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 ␣ 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 ␣ 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 ␣ 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.

Proliferation of fibroblasts is vital for adequate wound healing but is probably also involved in different hyperproliferative disorders such as atherosclerosis and cancer. The regeneration of tissue usually starts with coagulation, involving release of mitogenic and inflammatory factors from activated platelets. This study focuses on the role of eicosanoids in the proliferative effects of platelets on human fibroblasts. We show that the phospholipase A (2) inhibitor 7,7-dimethyl-5,8-eicosadienoic acid (DMDA), the combined cyclooxygenase (COX) and lipoxygenase (LOX) inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) and the LOX inhibitor 5,8,11-eicosatriynoic acid (ETI) block the platelet-induced proliferation of serum starved subconfluent human fibroblasts. Anti-proliferative effects were also obtained by specific inhibition of 5-LOX with 5,6-dehydro arachidonic acid (5,6-dAA), whereas the 12-LOX inhibitor cinnamyl-3,4-dihydroxy- a -cyanocinnamate (CDC) did not affect the platelet-stimulated growth of fibroblasts. The expression of 5-LOX was analyzed by reverse-transcriptase-mediated PCR (RT-PCR), Western blotting and HPLC. 5-LOX message and protein was detected in fibroblasts but not in platelets. Incubation with platelets markedly increased, already after one hour, the expression of 5-LOX in the fibroblast culture. The increased 5-LOX activity was associated with an elevated level of the 5-LOX metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) reaching its maximum after 1 - 2 hours of co-incubation of fibroblasts and platelets. The 5-HETE production was reduced by the inhibitors DMDA, ETYA and ETI. In conclusion, this study suggests that platelet-stimulated proliferation of fibroblasts is mediated by an increased 5-LOX activity, which supports recent findings indicating a crucial role for this enzyme in proliferative disorders such as atherosclerosis.

1 Our aim was to determine if antigen challenge stimulates sensory nerves and provokes the release of tachykinins. The involvement of histamine and bradykinin was studied by using speci®c receptor antagonists. Capsaicin-induced responses were also examined. Experiments were performed in vitro on tracheal and bronchial preparations from ovalbumin-sensitized guinea-pigs. 2 Characterization of ovalbumin-induced contraction, with regard to histamine and bradykinin, was carried out on airway ring preparations in the presence of phosphoramidon. The histamine H 1 receptor antagonist pyrilamine reduced allergen-induced bronchial contractions by about 30%, whereas the bradykinin B 2 receptor antagonist icatibant (Hoe 140) did not signi®cantly aect the response. Combined treatment with pyrilamine (1 mM) and icatibant (0.1 mM) reduced the contractions by about 80%, indicating a synergistic inhibitory action. Tracheal preparations were not signi®cantly aected by treatments, neither were capsaicin-induced contractions. 3 To study the out¯ow of tachykinins, we used a perfused bronchial-tube preparation, allowing simultaneous measurement of smooth muscle tension and mediator release. Neurokinin A-like immunoreactivity (NKA-LI) and substance P-like immunoreactivity (SP-LI) were determined by radioimmunoassay. 4 The results of the perfusion study showed an increased out¯ow of NKA-LI into the perfusate in response to ovalbumin (127% of basal) challenge. SP-LI determined in some of the samples showed a much lower amount (40 to 70 times lower) of SP-LI than NKA-LI. Treatment with icatibant and pyrilamine, separately and in combination, signi®cantly reduced the ovalbumin-induced NKA-LI out¯ow by 38%, 26% and 22%, respectively. 5 Capsaicin-induced out¯ow (124% of basal) was not signi®cantly aected by treatments (icatibant 121%, pyrilamine 107% and combined treatment 111% of basal). However, when pyrilamine was present the increased out¯ow was not statistically signi®cant. 6 In conclusion, we found that allergen provocation of guinea-pig bronchi caused an increased out¯ow of NKA-LI that was reduced by treatment with both pyrilamine and icatibant. These ®ndings demonstrate that the allergen-induced release of histamine and bradykinin stimulate sensory nerves and thereby increase out¯ow of tachykinins that contribute to the allergic reaction.

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 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 HA, the HA-binding surface receptor CD44 and focal adhesion kinase (FAK) in plateletinduced 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.

Excitatory nonadrenergic, noncholinergic (e-NANC) bronchoconstrictor responses have been shown to be mediated by the release of tachykinins from sensory nerves. We investigated whether bronchial-tube contractions evoked by electrical-field stimulation (EFS) or capsaicin coincided with the release of neurokinin A (NKA). We also studied the modulatory action of morphine and the ability of naloxone to affect these responses. We used a guinea pig bronchial-tube preparation denuded of epithelium. The method allows simultaneous measurement of smooth-muscle tension and mediator release. A significant release of NKA-LI, at 37.3% above the basal level, was detected in response to EFS. Morphine pretreatment was found to inhibit the release, and such inhibition was not prevented by naloxone. Contractile responses to EFS coincided with the NKA-LI release. Capsaicin stimulation evoked a significant release (35.4%) of NKA-LI, and this release was accompanied by contractions. Treatment with morphine decreased capsaicin-induced responses, and naloxone reversed the inhibitory effect. In conclusion, both capsaicin- and EFS-induced e-NANC responses were inhibited by morphine treatment. This indicates presynaptic inhibition of tachykinin release from sensory nerves. Furthermore, the ability of naloxone to reverse this inhibitory effect differed in capsaicin- and EFS-challenged preparations.