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  • 1.
    Asplund Persson, Anna
    et al.
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Larsson, Jenny
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    The nitrosoamine dephostatin interacts with nitric oxide/cGMP signalling and modulates cytosolic calcium responses in human plateletsManuscript (preprint) (Other academic)
    Abstract [en]

    1 The effects of the nitrosoamine dephostatin on cytosolic Ca2+ and nitric oxide (NO)/cGMP signallings in human platelets were investigated.

    2 Dephostatin has been characterised as a protein tyrosine phosphatase (PTP) inhibitor, and may on that account affect platelet responses. However, western blot analysis revealed that dephostatin (1-100 µM) did not increase tyrosine-specific protein phosphorylation.

    3 Dephostatin dose-dependently (0.003-3 µM) inhibited thrombin-, thrombin-receptor activating peptide-, and ADP-stimulated rises in cytosolic free Ca2+ concentration, [Ca2+]i. in fura-2-loaded platelets. Surprisingly, higher doses of dephostatin (10-30 µM) augmented thrombin-triggered Ca2+ response. This dual action of dephostatin mainly involved modulation of Ca2+ influx.

    4 The results revealed that dephostatin antagonised NO/cGMP- and prostaglandin E1/cAMP-mediated inhibition of [Ca2+]1. The action of the thrornbin-neutralising peptide hirudin was, however, unaffected.

    5 Dephostatin did not affect basal or NO-mediated rises in platelet cGMP content. On the other hand, dephostatin alone directly increased the phosphorylation of ser239 on vasodilator-stimulated phosphoprotein (VASP) and markedly enhanced NO/cGMP-dependent VASP phosphorylation.

    6 Cell functional studies revealed that dephostatin amplified NO-induced inhibition of platelet aggregation. Opposite to that, dephostatin diminished the inhibitory action of NO on phosphatidylserine exposure.

    7 In conclusion, the results revealed that dephostatin affects a wide range of mechanisms involved in Ca2+ homeostasis in platelets. These effects are apparently not due to inhibition of PTPs. However, enhancement of VASP phosphorylation represents another important molecular mechanism of dephostatin. Considering NO signalling, the results indicate that dephostatin may be an excellent tool when elucidating the relative importance of inhibition of Ca2+ versus VASP phosphorylation.

  • 2.
    Asplund Persson, Anna
    et al.
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Dual actions of dephostatin on the nitric oxide/cGMP-signalling pathway in porcine iliac arteries2005In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 521, no 1-3, p. 124-132Article in journal (Refereed)
    Abstract [en]

    We examined the effects of the nitrosoamine dephostatin on the nitric oxide (NO)/cyclic guanosine 3′,5′-monophosphate (cGMP)-signalling in porcine iliac arteries. Dephostatin has been characterised as a tyrosine phosphatase inhibitor, but Western blot analyses showed that dephostatin did not augment tyrosine phosphorylation of arterial proteins. However, dephostatin relaxed pre-contracted arteries, and this effect was antagonised by the soluble guanylyl cyclase inhibitor 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Furthermore, dephostatin increased the cGMP content and the serine phosphorylation of vasodilator-stimulated phosphoprotein. Dephostatin also inhibited the relaxation induced by acetylcholine and the NO-donor S-nitroso-N-acetyl-penicillamine (SNAP). In contrast, dephostatin did not affect the NO-dependent actions of 1,2,3,4-Oxatriazolium, 3-(3-chloro-2-metylphenyl)-5-[[(4methylphenyl)sulfonyl]amino]-hydroxide inner salt (GEA 3175). Measurement of NO revealed that dephostatin accelerated the consumption of NO. In conclusion, dephostatin exerts dual effects on the NO/cGMP-signalling pathway in iliac arteries. The drug actions included scavenging of NO, but also stimulation of cGMP production. These effects were not related to inhibition of tyrosine phosphatases.

  • 3.
    Asplund Persson, Anna K
    et al.
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Palmér, Louise
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Characterisation of GEA 3175 on human platelets: comparison with S-nitroso-N-acetyl-D,L-penicillamine2004In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 496, no 1-3, p. 1-9Article in journal (Refereed)
    Abstract [en]

    By comparing the effect of two nitric oxide (NO)-containing compounds, we found that S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not GEA 3175 (1,2,3,4-Oxatriazolium,3-(3-chloro-2-metylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt), released NO. Despite this, both drugs elevated cyclic guanosine 3′,5′-monophosphate (cGMP) levels in human platelets. However, SNAP was more effective after short exposure times (5 and 20 s). The compounds also inhibited thrombin-induced rises in cytosolic Ca2+. Time studies revealed that the action of SNAP rapidly declined by increasing the length of incubation (from 5 s to 30 min). This desensibilisation phenomenon mainly involved the release of Ca2+ from intracellular stores. In comparison, GEA 3175-induced inhibition of cytosolic Ca2+ signalling was much more long-lasting. The soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed the effect of GEA 3175 on cytosolic Ca2+. Consequently, this inhibition depends solely on the increase in cGMP. In summary, differences between GEA 3175 and SNAP were observed in NO releasing, cGMP elevating and Ca2+ suppressive properties.

  • 4.
    Asplund Persson, Anna
    et al.
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Zalavary, Stefan
    Linköping University, Faculty of Health Sciences.
    Lindström, Eva
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Whiss, Per A
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Cross-talk between adenosine and the oxatriazole derivative GEA 3175 in platelets2005In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 517, no 3, p. 149-157Article in journal (Refereed)
    Abstract [en]

    We examined the interplay between adenosine and the nitric oxide (NO)-containing oxatriazole derivative GEA 3175 in human platelets. The importance of cyclic guanosine 3′5′-monophosphate (cGMP)-inhibited phosphodiesterases (PDEs) was elucidated by treating the platelets with adenosine combined with either GEA 3175 or the PDE3-inhibitor milrinone. The drug combinations provoked similar cyclic adenosine 3′5′-monophosphate (cAMP) responses. On the contrary, cGMP levels were increased only in GEA 3175-treated platelets. Both drug combinations reduced P-selectin exposure, platelet adhesion and fibrinogen-binding. However, adenosine together with GEA 3175 was more effective in inhibiting platelet aggregation and ATP release. Thrombin-induced rises in cytosolic Ca2+ were suppressed by the two drug combinations. Adenosine administered with GEA 3175 was, however, more effective in reducing Ca2+ influx.

    In conclusion, the interaction between adenosine and GEA 3175 involves cGMP-mediated inhibition of PDE3. The results also imply that inhibition of Ca2+ influx represent another cGMP-specific mechanism that enhances the effect of adenosine.

  • 5.
    Bengtsson, Torbjörn
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Frydén, A
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Zalavary, S
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Whiss, Per A
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Orselius, Kristina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Grenegård, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Platelets enhence neutrophil locomotion: evidence for a role of P-selectin1999In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 59, p. 439-450Article in journal (Refereed)
  • 6.
    Bengtsson, Torbjörn
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Grenegård, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Grenegård, M
    Leucocyte activation by collagen-stimulated platelets in whole blood2002In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 62, no 6, p. 451-462Article in journal (Refereed)
    Abstract [en]

    Interaction between vascular cells plays an important role in the initial phases of the inflammatory process, but the mechanisms responsible for cell-cell communication are not fully understood. In this study, activation of leucocytes and platelets in heparinized whole blood was assessed using lumi-aggregometry. This technique enables simultaneous measurement of aggregation and oxygen radical production by monitoring impedance and luminol-amplified chemiluminescence (CL), respectively. Collagen induced aggregation and CL, depending on dose, and markedly enhanced subsequent aggregation and CL-response triggered by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Collagen stimulation of whole blood down- and upregulated the expression of L-selectin and CD11b, respectively. Monoclonal antibodies against sialyl LewisX and P-selectin caused a pronounced inhibition of the oxidative burst, triggered by collagen itself or by a combination of collagen and fMet-Leu-Phe. Furthermore, the Arg-Gly-Asp-Ser(RGDS)-peptide effectively inhibited collagentriggered aggregation and CL, and the subsequent enhancement of the fMet-Leu-Phe-induced responses. This suggests that fibrinogen plays a part in linking platelet GpIIb/IIIa with CD11b on the leucocyte surface. However, neither anti-CD11b nor the PI-peptide (containing the ?-chain motif in fibrinogen that interacts with CD11b) counteracted the stimulatory effects of activated platelets on leucocyte functions. The selectin- and integrin-antagonizing substances were ineffective on the CL-responses induced by fMet-Leu-Phe itself. This study suggests that, through selectin- and integrin-dependent interaction, activated platelets potentiate leucocyte aggregation and oxygen radical production, which might be important for the outcome of inflammatory reactions.

  • 7.
    Berg, Anna
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Redéen, Stefan
    Linköping University, Department of Biomedicine and Surgery, Surgery. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Ericson, Ann-Charlott
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sjöstrand, Sven-Erik
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Nitric oxide inhibits gastric acid secretion by increasing intraparietal cell levels of cGMP in isolated human gastric glands2005In: American Journal of Physiology - Gastrointestinal and Liver Physiology, ISSN 0193-1857, E-ISSN 1522-1547, Vol. 289, no 6, p. G1061-G1066Article in journal (Refereed)
    Abstract [en]

    We have previously identified cells containing the enzyme nitric oxide (NO) synthase (NOS) in the human gastric mucosa. Moreover, we have demonstrated that endogenous and exogenous NO has been shown to decrease histamine-stimulated acid secretion in isolated human gastric glands. The present investigation aimed to further determine whether this action of NO was mediated by the activation of guanylyl cyclase (GC) and subsequent production of cGMP. Isolated gastric glands were obtained after enzymatic digestion of biopsies taken from the oxyntic mucosa of healthy volunteers. Acid secretion was assessed by measuring [14C]aminopyrine accumulation, and the concentration of cGMP was determined by radioimmunoassay. In addition, immunohistochemistry was used to examine the localization of cGMP in mucosal preparations after stimulation with the NO donor S-nitroso-N-acetylpenicillamine (SNAP). SNAP (0.1 mM) was shown to decrease acid secretion stimulated by histamine (50 μM); this effect was accompanied by an increase in cGMP production, which was histologically localized to parietal cells. The membrane-permeable cGMP analog dibuturyl-cGMP (db-cGMP; 0.1–1 mM) dose dependently inhibited acid secretion. Additionally, the effect of SNAP was prevented by preincubating the glands with the GC inhibitor 4H-8-bromo-1,2,4-oxadiazolo[3,4-d]benz[b][1,4]oxazin-1-one (10 μM). We therefore suggest that NO in the human gastric mucosa is of physiological importance in regulating acid secretion. Furthermore, the results show that NO-induced inhibition of gastric acid secretion is a cGMP-dependent mechanism in the parietal cell involving the activation of GC.

  • 8.
    Boknäs, Niklas
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Faxälv, Lars
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Chemistry.
    Sanchez Centellas, Daniel
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Wallstedt, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Ramström, Sofia
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    University of Örebro, Sweden.
    Lindahl, Tomas
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Chemistry.
    Thrombin-induced platelet activation via PAR4: pivotal role for exosite II2014In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 112, no 3, p. 558-565Article in journal (Refereed)
    Abstract [en]

    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.

  • 9.
    Désilets, Stéphanie
    et al.
    Linköping University, Department of Medicine and Care.
    Whiss, Per A
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Eriksson, Andreas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Nilsson, Ulrika
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Grenegård, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Lysophosphatidic acid inhibits ADP-activated platelets2004In: 10th Annual Scandinavian Atherosclerosis Conference and International Meeting,2004, 2004Conference paper (Other academic)
  • 10.
    Edoff, Karin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Hildebrand, Claes
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Retrograde tracing and neuropeptide immunohistochemistry of sensory neurones projecting to the cartilaginous distal femoral epiphysis of young rats2000In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 299, no 2, p. 193-200Article in journal (Refereed)
    Abstract [en]

    Although cartilage is considered to be devoid of innervation, axons occur in the perichondrium and during development in cartilage canals, thereby having a relatively close spatial relationship to chondroblasts and chondrocytes. The present study locates the source of the sensory innervation of the femoral cartilaginous epiphyses of young rats and investigates whether the neuropeptide calcitonin gene-related peptide (CGRP) can influence chondrocytes. Retrograde tracing from the distal femoral epiphysis of young rats with Fast Blue (FB) showed labelled neuronal profiles in the L2-L5 dorsal root ganglia. Sample countings indicated that 50% of the FB-labelled neuronal profiles were located at the L3 level and 25% at the L4 level. The labelled neurones had diameters of 15-40 µm, with a peak at 25-30 µm. Immunohistochemistry showed that about 50% of the FB-labelled profiles contained CGRP. Together with the finding that CGRP influences bone cells to generate the second messenger cAMP, this result suggested the hypothesis that chondrocytes might be similarly influenced by CGRP. However, stimulation of cartilage slices with CGRP in vitro followed by an assay of the cAMP content did not provide support for this hypothesis. We conclude that primary sensory neurones containing CGRP project to the perichondrium and to cartilage canals of growing cartilage, and that exogenous CGRP does not elevate the cAMP content of cartilage slices in vitro.

  • 11.
    Elvers, Margitta
    et al.
    University of Tubingen, Germany .
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Khoshjabinzadeh, Hanieh
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Muenzer, Patrick
    University of Tubingen, Germany .
    Borst, Oliver
    University of Tubingen, Germany University of Tubingen, Germany .
    Tian, Huasong
    Columbia University, NY 10032 USA .
    Di Paolo, Gilbert
    Columbia University, NY 10032 USA .
    Lang, Florian
    University of Tubingen, Germany .
    Gawaz, Meinrad
    University of Tubingen, Germany .
    Lindahl, Tomas
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Chemistry.
    Fälker, Knut
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity2012In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 24, no 9, p. 1743-1752Article in journal (Refereed)
    Abstract [en]

    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 Ca2+ 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.

  • 12.
    Fälker, Knut
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry.
    Haglund, Linda
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Nylander, Martina
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindahl, Tomas
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Chemistry.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets2011In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 436, p. 469-480Article in journal (Refereed)
    Abstract [en]

    FARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(alpha 12/13) and G(alpha q) 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 Ca(2+) mobilization, PKC (protein kinase C) signalling and alpha-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 P2Y(12) receptor-induced G(alpha i) signalling accounted for the loss of the aggregation response, as mimicking G(alpha i/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 alpha-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.

  • 13.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Platelets: intracellular signalling and cellular interaction1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Activation of platelets is essential to prevent excessive blood loss at the site of vascular injury. These highly reactive cell fragments are also involved in several pathological conditions, in particular arterial thrombosis, which, in the coronary vessels, can lead to reduced blood flow, vessel occlusion and myocardial infarction.

