| 1. |
- Donner, Lili, et al.
(författare)
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Platelets contribute to amyloid-β aggregation in cerebral vessels through integrin αIIbβ3-induced outside-in signaling and clusterin release
- 2016
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Ingår i: Science Signaling. - Washington, USA : American Association for the Advancement of Science (A A A S). - 1945-0877 .- 1937-9145. ; 9:429
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Tidskriftsartikel (refereegranskat)abstract
- Cerebral amyloid angiopathy (CAA) is a vascular dysfunction disorder characterized by deposits of amyloid-β (Aβ) in the walls of cerebral vessels. CAA and Aβ deposition in the brain parenchyma contribute to dementia and Alzheimer's disease (AD). We investigated the contribution of platelets, which accumulate at vascular Aβ deposits, to CAA. We found that synthetic monomeric Aβ40 bound through its RHDS (Arg-His-Asp-Ser) sequence to integrin αIIbβ3, which is the receptor for the extracellular matrix protein fibrinogen, and stimulated the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin from platelets. Clusterin promoted the formation of fibrillar Aβ aggregates, and ADP acted through its receptors P2Y1 and P2Y12 on platelets to enhance integrin αIIbβ3 activation, further increasing the secretion of clusterin and Aβ40 binding to platelets. Platelets from patients with Glanzmann's thrombasthenia, a bleeding disorder in which platelets have little or dysfunctional αIIbβ3, indicated that the abundance of this integrin dictated Aβ-induced clusterin release and platelet-induced Aβ aggregation. The antiplatelet agent clopidogrel, which irreversibly inhibits P2Y12, inhibited Aβ aggregation in platelet cultures; in transgenic AD model mice, this drug reduced the amount of clusterin in the circulation and the incidence of CAA. Our findings indicate that activated platelets directly contribute to CAA by promoting the formation of Aβ aggregates and that Aβ, in turn, activates platelets, creating a feed-forward loop. Thus, antiplatelet therapy may alleviate fibril formation in cerebral vessels of AD patients.
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| 2. |
- Elvers, Margitta, et al.
(författare)
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A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity
- 2012
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Ingår i: Cellular Signalling. - New York, USA : Elsevier. - 0898-6568 .- 1873-3913. ; 24:9, s. 1743-52
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Tidskriftsartikel (refereegranskat)abstract
- Platelet aggregation, secretion and thrombus formation play a critical role in primary hemostasis to prevent excessive blood loss. On the other hand, uncontrolled platelet activation leads to pathological thrombus formation resulting in myocardial infarction or stroke. Stimulation of heterotrimeric G-proteins by soluble agonists or immunoreceptor tyrosine based activation motif-coupled receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI lead to the activation of phospholipases that cleave membrane phospholipids to generate soluble second messengers. Platelets contain the phospholipases (PL) D1 and D2 which catalyze the hydrolysis of phosphatidylcholine to generate the second messenger phosphatidic acid (PA). The production of PA is abrogated by primary alcohols that have been widely used for the analysis of PLD-mediated processes. However, it is not clear if primary alcohols effectively reduce PA generation or if they induce PLD-independent cellular effects. In the present study we made use of the specific PLD inhibitor 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) and show for the first time, that FIPI enhances platelet dense granule secretion and aggregation of human platelets. Further, FIPI has no effect on cytosolic Ca(2+) activity but needs proper Rho kinase signaling to mediate FIPI-induced effects on platelet activation. Upon FIPI treatment the phosphorylation of the PKC substrate pleckstrin was prominently enhanced suggesting that FIPI affects PKC-mediated secretion and aggregation in platelets. Similar effects of FIPI were observed in platelets from mouse wild-type and Pld1(-/-) mice pointing to a new role for PLD2 as a negative regulator of platelet sensitivity.
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| 3. |
- 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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| 4. |
- Fälker, Knut, 1971-, et al.
(författare)
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Adrenoceptor α2A signalling countervails the taming effects of synchronous cyclic nucleotide-elevation on thrombin-induced human platelet activation and aggregation
- 2019
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Ingår i: Cellular Signalling. - : Elsevier. - 0898-6568 .- 1873-3913. ; 59, s. 96-109
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Tidskriftsartikel (refereegranskat)abstract
- The healthy vascular endothelium constantly releases autacoids which cause an increase of intracellular cyclic nucleotides to tame platelets from inappropriate activation. Elevating cGMP and cAMP, in line with previous reports, cooperated in the inhibition of isolated human platelet intracellular calcium-mobilization, dense granules secretion, and aggregation provoked by thrombin. Further, platelet alpha granules secretion and, most relevant, integrin αIIaβ3 activation in response to thrombin are shown to be prominently affected by the combined elevation of cGMP and cAMP. Since stress-related sympathetic nervous activity is associated with an increase in thrombotic events, we investigated the impact of epinephrine in this setting. We found that the assessed signalling events and functional consequences were to various extents restored by epinephrine, resulting in full and sustained aggregation of isolated platelets. The restoring effects of epinephrine were abolished by either interfering with intracellular calcium-elevation or with PI3-K signalling. Finally, we show that in our experimental setting epinephrine likewise reconstitutes platelet aggregation in heparinized whole blood, which may indicate that this mechanism could also apply in vivo.
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| 5. |
- 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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| 6. |
- Fälker, Knut, 1971-, et al.
(författare)
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Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets
- 2011
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Ingår i: Biochemical Journal. - : Portland Press -- London. - 0264-6021 .- 1470-8728. ; 436:2, s. 469-480
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Tidskriftsartikel (refereegranskat)abstract
- PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca²⁺ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y₁₂ receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.
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| 7. |
- Fälker, Knut, et al.
