| 1. |
- Boknäs, Niklas, et al.
(författare)
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Thrombin-induced platelet activation via PAR4 : pivotal role for exosite II
- 2014
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Ingår i: Thrombosis and Haemostasis. - Stuttgart, Germany : Schattauer Gmbh. - 0340-6245 .- 2567-689X. ; 112:3, s. 558-565
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Tidskriftsartikel (refereegranskat)abstract
- Thrombin-induced platelet activation via PAR1 and PAR4 is an important event in haemostasis. Although the underlying mechanisms responsible for ensuring efficient PAR1 activation by thrombin have been extensively studied, the potential involvement of recognitions sites outside the active site of the protease in thrombin-induced PAR4 activation is largely unknown. In this study, we developed a new assay to assess the importance of exosite I and II for PAR4 activation with alpha- and gamma-thrombin. Surprisingly, we found that exosite II is critical for activation of PAR4. We also show that this dependency on exosite II likely represents a new mechanism, as it is unaffected by blockage of the previously known interaction between thrombin and glycoprotein Ib alpha.
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| 2. |
- Fälker, Knut, 1971-, et al.
(författare)
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ADP secretion and subsequent P2Y12 receptor signalling play a crucial role in thrombin-induced ERK2 activation in human platelets
- 2004
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Ingår i: Thrombosis and Haemostasis. - Stuttgart, Germany : Schattauer Gmbh. - 0340-6245 .- 2567-689X. ; 92:1, s. 114-23
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Tidskriftsartikel (refereegranskat)abstract
- Stimulating human platelets with thrombin induces the activation of the extracellular signal-regulated kinase 2 (ERK2). We demonstrate that this effect is highly dependent on ADP secretion and P2Y12 receptor signalling. AR-C69931MX (10 microM), a specific antagonist of the Gi-coupled P2Y12 ADP receptor, inhibits ERK2 activation induced by thrombin. Antagonists of the Gq-coupled P2Y1 ADP receptor, A3P5P (500 microM) and MRS2179 (100 microM), have no effect. ADP and its more potent analogue 2-methylthio-ADP alone (both up to 100 microM) do not induce ERK2 activation. Furthermore, we show that the inhibitory effect of AR-C69931MX on ERK2 activation induced by 0.1 U/ml thrombin as well as on platelet aggregation can be bypassed by epinephrine (1 and 10 microM), whereas epinephrine alone has no effect. Epinephrine acts on platelets mainly via alpha(2A)-adrenergic receptors, which, like P2Y12 receptors, couple to inhibitory G proteins. In addition, 2-methylthio-ADP as well as epinephrine provoke ERK2 activation at a thrombin concentration that alone has no detectable effect (0.05 U/ml). Thromboxane A2 (TXA2), which, like ADP, is released by activated platelets, acts as a positive feedback mediator. Stimulating the Gq-coupled TXA2 -receptor with U46619 (10 microM), which leads to ADP secretion and P2Y12 receptor-dependent platelet aggregation, also induces P2Y12-related ERK2 activation. The inhibition of U46619-induced ERK2 activation and platelet aggregation by AR-C69931MX are also rescued by epinephrine. Pretreatment with aspirin inhibits ERK2 activation induced by 0.1 U/ml thrombin, but has no effect at high concentrations of thrombin. The combination of U46619 and thrombin, at concentrations which alone have no effect, provokes ERK2 activation, suggesting that thrombin and released TXA2 act synergistically. Our data indicate that both primary signalling through Gq, which evokes ADP secretion, as well as subsequent coupling via Gi by the P2Y12 receptor are required for ERK2 activation.
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| 3. |
- Fälker, Knut, 1971-, et al.
(författare)
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P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets
- 2005
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Ingår i: Thrombosis and Haemostasis. - Stuttgart. - 0340-6245 .- 2567-689X. ; 93:5, s. 880-888
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Tidskriftsartikel (refereegranskat)abstract
- In thrombin-stimulated human platelets several proteins undergo rapid and transient changes in tyrosine phosphorylation. We demonstrate that a set of proteins of 27, 29, 31, 34, and 39 kDa is affected by released ADP and P2Y12 receptor signaling during platelet activation. AR-C69931MX, an antagonist of the Gi(2)-coupled P2Y12 ADP receptor, inhibits initial tyrosine phosphorylation of p27 and p31 and prevents subsequent dephosphorylation of p29, p34, and p39. Antagonists of the Gq-coupled P2Y1 ADP receptor have no effect. Precluding integrin alpha(IIb)beta(3) outside-in signaling with RGDS or S1197 does not affect the increase in tyrosine phosphorylation of the set of proteins but inhibits their subsequent dephosphorylation. Besides the ADP analogue 2-MeS-ADP, other platelet agonists such as collagen and the TXA(2)-mimetic U46619 also induce p27 and p31 tyrosine phosphorylation in a P2Y12 receptor-dependent manner. Tyrosine phosphorylation of p27 and p31 in response to collagen, but not thrombin, is prevented by aspirin and the TXA(2) receptor antagonist SQ29548, indicating that the effect of collagen strongly relies on TXA(2) signaling. Furthermore, epinephrine, acting via inhibitory Gz-coupled alpha(2A)-adrenoceptors, bypasses the inhibitory effect of AR-C69931MX on thrombin-induced p27 and p31 tyrosine phosphorylation. Finally, we demonstrate that tyrosine phosphorylation of p27 and p31 downstream of P2Y12 receptors is due to the inhibition of adenylyl cyclase but not phosphoinositide 3-kinase (PI 3-K) activation. Elevating cAMP levels with PGI(2) or forskolin precludes thrombin-induced p27 and p31 tyrosine phosphorylation. Moreover, direct inhibition of adenylyl cyclase by SQ22536 reverses the effect of AR-C69931MX. Our data indicate that the observed changes in tyrosine phosphorylation are the result of both primary Gq signaling, initiating the release of ADP, as well as subsequent P2Y12 receptor-mediated Gi coupling.
