liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Thrombin/ADP-induced platelet activation and drug intervention
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-31886Local ID: 17718ISBN: 91-7373-863-8 (print)OAI: oai:DiVA.org:liu-31886DiVA: diva2:252709
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
List of papers
1. Thrombin-induced platelet activation and its inhibition by anticoagulants with different modes of action
Open this publication in new window or tab >>Thrombin-induced platelet activation and its inhibition by anticoagulants with different modes of action
2003 (English)In: Blood Coagulation and Fibrinolysis, ISSN 0957-5235, E-ISSN 1473-5733, Vol. 14, no 2, 159-167 p.Article in journal (Refereed) Published
Abstract [en]

Thrombin-induced platelet activation involves cleavage of protease-activated receptors (PARs) 1 and 4, and interaction, via glycoprotein (Gp)Ibα, with the platelet GpIb/IX/V complex. This study investigated inhibition of platelet activation by thrombin inhibitors with different modes of action: two reversible direct thrombin inhibitors, melagatran and inogatran; hirudin, a tightly binding direct thrombin inhibitor; and two indirect thrombin inhibitors, heparin and dalteparin. Up-regulation of P-selectin (CD62P) and PAR-1 cleavage was measured in human whole blood, by flow cytometry. The thrombin concentration that induced 50% of maximum (EC50) PAR-1 cleavage was 0.028 nmol/l, while that of platelet activation (CD62P) was over two-fold higher (0.64 nmol/l). The EC50 of a PAR-1-independent component, defined as a further activating effect of thrombin on top of the maximum PAR-1-activating peptide (AP) effect, was 3.2 nmol/l. All anticoagulants were concentration-dependent inhibitors of thrombin-induced platelet activation and PAR-1 cleavage, but none inhibited PAR-1-AP or PAR-4-AP induced activation. Melagatran and inogatran were more potent inhibitors of CD62P up-regulation than of PAR-1 cleavage; conversely, hirudin, heparin and dalteparin were more potent inhibitors of PAR-1 cleavage.Thus, reversible direct thrombin inhibitors, such as melagatran, are potent inhibitors of thrombin-induced platelet activation, acting mainly by inhibition of a PAR-1-independent component.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84280 (URN)10.1097/00001721-200302000-00007 (DOI)
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2012-10-03Bibliographically approved
2. The relative importance of the ADP receptors, P2Y12 and P2Y1, in thrombin-induced platelet activation
Open this publication in new window or tab >>The relative importance of the ADP receptors, P2Y12 and P2Y1, in thrombin-induced platelet activation
2003 (English)In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 111, no 1-2, 65-73 p.Article in journal (Refereed) Published
Abstract [en]

The objective of this study was to test the relative importance of the two adenosine diphosphate (ADP) receptors P2Y1 and P2Y12 in thrombin-induced platelet activation using specific receptor antagonists. Blood from healthy volunteers was incubated with MRS2179, a reversible P2Y1 antagonist, or AR-C69931, a reversible P2Y12 antagonist prior to activation with different concentrations of ADP or thrombin. Platelet function in whole blood was assayed by flow cytometry using the antibody PAC-1 to estimate the expression of conformational active αIIbβ3, the fibrinogen receptor. Complete inhibition of P2Y12 or P2Y1 abolished the ADP response, but only inhibition of P2Y12 reduced the thrombin-induced response. The relative inhibition of the thrombin response by complete inhibition of P2Y12 was most pronounced at thrombin concentrations just enough for complete PAR1 cleavage, which is sufficient to release all ADP, giving 70–86% inhibition. Above this concentration, the relative importance of P2Y12 inhibition decreased due to activation of ADP independent pathways. This study supports P2Y12 as a drug target compared with P2Y1.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25241 (URN)10.1016/j.thromres.2003.08.021 (DOI)9680 (Local ID)9680 (Archive number)9680 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2015-03-13Bibliographically approved
3. Characterisation of species differences in the platelet ADP and thrombin response
Open this publication in new window or tab >>Characterisation of species differences in the platelet ADP and thrombin response
2006 (English)In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 117, no 5, 543-549 p.Article in journal (Refereed) Published
Abstract [en]

Introduction:

A number of animal models are used to study platelet-dependent diseases. In the present investigation, we have used a simple flow cytometry assay to evaluate platelet function in man, rat, mouse, guinea pig and dog.

Materials and methods:

Platelet activation was evaluated in diluted whole blood by measuring fibrinogen binding to activated platelets using a polyclonal anti-human fibrinogen antibody that cross-reacts with fibrinogen from all species tested. The assay was used to evaluate platelet function with respect to ADP and thrombin sensitivity. The relative importance of the two platelet ADP receptors and total ADP in the thrombin response was also studied by using receptor-specific antagonists and apyrase, respectively.

Results:

Mouse platelets were most sensitive to both agonists. Unlike in man and dog the maximal response to ADP was greater than to thrombin in mouse, rat and guinea pig. P2Y12 blockade was in all species equally effective as ADP removal in inhibiting thrombin-induced platelet activation whereas P2Y1 blockade was almost ineffective.

Conclusion:

The present study describes a simple platelet function test that can be used to evaluate platelet function in man, rat, mouse, guinea pig and dog. Platelets from the tested species differed in their sensitivity to ADP and thrombin. In contrast to human and canine platelets, mouse, rat and guinea pig platelets displayed a stronger maximal response to ADP than to thrombin. In terms of the relative contribution of P2Y1 and P2Y12 in the thrombin response, the P2Y12 receptor was the key receptor in all species and its blockade gave equal effect as total removal of ADP.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-36993 (URN)10.1016/j.thromres.2005.04.026 (DOI)33267 (Local ID)33267 (Archive number)33267 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2012-10-03Bibliographically approved
4. Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation
Open this publication in new window or tab >>Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-23582 (URN)10.1038/sj.bjp.0705885 (DOI)3066 (Local ID)3066 (Archive number)3066 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2015-03-13Bibliographically approved
5. Evaluation of platelet function, a method comparison
Open this publication in new window or tab >>Evaluation of platelet function, a method comparison
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-36171 (URN)10.1080/09537100500197448 (DOI)30328 (Local ID)30328 (Archive number)30328 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2012-10-03Bibliographically approved

Open Access in DiVA

No full text

By organisation
Clinical ChemistryFaculty of Health Sciences
Medical and Health Sciences

Search outside of DiVA

GoogleGoogle Scholar

Total: 235 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf