Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances.

Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-ß-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y12 antagonist cangrelor, while inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I2 (PGI2) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI2 or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y12 receptors is important for efficient platelet lysosomal exocytosis by thrombin.

Thyroid hormone does not induce maturation of embryonic chicken cardiomyocytes in vitro.

Fetal cardiac growth in mammalian models occurs primarily by cell proliferation (hyperplasia). However, most cardiomyocytes lose the ability to proliferate close to term and heart growth continues by increasing cell size (hypertrophy). In mammals, the thyroid hormone triiodothyronine (T3) is an important driver of this process. Chicken cardiomyocytes, however, keep their proliferating ability long after hatching but little information is available on the mechanisms controlling cell growth and myocyte maturation in the chicken heart. Our aim was to study the role of T3 on proliferation and differentiation of embryonic chicken cardiomyocytes (ECCM), enzymatically isolated from 19-day-old embryos and to compare the effects to those of insulin-like growth factor-1 (IGF-1) and phenylephrine (PE). Hyperplasia was measured using a proliferation assay (MTS) and hypertrophy/multinucleation was analyzed morphologically by phalloidin staining of F-actin and nuclear staining with DAPI. We show that IGF-1 induces a significant increase in ECCM proliferation (30%) which is absent with T3 and PE. PE induced both hypertrophy (61%) and multinucleation (41%) but IGF-1 or T3 did not. In conclusion, we show that T3 does not induce maturation or proliferation of cardiomyocytes, while IGF-1 induces cardiomyocyte proliferation and PE induces maturation of cardiomyocytes.

Inhibition of 12-lipoxygenase reduces platelet activation and prevents their mitogenic function.

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.

Hyaluronic acid influence on platelet-induced airway smooth muscle cell proliferation.

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.

Platelet membranes induce airway smooth muscle cell proliferation.

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 localized 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 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 GP-ASMC (129.9 ± 3.0 %) and H-ASMC (144.8 ± 12.2). However, neither supernatants from lysed or filtrated thrombin stimulated platelets induced ASMC proliferation to the same extent as the membrane preparation. We have previously shown that platelet-induced proliferation is dependent on 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.

Platelets stimulate airway smooth muscle cell proliferation through mechanisms involving 5-lipoxygenase and reactive oxygen species.

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 visualized 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 PLA(2)-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 co-incubation 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.