    The present research was focused on the anti-platelet properties, mechanisms of actions, and interactions of GEA 3175 and adenosine. The former a novel nitric oxidecontaining and cyclic GMP-elevating compound, the latter a cyclic AMP-elevating · purine nucleoside. Separately, the two substances significantly reduced the cytosolic , Ca2+ responses, but only marginally suppressed subsequent functional responses in thrombin-stimulated human platelets. However, coadministration of the compounds synergistically inhibited platelet aggregation and abolished dense granule secretion 1 and exposure of P-selectin, and also markedly reduced binding of fibrinogen to the i surface of platelets. The results imply that the stronger inhibition of platelet functions was due to potent blocking of the initial Ca2+ transient and abrogation of mechanisms involved in the influx of extracellular Ca2+. Together, the findings clearly manifest the importance of an interaction between different inhibitory intracellular pathways in order to completely suppress the activity of tbrombin activated platelets.

    Cell- and drug-comparative studies revealed that GEA 3175 provoked aremarkable long-acting inhibition of contractile responses and a concomitant i sustained increase in cyclic GMP levels in airway smooth muscle tissue. The mechanisms of relaxation involved iberiotoxin-sensitive K + channels and okadaic ' acid-sensitive phosphatases. Separately, GEA 3175 and adenosine markedly inhibited the respiratory burst in chemoattractant-activated neutrophil granulocytes. Opposite to the situation in platelets, the two compounds did not interact synergistically and coadministration had only a negligible effect on cytosolic Ca2+ signals in neutrophils. These data clearly demonstrate cell type-specific responses to cyclic GMP- and cyclic AMP-elevating agents.

    This thesis also delineates the regulatory effects of platelets on neutrophil cellular and intracellular responses. Simple mixture of suspensions of human platelets and neutrophils caused prominent changes in the neutrophil in regard to cytoskeletal arrangement, cytosolic Ca2+ responsiveness, and capacity to generate reactive oxygen species. Platelet-derived factor(s) induced a marked suppression of the respiratory burst in activated neutrophils, an effect attributed to peripheral accumulation of ! filamentous actin and enhanced release of endogenous adenosine from the neutrophil. The chemotactic-peptide-induced rise in cytosolic Ca2+ in neutrophils was dramatically amplified in the presence of platelets, and ATP released from the platelets may play a role in this priming phenomenon, However, additional experiments showed that the amplified Ca2+ response was virtually independent of the state of activation of the platelet, required extracellular Ca2+ ions, and was completely insensitive to the NO-donor GEA 3175. Further analysis strongly indicated that platelets affected the cyclic AMP-sensitive phase of the Ca2+ response in neutrophils. In conclusion, multiple mediators and mechanisms participate in the platelet-mediated modulation of neutrophil responses, and greater understanding of the complex mechanisms and consequences of the interactions between these blood cells may provide useful information for the design of pharmacological tools and methods to control the inflammatory reaction.

  • 14.
    Grenegård, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Pharmacology .
    The tyrosine phosphatase inhibitor dephostatin exerts multiple effects on cytosolic calcium in human platelets1999In: Thrombosis and Haemostasis, ISSN 0340-6245, p. 1121-Conference paper (Other academic)
  • 15.
    Grenegård, Magnus
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Reactive oxygen species derived from platelets reduce the action of nitric oxide2007Conference paper (Other academic)
  • 16.
    Grenegård, Magnus
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Reactive oxygen species derived from platelets reduce the action of nitric oxide2007Conference paper (Other academic)
  • 17.
    Grenegård, Magnus
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Asplund-Persson, Anna
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Mechanisms underlying platelet desensitisation towards nitric oxide2006Conference paper (Other academic)
  • 18.
    Grenegård, Magnus
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Vretenbrant-Öberg, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Nylander, Martina
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Désilets, Stéphanie
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Lindström, Eva G
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Larsson, Anders
    Department of Medical Sciences, University Hospital, Uppsala SE-75105, Sweden.
    Ramström, Ida
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Ramström, Sofia
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Lindahl, Tomas L
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
    The ATP-gated P2X1 receptor plays a pivotal role in activation of aspirin-treated platelets by thrombin and epinephrine2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 27, p. 18493-18504Article in journal (Refereed)
    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.

  • 19.
    Grenegård, Magnus
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Whiss, Per A
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Svensson, Samuel
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    The novel nitric oxide-donor GEA 3175 and adenosine inhibit intracellular responses in thrombin-activated platelets1997In: FEBS special meeting: Cell Signalling Mechanisms, From Membrane to Nucleus,1997, 1997Conference paper (Other academic)
  • 20.
    Gunnarsson, Peter
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Fornander, Louise
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Sialic acid residues play a pivotal role in alpha(1)-acid glycoprotein (AGP)-induced generation of reactive oxygen species in chemotactic peptide pre-activated neutrophil granulocytes2010In: INFLAMMATION RESEARCH, ISSN 1023-3830, Vol. 59, no 2, p. 89-95Article in journal (Refereed)
    Abstract [en]

    We have recently shown that terminal sialic acid residues are essential for alpha(1)-acid glycoprotein (AGP)-induced Ca2+ mobilization in neutrophils. The aim of the present study was to establish the importance of sialic acid residues on AGP in modulating human neutrophil functions, with emphasis on the generation of reactive oxygen species (ROS). ROS were measured by luminol-enhanced chemiluminescence in isolated human neutrophils. We found that AGP did not provoke ROS generation in resting or L-selectin presensitized neutrophils. Moreover, AGP did not affect the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS generation, but it slightly suppressed opsonized zymosan-induced responses. However, when the neutrophils were prestimulated with fMLP, the subsequent addition of AGP provoked a marked ROS response. Dose-response studies and time studies revealed that the ROS generating capacity of AGP was highest at a concentration of 0.05 mg/ml and when given 3-10 min after addition of fMLP. A desialylated form of AGP or pretreatment of neutrophils with 3- and 6-sialyllactose caused a substantially lower ROS response in neutrophils prestimulated with fMLP. Our data show that AGP can stimulate a second ROS response in fMLP preactivated neutrophils and that terminal sialic acid residues on AGP play a crucial role in this regard.

  • 21.
    Gunnarsson, Peter
    et al.
    Linköping University, Department of Medicine and Health Sciences. Linköping University, Faculty of Health Sciences.
    Levander, Louise
    Linköping University, Department of health and environment. Linköping University, Faculty of Health Sciences.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences. Linköping University, Faculty of Health Sciences.
    Sialic Acid Dependent and Independent Effects of alpha 1-Acid Glycoprotein (AGP) on Human Platelets2008In: 2008 Meeting of the Society for Glycobiology, 2008, Vol. 18, no 11, p. 990-990Conference paper (Other academic)
    Abstract [en]

    Objective: We have recently shown that terminal sialic acid residues are essential for α1-acidglycoprotein (AGP)-induced Ca2+ mobilization in neutrophils. The aim of the present studywas to establish the importance of sialic acid-residues on AGP in modulating humanneutrophil functions, with emphasis on the generation of reactive oxygen species (ROS).Material and methods: ROS were measured by luminol-enhanced chemiluminescence inisolated human neutrophils.

    Results: We found that AGP did not provoke ROS generation in resting or L-selectin presensitizedneutrophils. Moreover, AGP did not affect the N-formyl-methionyl-leucylphenylalanine(fMLP)-induced ROS generation but it slightly suppressed opsonized zymosaninducedresponses. However, when the neutrophils were pre-stimulated with fMLP, thefollowing addition of AGP provoked a marked ROS response. Dose-response studies and timestudies revealed that the ROS generating capacity of AGP was maximal at a concentration of0.05 mg/ml and when given 3-10 min after addition of fMLP. A desialylated form of AGP orpre-treatment of neutrophils with 3’- and 6’-sialyllactose caused a substantial lower ROSresponse in neutrophils pre-stimulated with fMLP.

    Conclusions: Our data show that AGP can stimulate a second ROS response in fMLP preactivatedneutrophils and that terminal sialic acid residues on AGP play a crucial role in thisregard.