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| 4. |
- Kälvegren, Hanna, et al.
(författare)
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Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X(1)-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y(1) and P2Y(12) receptor activation
- 2010
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Ingår i: Thrombosis and Haemostasis. - 0340-6245 .- 2567-689X. ; 103:2, s. 398-407
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Tidskriftsartikel (refereegranskat)abstract
- Toll-like receptor 2 (TLR2), which recognise and respond to conserved microbial pathogen-associated molecular patterns, is expressed on the platelet surface. Furthermore, it has recently been shown that the TLR2/1 agonist Pam(3)CSK(4) stimulates platelet activation. The aim of the present study was to clarify important signalling events in Pam(3)CSK(4)-induced platelet aggregation and secretion. Platelet interaction with Pam(3)CSK(4) and the TLR2/6 agonist MALP-2 was studied by analysing aggregation, ATP-secretion, [Ca2+](i) mobilisation and thromboxane B2 (TxB(2)) production. The results show that Pam(3)CSK(4) but not MALP-2 induces [Ca2+](i) increase, TxB(2) production, dense granule secretion and platelet aggregation. Preincubation of platelets with MALP-2 inhibited the Pam(3)CSK(4)-induced responses. The ATP-secretion and aggregation in Pam(3)CSK(4)-stimulated platelets was impeded by the purinergic P2X(1) inhibitor MRS 2159, the purinergic P2Y(1) and P2Y(12) antagonists MRS 2179 and cangrelor, the phospholipase C inhibitor U73122, the calcium chelator BAPT-AM and aspirin. The calcium mobilisation was lowered by MRS 2159, aspirin and U73122 whereas the TxB(2) production was antagonised by MRS 2159, aspirin and BAPT-AM. When investigating the involvement of the myeloid differentiation factor-88 (MyD88) -dependent pathway, we found that platelets express MyD88 and interleukin 1 receptor-associated kinase (IRAK-1), which are proteins important in TLR signalling. However, Pam(3)CSK(4) did not stimulate a rapid (within 10 minutes) phosphorylation of IRAK-1 in platelets. In conclusion, the results show that Pam(3)CSK(4)-induced platelet aggregation and secretion depends on a P2X(1)-mediated Ca2+ mobilisation, production of TxA(2) and ADP receptor activation. The findings in this study further support a role for platelets in sensing bacterial components.
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| 5. |
- Singh, Sukhi, 1990, et al.
(författare)
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Adrenaline Improves Platelet Reactivity in Ticagrelor-Treated Healthy Volunteers
- 2019
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Ingår i: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 119:5, s. 735-743
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Administration of agents that enhance platelet reactivity may reduce the perioperative bleeding risk in patients treated with the adenosine diphosphate (ADP)-receptor antagonist ticagrelor. Adrenaline potentiates ADP-induced aggregation and activation in blood samples from ticagrelor-treated patients, but it has not previously been evaluated in vivo.METHODS: Ten healthy male subjects were included in an interventional study. A loading dose of ticagrelor (180 mg) was administered, followed 2 hours later by a gradually increased intravenous adrenaline infusion (0.01, 0.05, 0.10 and 0.15 µg/kg/min; 15 minutes at each step). Blood pressure, heart rate, platelet aggregation (impedance aggregometry), platelet activation (flow cytometry), clot formation (rotational thromboelastometry) and adrenaline plasma concentration were determined before and after ticagrelor administration and at the end of each adrenaline step.RESULTS: = 0.007).CONCLUSION: Infusion of adrenaline at clinically relevant doses improves in vivo platelet reactivity and clot formation in ticagrelor-treated subjects. Adrenaline could thus potentially be used to prevent perioperative bleeding complications in ticagrelor-treated patients. Studies in patients are necessary to determine the clinical importance of our observations.TRIAL REGISTRY NUMBER: ClinicalTrials.gov NCT03441412.
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