(författare)
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Targeting Platelet G Protein-Coupled Receptors for Antithrombotic Therapy
- 2013
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Ingår i: Drug development research (Print). - : Wiley. - 0272-4391 .- 1098-2299. ; 74:7, s. 440-449
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Tidskriftsartikel (refereegranskat)abstract
- Platelets are small anucleated cells produced by bone marrow megakaryocytes that circulate in the blood as sentinels of vascular integrity. They play a pivotal role in the regulation of vascular homeostasis through adhesion to the injured vessel wall, aggregation, propagation of coagulation, and thrombus formation. Furthermore, platelets are also involved in fibrinolysis and the repair of the blood vessel wall, restoring blood flow and vascular integrity. Under pathophysiological conditions such as atherosclerosis, inappropriate platelet aggregation and clot formation can cause vascular occlusions, resulting in myocardial infarctions or stroke that, according to the World Health Organization, represent with more than 10% of worldwide death a major health risk (http://who.int/mediacentre/factsheets/fs310/en/). Over the last several decades, increasing efforts have been made to elucidate the cellular components, signaling pathways, and risk factors contributing to platelet activation with the main goal of providing a sound basis for the development of antiplatelet drugs and novel therapeutic treatment strategies. The family of seven transmembrane receptors, also designated G protein-coupled receptors (GPCRs), represented by approximately 800 members identified in the human genome represent the largest class of receptors and, hence, the richest source of targets for drug discovery. Here, we here provide an overview of the commonly applied therapies targeting platelet-GPCRs as well as a brief summary of novel approaches.
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| 8. |
- Fälker, Knut, 1971-, et al.
(författare)
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The Toll-like receptor 2/1 (TLR2/1) complex initiates human platelet activation via the src/Syk/LAT/PLC gamma 2 signalling cascade
- 2014
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Ingår i: Cellular Signalling. - New York, USA : Elsevier. - 0898-6568 .- 1873-3913. ; 26:2, s. 279-286
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Tidskriftsartikel (refereegranskat)abstract
- The specific TLR2/1 complex activator Pam3CSK4 has been shown to provoke prominent activation and aggregation of human non-nucleated platelets. As Pam3CSK4-evoked platelet activation does not employ the major signalling pathway established in nucleated immune cells, we investigated if the TLR2/1 complex on platelets may initiate signalling pathways known to be induced by physiological agonists such as collagen via GPVI or thrombin via PARs. We found that triggering TLR2/1 complex-signalling with Pam3CSK4, in common with that induced via GPVI, and in contrast to that provoked by PARS, involves tyrosine phosphorylation of the adaptor protein LAT as well as of PLC gamma 2 in a src- and Syk-dependent manner. In this respect, we provide evidence that Pam3CSK4 does not cross-activate GPVI. Further, by the use of platelets from a Glanzmann's thrombasthenia patient lacking beta(3), in contrast to findings in nucleated immune cells, we show that the initiation of platelet activation by Pam3CSK4 does not involve integrin beta(3) signalling; whereas the latter, subsequent to intermediate TXA2 synthesis and signalling, was found to be indispensable for proper dense granule secretion and full platelet aggregation. Together, our findings reveal that triggering the TLR2/1 complex with Pam3CSK4 initiates human platelet activation by engaging tyrosine kinases of the src family and Syk, the adaptor protein LAT, as well as the key mediator PLC gamma 2. (C) 2013 Elsevier Inc. All rights reserved.
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| 9. |
- Kardeby, Caroline, 1989-
(författare)
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Studies of platelet signalling and endothelial cell responses using unique synthetic drugs
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Haemostasis is a complex and tightly regulated process which protects us from bleeding. Platelets are essential for maintained haemostasis. Under normal conditions platelets are calmed by antithrombotic substances release by the endothelium. During vascular injury, the platelets will activate and form a haemostatic plug to prevent bleeding. Inflammatory processes like atherosclerosis can disturb the haemostatic balance and lead to severe consequences like myocardial infarction and stroke. Inhibition of platelets and coagulation are common treatments to prevent unwanted blood clot formation. There is a great need for increased knowledge on the mechanisms of thrombosis and characterisation of new substances with possible therapeutic potential. This thesis used unique synthetic drugs to study platelet signalling and endothelial responses. Paper I showed that both sulfated polysaccharides from seaweed and synthetic glycopolymers which mimic their chemical properties caused platelet activation.Paper II elucidated the molecular mechanism underlying platelet activation by sulfated glycopolymers and polysaccharides. We found that human platelet activation took place via the Platelet endothelial aggregation receptor 1 (PEAR1), while mouse platelet activation was mainly via C-type lectin-like receptor 2. Aggregation was supported by Glycoprotein Ibα in both species.Paper III showed the effect of synthetic glycopolymers and natural polysaccharides on cultured human endothelial cells. We found that both the glycopolymers and polysaccharides caused a proinflammatory response after 24h.In Paper IV, the effect of a synthetic purine analogue with a nitrate ester motif was studied. We found that the purine analogue reduced platelet functions by inhibiting Rho-associated protein kinase (ROCK).This thesis describes unique synthetic drugs that can be used for further studies of the mechanisms underlying the biological processes of thrombosis and inflammation. The synthetic glycopolymers can be used to further elucidate the physiological role of PEAR1, a potential future therapeutic target.
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| 10. |
- Kardeby, Caroline, 1989-, et al.
(författare)
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Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα
- 2019
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Ingår i: Blood Advances. - : American Society of Hematology. - 2473-9529 .- 2473-9537. ; 3:3, s. 275-287
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Tidskriftsartikel (refereegranskat)abstract
- Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)-dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)-like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.
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