  • 22.
    Gunnarsson, Peter
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Levander, Louise
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Påhlsson, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    The acute-phase protein alpha 1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (siglecs)2007In: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, ISSN 1530-6860, Vol. 21, no 14, p. 4059-4069Article in journal (Refereed)
    Abstract [en]

    We studied whether the acute-phase protein alpha1-acid glycoprotein (AGP) induces rises in [Ca2+]i in neutrophils and sought to identify the corresponding AGP receptor (or receptors). We found that AGP elicited a minimal rise in [Ca2+]i in Fura-2-loaded neutrophils, and this response was markedly enhanced by pretreatment with anti-L-selectin antibodies. (The EC50 value of the AGP-induced Ca2+ response was 9 microg/ml.) Activation of phospholipase-C, Src tyrosine kinases, and PI3 kinases proved to be essential for the AGP-mediated increase in [Ca2+]i, whereas the p38 MAPK and SYK signaling pathways were not involved. Furthermore, antibodies against sialic acid binding, immunoglobulin-like lectin 5 (Siglec-5) and oligosaccharide 3'-sialyl-lactose both antagonized the AGP-induced response and caused an immediate increase in [Ca2+]i in anti-L-selectin-treated neutrophils, which indicates a signaling capacity of Siglec-5. We used modified forms of AGP (treated with mild periodate or neuraminidase) to establish the importance of sialic acid residues. The modified forms of AGP caused a much smaller rise in [Ca2+]i than did unaltered AGP. Affinity chromatography confirmed that unchanged AGP, but not neuraminidase-treated AGP, bound to Siglec-5. Our report provides the first evidence for a signaling capacity by AGP through a defined receptor. Pre-engagement of L-selectin significantly enhanced this signaling capacity.

  • 23.
    Gunnarsson, Peter
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Levander, Louise
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    α1-acid glycoprotein (AGP)-induced platelet shape change involvesthe Rho/Rho kinase signalling pathway2009In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 102, no 4, p. 694-703Article in journal (Refereed)
    Abstract [en]

    α1-acid glycoprotein (AGP) is an acute-phase protein that contributes to inflammation processes. The role of AGP in platelet activation and thrombosis is, however, largely unknown. Therefore, we thoroughly investigated the effects of AGP on human platelets. Platelets were isolated from healthy volunteers and subsequently exposed to AGP. Platelet responses were monitored as change in light transmission, intracellular calcium concentration, light microscopy and protein phosphorylation by Western blot. We found that AGP induced platelet shape change independently of a second release of adenine nucleotides or thromboxane A2, and that effect was abolished by endotheliumderived platelet inhibitors such as nitric oxide (NO) and adenosine. Furthermore, AGP triggered a minor calcium response and a pronounced Rho/Rho-kinase-dependent increase in Thr696 phosphorylation of myosin phosphatase target subunit 1 (MYPT1). Moreover, the Rho/Rho-kinase inhibitor Y-27632 significantly decreased the AGP-induced shape change. The results also showed that the AGP-elicited shape change was antagonised by pretreatment with low doses of collagen and thrombospondin- 1. Our results describe a novel mechanism by which AGP stimulates platelet shape change via activation of the Rho/Rhokinase signalling pathway. Physiological important platelet inhibitors, such as NO, completely counterbalance the effect of AGP. Hence, the present study indicates that AGP directly contributes to platelet activation, which in turn might have an impact in physiological haemostasis and/or pathological thrombosis.

  • 24.
    Junker, J P E
    et al.
    Harvard University.
    Lonnqvist, Susanna
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Karlsson, L K
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Grenegard, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Plastic Surgery, Hand surgery UHL.
    Endothelial differentiation of human dermal fibroblasts in JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, vol 6, issue SI, pp 149-1492012In: JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, John Wiley and Sons , 2012, Vol. 6, no SI, p. 149-149Conference paper (Refereed)
    Abstract [en]

    n/a

  • 25.
    Junker, J P
    et al.
    Harvard University.
    Lönnqvist, Susanna
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Karlsson, L K
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Plastic Surgery, Hand Surgery and Burns. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Plastic Surgery, Hand surgery UHL.
    ENDOTHELIAL DIFFERENTIATION OF HUMAN DERMAL FIBROBLASTS in WOUND REPAIR AND REGENERATION, vol 20, issue 2, pp A27-A272012In: WOUND REPAIR AND REGENERATION, Wiley-Blackwell , 2012, Vol. 20, no 2, p. A27-A27Conference paper (Refereed)
    Abstract [en]

    n/a

  • 26.
    Junker, Johan
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Lönnqvist, Susanna
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    Rakar, Jonathan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    Karlsson, Lisa K.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Hand and Plastic Surgery.
    Differentiation of human dermal fibroblasts towards endothelial cells2013In: Differentiation, ISSN 0301-4681, E-ISSN 1432-0436, Vol. 85, no 3, p. 67-77Article in journal (Refereed)
    Abstract [en]

    The ultimate goal of vascular tissue engineering is the production of functional grafts for clinical use. Difficulties acquiring autologous endothelial cells have motivated the search for alternative cell sources. Differentiation of dermal fibroblasts towards several mesenchymal lineages as well as endothelial cells has been proposed. The aim of the present study was to investigate the endothelial differentiation capacity of human dermal fibroblasts on a gene expression, protein expression and functional physiological level. Endothelial differentiation of fibroblasts was induced by culturing cells in 30% human serum, but not in fetal calf serum. Expression of proteins and genes relevant for endothelial function and differentiation was increased after induction. Furthermore, fibroblasts exposed to 30% human serum displayed increased uptake of low-density lipoprotein and formation of capillary-like networks. The results of this study may have an impact on cell sourcing for vascular tissue engineering, and the development of methods for vascularization of autologous tissue engineered constructs.

  • 27.
    Karlsson, Lisa K.
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Junker, Johan
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine, Burn Unit . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Human Dermal Fibroblasts and Single-Cell Clone Fibroblasts Have theCapacity to Alter Their Phenotype Towardsan Endothelial-Like Cell type2009In: European Cells and Materials, ISSN 1473-2262, E-ISSN 1473-2262Article in journal (Other academic)
    Abstract [en]

    We investigated the capacity of normal human dermal fibroblasts to alter their phenotype into an endothelialcell-like phenotype. By utilising in vitro cell culture models, the part played by different types of serum andmedium constituents in inducing a phenotypic change of fibroblasts was investigated. The experiments usedprimary cultures of human endothelial cells, human dermal fibroblasts and single-cell clone fibroblasts. Thelatter cell type was obtained by clonal expansion using a micromanipulator technique. The results showed thatthe presence of human serum in the cell culture medium caused both types of fibroblasts to express vonWillebrand factor, to incorporate fluorochrome-labelled LDL, and to start forming capillary-like networks in asimilar way to endothelial cells. The phenotypic shift was detectable after 4 days of cell culture and reached amaximum after 7-10 days. To our knowledge this is the first report to describe differentiation of humanfibroblasts towards an endothelial cell-like phenotype. The results also show that the underlying mechanism ofthe phenotypic shift is a change in gene expression in the dermal fibroblasts and not fusion between different celltypes. Collectively, the present results indicate that human dermal fibroblasts may be a novel cell source forcreating vascular endothelium.

