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Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation
Department of Molecular Pharmacology, Preclinical R and D, AstraZeneca R and D, Mölndal, Sweden.
Department of Molecular Pharmacology, Preclinical R and D, AstraZeneca R and D, Mölndal, Sweden.
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0002-1920-3962
Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2004 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 142, no 8, 1325-1331 p.Article in journal (Refereed) Published
Abstract [en]
  • The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y12 and P2Y1 inhibition and P2Y12 and thrombin inhibition, on ADP- and thrombin-induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both Gαq and Gαi signalling.

  • Blood from healthy volunteers was preincubated with AR-C69931MX, a reversible P2Y12 antagonist; MRS2179, a reversible P2Y1 antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC-1 to estimate the expression of active αIIbβ3 (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel.

  • A synergistic effect regarding inhibition of ADP-induced platelet activation (10 μM) was obtained with different combinations of AR-C69931MX and MRS2179.

  • Inhibition of thrombin-induced platelet activation (2 nM) with combinations of AR-C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect.

  • To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described.

  • Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well-conducted clinical studies.

Place, publisher, year, edition, pages
2004. Vol. 142, no 8, 1325-1331 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-23582DOI: 10.1038/sj.bjp.0705885Local ID: 3066OAI: diva2:243897
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2015-03-13Bibliographically 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.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 885
National Category
Medical and Health Sciences
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)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-03Bibliographically approved

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