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The role of platelet thrombin receptors PAR1 and PAR4 in platelet activation
Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platelets play a pivotal role in coagulation and haemostasis. Their most prominent task is to seal damaged blood vessels by the formation of a platelet plug at the damaged area. Once the injury is covered, platelets retract the coagulum to close the wound and allow the blood to flow freely in the vessel. Platelets are strongly activated by the essential enzyme thrombin, formed in the coagulation cascade. Activation of the platelet thrombin receptors PAR1 and PAR4 leads to shape change, secretion of granule content, and aggregation, all of which can be accomplished by each receptor individually. However more and more findings indicate that there are differences between the receptors and that they have different physiological functions.

This thesis presents studies performed to elucidate the relative role of PAR1 and PAR4 in platelet activation and coagulation.

We have studied the effects on platelet activation and coagulation, and revealed a possible physiological role for PAR4 in the stabilisation of the coagulum. We also investigated the relative role of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine with and without inhibition of COX-1. We demonstrated that PAR4 interacts with adrenergic receptors and causes an aggregation of platelets dependent on released ATP and its receptor P2X1, thereby circumventing the inhibition by aspirin. Not only is this an interesting specific role for PAR4, but it may also be of clinical importance considering that COX-1 inhibition is the most common treatment for patients with cardiovascular disease to prevent thrombosis.

We show that the number of PAR1 receptors varied between donors and that this variation was correlated to the response on receptor activation. The number of PAR1 receptors on the platelet surface was decreased after PAR1 stimulation but increased after stimulation of other receptors.

In a final attempt to elucidate the nature of PAR1 and PAR4 we used mathematics to evaluate the effect of co-stimulation of the receptors. We found a strong synergistic effect for both platelet activation and aggregation. This indicates that PAR1 and PAR4 interact in a yet unknown way to regulate or amplify the effect of each other rather than merely transmitting the incoming signal the same way.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2009. , 70 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1156
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-51935ISBN: 978-91-7393-505-0 (print)OAI: oai:DiVA.org:liu-51935DiVA: diva2:278163
Public defence
2009-12-04, Linden, Hälsouniversitetet, Campus US, Linköpings Universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2009-11-24 Created: 2009-11-24 Last updated: 2015-03-13Bibliographically approved
List of papers
1. Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development
Open this publication in new window or tab >>Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development
2007 (English)In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 97, no 3, 417-424 p.Article in journal (Refereed) Published
Abstract [en]

Thrombin is a pivotal enzyme formed in the coagulation cascade and an important and potent platelet activator. The two protease-activated thrombin receptors on human platelets are denoted PARI and PAR4. The physiological relevance of PAR4 is still unclear, as both aggregation and secretion can be accomplished by PAR1 activation alone. In the present study we have investigated the role of PARS in platelet activation, blood coagulation, clot elasticity and fibrinolysis. Flow cytometry, free oscillation rheometry and thrombin generation measurements were used to analyze blood or platelet-rich plasma from healthy individuals. Maximum PAR1 activation with the peptide SFLLRN gave fewer fibrinogen-binding platelets with lower mean fluorescent intensity than maximum PAR4 activation with AYPGKF. Inhibition of any of the receptors prolonged clotting times. However, PAR1 is more important for fibrinolysis, inhibition of this receptor prolonged all the steps in the fibrinolytic process. Clot elasticity decreased significantly when the PAR4 receptor was inhibited. In the thrombin generation measurements, PAR4 inhibition delayed the thrombin generation start and peak, but did not affect the total amount of thrombin generated. PAR1 inhibition had no significant impact on thrombin generation. We found that PAR4 is most likely activated by low concentrations of thrombin during the initial phase of thrombin generation and is of importance to the clotting time. Furthermore, we suggest that the PAR4 receptor may have a physiological role in the stabilisation of the coagulum.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-39478 (URN)10.1160/TH06-07-0397 (DOI)48850 (Local ID)48850 (Archive number)48850 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
2. Platelet PAR1 receptor density-Correlation to platelet activation response and changes in exposure after platelet activation
Open this publication in new window or tab >>Platelet PAR1 receptor density-Correlation to platelet activation response and changes in exposure after platelet activation
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2008 (English)In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 121, no 5, 681-688 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: A polymorphism (-14 A/T) affecting PAR1 expression on the platelet surface has recently been identified. A two-fold variation in receptor density, which correlated with the platelet response to PAR1-activating peptide (PAR1-AP), has been reported. Materials and methods: We used flow cytometry to measure the correlation between the number of PAR1 receptors and platelet activation. We also measured the changes in receptor exposure after platelet activation with PAR1-AP, ADP, PAR4-AP or a collagen-related peptide (CRP). Results: In our study, the PAR1 receptor number varied almost four-fold, from 547 to 2063 copies/platelet (mean ± S.D. 1276 ± 320, n = 70). The number of PAR1 receptors on resting platelets correlated to platelet fibrinogen binding and P-selectin expression following platelet activation with PAR1-AP (r2 = 0.30, p < 0.01 and r2 = 0.15, p < 0.05, respectively, n = 36). The correlation was not improved by exclusion of the ADP-component from the PAR1-AP-induced response. We found a trend, but no statistically significant differences in PAR1 receptor number and platelet reactivity between A/A individuals and T/A or T/T individuals. Ex vivo activation with PAR1-AP decreased PAR1 surface exposure to 71 ± 19% of the exposure on resting platelets (mean ± S.D., p < 0.01, n = 19), while activation by ADP, PAR4-AP or CRP significantly increased the exposure, to 151 ± 27%, 120 ± 21% and 138 ± 25%, respectively (n = 11, 11 and 10). Conclusions: This study shows a large variation in PAR1 receptor number in healthy individuals, a variation correlated to the platelet activation response. We found a significant reduction in PAR1 surface exposure after adding PAR1-AP, while activation with ADP, PAR4-AP or CRP increased the exposure.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-43417 (URN)10.1016/j.thromres.2007.06.010 (DOI)73781 (Local ID)73781 (Archive number)73781 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
3. The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine
Open this publication in new window or tab >>The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 27, 18493-18504 p.Article in journal (Refereed) Published
Abstract [en]

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20726 (URN)10.1074/jbc.M800358200 (DOI)18480058 (PubMedID)
Available from: 2009-09-18 Created: 2009-09-18 Last updated: 2017-12-13Bibliographically approved
4. Platelet thrombin receptors PAR1 and PAR4 act in a synergistic manner
Open this publication in new window or tab >>Platelet thrombin receptors PAR1 and PAR4 act in a synergistic manner
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

n/a

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-51932 (URN)
Available from: 2009-11-24 Created: 2009-11-24 Last updated: 2015-03-13Bibliographically approved

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Vretenbrant Öberg, Karin

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