A Novel Pharmacodynamic Perfusion Chamber Assay Reveals Superior and Unique Antithrombotic Activities of a Direct-Acting P2Y12 Antagonist, PRT128, Relative to Clopidogrel.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 900-900 ◽  
Author(s):  
Pamela Conley ◽  
Marzena Jurek ◽  
Gillian Stephens ◽  
Ming He ◽  
Alok Finn ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Antiplatelet Agents ◽  
Platelet Inhibition ◽  
Light Transmittance ◽  
Diabetic Patients ◽  
Perfusion Chamber ◽  
Direct Acting ◽  
P2y12 Antagonist

Abstract Pharmacodynamic evaluation of novel antiplatelet agents in clinical trials has traditionally proven problematic due to the inaccessibility of assays to measure the effects on platelet thrombosis in human blood. The present study utilized real-time perfusion chamber technology (RTTP) to compare the antithrombotic activities of PRT128, an orally available, direct-acting, reversible P2Y12 antagonist, to those achieved by chronic dosing of humans with clopidogrel (C, 75 mg/day, ± aspirin (A)). PRT128 was added in vitro to blood collected from healthy volunteers (n=20) at baseline (no treatment) and following A treatment, allowing comparison to C (±A) treatments. Blood samples were monitored using the RTTP assay, where thrombus formation was initiated by a combination of a collagen-coated surface and physiologic shear. 1.25 mM PRT128 provided equivalent inhibition of the thrombotic process achieved by chronic dosing of C (75 mg/d, 14 d)(±A, 325 mg/d, 7 d): higher concentrations (2.5, 5 mM) of PRT128 provided superior inhibition. Although neither P2Y12 antagonist alone affected the initial rate of thrombus formation, both destabilized growing thrombi, to a greater extent in the presence of aspirin, with larger effects achieved by the direct P2Y12 antagonist. Light transmittance platelet aggregation (PA) assays using platelet rich plasma (initiated by ADP (10 mM) or collagen (4 mg/ml)) indicated that the concentration of PRT128 which provided similar inhibition to C (±A) was 0.5–3 mM, while higher concentrations achieved greater inhibition. The RTTP assay was also used to determine the levels of inhibition of thrombosis achieved by C plus A treatment in 5 diabetic patients undergoing stent-placement. Interestingly, 2 of the 5 patients displayed a profile typical of a non-responder (i.e., no observed thrombus destabilization). However, when 2.5 or 5 mM PRT128 was added in vitro to blood from these patients, a clear antithrombotic effect was observed, indicating that the lack of benefit of C plus A therapy in these patients was due to C nonresponsiveness which could be circumvented by the direct-acting antagonist PRT128. Finally, a unique feature of a direct-acting P2Y12 antagonist was revealed using the RTTP assay. Thrombi formed by the perfusion of untreated whole blood for 3 minutes through the perfusion chamber were subsequently exposed to a perfusion of blood treated with PRT128. The second perfusion destabilized thrombi, illustrating the role of ADP acting on P2Y12 in maintaining thrombus stability. In summary, through use of novel perfusion chamber technology, we demonstrate that (1) PRT128, a direct-acting P2Y12 antagonist, can provide superior levels of platelet inhibition than achieved by C (±A), (2) PRT128 can overcome the limitations of C in diabetic patients undergoing PCI who appeared to be C non-responders, (3) direct-acting P2Y12 antagonism can induce destabilization of existing thrombi, and (4) the RTTP assay provides a unique tool for dose selection and evaluation of clinical indications for the further development of PRT128 and other novel antiplatelet agents.

The influence of variation in the P2Y12 receptor gene on in vitro platelet inhibition with the direct P2Y12 antagonist cangrelor

2010 ◽  
Vol 103 (02) ◽  
pp. 379-386 ◽  
Author(s):  
Jochem van Werkum ◽  
Goran Rude ◽  
Frank Leebeek ◽  
Adrian Kruit ◽  
Christian Hackeng ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Receptor Gene ◽  
Platelet Inhibition ◽  
P2y12 Receptor ◽  
P2y12 Inhibitor ◽  
P2y12 Inhibitors ◽  
P2y12 Antagonist ◽  
Single Snps

SummaryNovel P2Y12 inhibitors are in development to overcome the occurrence of atherothrombotic events associated with poor responsiveness to the widely used P2Y12 inhibitor clopidogrel. Cangrelor is an intravenously administered P2Y12 inhibitor that does not need metabolic conversion to an active metabolite for its antiplatelet action, and as a consequence exhibits a more potent and consistent antiplatelet profile as compared to clopidogrel. It was the objective of this study to determine the contribution of variation in the P2Y12 receptor gene to platelet aggregation after in vitro partial P2Y12 receptor blockade with the direct antagonist cangrelor. Optical aggregometry was performed at baseline and after in vitro addition of 0.05 and 0.25 μM cangrelor to the platelet-rich plasma of 254 healthy subjects. Five haplotype-tagging (ht)-SNPs covering the entire P2Y12 receptor gene were genotyped (rs6798347C>t, rs6787801T>c, rs9859552C>a, rs6801273A>g and rs2046934T>c [T744C]) and haplotypes were inferred. The minor c allele of SNP rs6787801 was associated with a 5% lower 20 μM ADP-induced peak platelet aggregation (0.05 μM cangrelor, p<0.05). Aa homozygotes for SNP rs9859552 showed 20% and 17% less inhibition of platelet aggregation with cangrelor when compared to CC homozygotes (0.05 and 0.25 μM cangrelor respectively; p<0.05). Results of the haplotype analyses were consistent with those of the single SNPs. Polymorphisms of the P2Y12 receptor gene contribute significantly to the interindividual variability in platelet inhibition after partial in vitro blockade with the P2Y12 antagonist cangrelor.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

Modified Technique for Measuring Platelet Aggregation

1997 ◽  
Vol 3 (3) ◽  
pp. 196-202 ◽  
Author(s):  
Alexander Kaplan ◽  
Svetlana Kaplan ◽  
Karen F. Marcoe ◽  
Lester R. Sauvage ◽  
William P. Hammond
Keyword(s):  
Platelet Aggregation ◽  
Citric Acid ◽  
Platelet Rich Plasma ◽  
Antiplatelet Agents ◽  
Current Approach ◽  
Molar Ratio ◽  
Maximum Height ◽  
Modified Technique ◽  
Aggregation Pattern

The turbidimetric method of Bom is the current approach for assessing the aggregation behavior of platelets. It has been of modest practical value, due to difficulty in standardizing laboratory techniques for plasma preparation and inadequate quantification of the aggregation process. We report a new technique of sedimented platelet rich plasma (SPRP) preparation that reduces the irregularities caused by factors associated with procuring and preparing blood samples and permits a more flexible protocol for laboratory practice. We quantified results with a platelet aggregation score, which is calculated by multiplying the ratio of the height of the initial wave of aggregation to the maximum height of the aggregation pattern by the total area under the aggregation curve and by the ratio of the whole blood platelet count and the number of platelets in the sample. Comparative analysis of platelet aggregation scores (n = 95) obtained with both plasma preparation techniques using a paired t test demonstrated no statistical differences ( t = 1.368, p = 0.174). To demonstrate the application of this modified method to evaluation of antiplatelet agents, the effects of aspirin and aspirin combined with citric acid on platelet aggregation were studied in vitro. The antiaggregatory effect of aspirin combined with citric acid was dependent on the pH and on their molar ratio, and was greater than the effect of aspirin alone. The SPRP protocol with platelet aggregation scoring methodology could be a valid alternative for measurement of the platelets' propensity to aggregate and the effect of antithrombotic treatments. Key Words: Platelet aggregation— Aspirin—Citric acid.

Platelet inhibition by aspirin is diminished in patients during carotid surgery: a form of transient aspirin resistance?

2004 ◽  
Vol 92 (07) ◽  
pp. 89-96 ◽  
Author(s):  
David Payne ◽  
Chris Jones ◽  
Paul Hayes ◽  
Sally Webster ◽  
A. Naylor ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Platelet Inhibition ◽  
Aspirin Resistance ◽  
Arachidonic Acid Metabolism ◽  
Significant Rise ◽  
Control Group

SummaryThe majority of patients who suffer peri-operative thromboembolic complication while undergoing vascular procedures do so despite taking aspirin. This study examined the antiplatelet effect of aspirin during surgery in patients undergoing carotid endarterectomy (CEA). Fifty patients undergoing CEA were standardised to 150 mg aspirin daily for ≥2 weeks. Platelet aggregation in response to arachidonic acid (AA) was measured in platelet rich plasma prepared from blood taken prior to, during, and at the end of surgery. Spontaneous platelet aggregation was also studied, as was the role of physiological agonists (ADP, collagen, thrombin, and epinephrine) in mediating the in vivo and in vitro responses to AA. Eighteen patients undergoing leg angioplasty, also on 150 mg aspirin, without general anaesthesia, served as a control group. In the CEA patients aggregation induced by AA (5 mM) increased significantly from 7.6 ± 5.5% pre-surgery to 50.8 ± 29.5% at the end of surgery (p <0.0001). Aggregation to AA was even greater in samples taken mid-surgery from a sub-set of patients (73.8 ± 7.2%; p = 0.0001), but fell to 45.9 ± 7.4% by the end of surgery. The increased aggregation in response to AA was not due to intra-operative release of physiological platelet agonists since addition of agents that block/neutralise the effects of ADP (apyrase; 4 µg/ml), thrombin (hirudin; 10 units/ml), or epinephrine (yohimbine; 10 µM/l) to the samples taken at the end of surgery did not block the increased aggregation.The patients undergoing angioplasty also showed a significant rise in the response to AA (5 mM), from 5.6 ± 5.5% pre-angioplasty to 32.4 ± 24.9% at the end of the procedure (p <0.0001), which fell significantly to 11.0 ± 8.1% 4 hours later. The antiplatelet activity of aspirin, mediated by blockade of platelet arachidonic acid metabolism, diminished significantly during surgery, but was partially restored by the end of the procedure without additional aspirin treatment.This rapidly inducible and transient effect may explain why some patients undergoing cardiovascular surgery remain at risk of peri-operative stroke and myocardial infarction.