  • 28.
    Karlsson, Lisa K
    et al.
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Junker, Johan P E
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Kratz, Gunnar
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Human Dermal Fibroblasts: A Potential Cell Source for Endothelialization of Vascular Grafts2009In: Annals of Vascular Surgery, ISSN 0890-5096, E-ISSN 1615-5947, Vol. 23, no 5, p. 663-674Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Recently, there has been an intense ongoing search for suitable cell sources for vascular tissue engineering. Previous studies report that cells with multilineage potential have been found within the connective stroma of the skin. In line with this, preliminary data from our group suggest that human dermal fibroblasts have the capacity to alter their phenotype into an endothelial cell-like phenotype in vitro. As a first step in using these cells in vascular tissue engineering, we investigated their ability to form an endothelial cell-like layer on a scaffold in vitro. Furthermore, we studied the possibility of seeding dermal fibroblasts on a scaffold and later commencing with induction toward an endothelial cell-like phenotype. METHODS: Cells cultured in either normal fibroblast medium or endothelial induction medium were seeded on a gelatin-based scaffold. To study the organization of cells, routine staining was performed. Differentiation was confirmed by Western blotting and immunohistochemistry with antibodies directed toward molecules commonly used to identify endothelial cells. RESULTS AND CONCLUSION: Our data support that human dermal fibroblasts differentiated toward endothelial cell-like cells prior to seeding showed histological resemblance to mature endothelial cells, while fibroblasts seeded and later induced into endothelial differentiation grew in multilayer. However, expression of various surface molecules indicative of an endothelial phenotype was seen using both techniques. In conclusion, the results presented in this study indicate that human dermal fibroblasts differentiated toward an endothelial cell-like phenotype may be a novel cell source for endothelialization of vascular grafts.

  • 29.
    Kälvegren, Hanna
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Health Sciences.
    Andersson, Johanna
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Platelet activation triggered by Chlamydia pneumoniae is antagonized by 12-lipoxygenase inhibitors but not cyclooxygenase inhibitors.2007In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 566, no 1-3, p. 20-27Article in journal (Refereed)
    Abstract [en]

    Chlamydia pneumoniae is a respiratory pathogen that has been linked to cardiovascular disease. We have recently shown that C. pneumoniae activates platelets, leading to oxidation of low-density lipoproteins. The aim of the present study was to evaluate the inhibitory effects of different pharmacological agents on platelet aggregation and secretion induced by C. pneumoniae.

    Platelet interaction with C. pneumoniae was studied by analyzing platelet aggregation and ATP-secretion with Lumi-aggregometry.

    Platelet aggregation and ATP-secretion induced by C. pneumoniae was markedly inhibited by the NO-donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), an effect that was counteracted by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Pre-treatment of platelets with the 12-lipoxygenase (12-LOX) inhibitors cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and 5,6,7-trikydroxyflavone (baicalein) completely blocked the activation, whereas the cyclooxygenase (COX) inhibitors 2-acetyloxybenzoic acid (aspirin) and (8E)-8-[hydroxy-(pyridin-2-ylamino)methylidene]-9-methyl-10,10-dioxo-10$l^(6)thia-9-azabicyclo[4.4.0]deca-1,3,5-trien-7-one (piroxicam) had no inhibitory effects. Opposite to C. pneumoniae-induced activation, platelets stimulated by collagen were inhibited by the COX-inhibitors but were unaffected by the 12-LOX-inhibitors. The platelet activating factor (PAF) antagonist Ginkgolide B blocked the C. pneumoniae-induced platelet activation, whereas the responses to collagen were unaffected. Furthermore, the P2Y1 and P2Y12 purinergic receptor antagonists 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS2179) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (cangrelor) inhibited the aggregation and secretion caused by C. pneumoniae.

    It is well-known that the efficacy of COX inhibitors in the prevention and treatment of cardiovascular disease varies between different patients, and that patients with low responses to aspirin have a higher risk to encounter cardiovascular events. The findings in this study showing that platelets stimulated by C. pneumoniae are unaffected by COX inhibitors but sensitive to 12-LOX inhibitors, may thus be of importance in future management of atherosclerosis and thrombosis.

  • 30.
    Kälvegren, Hanna
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Bylin, Helena
    Linköping University, Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine . Linköping University, Faculty of Health Sciences.
    Leanderson, Per
    Linköping University, Department of Clinical and Experimental Medicine, Occupational and Environmental Medicine . Linköping University, Faculty of Health Sciences.
    Richter, Arina
    Linköping University, Department of Medicine and Health Sciences, Cardiology . Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Chlamydia pneumoniae induces nitric oxide synthase and lipoxygenase-dependent production of reactive oxygen species in platelets — effects on oxidation of low-density lipoproteins.2005In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 94, no 2, p. 327-335Article in journal (Refereed)
    Abstract [en]

    There is increasing evidence that Chlamydia pneumoniae is linked to atherosclerosis and thrombosis. In this regard, we have recently shown that C. pneumoniae stimulates platelet aggregation and secretion, which may play an important role in the progress of atherosclerosis and in thrombotic vascular occlusion. The aims of the present study were to investigate the effects of C. pneumoniae on platelet-mediated formation of reactive oxygen species (ROS) and oxidation of low-density lipoprotein (LDL) in vitro. ROS production was registered as changes in 2´,7`-dichlorofluorescin- fluorescence in platelets with flow cytometry. LDL-oxidation was determined by measuring thiobarbituric acid reactive substances (TBARs). We found that C. pneumoniae stimulated platelet production of ROS.Polymyxin B treatment of C. pneumoniae, but not elevated temperature, abolished the stimulatory effects on platelet ROS- production, which suggests that chlamydial lipopolysaccharide has an important role. In hibition of nitric oxide synthase with nitro-L-arginine, lipoxygenase with 5,8,11-eicosatriynoic acid and protein kinase C with GF 109203X significantly lowered the production of radicals. In contrast, inhibition of NADPH-oxidase with di-phenyleneiodonium (DPI) did not affect the C. pneumoniae induced ROS-production. These findings suggest that the activities of nitric oxide synthase and lipoxygenase are the sources for ROS and that the generation is dependent of the activity of protein kinase C.The C. pneumoniae-induced ROS-production in platelets was associated with an extensive oxidation of LDL, which was significantly higher compared to the effect obtained by separate exposure of LDL to C. pneumoniae or platelets. In conclusion, C. pneumoniae interaction with platelets leading to aggregation, ROS-production and oxidative damage on LDL, may play a crucial role in the development of atherosclerotic cardiovascular disease.

  • 31.
    Kälvegren, Hanna
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Health Sciences.
    Skoglund, Caroline
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Helldahl, Christian
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lerm, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Toll-like receptor 2 stimulation of platelets is mediated by purinergic P2X1-dependent Ca2+ mobilisation, cyclooxygenase and purinergic P2Y1 and P2Y12 receptor activation2010In: Thrombosis and Haemostasis, ISSN 0340-6245, Vol. 103, no 2, p. 398-407Article in journal (Refereed)
    Abstract [en]

    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.

  • 32.
    Larsson, Jenny
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Sonesson, Linus
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lundqvist-Gustafsson, Helen
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Platelet-mediated inhibition of polymorphonuclear neutrophil apoptosis principally involves membranes structures: role of sialyl-Lewisχ epitopes and CD18Manuscript (preprint) (Other academic)
    Abstract [en]

    Objective and design: To clarify the long-time effects of platelets on polymorphonuclear neutrophils (PMNs) apoptosis, with particular emphasis on the involvement of cell adhesion molecules (CAMs).

    Material: Isolated human platelets and PMNs.

    Treatment: PMNs were treated with antibodies towards adhesion molecules CD18 (2 µg/ml), sialyl-Lewisχ (2 µg/ml) or P-selectin glycoprotein ligand 1 (5 µg/ml) and then incubated in the absence or presence of resting, thrombin-activated or inhibited platelets (PMN:platelet ratio of 1:50), platelet membrane (0.7 mg/ml; equivalent to the 1:50 ratio), or supematant from thrombin-activated platelets.

