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Kardeby, C., Paramel Varghese, G., Pournara, D., Fotopoulou, T., Sirsjö, A., Koufaki, M., . . . Grenegård, M. (2019). A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition. European Journal of Pharmacology, 857, Article ID 172428.
Open this publication in new window or tab >>A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition
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2019 (English)In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 857, article id 172428Article in journal (Refereed) Published
Abstract [en]

Natural purines like ATP, ADP and adenosine have crucial roles in platelet physiology. This knowledge has been significant in drug development and today ADP receptor antagonists are widely used for prevention of thrombotic events following myocardial infarction and ischaemic stroke.

Recent studies have shown that a purine analogue bearing nitrate ester group (denoted MK128) has anti-inflammatory effects probably due to its ability to donate nitric oxide (NO). However, other pharmacological mechanisms may contribute to the observed effect. The aim of the present study was to establish the anti-platelet activity and elucidate the underlying molecular mechanism(s) of the purine analogue MK128.

We found that MK128 reduced aggregation and secretion induced by the thrombin receptor agonist SFLLRN and nearly abolished aggregation and secretion induced by thromboxane A2 (TxA2) and collagen receptor agonists. The inhibition took place despite blockage of the NO/cGMP signalling system. Furthermore, interaction between MK128 and platelet purinergic receptors did not explain the observed inhibition. Instead, we found that MK128 concentration-dependently inhibited Rho-associated kinase (ROCK), which led to decreased ROCK-dependent myosin phosphatase target subunit (MYPT)-1 phosphorylation and suppression of platelet functional responses.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Nitric oxide, Platelet inhibitor, Purinergic receptors, Rho associated protein kinase, Thromboxane A2
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:oru:diva-74650 (URN)10.1016/j.ejphar.2019.172428 (DOI)000472711200011 ()31175850 (PubMedID)2-s2.0-85066786586 (Scopus ID)
Funder
Knowledge Foundation
Note

Funding Agency:

Onassis Foundation

Available from: 2019-06-10 Created: 2019-06-10 Last updated: 2019-11-14Bibliographically approved
Fälker, K., Ljungberg, L., Kardeby, C., Lindkvist, M., Sirsjö, A. & Grenegård, M. (2019). Adrenoceptor α2A signalling countervails the taming effects of synchronous cyclic nucleotide-elevation on thrombin-induced human platelet activation and aggregation. Cellular Signalling, 59, 96-109
Open this publication in new window or tab >>Adrenoceptor α2A signalling countervails the taming effects of synchronous cyclic nucleotide-elevation on thrombin-induced human platelet activation and aggregation
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2019 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 59, p. 96-109Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Cyclic nucleotide, Epinephrine, Human platelets, Nitric oxide, Prostacyclin, α(2A) adrenoceptor
National Category
Physiology Cell and Molecular Biology
Identifiers
urn:nbn:se:oru:diva-73421 (URN)10.1016/j.cellsig.2019.03.019 (DOI)000468251000010 ()30926386 (PubMedID)2-s2.0-85063486315 (Scopus ID)
Funder
AFA Insurance, 130275Knowledge Foundation, 20150240
Available from: 2019-04-04 Created: 2019-04-04 Last updated: 2019-06-19Bibliographically approved
Kardeby, C. (2019). Studies of platelet signalling and endothelial cell responses using unique synthetic drugs. (Doctoral dissertation). Örebro: Örebro University
Open this publication in new window or tab >>Studies of platelet signalling and endothelial cell responses using unique synthetic drugs
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Örebro: Örebro University, 2019. p. 64
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 195
Keywords
Haemostasis, glycopolymers, purine analogue, PEAR1, GPIbα, CLEC-2, inflammation, ROCK
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-73147 (URN)978-91-7529-287-8 (ISBN)
Public defence
2019-05-29, Örebro universitet, Campus USÖ, hörsal C3, Södra Grev Rosengatan 32, Örebro, 13:00 (English)
Opponent
Supervisors
Available from: 2019-03-14 Created: 2019-03-14 Last updated: 2019-05-06Bibliographically approved
Kardeby, C., Fälker, K., Haining, E. J., Criel, M., Lindkvist, M., Barroso, R., . . . Grenegård, M. (2019). Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα. Blood advances, 3(3), 275-287
Open this publication in new window or tab >>Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα
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2019 (English)In: Blood advances, ISSN 2473-9529, Vol. 3, no 3, p. 275-287Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Society of Hematology, 2019
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Hematology
Identifiers
urn:nbn:se:oru:diva-72478 (URN)10.1182/bloodadvances.2018024950 (DOI)000458442500007 ()30700416 (PubMedID)2-s2.0-85060943358 (Scopus ID)
Funder
Knowledge Foundation
Note

