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Effects of inhibition of P2Y1 and P2Y12 on whole blood clotting, coagulum elasticity and fibrinolysis resistance studied with free oscillation rheometry
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0002-1920-3962
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2003 (English)In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 109, no 5-6, 315-322 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: In vivo, initial platelet activation is likely caused by platelet contacts with collagen in the subendothelium or from the small amounts of thrombin formed by the tissue factor/factor VIIa complex. Our aim was to study the coagulative role of ADP released by the platelets after activation with strong stimuli such as collagen and/or thrombin, and the relative importance of the platelet ADP receptors P2Y1 and P2Y12.

Materials and methods: We used 10 Hz free oscillation rheometry to measure clotting time, clot elasticity and fibrinolysis resistance of non-anticoagulated whole blood. The platelets were activated with a collagen-related peptide (CRP), with the PAR1 thrombin receptor activating peptide TRAP-6 or by thrombin, the latter generated by small amounts of thromboplastin. To inhibit the platelet ADP receptors, we used the P2Y1 antagonist MRS2179 and the P2Y12 antagonist AR-C69931MX.

Results: Both antagonists significantly retarded the clotting induced by CRP. The effects were most pronounced with AR-C69931MX. For TRAP-6, the same trend was seen, but the retardation was only significant with AR-C69931MX. Clotting induced by small amounts of thromboplastin was not affected by any ADP-receptor antagonist. Addition of both antagonists did not change the results as compared to samples with AR-C69931MX alone. Nor did the antagonists, one at a time or in concert, effect fibrinolysis or the elastic properties of the clot.

Conclusion: We conclude that ADP-receptor inhibition prolongs the clotting time for whole blood activated by CRP, but that it does not affect the properties of the subsequently formed coagulum.

Place, publisher, year, edition, pages
2003. Vol. 109, no 5-6, 315-322 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-25244DOI: 10.1016/S0049-3848(03)00254-8Local ID: 9683OAI: diva2:245572
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2015-03-13
In thesis
1. The role of platelets in whole blood coagulation
Open this publication in new window or tab >>The role of platelets in whole blood coagulation
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The body's first line of defence against bleeding is the platelets, small cell fragments with multiple roles in the coagulation process. The platelets are patrolling our blood vessels, looking for damage in the endothelial cell layer, to which they attach and transform, recruit more platelets and assemble the plasma coagulation proteases on their surfaces. Cleavage of the plasma coagulation factors then leads to the formation of a fibrin network sealing the damage.

This thesis present studies of the role of platelets in whole blood coagulation, using methods which enable studies in a whole blood environment, flow cytometry and free oscillation rheometry (FOR). FOR proved to be equivalent in accuracy to visual inspection in detecting clotting, but allowing for simultaneous analysis of a large number of blood samples. A flow cytometry method for measuring the contents and release capacity of platelet dense granules was also developed.

FOR was used to study platelets in coagulation. A quick removal of all blood cells was shown to be enough to keep the plasma anticoagulated. Activation of platelets by addition of collagen or a thrombin receptor agonist peptide (TRAP-6) shortened the whole blood clotting time, indicating the importance of platelets for the acceleration of coagulation. Inhibitors of the platelet fibrinogen receptor prevented clot retraction and prolonged the clotting time with TRAP-6. In collagen-stimulated samples, however, MK-852 accelerated clotting but delayed completion of clotting, while abciximab prolonged both clotting time and completion of clotting. Inhibitors to the platelet ADP receptors prolonged the clotting time, but did not affect the coagulum elasticity or the resistance against fibrinolysis. The same results were seen in a patient with a defective release of dense granule contents (which normally includes ADP). When comparing the platelet surface exposure of phosphatidylserine (PS) with the actual shortening of the clotting time, we found that despite the large decrease in clotting times seen with collagen or TRAP-6, only low numbers of PS-exposing platelets were detected. Induction of a similar PS exposure by the calcium ionophore A23187 could not decrease the clotting times to the same extent, indicating that PS exposure is only one of the mechanisms involved in platelet binding of plasma coagulation factors. Addition of annexin V, a substance that binds to PS, hampered, but could not abolish, coagulation. Artificial PS-containing lipid vesicles could not replace platelets, as they did not affect the coagulation of blood or plasma. To see which coagulation pathway that was involved in platelet dependent blood coagulation, inhibitors to factor XII or tissue factor (TF) were added to the blood prior to the platelet agonist Inhibition of factor xn increased the clotting time from about 10 to 15 minutes. Inhibition of TF was without effect. The results harmonised with the coagulation behaviour of blood congenitally deficient in factor XII or VII. Inhibition of factor XI caused a two to three fold increase in clotting time.

We conclude that plasma does not necessarily coagulate if all blood cells are quickly removed. Activated platelets accelerate the coagulation of native whole blood ex vivo in a process which is independent of TF. Platelet PS, factor XI and factor XII all promote this coagulation, but are not essential This suggests that there might exist a platelet dependent blood coagulation pathway differing from the established pathways. Drugs affecting the platelet fibrinogen and ADP receptors affect the coagulation of the blood samples which might be detected by FOR. Our studies suggest the possibility of using FOR to measure the "platelet reactivity potential".

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 86 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 776
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-25703 (URN)10079 (Local ID)91-7373-535-3 (ISBN)10079 (Archive number)10079 (OAI)
Public defence
2003-03-14, Aulan, Administrationshuset, Hälsouniversitet, Linköping, 09:00 (Swedish)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2015-03-13Bibliographically approved

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