scholarly journals A novel nucleotide-based thrombin inhibitor inhibits clot-bound thrombin and reduces arterial platelet thrombus formation

Blood ◽  
1994 ◽  
Vol 83 (3) ◽  
pp. 677-682 ◽  
Author(s):  
WX Li ◽  
AV Kaplan ◽  
GW Grant ◽  
JJ Toole ◽  
LL Leung
Keyword(s):  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Thrombin Inhibitors ◽  
Thrombin Inhibitor ◽  
Shear Rates ◽  
High Shear Rates ◽  
Dependent Inhibition ◽  
Platelet Thrombus

A novel thrombin inhibitor based on single-stranded (ss) deoxynucleotides with the sequence GGTTGGTGTGGTTGG (thrombin aptamer) has been recently discovered. In this study, we tested its efficacy in inhibiting clot-bound thrombin activity and platelet thrombus formation in an ex vivo whole artery angioplasty model. The thrombin aptamer showed a specific dose-dependent inhibition of thrombin-induced platelet aggregation (0.5 U/mL) in human platelet-rich plasma, with an IC50 of approximately 70 to 80 nmol/L. In an in vitro clot-bound thrombin assay system, heparin, used at clinically relevant concentrations of 0.2 U/mL and 0.4 U/mL, was ineffective in inhibiting clot-bound thrombin (6.5% and 34.9% inhibition at 0.2 U/mL and 0.4 U/mL, respectively). In contrast, the thrombin aptamer at an equivalent anticoagulant concentration inhibited clot-bound thrombin (79.7% inhibition). In an ex vivo whole artery angioplasty model, the thrombin aptamer markedly suppressed the generation of fibrinopeptide A (FPA), whereas heparin at 2 U/mL was ineffective. Compared with a scrambled ssDNA control, the thrombin aptamer reduced platelet deposition by 34.5% +/- 5% (mean +/- SEM, n = 4, P = .09) at low shear rates (approximately 200 s-1) and 61.3% +/- 11% (mean +/- SEM, n = 4, P = .05) at high shear rates (approximately 850 s-1). Thrombin aptamers based on ssDNA molecules represent a new class of thrombin inhibitors with potent anticoagulant and antithrombotic properties.

Studies on the Effect of Platelet Inhibitors on Platelet Adhesion to Collagen and Collagen-Induced Human Platelet Activation

1985 ◽  
Vol 53 (03) ◽  
pp. 337-342 ◽  
Author(s):  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Antiplatelet Agents ◽  
Calmodulin Antagonist ◽  
Release Reaction ◽  
Low Concentrations

SummaryThe effect of pyridoxal 5’-phosphate (PALP) and trifluoperazine (TFPZ), the calmodulin antagonist, on in vitro platelet adhesion to collagen and collagen-induced platelet activation was studied using platelet-rich-plasma (PRP) or washed platelets (WPL). Platelet aggregation and [14C]-5HT release induced by “threshold” or low concentrations of collagen (0.6 μg/ ml) in PRP were completely abolished by PALP (24 mM), TFPZ (250 μM) as well as indomethacin (10 μM). At higher concentrations of collagen (10–15 μg/ml) in PRP and WPL, the use of stirred and unstirred platelets treated with collagen enabled a distinction to be made between aggregation and adhesion- mediated release reaction. Platelet aggregation and the aggregation-mediated release reaction induced by these concentrations of collagen in stirred platelets were completely abolished by PALP, TFPZ and indomethacin although neither adhesion to collagen nor the adhesion-mediated release reaction of unstirred platelets was significantly affected by these inhibitors. Interestingly, both adhesion and the adhesion-mediated release reaction were abolished by concentrations of PALP 10–40 fold higher than those required to abolish aggregation. Collagen-induced platelet aggregation, but not platelet adhesion, was inhibited in resuspended platelets pretreated with PALP and NaBH4 indicating a separation in the membrane sites involved in aggregation and adhesion. The results further emphasize the distinction between adhesion and aggregation-mediated events with regards to collagen with the latter being more susceptible to inhibition by antiplatelet agents such as PALP and TFPZ.