Funding Agencies:

BHF  PG/16/53/32242  RG/13/18/30563 

Deutsche Forschungsgemeinschaft  DFG: Eb 177/14-1 

Fonds voor Wetenschappelijk Onderzoek Vlaanderen grant  G0A6514N 

Available from: 2019-02-14 Created: 2019-02-14 Last updated: 2019-06-18Bibliographically approved
Welander, E., Åström, M., Enonge Fotabe, L., Kardeby, C., Tina, E., Elgbratt, K., . . . Ivarsson, M. (2018). Integrated analysis indicates reciprocal immune response dysregulations between bone marrow multipotent stromal cells and granulocytes at the mRNA but not at the protein level in myelofibrosis. In: : . Paper presented at Fortbildningsdagar i hematologi, Umeå, 3-5 Oktober, 2018.
Open this publication in new window or tab >>Integrated analysis indicates reciprocal immune response dysregulations between bone marrow multipotent stromal cells and granulocytes at the mRNA but not at the protein level in myelofibrosis
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2018 (English)Conference paper, Poster (with or without abstract) (Refereed)
National Category
Hematology
Identifiers
urn:nbn:se:oru:diva-69350 (URN)
Conference
Fortbildningsdagar i hematologi, Umeå, 3-5 Oktober, 2018
Available from: 2018-10-08 Created: 2018-10-08 Last updated: 2019-03-26Bibliographically approved
Tengdelius, M., Kardeby, C., Fälker, K., Griffith, M., Påhlsson, P., Konradsson, P. & Grenegård, M. (2017). Fucoidan-Mimetic Glycopolymers as Tools for Studying Molecular and Cellular Responses in Human Blood Platelets. Macromolecular Bioscience, 17(2), Article ID UNSP 1600257.
Open this publication in new window or tab >>Fucoidan-Mimetic Glycopolymers as Tools for Studying Molecular and Cellular Responses in Human Blood Platelets
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2017 (English)In: Macromolecular Bioscience, ISSN 1616-5187, E-ISSN 1616-5195, Vol. 17, no 2, article id UNSP 1600257Article in journal (Refereed) Published
Abstract [en]

The marine sulfated polysaccharide fucoidan displays superior ability to induce platelet aggregation compared to other sulfated polysaccharides. As such, it is an attractive tool for studying molecular and cellular responses in activated platelets. The heterogeneous structure, however, poses a problem in such applications. This study describes the synthesis of sulfated α-l-fucoside-pendant poly(methacryl amides) with homogeneous structures. By using both thiol-mediated chain transfer and reversible addition-fragmentation chain transfer polymerization techniques, glycopolymers with different chain lengths are obtained. These glycopolymers show platelet aggregation response and surface changes similar to those of fucoidan, and cause platelet activation through intracellular signaling as shown by extensive protein tyrosine phosphorylation. As the platelet activating properties of the glycopolymers strongly mimic those of fucoidan, this study concludes these fucoidan-mimetic glycopolymers are unique tools for studying molecular and cellular responses in human blood platelets.

