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Evaluation of platelet function, a method comparison
Department of Molecular Pharmacology, Preclinical R&D, AstraZeneca R&D, Mölndal, Sweden.
Department of Molecular Pharmacology, Preclinical R&D, AstraZeneca R&D, Mölndal, Sweden.
Department of Integrative Pharmacology, Preclinical R&D, AstraZeneca R&D, Mölndal, Sweden.
Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2006 (English)In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 17, no 1, 49-55 p.Article in journal (Refereed) Published
Abstract [en]

Platelet function can be studied using many different methods why it is of interest to understand how data from different assays relate to each other. In the present study we compare two methods suitable for screening purposes with two established although laborious methods, impedance aggregometry and platelet-rich plasma (PRP) aggregation. The alternative assays tested were: (i) exposure of active αIIbβ3, in diluted whole blood and (ii) whole blood aggregation assessed by residual platelet counting. The fibrinogen receptor activation assay was found to have the lower variability, higher sensitivity to ADP, and higher signal to noise ratio compared with residual platelet counting. The sensitivity and response profile of the fibrinogen receptor activation assay and residual platelet counting were more similar to PRP aggregation than to impedance aggregometry, whereas impedance aggregometry displayed lower sensitivity to ADP. The two alternative assays correlated well with PRP aggregation as well as with each other. The fibrinogen receptor activation assay displayed the highest potency for AR-C69931MX, possibly due to a lower protein content compared with residual platelet counting. The two studied assays compare well with the more established assays, and are thus both good alternatives for platelet function testing and evaluation of new potential platelet antagonists.

Place, publisher, year, edition, pages
2006. Vol. 17, no 1, 49-55 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-36171DOI: 10.1080/09537100500197448Local ID: 30328OAI: oai:DiVA.org:liu-36171DiVA: diva2:257019
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Thrombin/ADP-induced platelet activation and drug intervention
Open this publication in new window or tab >>Thrombin/ADP-induced platelet activation and drug intervention
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood platelets are key players in haemostasis, the system that maintains an intact circulation. Circulating platelets are constantly on guard for vessel wall injury and will momentarily adhere to exposed subendothelial proteins, mainly collagens, at site of the injury. In parallel with platelet adhesion, tissue damage will also lead to the exposure of tissue factor, which will initiate thrombin generation. Platelet adhesion to collagen and the formation of thrombin will work in concert to induce the formation of a lose platelet plug as a result of platelet activation. This process is amplified by platelet agonists, mainly ADP, released from platelets themselves upon activation. The lose platelet plug is then stabilised by the formation of an armouring network of fibrin as a result of a burst in thrombin generation, dependent on the assembly of coagulation factors on the surface of activated platelets.

This thesis presents studies on thrombin- and ADP-induced platelet activation, and drug intervention in vitro, mainly using tlow cytometry as a tool for platelet function evaluation in diluted whole blood. Thrombin-induced platelet activation is composed of an initial response to low, and a secondary response to high concentrations of the agonist. Low concentrations will result in the activation of the high affinity thrombin receptor [protease activated receptor 1 (PAR1)], which in turn is sufficient to induce ADP release. Higher concentrations are needed to activate the low affinity PAR4. The platelet response to thrombin is thus the combined effect of signalling from PAR1, PAR4 and the ADP receptors, P2Y1 and P2Y12.

Reversible and irreversible or tight binding thrombin inhibitors differentially inhibited thrombin-induced platelet activation. Reversible inhibitors inhibited the thrombin response via a potent indirect inhibition of PAR4, and a partial inhibition of PAR1. Irreversible or tight binding inhibitors were potent indirect inhibitors of both PAR4 and PAR1. This can be explained by the different aftinity of thrombin for the two receptors and the different affinity of the inhibitors for thrombin. It was possible to completely block the ADP component in the thrombin response by inhibition of P2Y12, which resulted in a 15-80% relative inhibition depending on the thrombin concentration, whereas P2Y1 inhibition was ineffective. This was true not only in man but also in dog, mouse, rat and guinea pig. Blockade of P2Y12 gave in all species an effect equal to total removal of ADP. The relative inhibitory effect was most pronounced at low thrombin concentrations just enough to cleave PAR1 and thus release all ADP. The difference in effect between PSY1 and PSY12 inhibition is likely explained by the need for both a Gαi and Gαq signal in order to obtain potent platelet activation and that the only strong Gαq signal after thrombin stimulation is via PSY12. PSY1 on the other hand couples to Gαq, which is also activated by thrombin it-self via both PARI and PAR4. PSY1 dependent Gαq-signalling is therefore more or less redundant when thrombin is used as agonist. Finally, by a concomitant inhibition of a Gαq and a Gαisignalling true synergistic inhibition of both the thrombin and ADP response could be achieved.

We conclude that reversible thrombin inhibitors are potent platelet inhibitors by indirect inhibition of primarily PAR4 and to a less extent PAR1. Thrombin-induced platelet activation can also be potently inhibited by PSY12 antagonists, especially at low thrombin concentrations, just enough to cleave PAR1 and release all ADP. Synergistic inhibition of thrombin- and ADP induced platelet activation can be achieved by a concomitant inhibition of thrombin/P2Y12 and PSY1/P2Y12, respectively.

Place, publisher, year, edition, pages
Göteborg: Intellecta Docusys, 2005. 76 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 885
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-31886 (URN)17718 (Local ID)91-7373-863-8 (ISBN)17718 (Archive number)17718 (OAI)
Public defence
2005-03-17, Elsa Brändströmsalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-03Bibliographically approved

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