francesca Nuzzo ◽  
Claudia Radu ◽  
Marco Baralle ◽  
Luca Spiezia ◽  
Tilman M. Hackeng ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Factor V ◽  
Splicing Mutation ◽  
Splicing Defect ◽  
Factor V Deficiency ◽  
Key Points ◽  
Life Threatening

Key Points Homozygosity for the F5 c.1296+268A>G splicing mutation causes life-threatening factor V deficiency. Mutation-specific antisense molecules can correct this splicing defect and restore factor V synthesis in the patient’s megakaryocytes.

scholarly journals Interleukin-4 Signaling Plays a Major Role in Urogenital Schistosomiasis-Associated Bladder Pathogenesis

2019 ◽  
Vol 88 (3) ◽  
Author(s):  
Evaristus C. Mbanefo ◽  
Chi-Ling Fu ◽  
Christina P. Ho ◽  
Loc Le ◽  
Kenji Ishida ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Signaling Pathway ◽  
Ex Vivo ◽  
Bladder Wall ◽  
Interleukin 4 ◽  
Wild Type ◽  
Urogenital Schistosomiasis ◽  
Content Type ◽  
Helminth Infections

ABSTRACT Interleukin-4 (IL-4) is crucial in many helminth infections, but its role in urogenital schistosomiasis, infection with Schistosoma haematobium worms, remains poorly understood due to a historical lack of animal models. The bladder pathology of urogenital schistosomiasis is caused by immune responses to eggs deposited in the bladder wall. A range of pathology occurs, including urothelial hyperplasia and cancer, but associated mechanisms and links to IL-4 are largely unknown. We modeled urogenital schistosomiasis by injecting the bladder walls of IL-4 receptor-alpha knockout (Il4ra−/−) and wild-type mice with S. haematobium eggs. Readouts included bladder histology and ex vivo assessments of urothelial proliferation, cell cycle, and ploidy status. We also quantified the effects of exogenous IL-4 on urothelial cell proliferation in vitro, including cell cycle status and phosphorylation patterns of major downstream regulators in the IL-4 signaling pathway. There was a significant decrease in the intensity of granulomatous responses to bladder-wall-injected S. haematobium eggs in Il4ra−/− versus wild-type mice. S. haematobium egg injection triggered significant urothelial proliferation, including evidence of urothelial hyper-diploidy and cell cycle skewing in wild-type but not Il4ra−/− mice. Urothelial exposure to IL-4 in vitro led to cell cycle polarization and increased phosphorylation of AKT. Our results show that IL-4 signaling is required for key pathogenic features of urogenital schistosomiasis and that particular aspects of this signaling pathway may exert these effects directly on the urothelium. These findings point to potential mechanisms by which urogenital schistosomiasis promotes bladder carcinogenesis.

In vitro and ex vivo effect of cimetidine on human platelet aggregation

1986 ◽  
Vol 42 (1) ◽  
pp. 113-114
Author(s):  
B. Gachályi ◽  
K. Tihanyi ◽  
Á. Vas ◽  
B. Nádas ◽  
A. Káldor
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Ex Vivo ◽  
Human Platelet Aggregation

Novel 12-LOX Inhibitor ML355 Attenuates Platelet Reactivity and Impairs Thrombus Growth, Stability and Vessel Occlusion In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3442-3442 ◽  
Author(s):  
Reheman Adili ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Vessel Occlusion ◽  
Growth Stability

Abstract Background: Adequate platelet reactivity is required for platelet adhesion and aggregation at the site of vascular injury to maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi, the predominate underlying cause of myocardial infarction and stroke. While current anti-platelet treatments limit platelet function, they often result in an increased risk of bleeding. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated by our lab and others to regulate PAR4 and GPVI-mediated platelet reactivity suggesting a role of 12-LOX in regulation of vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Aims: To determine how 12-LOX regulates thrombus formation in vivo and whether platelet 12-LOX is an effective target for anti-platelet therapeutics, wild-type (WT) or 12-LOX deficient (12-LOX-/-) mice were treated with or without the 12-LOX inhibitor, ML355, and were assessed for inhibitory effects on platelet activation in vitro, ex-vivo and in vivo. Methods: The effect of the novel 12-LOX inhibitor ML355 on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber. In vivo thrombus formation and vessel occlusion in small and large vessels were studied in 12-LOX-/-, WT mice and mice treated with ML355 using intravital microscopy using the FeCl3 injury models. Results: Using in vitro platelet aggregation assays, ML355 dose dependently inhibited thrombin, PAR1-AP, and PAR4-AP-induced aggregation in washed human platelets. Interestingly, the negative regulatory effects of ML355 inhibition of 12-LOX can be overcome by high concentration of thrombin. Additionally, ML355 was able to attenuate ADP-induced platelet aggregation both in platelet-rich-plasma and whole blood. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX-/- mice was impaired in FeCl3-induced mesenteric or carotid artery thrombosis models. Thrombi in 12-LOX-/- mice were unstable and frequently form emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The highly selective 12-LOX inhibitor ML355 inhibits platelets aggregation induced by various platelet agonists and ML355 inhibition of platelet function is not agonist specific. Platelet function at high shear in ex vivo conditions in both mice and human was attenuated in the presence of ML355. Thrombus growth, stability, and vessel occlusion was impaired in mice deficient for 12-LOX. Finally, the highly selective 12-LOX inhibitor ML355 attenuates thrombus formation and prevents vessel occlusion in vivo. Our data strongly indicates 12- LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics. Disclosures No relevant conflicts of interest to declare.

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