    Methods: Measurement of DNA fragmentation using flow cytometry, microscopical evaluation of adhesion and cell death. Light transmission analysis for recording platelet aggregation.

    Results: Activated, and to lesser extent, resting platelets prevented spontaneous PMN apoptosis. Comparable effects were detected by using platelet membrane. Platelet-mediated suppression of PMN apoptosis and PMN-platelet adhesion were reversed by pretreatment with antibodies directed towards the adhesive structures sialyl-Lewisχ (p<0.001).

    Conclusions: Our results point to a central role of CAMs in plateletinduced inhibition of PMN apoptosis. Furthermore, the results add new evidences for a close association between the hemostatic and the inflammatory systems.

  • 33.
    Levander, Louise
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Ryden, I
    Kalmar County Hospital.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Effects of alpha 1-acid Glycoprotein Fucosylation on its Ca2+ Mobilizing Capacity in Neutrophils2009In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 69, no 5, p. 412-420Article in journal (Refereed)
    Abstract [en]

    We recently showed that the acute-phase protein alpha(1)-acid glycoprotein (AGP) induces rises in cytosolic calcium concentration, [Ca2+](i,) in neutrophils through sialic acid dependent interactions with the neutrophil receptors siglec-5 and/or siglec-14. Whereas both siglec-5 and siglec-14 have a relatively broad specificity for sialylated oligosaccharide structures, including both structures with terminal alpha 2-3 or alpha 2-6 linked sialic acid, there is a markedly reduced affinity to the fucosylated epitope sialyl Lewis x (SLe(x)). Increased fucosylation, leading to increased expression of SLe(x) on AGP is commonly associated with inflammatory conditions. In the present study, we investigated whether an increased SLe(x) expression would affect the Ca2+-mobilizing effect of AGP. AGP with elevated fucose content isolated from patients with untreated chronic joint inflammation showed a decreased [Ca2+](i) modulatory effect on neutrophils compared to normally fucosylated AGP. Furthermore a hyperfucosylated AGP form produced by in vitro fucosylation, that consequently had an elevated expression of SLe(x), could not elicit a [Ca2+](i) increase in neutrophils. The role of the carbohydrate portion of AGP in modulating neutrophil responses was further strengthened by showing that synthetic glycoconjugates carrying oligosaccharides with terminal alpha 2-3 or alpha 2-6 linked sialic acid were able to mimic the Ca2+-mobilizing effect of AGP whereas a synthetic glycoconjugate carrying SLe(x) was not. Based on these data, we conclude that increased fucosylation can alter the ability of AGP to induce neutrophil signalling and further supports an important role of the oligosaccharide chains of AGP in the modulation of leukocyte functions during an inflammatory process.

  • 34.
    Levander, Louise
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Alpha-1-acid glycoprotein interacts with the neutrophilproteins S100A8 and S100A9Manuscript (preprint) (Other academic)
    Abstract [en]

    The acute phase protein α1-acid glycoprotein (AGP) has been indicated tobind to neutrophils and to affect neutrophil functions. In the presentinvestigation neutrophil proteins interacting with AGP was studied. Twolow molecular weight proteins were isolated from neutrophil lysates byaffinity chromatography on a column with immobilized AGP. The proteinswere identified as the calcium-binding, myeloid related proteins S100A8and S100A9 using mass spectrometry and Western blot analyses. Theinteraction was further confirmed using a biotin affinity-tagging procedure.The interaction between AGP and S100A8/A9 was sensitive to EDTAindicating a calcium-dependent binding. Desialylation andhyperfucosylation of AGP did not affect its binding to S100A8/A9.

  • 35.
    Levander, Louise
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Gunnarsson, Peter
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Påhlsson, Peter
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Alpha1-acid glycoprotein is a carrier of hydroxyeicosatetraenoic acids – role in calcium mobilization ofpolymorphonuclear granulocytesManuscript (preprint) (Other academic)
    Abstract [en]

    We have previously shown that α1-acid glycoprotein (AGP) induces rises incytosolic calcium concentration, [Ca2+]i, in human polymorphonuclear granulocytes(PMN) and that this effect is enhanced by prior pre-sensitization of PMN with theanti L-selectin antibody DREG-56. AGP is a known carrier of several lipophilicsubstances. This study was designed to determine whether lipids bound to AGP areinvolved in the induction of [Ca2+]i mobilization in PMN. We found that delipidatedAGP elicited a smaller rise in [Ca2+]i in DREG-56 pretreated PMN compared tonative AGP and that lipids extracted from AGP provoked an increase in [Ca2+]i thatwas potentiated by L-selectin pre-engagement with DREG-56. Similarly to whatwas previously found for AGP, the increase in [Ca2+]i produced by the AGP lipidextract involved activation of src-tyrosine kinases and PI3-kinases. The AGP lipidextract was analyzed by high-performance thin layer chromatography. Individualbands were extracted from the plate and their Ca2+ mobilizing activity wasanalyzed. One band contained activity and was further analyzed by electro-spraytandem mass spectrometry. The active band contained a mixture of hydroxyeicosatetraenoic acids (HETEs) with 12-HETE being one of the major components.Pharmacological studies indicated that the AGP lipid extract acted through theleukotriene B4-receptor type II, BLT2. This study supports the hypothesis that someof the immunomodulatory properties that have been attributed to AGP may beconnected to lipids carried by this plasma protein.

  • 36.
    Nilsson, Ulrika K.
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Berg, Göran
    Linköping University, Department of Clinical and Experimental Medicine, Obstetrics and gynecology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Paediatrics and Gynecology and Obstetrics, Department of Gynecology and Obstetrics in Linköping.
    Svensson, Samuel P.S.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Different proliferative responses of Gi/o-protein-coupled receptors in human myometrial smooth muscle cells: a possible role of calcium1998In: Journal of Molecular Neuroscience, ISSN 0895-8696, Vol. 11, no 1, p. 11-21Article in journal (Refereed)
    Abstract [en]

    The majority of studies investigating the proliferative effect of Gi/o-protein-coupled receptor agonists are performed in recombinant receptor systems or cell lines. In these systems the relative stoichiometry of receptors compared to other cell components might be changed, which may lead to anomalies in cellular responses in contrast to natural occurring systems. In the present study, we have used primary cultures of smooth muscle cells (SMCs) isolated from human myometrium to characterize the proliferative effects of agonists binding to two different G protein-coupled receptors. Treatment of quiescent SMCs with lysophosphatidic acid (LPA) and noradrenaline resulted in significant increases in [3H]thymidine incorporation. However, LPA was almost four times more effective than noradrenaline in this respect. The proliferative effects of the agonists could be completely blocked by pertussis toxin, indicating that the response are mediated through Gi/o-proteins. The selective α2-adrenergic receptor (α2-AR) antagonist yohimbine dose-dependently reduced the effect of noradrenaline suggesting that the proliferative response was mediated through α2-ARs. The proliferative effects induced by LPA and noradrenaline was markedly reduced in SMCs treated with the tyrosine kinase inhibitor genistein and the cAMP elevating compound forskolin. However, LPA but not noradrenaline induced rapid rises in the cytosolic free Ca2+ concentration [Ca2+]i. The ability to increase Ca2+ might be one explanation why LPA produce a more pronounced proliferative response than noradrenaline in primary cultures of human myometrial SMCs.