Effect of fibrinogen concentration and platelet count on the inhibitory effect of abciximab and tirofiban

2003 ◽  
Vol 89 (02) ◽  
pp. 348-354 ◽  
Author(s):  
Giulia Renda ◽  
Bianca Rocca ◽  
Roberto Crocchiolo ◽  
Raimondo De Cristofaro ◽  
Raffaele Landolfi
Keyword(s):  
Platelet Count ◽  
Platelet Rich Plasma ◽  
Classical Model ◽  
Tight Binding ◽  
Receptor Occupancy ◽  
Platelet Inhibition ◽  
Inhibitory Effect ◽  
Fibrinogen Concentration ◽  
Wide Variability

SummaryGlycoprotein (Gp) IIb/IIIa blockers are powerful antithrombotic agents, but display a wide variability of their effect using weight-adjusted dosing. We investigated whether some hemostatic variables affected in vitro platelet inhibition exerted by the antibody abciximab and the peptidomimetic tirofiban. High fibrinogen concentrations reduced platelet inhibition by tirofiban, assessed in whole blood (r = –0.85, n = 10, p <0.01) and in platelet rich plasma (r = –0.89, n = 10, p <0.01) through PFA 100 and Born’s aggregometry assay, respectively. Both drugs were unaffected by von Willebrand factor levels, while platelet count was inversely related to their inhibitory effect (tirofiban: r = –0.9, n = 7, p <0.01; abciximab: r = –0.81, n = 9, p <0.01). Analysis of GpIIb-IIIa blockade showed that receptor occupancy at a fixed abciximab dose was inversely related to platelet counts. These experimental data were in agreement with the classical model of receptor saturation by a tight binding inhibitor.A role for fibrinogen and/or platelet count in influencing the antithrombotic properties of platelet GpIIb-IIIa antagonists can be hypothesized.

Anti-thrombotic effects of selective estrogen receptor modulator tamoxifen

2011 ◽  
Vol 106 (10) ◽  
pp. 624-635 ◽  
Author(s):  
Manasa Nayak ◽  
Sunil Singh ◽  
Arnab Roy ◽  
Vivek Prakash ◽  
Anand Kumar ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Present Report ◽  
Protein S ◽  
Collagen Matrix ◽  
Cancer Drug ◽  
Receptor Modulator

SummaryTamoxifen is a known anti-cancer drug and established estrogen receptor modulator. Few clinical studies have earlier implicated the drug in thrombotic complications attributable to lower anti-thrombin and protein S levels in plasma. However, action of tamoxifen on platelet signalling machinery has not been elucidated in detail. In the present report we show that tamoxifen is endowed with significant inhibitory property against human platelet aggregation. From a series of in vivo and in vitro studies tamoxifen was found to inhibit almost all platelet functions, prolong tail bleeding time in mouse and profoundly prevent thrombus formation at injured arterial wall in mice, as well as on collagen matrix perfused with platelet-rich plasma under arterial shear against the vehicle dimethylsulfoxide (DMSO). These findings strongly suggest that tamoxifen significantly downregulates platelet responses and holds potential as a promising anti-platelet / anti-thrombotic agent.

scholarly journals Antiplatelet Activity of Coumarins: In Vitro Assays on COX-1

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 3036
Author(s):  
Cristina Zaragozá ◽  
Francisco Zaragozá ◽  
Irene Gayo-Abeleira ◽  
Lucinda Villaescusa
Keyword(s):  
Platelet Rich Plasma ◽  
Antiplatelet Agents ◽  
Developed Countries ◽  
In Vitro Assays ◽  
Atherosclerotic Cardiovascular Disease ◽  
Antiplatelet Activity ◽  
Antiplatelet Aggregation ◽  
Aggregation Inhibition ◽  
Biochemical Mechanisms

Atherosclerotic cardiovascular disease is the leading cause of death in developed countries. Therefore, there is an increasing interest in developing new potent and safe antiplatelet agents. Coumarins are a family of polyphenolic compounds with several pharmacological activities, including platelet aggregation inhibition. However, their antiplatelet mechanism of action needs to be further elucidated. The aim of this study is to provide insight into the biochemical mechanisms involved in this activity, as well as to establish a structure–activity relationship for these compounds. With this purpose, the antiplatelet aggregation activities of coumarin, esculetin and esculin were determined in vitro in human whole blood and platelet-rich plasma, to set the potential interference with the arachidonic acid cascade. Here, the platelet COX activity was evaluated from 0.75 mM to 6.5 mM concentration by measuring the levels of metabolites derived from its activity (MDA and TXB2), together with colorimetric assays performed with the pure recombinant enzyme. Our results evidenced that the coumarin aglycones present the greatest antiplatelet activity at 5 mM and 6.5 mM on aggregometry experiments and inhibiting MDA levels.

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