Place, publisher, year, edition, pages
Weinheim, Germany: Wiley-VCH Verlagsgesellschaft, 2017
Keywords
biological applications of polymers; biomimetic; radical polymerization; reversible addition fragmentation chain transfer; structure-property relations
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:oru:diva-52179 (URN)10.1002/mabi.201600257 (DOI)000394592600012 ()27616165 (PubMedID)2-s2.0-84987653303 (Scopus ID)
Note

Funding Agency:

AFA Insurance, VR Treatments of the Future grant

Available from: 2016-09-21 Created: 2016-09-14 Last updated: 2019-05-06Bibliographically approved
Koskela von Sydow, A., Janbaz, C., Kardeby, C., Repsilber, D. & Ivarsson, M. (2016). IL-1α Counteract TGF-β Regulated Genes and Pathways in Human Fibroblasts. Journal of Cellular Biochemistry, 117(7), 1622-1632
Open this publication in new window or tab >>IL-1α Counteract TGF-β Regulated Genes and Pathways in Human Fibroblasts
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2016 (English)In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 117, no 7, p. 1622-1632Article in journal (Refereed) Published
Abstract [en]

Dysregulated wound healing is commonly associated with excessive fibrosis. Connective tissue growth factor (CTGF/CCN2) is characteristically overexpressed in fibrotic diseases and stimulated by transforming growth factor-β (TGF-β) in dermal fibroblasts. We previously showed that interleukin-1 (IL-1α) counteracts TGF-β-stimulated CTGF mRNA and protein expression in these cells. The aim of this study was to explore the effects of IL-1α on further genes and pathways in TGF-β regulated fibroblasts. Transcriptional microarray and multiple comparison analysis showed that the antagonizing effects of IL-1α was much more prominent than the synergistic effects, both with respect to number of genes and extent of changes in gene expression. Moreover, comparing canonical pathways by gene set enrichment analysis and the Ingenuity Pathway Analysis tool revealed that IL-1α counteracted TGF-β in the top six most confident pathways regulated by both cytokines. Interferon and IL-1 signaling, as well as two pathways involved in apoptosis signaling were suppressed by TGF-β and activated by IL-1α. Pathways involving actin remodeling and focal adhesion dynamics were activated by TGF-β and suppressed by IL-1α. Analyzing upstream regulators in part corroborate the comparison of canonical pathways and added cell cycle regulators as another functional group regulated by IL-1α. Finally, gene set enrichment analysis of fibrosis-related genes indicated that IL-1 moderately counteracts the collective effect of TGF-β on these genes. Microarray results were validated by qPCR. Taken together, the results indicate prominent antagonistic effects of IL-1α on TGF-β regulated interferon signaling, as well as on a wide variety of other genes and pathways in fibroblasts. This article is protected by copyright. All rights reserved.

Place, publisher, year, edition, pages
Hoboken, USA: Wiley-Blackwell, 2016
Keywords
Connective tissue growth factor, transforming growth factor-beta, interleukin-1, interferon, fibroblast, fibrosis and ingenuity pathway analysis
National Category
Other Basic Medicine Cell Biology Biochemistry and Molecular Biology
Research subject
Cell Research
Identifiers
urn:nbn:se:oru:diva-48463 (URN)10.1002/jcb.25455 (DOI)000375916800014 ()26629874 (PubMedID)2-s2.0-84964773875 (Scopus ID)
Note

Funding Agencies:

Örebro County Council Research Committee of the Örebro University Hospital OLL-550071

Nyckelfonden AE56340

Available from: 2016-02-22 Created: 2016-02-22 Last updated: 2019-03-26Bibliographically approved
Kardeby, C., Paramel Varghese, G., Pournara, D., Fotopoulou, T., Sirsjö, A., Koufaki, M., . . . Grenegård, M.A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition.
Open this publication in new window or tab >>A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition
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(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-74034 (URN)
Available from: 2019-05-06 Created: 2019-05-06 Last updated: 2019-05-06Bibliographically approved
Kardeby, C., Sirsjö, A., Ljungberg, L. & Grenegård, M.Sulfated glycopolymers and polysaccharides regulate inflammation-related proteins in human vascular endothelial cells.
Open this publication in new window or tab >>Sulfated glycopolymers and polysaccharides regulate inflammation-related proteins in human vascular endothelial cells
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-74033 (URN)
Available from: 2019-05-06 Created: 2019-05-06 Last updated: 2019-05-06Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-5025-9454

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