  • 37.
    Nilsson, Ulrika K.
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Svensson, Samuel P.S.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Synergistic activation of human platelets by adrenaline and lysophosphatidic acid2002In: Haematologica, ISSN 0390-6078, Vol. 87, no 7, p. 730-739Article in journal (Refereed)
    Abstract [en]

    BACKGROUND AND OBJECTIVES: Platelet reactivity is regulated by various important bioactive and physiologic substances. The objective of this study was to characterize lysophosphatidic acid (LPA)-triggered responses in human platelets. In addition, the effect of LPA was compared with that of other activators and possible synergistic interactions were evaluated.

    DESIGN AND METHODS: LPA-triggered cytosolic Ca(2+) responses were measured using fura-2-loaded platelets in a spectrofluorometer. Furthermore, platelet aggregation and secretion were analyzed in a lumi-aggregometer and protein tyrosine phosphorylation was detected with the Western blot technique.

    RESULTS: LPA dose-dependently increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in platelets. This response involved both influx of extracellular Ca(2+) and release of Ca(2+) from intracellular stores. However, in comparison with other platelet agonists, i.e. thrombin and adenosine 5'-diphosphate (ADP), LPA was a very weak Ca(2+)-elevating agent. Furthermore, we observed that the LPA-induced rise in [Ca(2+)](i) was markedly suppressed by cyclic nucleotide-elevating agents. In functional studies, LPA failed to stimulate platelet aggregation and secretion. However, in combination with adrenaline, another weak platelet agonist, LPA could induce an irreversible and complete aggregatory response. There was an individual variation in aggregatory response and tyrosine phosphorylation when LPA and adrenaline were combined. These agents induced a powerful response on platelets from some individuals, but had a weak or no effect on others.

    INTERPRETATION AND CONCLUSIONS: The present study shows, for the first time, that isolated platelets from some healthy blood donors respond synergistically to a combination of LPA and adrenaline. Platelet activation is a key step in distinguishing normal hemostasis from pathologic hemostasis. Increased knowledge about this mechanism might help to predict individual responses and provide new insights into molecular mechanisms responsible for pathologic thrombosis.

  • 38.
    Nylander, Martina
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Fälker, Knut
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Ramström, Sofia
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindahl, Tomas
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Chemistry.
    Release of ADP or PAR4 Activation is Required to Sustain Thrombin-induced Platelet AggregationManuscript (preprint) (Other academic)
    Abstract [en]

    Thrombin activates human platelets through cleavage of two G-protein-coupled proteaseactivatedreceptors (PARs) denoted PAR1 and PAR4. The aim of this study was to investigatedifferences in PAR1 and PAR4 signaling regarding formation and stability of plateletaggregates. We show that weak PAR1-mediated aggregation is reversible, whereas PAR4-mediated aggregation, weak or strong, is always sustained. PAR1-induced plateletaggregation is decreased and more reversible in the presence of the P2Y12 antagonistcangrelor. However, the effects by cangrelor can be concentration-dependently reversed byconcomitant PAR4 activation in a PI3-kinase-dependent manner. In contrast; in PAR4-APstimulated platelets, aggregation is reduced by cangrelor or inhibition of PI3-kinase byLY294002 but remains irreversible. However, a combined inhibition of PI3-K and P2Y12results in reduced and reversible aggregation. In the light of recently published data on PAR1desensitization, we suggest that the physiological role of the differences between PAR1 andPAR4 activation on aggregate and clot stability could be to fine-tune the response tothrombin. A repeated or continuous very low thrombin generation will desensitize PAR1 andeven if small platelet aggregates are formed they will dissolve, preventing inappropriatethrombus formation. At higher concentrations of thrombin, PAR4 will become activatedabrogating desensitization of PAR1 and enforcing stability of platelet aggregates.

  • 39.
    Nylander, Martina
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Lindahl, Tomas L.
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Chemistry. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Chemistry.
    Bengtsson, Torbjörn
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    The periodontal pathogen Porphyromonas gingivalis sensitises human blood platelets to epinephrine2008In: Platelets, ISSN 1369-1635, Vol. 19, no 5, p. 352-358Article in journal (Refereed)
    Abstract [en]

    Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct connection between periodontitis and stress, and describe a novel mechanism that may contribute to pathological platelet activation.

  • 40.
    Peng, Xiang
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Jinan University, Guangdong, China.
    Ramström, Sofia
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Kurz, Tino
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. University of Örebro, Sweden.
    Segelmark, Mårten
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Nephrology.
    The neutrophil serine protease PR3 induces shape change of platelets via the Rho/Rho kinase and Ca2+ signaling pathways2014In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 134, no 2, p. 418-425Article in journal (Refereed)
    Abstract [en]

    Introduction: Proteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated. Methods: The experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry. Results: PR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca2+ mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca2+ chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it. Conclusion: The data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca2+ signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.

  • 41.
    Svensson, Ann-Charlotte B.
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva G.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Platelet-induced 5-LOX activity is associated with ROS-dependent ASMC proliferation2007Conference paper (Refereed)
  • 42.
    Svensson, Ann-Charlotte B.
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva G.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Platelet fragments, like platelets, induce airway smooth muscle cell proliferation through mechanisms dependent on ros and 5-lox2007Conference paper (Refereed)
  • 43.
    Svensson, Ann-Charlotte
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Bengtsson, Torbjörn
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Grenegård, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Lindström, Eva
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Platelets induce airway smooth muscle cell proliferation2005In: Young Investigators Symposium,2005, 2005Conference paper (Other academic)
  • 44.
    Svensson, Ann-Charlotte
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Bengtsson, Torbjörn
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Grenegård, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Lindström, Eva
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Pharmacology.
    Platelets induce proliferation of airway smooth muscle cells through mechanisms dependent on ROS and 5-LOX metabolites2005In: European respiratory Society Annual Congress,2005, 2005Conference paper (Other academic)
  • 45.
    Svensson Holm, Ann-Charlotte B.
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Department of Biomedicine, School of Health and Medical Sciences, Örebro University, Sweden.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Lindström, Eva G.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species2008In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 19, no 7, p. 528-536Article in journal (Refereed)
    Abstract [en]

    Continuous recruitment and inappropriate activity of platelets in the airways may contribute to airway remodeling, a characteristic feature of inflammatory airway diseases that includes increased proliferation of the smooth muscle.

    The aim of the present investigation was to examine the effect of platelets on proliferation of airway smooth muscle cells (ASMC) in culture and to determine the possible role of 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) in this context.

    ASMC obtained from guinea pigs were cultured and co-incubated with washed platelets for 24 hours. Thereafter, the proliferation was measured with the MTS-assay, the results were also verified by using thymidine incorporation, DNA measurements and manual counting. The interaction between platelets and ASMC was visualised with fluorescence microscopy.

    We found that platelets bind to the ASMC and the presence of platelets caused a significant dose-dependent increase in ASMC proliferation. Co-incubation of ASMC with platelets also increased ROS-production, detected by the fluorescent probe DCFDA. Furthermore, the platelet-induced proliferation was reduced in the presence of the NADPH-oxidase inhibitors DPI and apocynin.

    A possible role of 5-LOX in platelet-induced proliferation and ROS-generation was evaluated by using the 5-LOX inhibitor AA-861 and the PLA2-inhibitor ATK. The results showed that inhibition of these enzymes significantly reduced the platelet-induced proliferation. Moreover, Western blot analysis revealed that the ASMC but not the platelets express 5-LOX.

    In addition, our experiments revealed that the presence of AA-861 and ATK significantly inhibited the ROS-production generated upon coincubation of platelets and ASMC.

    In conclusion, we show that platelets have a marked capacity to induce ASMC proliferation. Furthermore, our study indicates that the interaction between platelets and ASMC leads to activation of 5-LOX in the ASMC followed by an increased ROS-production, events resulting in enhanced ASMC proliferation. The new findings are of importance in understanding possible mechanisms contributing to airway remodeling.

  • 46.
    Svensson Holm, Ann-Charlotte B.
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Department of Biomedicine, School of Health and Medical Sciences, Örebro University, Sweden.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva G.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Platelet membranes induce airway smooth muscle cellproliferation2011In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 22, no 1, p. 45-55Article in journal (Refereed)
    Abstract [en]

    The role of platelets in airway disease is poorly understood although they have been suggested to influence on proliferation of airway smooth muscle cells (ASMC). Platelets have been found localised in the airways in autopsy material from asthmatic patients and have been implicated in airway remodeling. The aim of the present study was to investigate the effects of various platelet fractions on proliferation of ASMC obtained from guinea pigs (GP-ASMC) and humans (H-ASMC). Proliferation of ASMC was measured by the MTS-assay and the results were confirmed by measurements of the DNA content. A key observation was that the platelet membrane preparations induced a significant increase in the proliferation of both GPASMC (129.9 ± 3.0 %) and H-ASMC (144.8 ± 12.2). However, neither supernatants obtained from lysed nor filtrate from thrombin stimulated platelets did induce ASMC proliferation to the same extent as the membrane preparation. We have previously shown the platelet-induced proliferation is dependent on the 5-lipoxygenase (5-LOX) and reactive oxygen species (ROS) pathways. In the present work we established that platelet membrane-induced ASMC proliferation was reduced in the presence of the NADPH oxidase inhibitor DPI and the 5-LOX inhibitor AA-861. In conclusion, our results showed that platelet  membranes significantly induced ASMC proliferation, demonstrating that the mitogenic effect of platelets and platelet membranes on ASMC is mainly due to membrane-associated factors. The effects of platelet membranes were evident on both GP-ASMC and H-ASMC and involved 5-LOX and ROS. These new findings are of importance in understanding the mechanisms contributing to airway remodeling and may contribute to the development of new pharmacological tools in the treatment of inflammatory airway diseases.

  • 47.
    Svensson Holm, Ann-Charlotte B.
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Department of Biomedicine, School of Health and Medical Sciences, Örebro University, Sweden.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Lindström, Eva G.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Platelets bind to hyaluronic acid through CD44 and induce a focal adhesion kinase dependent airway smooth muscle cell proliferation2008Manuscript (preprint) (Other academic)
    Abstract [en]

    Platelets have been implicated as important players in the remodeling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of the extracellular matrix component hyaluronic acid (HA), the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. The ability of ASMC to synthesize HA was investigated by fluorescent staining using biotinylated HA-binding protein and streptavidin conjugate. In addition, the interaction between ASMC and platelets was studied by fluorescent staining of the F-actin. We found that ASMC produced HA and that a CD44 blocking antibody and the hyaluronic acid synthase inhibitor 4-Methylumbelliferone (4-MU) inhibited platelet binding to the area surrounding the ASMC. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and 4-MU inhibited platelet-induced ASMC proliferation. We also found that co-culture of ASMC and platelets resulted in increased phosphorylation of FAK as detected by Western blot analysis. Furthermore, the FAKinhibitor PF 573228 inhibited platelet-induced ASMC proliferation. In conclusion, our findings demonstrate that HA, CD44 and FAK contribute to the increased ASMC proliferation caused by platelets. This event is initiated by an interaction between platelets CD44 and HA produced by the ASMC. These new findings may be important in understanding the interplay between ECM, platelets and ASMC in the remodeling process. In conclusion, our results demonstrate that FAK is phosphorylated and on that account activated during the CD44-dependent platelet/ASMC interaction and this contributes to proliferation of the ASMC. These new findings may be important in understanding the interplay between ECM, platelets and ASMC in the remodeling process.

  • 48.
    Svensson Holm, Ann-Charlotte
    et al.
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    The role of reactive oxygen species and 5-lipoxygenase in platelet-induced airway smooth muscle cell proliferation2006Conference paper (Refereed)
  • 49.
    Svensson Holm, Ann-Charlotte
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Bengtsson, Torbjörn
    Örebro University.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindström, Eva G
    Linköping University, Department of Medical and Health Sciences, Pharmacology. Linköping University, Faculty of Health Sciences.
    Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation2012In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 318, no 5, p. 632-640Article in journal (Refereed)
    Abstract [en]

    Hyaluronic acid (HA) is one of the main components of the extracellular matrix (ECM) and is expressed throughout the body including the lung and mostly in areas surrounding proliferating and migrating cells. Furthermore, platelets have been implicated as important players in the airway remodelling process, e.g. due to their ability to induce airway smooth muscle cell (ASMC) proliferation. The aim of the present study was to investigate the role of HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in platelet-induced ASMC proliferation. Proliferation of ASMC was measured using the MTS-assay, and we found that the CD44 blocking antibody and the HA synthase inhibitor 4-Methylumbelliferone (4-MU) significantly inhibited platelet-induced ASMC proliferation. The interaction between ASMC and platelets was studied by fluorescent staining of F-actin. In addition, the ability of ASMC to synthesise HA was investigated by fluorescent staining using biotinylated HA-binding protein and a streptavidin conjugate. We observed that ASMC produced HA and that a CD44 blocking antibody and 4-MU significantly inhibited platelet binding to the area surrounding the ASMC. Furthermore, the FAK-inhibitor PF 573228 inhibited platelet-induced ASMC proliferation. Co-culture of ASMC and platelets also resulted in increased phosphorylation of FAK as detected by Western blot analysis. In addition, 4-MU significantly inhibited the increased FAK-phosphorylation. In conclusion, our findings demonstrate that ECM has the ability to influence platelet-induced ASMC proliferation. Specifically, we propose that HA produced by ASMC is recognised by platelet CD44. The platelet/HA interaction is followed by FAK activation and increased proliferation of co-cultured ASMC. We also suggest that the mitogenic effect of platelets represents a potential important and novel mechanism that may contribute to airway remodelling.

  • 50.
    Svensson Holm, Ann-Charlotte
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Örebro University, Sweden.
    Ollinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pathology and Clinical Genetics.
    Lindström, Eva
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Inhibition of 12-lipoxygenase reduces platelet activation and prevents their mitogenic function2014In: Platelets, ISSN 0953-7104, E-ISSN 1369-1635, Vol. 25, no 2, p. 111-117Article in journal (Refereed)
    Abstract [en]

    The aim of the present study was to investigate the role of 12-lipoxygenase (12-LOX) on platelet-induced airway smooth muscle cell (ASMC) proliferation. Co-incubation of platelets and ASMC caused platelet activation as determined by morphological changes. Simultaneously, reactive oxygen species (ROS)-generation was detected and ASMC proliferation (measured by using the MTS assay) increased significantly. Furthermore, we found that the 12-LOX inhibitors cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC) and Baicalein prevented platelet activation in a co-cultures of platelets and ASMC. The inhibitory effect of CDC and Baicalein on platelets was also registered in a pure platelet preparation. Specifically, the 12-LOX inhibitors reduced collagen-induced platelet aggregation both in the presence and absence of external added fibrinogen. Importantly, platelet-induced ASMC proliferation and ROS production generated during the platelet/ASMC interaction was significantly inhibited in the presence of 12-LOX inhibitors. In conclusion, our findings reveal that 12-LOX is crucial for the observed enhancement of ASMC proliferation in co-cultures of platelets and ASMC. The present result suggests that 12-LOX activity is important in the initial step of platelet/ASMC interaction and platelet activation. Such action of 12-LOX represents a potential important mechanism that may contribute to platelet-induced airway remodelling.

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